WO2017010945A1

WO2017010945A1 - Microencapsulation of compounds into natural spores and pollen grains

Info

Publication number: WO2017010945A1
Application number: PCT/SG2016/050333
Authority: WO
Inventors: Nam-Joon Cho; Michael Graeme POTROZ; Raghavendra Chaluvayya MUNDARGI; Jae Hyeon Park; Joshua Alexander JACKMAN
Original assignee: Nanyang Technological University
Priority date: 2015-07-16
Filing date: 2016-07-15
Publication date: 2017-01-19
Also published as: TW201717889A

Abstract

In one aspect, provided herein are whole spores engineered to capsulate a compound(s) or substance(s). In certain embodiments, the whole spore encapsulating the compound(s) or substance(s) is coated with or co-encapsulated with a hydrogel or other agent(s) to control the rate release of the compound(s) or substance(s) from the spore. In another aspect, provided herein are methods of producing whole spores encapsulating a compound(s) or substance(s). In another aspect, provided herein are formulations comprising either a whole spore, or a whole spore encapsulating a compound(s) or substance(s), and uses of those formulations.

Description

MICROENCAPSULATION OF COMPOUNDS INTO NATURAL SPORES AND

POLLEN GRAINS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/193,307, filed July 16, 2015, and U.S. Provisional Application No. 62/263,192 filed December 4, 2015, which are incorporated herein by reference in their entirety.

1. FIELD

[0002] In one aspect, provided herein are whole spores engineered to capsulate a compound(s) or substance(s). The compound or substance may be, e.g., a therapeutic agent, herb, nutraceutical, food substance, food supplement, herbicide, pesticide, cosmetic (e.g., a fragrance), disinfectant, cleaning agent, diagnostic agent, ink, antimicrobial substance, fuel. In certain embodiments, the whole spore encapsulating the compound(s) or substance(s) is coated with or co-encapsulated with a hydrogel or other agent(s) to control the rate of release of the compound(s) or substance(s) from the spore. In another aspect, provided herein are methods of producing whole spores encapsulating a compound(s) or substance(s). In certain embodiments, the method further comprises coating the whole spore, or encapsulating the compound(s) or substance(s) with a hydrogel or other agent(s) to control the rate of release of the compound(s) or substance(s) from the spore. In another aspect, provided herein are formulations comprising either a whole spore, or a whole spore encapsulating a compound(s) or substance(s), and uses of those formulations.

2. BACKGROUND

[0003] Plant based spores, algae, and pollen grains represent a form of natural

encapsulation, and a wide range of specific plant species which produce these spores and pollen grains are commonly found in the natural world. Such natural packaging means are effective in protecting sensitive biological materials from environmental extremes in the form of prolonged desiccation, UV exposure, and predatory organisms. A range of plants produce spores as a form of seed, which contains all the genetic material necessary to produce a new plant. Such spores provide a ready-made capsule scaffold with high structural uniformity and a large internal cavity which may be used to encapsulate a wide range of materials. Human consumption of natural spores and pollen grains as biosupplements, homoeopathy medicine pave a way to explore these materials for encapsulation applications specific to therapeutic loading and release. For example, lycopodium clavatum is one species of the genus Lycopodium which produces spores and which has been identified to contain a range of promising phytochemicals for therapeutic applications ranging from stomach ailments to Alzheimer's disease. Lycopodium spores provide a robust capsule structure and are commercially available in large quantities across globe and these spores are often used in traditional herbal medicine with a wide range of therapeutic benefits including improved osteogenesis, cognitive function, treatment of gastrointestinal disorders,

hepatoprotective activity, and antioxidative properties. Recent studies demonstrated the use of processed lycopodium shells for encapsulation, however, the production of exine empty capsules (sporopollenin) requires the prolonged processing of natural spores with extreme chemical treatments at elevated temperatures, such that these resulting capsules are devoid of all other biological materials. In many applications, this extensive processing may be unnecessary and potential therapeutic benefits may be lost.

[0004] A major challenge in producing microencapsulated products is ensuring size monodispersity, which can have a large effect on drug release characteristics with respect to an intended target organ. In addition to size monodispersity, having well-defined microstructures plays an important role in exploring widespread applications. Most conventional materials processing techniques used for encapsulation such as emulsion solvent evaporation, spray drying, and chemical conjugation fail to reliably provide either size monodispersity or well- defined microstructures. Although prior arts reported the use of processed empty exine microcapsules of spores and pollen for the encapsulation of drugs, vaccines, and MRI contrast agents. Producing these empty capsules is very tedious involving harsh chemical processing for prolonged days highly affecting the industrial costing inclusive of manpower, process and time duration. Thus, there is a need for new methods for microencapsulating various compounds and substances.

2.1 CAMELLIA OIL

[0005] Camellia oil, also known as tea seed oil is the actual green tea oil. Tea seed oil is a wonderfully healthy in more ways than one. It is great for cooking, and from nutritional point of view. Tea seed oil is used in a number of beauty products. This oil has been used as a cooking for centuries in Southern China and they make many more uses with it. The oil helps to prevent and smooth wrinkles and stretch marks. It is also used to strengthen and promote healthy growth of fingernails by massaging the oil into the nail. This product is also suitable for the formulation of cosmetic products designed to condition hair, and to treat and prevent hair damages. [0006] Camellia oil is extracted from the seeds of the tea plant. That makes it the real tea oil. Tea tree oil on the other hand does not come from the tea plant. It comes from the tree called Melaleuca alternifolia, which is native to Australia. There are some varieties of Camellia oil.

[0007] · Camellia japonica oil - Also known as Japanese tea oil. However, this plant does not produce tea leaves. It is a flowering plant with red blooms. Its oil is known as tsubaki oil and it is used heavily in cosmetic applications.

[0008] · Camellia sinensis oil - This is the tea seed oil.

[0009] · Camellia oleifera oil - This is known as tea oil or Camellia oil.

[0010] The oil is extracted using solvent extraction or cold processing. One might hear about cold filtered oil, but that does not mean cold pressed oil. If the contents used to make oil are heated prior to oil extraction, it may change the chemical composition and properties of nutrients in that oil, which is often not natural. However, tea tree oil, the increasingly popular remedy for everything from spots to insect bites and vapour rubs, is under threat of being banned by the European Union. The EU has said that even small amounts of the undiluted oil could be unsafe and unstable after clinical trials found users risked rashes and allergies.

[0011] Cosmetic products, such as shampoo and bath oils, that use the oil in concentrations of less than 1 per cent are safe. But the toiletries and cosmetics firms that produce the neat form of the natural remedy have been given until June to convince a panel of scientists that the oil is safe to sell to the public. "Because essential oils are natural products, the public often assumes they must be safe," says Frances Fewell, director of the Institute for Complementary Medicine. "You should never apply any sort of essential oil directly to the skin without diluting it first in a suitable carrier oil. Tea tree oil has become very popular, and many people have started applying it directly to deal with acne and skin infections. In fact this is a very aggressive oil. The skin can dry out, blister or form a rash." In a strongly worded report, the EU's Scientific Committee on Consumer Products has said it has serious concerns about the neat oil which, it found, is 'a severe irritant' to the skin and 'degraded rapidly' if exposed to air, light and heat.

[0012] Conventional encapsulation and microencapsulation methods include alignate encapsulation, polyoxymethylene urea microencapsulation, and complex coacervation (gelatin) microencapsulation. In the aforementioned cases, the bead itself is not therapeutic and requires synthesis. In nearly all cases, the use of synthetic microparticles in personal care products is being banned so these solutions are no longer viable and are technically sub-optimal. [0013] There is a need for new solutions that combine the health benefits of Camellia oil with a better delivery option and combined synergistic effect of Camellia oil and other natural substances found in Camellia plant-based species.

2.2 MICROBEADS

[0014] Today, a significant number of personal care products such as scrubs and toothpastes are known to contain thousands of minuscule balls of plastic called microplastics, or more specifically, microbeads. Over the years, microbeads have replaced traditional,

biodegradable alternatives such as ground nut shells, and salt crystals. The microbeads used in personal care products are mainly made of polyethylene (PE), but can be also be made of polypropylene (PP), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA) and nylon. Where products are washed down the drain after use, microbeads flow through sewer systems around the world before making their way into rivers and canals and ultimately, straight into the seas and oceans, where they contribute to the plastic soup. Typically, microplastics are defined as: plastic pieces or fibres measuring less than 5 mm. The microbeads found in personal care products are almost always smaller than 1 mm

[0015] A major problem that continues to exist in the world is the amount of plastic debris in the world's oceans and other marine environments. Plastic is produced in large quantities each year primarily because its applications in the modern world are infinite. According to the New York Times, approximately 300 million tons of plastic are produced globally each year. Due to its variety of uses and relatively low cost, plastic production is going to continue to increase for the future. It has reached the point where plastic pollution is accounting for an estimated $13 billion dollars of damage. In addition, micro-plastic beads have become a significant issue in the world's marine environments.

[0016] At the same time, microplastics are a large industry that often goes unnoticed. They are used in many everyday items such as switches, sensors, and in lighting. Additionally, they are used as exfoliants in everyday cosmetic products such as face wash, moisturizer, lipstick, and toothpaste. The term "microplastics" specifically refers to small pieces of plastic material that are found in the marine environment. In general, microplastics range in size from a few μπι to 500 μπι (or 0.50 mm), which is almost microscopic. Microplastics can originate from a variety of sources, including the production of plastic microbeads often found in cosmetics. [0017] Cosmetic products containing microbeads are popular all around the world because consumers enjoy the clean feeling that they provide. In fact, it has been reported that over half of women use four or more beauty products a day, and women globally spend $426 billion a year, a large portion of which consists of products containing microbeads. Microbeads act as good exfoliants because they can be shaped into small spheres which are effective at removing excess oil and dirt on the surface of skin without being harsh or stripping the skin of its essential oils. It is important to note that a typical facial scrub contains approximately 350,000 microbeads. However, these microbeads, made of synthetic polymers like polyethylene and/or polypropylene plastic, are having an adverse effect on the environment.

[0018] Many states have introduced and even passed legislation banning the sale of cosmetics containing microbeads in the state. In 2014, Illinois banned the sale of soaps and shower gels containing plastic microbeads, and California and New York have both introduced similar bills. An article describing the new bill passed in Illinois reports, "The new law requires synthetic microbeads to be removed from manufacturing by the end of 2018 and bans the sale of items containing the beads by the close of 2019". This bill is the first big step into eliminating synthetic microbeads entirely and has also given the issue national recognition. Cosmetics companies are backing the bans and vowing to support the transition to natural exfoliants, indicating there is a potentially large market for a suitable replacement. Herein, we disclose the use of natural microbeads in cosmetics, namely plant-based spores and pollen grains from a wide range of specific plant species.

[0019] The majority of personal care products contain traditional plastic microbeads, which are composed of organic polymers of polyethylene and/or polypropylene compounds.

Unfortunately, these plastics are non-degradable, which contributes to the problem of pollution in aquatic systems. However, one solution is to incorporate the use of biodegradable plastics in the production of microbeads. The process to make these plastics into spherically shaped microbeads is very similar to that of synthetic microbeads. In addition to microbeads varying in composition, size, and shape, they also differ in hardness. The hardness depends on the particular application, however they should be sufficiently hard so that they cleanse the skin as desired. At the current rate of plastic pollution, traditional polypropylene and polyethylene microbeads are no longer an appropriate option for cosmetic products. Though they are inexpensive to produce, their effect on the environment cancels out the cost benefits and proves them to be an unsustainable exfoliant. However, biodegradable plastics are currently very expensive to produce, hence the reason it is not a widely used plastic. Hence, there is a need for an affordable and easy-to-obtain natural microbead substitute.

3. SUMMARY

[0020] In one aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest. In a specific embodiment, a whole spore described herein is engineered to encapsulate a compound or substance of interest and coated with an agent to facilitate controlled release of the compound or substance of interest from the whole spore. In a particular embodiment, an whole spore described herein is engineered to encapsulate a compound or substance of interest and an agent to facilitate controlled release of the compound or substance of interest from the whole spore

[0021] In one embodiment, an whole spore is a Abies spore, a Agrocybe spore, a

Aspergillus niger spore, a Bacillus subtilis spore, a Cantharellus minor spore, a Epicoccum spore, a Cuburbita spore, a Cucurbitapapo spore, a Ganomerma spore, a Lycopodium clavatum spore, a Myosotis spore, a Penicillium spore, a Periconia spore, a ryegrass spore, a Timothy grass spore, a maize spore, a hemp spore, a rape hemp spore, a wheat spore, a Urtica dioica spore, a sunflower spore, a corn spore, a pine spore, a cattail spore, a rape spore, a dandelion spore, a rye spore, a Baccharis spore, a Chorella, a Camellia spore, a ragweed spore, a mulberry spore, or a pecan spore.

[0022] In another embodiment, an whole spore has a size in the range of 0.5 μπι to 300 μπι. In another embodiment, an whole spore has a size in the range of 40 μπι to 100 μπι. In another embodiment, an whole spore has a size in the range of 1 μπι to 40 μπι. In another embodiment, an whole spore has a size in the range of 1 μπι to 80 μπι.

[0023] In one embodiment, the compound or substance of interest is a therapeutic agent. In some embodiment, the therapeutic agent is a small organic molecule, a peptide, a nucleic acid, a protein, a polymer, a biologies, a medicinal preparation of proteins, a herbal medicine, an inorganic compound, an organometallic compound, lithium, a platinum-based agent, or gallium. In another embodiment, the compound or substance of interest is an oil. In another embodiment, the compound or substance of interest is a fragrance. In another embodiment, the compound or substance of interest is a cleaning agent. In another embodiment, the compound or substance of interest is a disinfectant agent. In another embodiment, the compound or substance of interest is a pesticide. In another embodiment, the compound or substance of interest is a herb. In another embodiment, the compound or substance of interest is a food ingredient. In one embodiment, the food ingredient is a caffeine. In another embodiment, the compound or substance of interest is a herbicide. In another embodiment, the compound or substance of interest is a fuel.

[0024] In another aspect, provided herein is a formulation comprising the whole spore and a diluent or carrier.

[0025] In another aspect, provided herein is a formulation comprising the whole spore and a diluents or pharmaceutically acceptable carrier. In one embodiment, the formulation is for topical administration. In another embodiment, the formulation is for parenteral administration.

[0026] In another aspect, provided herein is a method for of treating a disease or condition in a subject, comprising the formulation, wherein the therapeutic agent encapsulated in the whole spore is beneficial for treating the disease or condition.

[0027] In another aspect, provided herein is a cosmetic product or personal care product comprising the whole spore. In another aspect, provided herein is a food or drink product comprising the whole spore. In yet another aspect, provided herein is a herbal product comprising the whole spore. In another aspect, provided herein is a pesticide. In another aspect, provided herein is a herbicide.

[0028] In another aspect, provided herein is a method for masking the taste of a compound or substance, comprising encapsulating the compound or substance in a whole spore and formulating that in a drink or food product. In one embodiment, the encapsulation comprises contacting the compound or substance with the whole spore. In another embodiment, the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure. In some embodiments, the method further comprises coating the whole spore with agent for controlling the release of the compound or substance from the spore.

[0029] In another aspect, provided herein is a method of improving the stability of a compound or substance, comprising encapsulating the compound or substance in a naturally occurring whole spore. In some embodiments, the encapsulation comprises contacting the compound or substance with the whole spore. In other embodiments, the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure. In some embodiments, the method further comprises coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore. [0030] In another aspect, provided herein is a method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore.

[0031] In another aspect, provided herein is a method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore engineered to encapsulate a compound or substance that is beneficial or useful in a cosmetic or personal care product.

[0032] In another aspect, provided herein is a method of reducing the toxicity of a compound or substance, comprising encapsulating the compound or substance in a naturally occurring whole spore. In one embodiment, the the encapsulation comprises contacting the compound or substance with the whole spore. In another embodiment, the encapsulation comprises contacting the compound or substance with the whole spore. In yet another embodiment, the method further comprises coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore.

[0033] In another aspect, provided herein is a method of preparing a formulation comprising a compound or substance of interest and the whole spore, comprising encapsulating the compound or substance of interest in the whole spore.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGS. 1A-1F. Schematic of natural lycopodium spores and processing techniques to encapsulate biomacromolecules. FIG. 1A: Spore microstructure depicting uniform ridges distributed on the surface with natural sporoplasm constituents contained inside, these spores originate from a vascular plant with spirally arranged leaves. FIG. IB: Natural spores suspended in a biomacromolecule solution for the uptake of macromolecules, the enlarged insert depicts macromolecule entry via nanochannels located within the lycopodium microstructure. FIG. 1C: Spores encapsulating biomacromolecules are indicated along with the natural sporoplasm constituents. The three different microencapsulation techniques used are represented. FIG. ID: Passive macromolecule loading technique involving the incubation of natural spores in the aqueous macromolecule solution at 4°C under stirring at 500 rpm. FIG IE: Compression technique involving the compression of a dry spore powder and incubating the resulting spore tablet in the macromolecule solution for the uptake of macromolecules by the spores. FIG IF. Vacuum loading technique involving the application of a vacuum to a suspension containing natural spores and macromolecules, whereby the biomacromolecules enter the spores through the nanochannels located within the surface microstructures of natural spores.

[0035] FIG. 2A-2D. FlowCam measurements: Polymer microspheres standard (50 ± 1 μπι) (Thermoscientific, USA). FIG. 2A: Representative histogram of equivalent spherical diameter vs. frequency using 1000 highly focused image analysis after measurement with particle count of 5000 with ESD of 49.65 ± 0.91 μηι. FIG. 2B: Representative graph from histogram of circularity vs. frequency indicating microspheres very near to ideal circle value (1). FIG. 2C: Histogram of edge gradient vs. frequency indicating highly focused microspheres. FIG. 2D: Representative image of microspheres at 20 X magnification with FC200 flow cell at flow rate of 0.1 ml/min.

[0036] FIGS. 3A-3H. Characterization of natural lycopodium spores before and after biomacromolecule loading by FlowCam^®: Size and circularity by dynamic imaging particle analysis (DIPA, FlowCam^®) with a particle count of 10,000 spores before and after BSA- loading. Representative graphs from curve fitting to histograms for equivalent spherical diameter, circularity and edge gradient for spores before and after encapsulation of

biomacromolecules are presented as Natural spores (FIG 3A), Passive loading (FIG 3B), Compression loading (FIG 3C) and Vacuum loading techniques (FIG. 3D). FIGS. 3E, 3F, 3G, and 3H represent natural spores before loading, as well as, passive, compression, and vacuum loading techniques captured by FlowCam^® at 20x magnification, respectively.

[0037] FIG. 4 Characterization of natural lycopodium spores before and after

biomacromolecule loading by scanning electron microscopy (SEM): SEM Images FIG. 4 A, FIG. 4 B, FIG. 4C, and FIG. 4D, respectively, represent natural spores before loading, as well as, passive, compression, and vacuum loading techniques captured by FESEM (JEOL, Japan).

[0038] FIGS. 5A-5D. Confocal microscopy analysis of natural lycopodium spores before and after biomacromolecule loading: CLSM images in the row of FIG. 5A are natural lycopodium spores before BSA-loading. These natural spores exhibit autofluorescence due to the presence of terpenoid, phenolic, and carotenoid molecules. The spore's natural sporoplasm constituents are observed as microglobules inside the spore in both the blue and red channel along with the overlaid image of the natural spore without biomacromolecule loading, and there is also a clear absence of any green fluorescence. The row of FIG. 5B depicts BSA-loaded spores using the passive loading technique. The row of FIG. 5C depicts BSA-loaded spores using the compression loading technique. The row of FIG. 5D depicts BSA-loaded spores using the vacuum loading technique. All of these spore microparticles exhibit a green colour due to the presence of FITC-BSA in the green channel, and the overlaid images indicate the presence of spore constituents along with encapsulated biomacromolecules. (Scale bars are 10 μπι).

[0039] FIGS. 6A-6B. Z-stack images from confocal laser scanning microscopy (CLSM) showing 35 optical sections of an L. clavatum spore after FITC-BSA loading (FIG. 6A) and before FITC-BSA loading (FIG. 6B).

[0040] FIGS. 7A-7D. In-vitro release profiles of biomacromolecules-loaded natural lycopodium spores: Cumulative release profiles of BSA-loaded spores by passive, compression and vacuum loading in FIG. 7A: Simulated gastric fluid (SGF), pH 1.2 media, and FIG. 4B: Simulated intestinal fluid (SIF), pH 7.4 media. Further optimisation of tunable release was achieved using vacuum loaded spores and cumulative release profiles of vacuum loaded spores after different alginate coatings are presented in FIG. 7C: SGF, pH 1.2 media, and FIG. 4D: SIF, pH 7.4 media. All in-vitro release studies were performed in triplicate (n=3) and average values with standard deviations are presented.

[0041] FIGS. 8A-8C. CLSM images after FITC-BSA release from natural spores prepared by different techniques in pH 7.4 media: the row of FIG. 8A: Passive loading technique; the row of FIG. 8B: Compression loading technique; and the row of FIG. 8C: Vacuum loading technique, (scale bars are 10 μπι).

[0042] FIGS. 9A-9C. Scanning electron microscopic images of L. clavatum spores after coating. Images represent 0.5% alginate coated spores (FIG. 9A), 1% alginate coated spores (FIG. 9B), and 2% alginate coated spores (FIG. 9C), respectively.

[0043] FIGS. 10A-10D. Schematic of natural sunflower pollen grains processing to encapsulate macromolecules by different techniques. FIG. 10A: Dried natural pollen grains exhibiting a characteristic oval shape with uniform spikes on the surface. FIG. 10B: Pollen grains suspended in an aqueous solution of macromolecules for encapsulation by passive, compression and vacuum techniques. FIG. IOC: A fully hydrated natural pollen grain loaded with macromolecules are indicated along with the original pollen contents. FIG. 10D: A fully hydrated natural pollen grain after the release of macromolecules from the natural pores within the pollen grain walls. [0044] FIGS. 1 lA-11C. Characterization of natural sunflower pollen before and after BSA-loading by FlowCam : Size and circularity by dynamic imaging particle analysis (DIPA, FlowCam^®) with a particle count of 10,000 pollen grains before and after BSA-loading.

Representative graphs from curve fitting to histograms of (FIG. 11 A) Equivalent spherical diameter vs. Frequency, (FIG. 11B) Circularity vs. Frequency, and (FIG. 11C) Edge gradient vs. Frequency.

[0045] FIGS. 12A-12D. Characterization of natural sunflower pollen before and after BSA-loading by SEM: Images in FIGS. 12A, 12B, 12C, and 12D represent natural pollen grains before loading as well as, after passive, compression, and vacuum loading techniques captured in FlowCam^® at 20x magnification, respectively.

[0046] FIGS. 13A-13D. Characterization of natural sunflower pollen before and after BSA-loading by SEM: FIG. 13 A represents natural pollen grains before loading and the images in FIGS. 13B, 13C, and 13D, respectively, indicate macromolecule loaded pollen grains by passive, compression, and vacuum loading techniques captured by FESEM (JEOL, Japan).

[0047] FIGS. 14A-14D. Confocal microscopy analysis of natural sunflower pollen grains before and after macromolecule loading: CLSM images in the row of FIG. 14A are natural sunflower pollen grains before BSA-loading. In the row of FIG. 14B, BSA-loaded pollen grains using the passive loading technique. In the row of FIG. 14C, BSA-loaded pollen grains using the compression loading technique. In the row of FIG. 14D, BSA-loaded pollen grains using the vacuum loading technique. (Scale bars are 10 μπι).

[0048] FIGS. 15A-15B. Z-stack images from confocal laser scanning microscopy showing 50 optical sections of a pollen grain after FITC-BSA loading (FIG. 15A), and before FITC-BSA loading (FIG. 15B).

[0049] FIGS. 16A-16C. In-vitro release profiles: Simulated intestinal fluid, pH 7.4 media (FIG. 16A), Simulated gastric fluid, pH 1.2 media (FIG. 16B), Release profile of BSA-loaded pollen grains coated with alginate (FIG. 16 C).

[0050] FIGS. 17A-17D. Scanning electron microscope images of pollen grains after alginate coating. Images in the rows of FIGS. 17A and 17B represent 0.1 % alginate coated pollen grains after 1 min and 10 min incubation times and images in the rows of FIG. 17C and FIG. 17D represent 0.5 % alginate coated pollen grains after 1 min and 10 min incubation times. [0051] FIGS. 18A-18D. Scanning electron microscope images of pollen grains before and after 2 % alginate coating. FIGS. 18A and 18B: Natural pollen grain before coating process. FIG. 18C: Intact pollen grain after alginate coating. FIG. 18D: Represents pollen surface covered with alginate.

[0052] FIGS. 19A-19C. CLSM images after FITC-BSA release from pollen grains prepared by different techniques in pH 7.4 media: Passive technique (row in FIG. 19A),

Compression technique (row in FIG. 19B), and Vacuum technique (row in FIG. 19C). (scale bars are 10 μπι).

[0053] FIGS. 20A-20C. Characterization of 5-FU loaded L. clavatum spore formulations. Diameter, circularity, aspect ratio and edge gradient were analyzed by dynamic imaging particle analysis (DIPA) with a 1000 particle count. Representative graphs with standard deviation from three measurements and curve fitting to histograms are presented as diameter vs. frequency (FIG. 20A), circularity vs. frequency (FIG. 20B), aspect ratio v. frequency (FIG. 20C), and edge gradient vs. frequency (FIG. 20D).

[0054] FIGS. 21A-21D. Dynamic imaging particle analysis images of 5-FU loaded L. clavatum spores. Images in FIGS. 21A, 21B, 21C, and 21D represent L. clavatum spores before and after 5-FU loading by passive, compressive, and vacuum loading techniques, respectively.

[0055] FIGS. 22A-22D. Characterization of 5-FU loaded L. clavatum spores by SEM. SEM images in FIGS. 22A, 22B, 22C and 22D represent L. clavatum spores before loading (FIG. 22A) and after loading by passive (FIG. 22B), compression (FIG. 22C), and vacuum (FIG. 22D) loading techniques.

[0056] FIGS. 23A-23B. Characterization of Eudragit RS 100-coated L. clavatum spores by SEM. FIG. 23A: 5-FU loaded spores after coating with 2.5% Eudragit RS 100. FIG. 23B: 5-FU loaded spores after coating with 10% Eudragit RS 100.

[0057] FIGS. 24A-24C. In vitro release profiles of 5-FU loaded L. clavatum spores.

Cumulative release profiles of 5-FU loaded spores by passive, compression, and vacuum loading (FIG 24 A) in simulated gastric fluid (SGF pH 1.2) and (FIG. 24B) simulated intestinal fluid (SIF), pH 7.4 phosphate buffered saline. FIG. 24C: Controlled release of 5-FU from vacuum- loaded spores after Eudragit RSI 00 coating in SGF and SIF, and inset indicates 5-FU release in SGF in 2 hours. All in vitro release studies were performed in triplicate (n=3) and average values with standard deviation are presented. [0058] FIGS. 25A-25C. Characterization of BSA loading in natural pine pollen based on conventional vacuum loading protocols. FIG. 25A: SEM images depicting surface cleanliness of BSA loaded pine pollen in relation to washing. FIG. 25B: Encapsulation efficiency and loading efficiency in relation to washing. FIG. 25C: CLSM images depicting localization of FITC-BSA.

[0059] FIG. 26. CLSM images depicting short-term passive loading trends for FITC-BSA in natural pine pollen.

[0060] FIG. 27. Long-term passive loading trends for BSA / FITC-BSA in natural pine pollen. CLSM images depicting uptake and localization of FITC-BSA during passive loading.

[0061] FIG. 28. Confocal laser scanning microscopy (CLSM) depicting short-term passive loading trends for FITC-BSA in natural camellia pollen.

[0062] FIGS. 29 A- 29B. Confocal laser scanning microscopy (CLSM) analysis of L. clavatum spores before and after calcein loading. FIG. 29A: CLSM images in the first row indicate spores with sporoplasm. FIG. 29B: The CLSM images in the second row indicate calcein loading into spores. Scale bars are 10 μπι.

[0063] FIG 30. Confocal laser scanning microscopy (CLSM) images of Camellia seed oil and nile read dye blend in Camellia pollen based formulation.

[0064] FIGS. 31A-31B. Size and morphological characterization of Camellia pollen grains and sporopollenin exine capsules (SECs). FIG. 31 A: FlowCam analysis of Camellia pollen and SECs. FIG. 3 IB: FlowCam analysis of Camellia pollen (left) and SEC (right).

[0065] FIGS. 32A-32B. Characterization of caffeine (CF)-loaded L. clavatum spores by SEM. FIG. 32A: Spores before CF-loading. FIG. 32B: Spores after CF-loading with coencapsulant ERS. Scale bars are 10 μηι.

[0066] FIGS. 33A-33B. Confocal laser imaging microscopy (CLSM) analysis of L.

clavatum spores before and after calcein-CF loading. CLSM images in the FIG. 33A depict spores with sporoplasm. FIG. 33B depicts CF-Calcein loading into spores with coencapsulant ERS.

[0067] FIG. 34. In vitro release profiles of caffeine (CF) from L. clavatum spore formulations. Spores-CF physical mixture and CF-loaded with co-encapsulant ERS in simulated salvia fluid (SSF). [0068] FIGS 35A-35B. Taste masking evaluation of caffeine formulations. FIG. 35A: Bitterness score from human volunteers during bitterness threshold test. FIG. 35B: Human volunteer score with CF formulated with a physical mixture L. clavatum spores and ERS.

[0069] FIG. 36. Contact angle data for UV-Ozone treated camellia pollen showing a decrease in contact angle with increasing UV-Ozone treatment duration.

[0070] FIG. 37. Scanning electron microscopy images of Camellia pollen grains treated or untreated with UV/Ozone. Treated means that the pollen has been defatted and treated with UV- Ozone exposure.

[0071] FIG. 38. Aqueous suspensions of untreated and of UV-Ozone treated Camellia pollen.

[0072] FIG. 39. Scanning electron microscopy images of untreated and UV-Ozone treated Camellia SECs. Treated means that the pollen has been defatted and treated with UV-Ozone exposure.

[0073] FIG. 40. Aqueous suspensions of untreated and of UV-Ozone treated Camellia SECs.

[0074] FIG. 41A-41C. Aqueous suspensions. FIG. 41A: Camellia seed oil and water. FIG. 41B: Camellia SECs oil loaded, ethanol washed and UV-Ozone treated. FIG. 41C:

Camellia SECs.

[0075] FIG. 42. Macromolecular encapsulation in Camellia pollen grains and SECs.

[0076] FIG. 43. Process schematic to obtain defatted natural pollen grains.

[0077] FIG. 44. SEM images of whole spore microbead examples.

5. DETAILED DESCRIPTION

[0078] In one aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest. In another aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest and coated with an agent to facilitate controlled release of the compound or substance from the whole spores. In another aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest and an agent with the compound or substance to facilitate controlled release of the compound or substance from the whole spores.

[0079] In a specific embodiment, a whole spore engineered to encapsulate a compound or substance of interest has no significant amount of the compound or substance adhered to the surface of the whole spore. "No significant amount" in this context means that the exterior of the whole spore maintains its natural architectural features and surface appearance on a microscale (e.g., surface roughness) as assessed with a degree of high confidence using standard measuring techniques known in the art, for example, scanning electron microscopy. In an alternative embodiment, a whole spore engineered to encapsulate a compound or substance of interest with a percentage of the compound or substance adhered to the surface of the whole spore. For example, in one embodiment, the whole spore engineered to encapsulate a compound or substance of interest has more than 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the compound or substance adhered to the surface of the whole spore.

[0080] In a specific embodiment, a whole spore engineered to encapsulate a compound or substance of interest maintains the general size, shape and morphology of the whole spore without encapsulation of the compound or substance. In another embodiment, a whole spore engineered to encapsulate a compound or substance of interest maintains the general morphology of the whole spore without encapsulation of the compound or substance. In another

embodiment, a whole spore engineered to encapsulate a compound or substance of interest maintains the general size of the whole spore without encapsulation of the compound or substance. In another embodiment, a whole spore engineered to encapsulate a compound or substance of interest maintains the general shape of the whole spore without encapsulation of the compound or substance. In some embodiments, a whole spore engineered to encapsulate a compound or substance of interest swells in size relative to the whole spore without

encapsulation. For example, in one embodiment, the whole spore engineered to encapsulate a compound or substance of interest swells to more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 125%, 150%, 175% or 200% the size of the whole spore without encapsulation of the compound or substance.

[0081] In some embodiments, a whole spore is engineered to encapsulate a compound or substance of interest to localize the effect of the compound or substance to the site where the whole spore is applied. In certain embodiments, a whole spore is used to encapsulate a compound or substance of interest to protect the compound or substance from harsh

environmental conditions, for example, exposure to UV light, or from degradation to retain the compound or substance's efficacy. In some embodiments, a whole spore is used to encapsulate a compound or substance of interest in order to stabilize the compound or substance. In certain embodiments, a whole spore is used to encapsulate a compound or substance of interest to reduce the toxicity of the compound or substance in a subject. In some embodiments, a whole spore is used to encapsulate a compound or substance of interest to mask the taste of the compound or substance. In certain embodiments, a whole spore is used to encapsulate a compound or substance of interest in order to control the release of the compound or substance. In a specific embodiment, a whole spore is used to co-encapsulate a compound or substance of interest and an agent that allows for a modified release rate of the compound or substance. In another specific embodiment, a whole spore is used to encapsulate a compound or substance of interest and is coated with an agent that allows for a modified release rate of the compound or substance. For example, biologically active substances, such as potent pesticides, herbicides, and fertilizers that are used in agriculture, require methods that allow for their stability and release rates to be modified to minimize their environmental impact.

[0082] For applications in medicine, cosmetics, and food, enhanced effects may be obtained through the encapsulation of synergistic compounds, and overall, the use of whole spores provides significant benefits in terms of processing complexity and costs for a wide range of applications. This approach drastically reduces processing requirements for materials encapsulation in exine -reinforced microcapsules, while also taking advantage of the innate therapeutic benefits of whole spores (e.g., natural pollen).

[0083] In particular, there are a number of benefits to the use of whole spores over exine shells for encapsulation of a compound or substance of interest. For example, the use of whole spores can avoid the additional costs involved in chemical extraction of the exine shells from spore. In addition, chemically extracted exine shells need to undergo regulatory approval prior to use as an additive in products for oral consumption, whereas naturally occurring spores are commonly available in existing products (e.g., heath food products) for oral consumption.

Further, whole spores derived from naturally occurring sources are readily available, at low cost, and in large quantities. While whole spores derived from naturally occurring sources typically sell for about $20 to $30 per kg, extracting exine capsules raises the production costs to about $3500 to $35,000 per kg. Commercially available extracted exine capsules sell typically for about $200,000 per kg. [0084] Other advantages and improvements of whole spores over existing methods, devices or materials, for example, include:

[0085] · Reduced side effects compared to non-encapsulated Camellia oil.

[0086] · Camellia oil can be encapsulated in aqueous formulations.

[0087] · Protects Camellia oil from environmental degradation.

[0088] · Controlled release of oil over time. The oil can diffuse through the natural pollen pores over time in a controlled way. Also, the pollen grain whole spore itself will gradually dissolve in biological fluids, leading to stable and full release.

[0089] · Synergetic health benefits of Camellia pollen whole spore and Camellia oil.

[0090] Plant-based spores and pollen grains from different types of specific plant species are microscale particles that are naturally produced, abundant in renewable supply, highly monodisperse per species, mechanically strong, chemically resilient, biodegradable,

biocompatible, and have varying species-specific surface roughness. Moreover, there is a wide size range for all the pollen species which matches the industrial needs of microbeads. Because the plant-based spores and pollen grains are naturally manufactured, there are minimal production costs and, in light of the attractive set of properties, microscale plant-based spores and pollen grains are very attractive replacements for plastic microbeads in cosmetics. These considerations are important because cost and ease of production are the two biggest issues concerning the search for microbead replacements.

[0091] Additional advantages and improvements of whole spores over existing methods, devices or materials, for example, include:

[0092] · Natural production by plants occurs globally and yields very large quantities of microparticles. Further simple processing can yield protein-free microcapsules with less than 6 wt% protein if desired

[0093] · Each plant species produces highly monodisperse microparticles for tunable size depending on the application need.

[0094] · The range of plant-based spores and pollen grains covers a wide size range that meets the industry demands.

[0095] · The plant-based spores and pollen grains are biodegradable and permit organic recycling. [0096] · Hydrophilic/hydrophobic properties of the surface coating can be controlled in order to support water filtration and prevent clogging.

[0097] · The surface roughness of spores and grains from different species is variable and can be used in different cosmetic applications.

[0098] Personal care products such as lip balm, deodorant, eyeliner, lipstick, lotion, mouthwash, shampoo, conditioner, make up, shaving cream, toothpaste, and numerous others are all are used on the human body for personal hygiene and/or beatification. Many of these products contain synthetic plastics, which have already been proven to negatively affect the environment. Incorporating biodegradable and/or natural microbeads into personal care products will reduce aquatic pollution while serving the same purpose as synthetic plastics. Plant-based spores and pollen grains have very similar properties to plastic microbeads, which give them potential as a natural microbead in cosmetics. Indeed, microbeads must be spherically shaped in order to properly exfoliate and cleanse the skin of the consumer. The non-biodegradable plastics that are currently being used in personal care products range in size anywhere from 10 μπι to 100 μπι (0.01 mm-0.10 mm). All of these features lend plant-based spores and pollen grains great potential as substitutes for the aforementioned commercial applications.

5.1 WHOLE SPORES

[0099] As used herein, the term "whole" in the context of a spore means a spore that comprises an exine shell, an intine layer and cytoplasmic organelles therein. The term "whole" in the context of a spore excludes any spore consisting of or consisting essentially of only an exine shell or a fragment thereof. In other words, a whole spore contains additional components that are missing from an exine shell alone. In a specific embodiment, a whole spore retains more than 50% (preferably, more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the main components of a spore, e.g., extine shell, intine layer, and cytoplasmic organelles. In some embodiments, the whole spore comprises an exine shell, an intine layer, cytoplasmic organelles and other components found in nature associated with a spore (e.g., endexine, nexine, proteins, lipids, nucleic acids, etc.).

[00100] In certain embodiments, a whole spore has a size in the range of 0.5 μπι to 300 μπι. In some embodiments, a whole spore has a size in the range of 1 μπι to 100 μπι. In certain embodiments, a whole spore has a size in the range of 10 μπι to 100 μπι. In some embodiments, a whole spore has a size in the range of 3 μπι to 80 μπι. In certain embodiments, a whole spore has a size in the range of 0.5 μηι to 40 μπι. In some embodiments, a whole spore has a size in the range of 40 μπι to 300 μπι. In certain embodiments, a whole spore has a size in the range of 0.5 μπι to 300 μπι. In some embodiments, a whole spore has a size in the range of 40 μπι to 100 μπι. In certain embodiments, a whole spore has a size in the range of 1 μπι to 40 μπι.

[00101] In certain embodiments, a whole spore comprises a component(s) that is beneficial to a subject. In some embodiments, a whole spore comprises a component(s) that is of therapeutic value. In a specific embodiment, a whole spore has detoxification properties.

[00102] As used herein, the term "exine shell" means the acetolysis-resistant biopolymeric (e.g., sporopollenin) outer coating of a spore or pollen grain. The exine shell of a spore can be isolated by techniques known in the art, including, e.g., successive treatments with organic solvents, alkali, acid and/or enzymes so as to remove the other components of the spore, such as the cellulosic initine layer and lipid, protein and nucleic acid components that may be attached to or contained within the exine shell. The exine shell takes the form of an essentially hollow capsule, which typically comprises sporopollenin.

[00103] As used herein, the term "spore" refers not only to true spores, commonly defined as a unit of asexual reproduction, including endospores, and as such as are produced by nonflowering plants, bacteria, fungi, algae, ferns, and mosses, but also pollen grains, commonly defined as a unit of sexual reproduction, and as such are produced by seed-bearing plants (spermatophytes). Unless stated otherwise, the terms "pollen grains" and "pollens" are used herein interchangeably.

[00104] In a specific embodiment, the spore is pollen. In one embodiment, the spore is bee pollen, tree pollen, flower pollen, pine pollen or grass pollen. In another embodiment, the spore is a plant spore. In another embodiment, the spore is a fungal spore. In another embodiment, the spore is a bacterial spore. In some embodiments, the spore is an Abies spore, Agrocybe spore, Aspergillus niger spore, Bacillus subtilis spore, Cantharellus minor spore, Epicoccum spore, Cuburbita spore, Cucurbitapapo spore, Ganomerma spore, Lycopodium clavatum spore, Myosotis spore, Penicillium spore, Periconia spore, ryegrass spore, Timothy grass spore, maize spore, hemp spore, rape hemp spore, wheat spore, Urtica dioica spore, sunflower spore {e.g., Helianthus annuus spore), pine spore {e.g., Pinus taeda spore), corn spore {e.g., Zea mays spore), cattail spore {e.g., Typha angustifolia spore), rape spore {e.g., Brassica napus spore), dandelion spore {e.g., Taraxacum offinale spore), rye spore {e.g., Secale cereale spore), Eastern Baccharis spore (e.g., Baccharis halimifolia spore), Chorella spore (e.g., Chorella sorkiniana spore), Japanese camellia spore (e.g., Camellia japonica spore), ragweed spore (e.g., Ambrosia artemisi olia spore), mulberry spore (e.g., Moms nigra spore), or pecan spore (e.g., Carya illinoinesis spore).Examples of spores and pollen grains and their corresponding sizes (diameter in mm) are listed in Table 1.

[00105] Table 1. Examples of spores and pollen grains

Myosotis spore 2.4-5 25 Penicillium spore 3-5

26 Periconia spore 16-18

27 Ryegrass pollen 21

grain

28 Timothy pollen 22

grain

29 Maize pollen grain 80

30 Hemp pollen grain 24

31 Rape hemp pollen 25

grain

32 Wheat pollen grain 23

33 Urtica dioica spore 10-12

[00106] In a specific embodiment, a whole spore used in accordance with the disclosure herein is a naturally occurring spore. As used herein, the term "naturally occurring" in the context of a spore means that the spore is produced by a living organism found in nature. In certain embodiments, a whole spore used in accordance with the disclosure herein is derived from a naturally occurring source(s). As used herein, the term "naturally occurring source(s)" is a living organism found in nature. In some embodiments, the naturally occurring source(s) is a plant(s), bacteria, fungi, algae, fern(s), moss(es) or other spore-producing organism(s), whether prokaryotic or eukaryotic. In one embodiment, the naturally occurring organism is a plant(s), fern(s) or moss(es). In another embodiment, the naturally occurring organism is a bacteria. In another embodiment, the naturally occurring organism is algae. In another embodiment, the naturally occurring organism is fungi. In some embodiments, the naturally occurring organism is Abies, Agrocybe, Aspergillus niger, Bacillus subtilis, Cantharellus minor, Epicoccum,

Cuburbita, Cucurbitapapo , Ganomerma, Lycopodium clavatum, Myosotis, Penicillium,

Periconia, ryegrass, Timothy grass, maize, hemp, rape hemp, wheat, Urtica dioica, sunflower {e.g., Helianthus annuus), pine {e.g., Pinus taeda), corn {e.g., Zea mays), cattail {e.g., Typha angustifolia), rape {e.g., Brassica napus), dandelion {e.g., Taraxacum offinale), rye {e.g., Secale cereale), Eastern Baccharis (e.g., Baccharis halimifolia), Chorella (e.g., Chorella sorkiniana), Japanese camellia (e.g., Camellia japonica), ragweed (e.g., Ambrosia artemisifolia), mulberry (e.g., Moms nigra), or pecan (e.g., Carya illinoinesis). In a specific embodiment, the naturally occurring organism is a species described in the Example Section, infra. In one specific embodiment, the naturally occurring organism is Lycopodium clavatum or another species from the same family. In another specific embodiment, the naturally occurring organism is camellia. In another specific embodiment, the naturally occurring organism is pine.

[00107] In certain embodiments, a whole spore used in accordance with the disclosure herein is derived from a genetically engineered living organism that produces spores. In some embodiments, the genetically engineered living organism is a plant(s), bacteria, fungi, algae, fern(s), moss(es) or other spore-producing organism(s), whether prokaryotic or eukaryotic. In one embodiment, the genetically engineered living organism is a plant(s), fern(s) or moss(es). In another embodiment, the genetically engineered living organism is a bacteria. In another embodiment, the genetically engineered living organism is algae. In another embodiment, the genetically engineered living organism is fungi. In some embodiments, the genetically engineered living organism is a genetically engineered version of Abies, Agrocybe, Aspergillus niger, Bacillus subtilis, Cantharellus minor, Epicoccum, Cuburbita, Cucurbitapapo ,

Ganomerma, Lycopodium clavatum, Myosotis, Penicillium, Periconia, ryegrass, Timothy grass, maize, hemp, rape hemp, wheat, Urtica dioica, sunflower (e.g., Helianthus annuus), pine (e.g., Pinus taeda), corn (e.g., Zea mays), cattail (e.g., Typha angustifolia), rape (e.g., Brassica napus), dandelion (e.g., Taraxacum offinale), rye (e.g., Secale cereale), Eastern Baccharis (e.g.,

Baccharis halimifolia), Chorella (e.g., Chorella sorkiniana), Japanese camellia (e.g., Camellia japonica), ragweed (e.g., Ambrosia artemisifolia), mulberry (e.g., Moms nigra), or pecan (e.g., Carya illinoinesis). In a specific embodiment, the genetically engineered living organism is a genetically engineered version of a species described in the Example Section, infra. In one specific embodiment, the genetically engineered living organism is a genetically engineered version of Lycopodium clavatum or another species from the same family. In another specific embodiment, the genetically engineered living organism is a genetically engineered version of camellia. In another specific embodiment, the genetically engineered living organism is a genetically engineered version of pine pollen.

[00108] The genetically engineered organism may be a naturally occurring organism that has been genetically engineered to have a beneficial property. In one embodiment, the genetically engineered organism is a naturally occurring organism that has been genetically engineered to reduce production one or more allergens (e.g. , allergenic proteins). In another embodiment, the genetically engineered organism is a naturally occurring organism that has been genetically engineered to have an altered form of a protein known to be an allergen. In another embodiment, the genetically engineered organism is a naturally occurring organism that has been genetically engineered to produce a higher than normal amount of spores.

[00109] In certain embodiments, a whole spore used in accordance with the disclosure herein is isolated from a naturally occurring source or a genetically engineered organism that produces spores. In some embodiments, the whole spore is isolated from a biological matrix containing non-spore contaminants as well. Examples for non-spore contaminants include, but are not limited to plant-based or natural debris such as fragments of soil, stone, branches, leaves, flower petals, waxes, resins, nectar, and the like. Techniques for isolating of a whole spore are known to those of skill in the art and include, e.g. , sieving the matrix to isolate the spores and remove the non-spore contaminants. In some embodiments, the isolation of a whole spore includes cleaning the spore of contaminants and cleaning any surface-adhered compounds of the spore. In some embodiments, the isolated whole spore is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% free of non-spore contaminants.

[00110] In some embodiments, a whole spore used in accordance with the disclosure herein has been modified structurally. The whole spore may be modified after isolation from a naturally occurring source or a genetically engineered organism that produces spores. In certain embodiments, one, two, three or all of the following structural features of a whole spore may be modified: (i) the surface of the spore may modified (e.g., the surface roughness may be altered), (ii) the size of the spore may be modified, (iv) the shape of the spore may be modified and/or (v) the spore's structural robustness may be modified (e.g., the spore's resistance to mechanical pressure has been strengthened or weakened). Structural features of the spore may be modified using any technique known in the art so long as the components of the whole spore remain. For example, structural features of a spore may be modified by exposure to a buffer, a certain pH or pH range, or a certain temperature or temperature range. See, e.g. , Section 5.4 and the Example Section, infra, for methods for modifying the structural features of a whole spore as well as whole spores that have undergone such modifications.

[00111] In certain embodiments, a whole spore used in accordance with the disclosure herein has been subjected to processing either prior to, during or post-isolation from a naturally occurring source or a genetically engineered organism that produces spores. The spore may be processed in any way so long as the components of a whole spore remain. In some

embodiments, a whole spore used in accordance with the disclosure herein has been subjected to exposure to a solvent. In certain embodiments, the solvent is an organic or inorganic solvent. In some embodiments, the organic solvent is methyl ether, ethyl ether, diethyl ether, acetone, ethanol, methanol, N-methyl pyrrolidone, dimethyl formamide, dichlorome thane, ethylene glycol dimethyl ether, dimethylformamide, methyl sulfoxide, ethyl acetate, trifluoroacetic acid, tetrahydrofuran, any likewise organic solvent, or combinations thereof. In certain embodiments, the solvent is water, and the processing of the spore includes washing the spore after the whole spore has been isolated.

[00112] In certain embodiments, a whole spore used in accordance with the disclosure herein has been subjected to a washing step. The washing step may occur prior to or after the spore has been subjected to a treatment, such as a chemical treatment (e.g., a solvent).

Alternatively or in addition, the washing step may occur after isolation of the whole spore from a naturally occurring source or a genetically engineered organism that produces spores. In some embodiments, the washing includes removing surface adhered contaminants and/or naturally occurring surface adhered lipid-like compounds, typically referred to as pollenkitt, from the spore. In some embodiments, a whole spore used in accordance with the disclosure is defattened to minimize the spore's allergenicity. A "defattened" spore(s) refers to a spore(s) that has its surface proteins or other surface adhered contaminants removed. In some embodiments, defattened spores are obtained by washing the spores in organic solvent, for example, ethyl ether.

[00113] In some embodiments, as illustrated in FIG. 43, camellia oil and camellia pollen grains or derivatives thereof are dissolved in water or other aqueous suspension at an oikpollen mass ratio of 10: 1 or lower. The oil and dry pollen grains can be mixed until a homogenous slurry is formed. In a preferred embodiment, the oil is encapsulated inside the pollen grains which can be achieved by freeze-drying of the sample or other loading method known in the art. The pollen grains can also be treated with ultraviolet (UV) light and ozone in order to render them hydrophilic and therefore soluble in aqueous suspensions. The preferred UV light and ozone treatment occurs at atmospheric pressure with UV light generated at both 185 nm and 254 nm wavelengths. As a result, the oil encapsulated inside the pollen grains can be mixed with water in order to permit aqueous suspensions of Camellia green tea oil or other oil of choice. In a preferred embodiment, the pollen grains are first treated with UV-ozone before loading occurs in order to optimize loading.

[00114] In some embodiments, a whole spore used in accordance with the disclosure herein has not been subjected to processing either prior to, during or post-isolation from a naturally occurring source or a genetically engineered organism that produces spores. In certain embodiments, a whole spore used in accordance with the disclosure herein has not been subjected to exposure to a solvent, such as an organic solvent (e.g., methyl ether, ethyl ether, diethyl ether, acetone, ethanol, methanol, N-methyl pyrrolidone, dimethyl formamide, dichloromethane, ethylene glycol dimethyl ether, dimethylformamide, methyl sulfoxide, ethyl acetate, trifluoroacetic acid, tetrahydrofuran, any likewise organic solvent, or combinations thereof) or inorganic solvent.

[00115] In a specific embodiment, a whole spore used in accordance with the disclosure herein is not considered allergic. For example, generally, pollen allergies are due to a high level of airborne exposure to pollen combined with a genetic tendency of the allergic subject. Pollen grains used in food products typically result in minimal cases of allergic response, since they are ingested orally, rather than through the respiratory system. Furthermore, pollens are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.

[00116] In certain embodiments, a whole spore selected for encapsulation depends upon, inter alia, the compound or substance to be encapsulated in the whole spore, the formulation comprising the whole spore, and the intended use of the formulation. In a specific embodiment, a whole spore larger than 40 μπι is used to encapsulate a food product or component thereof, or an herbal medicine. In one embodiment, pine pollen, corn pollen, or rye pollen is used as a whole spore to encapsulate a food product or component thereof, or an herbal medicine.

[00117] In certain embodiments, a whole spore selected for encapsulation depends upon, inter alia, the compound or substance to encapsulated in the whole spore, the formulation comprising the whole spore, and the intended use of the formulation. In a specific embodiment, a whole spore smaller than 40 μπι (but larger than zero) is used to encapsulate a therapeutic. In one embodiment, a Lycopodium clavatum spore, sunflower pollen, or Camellia pollen is used as the whole spore to encapsulate a therapeutic.

[00118] In specific embodiments, sunflower pollen is used as the whole spore to encapsulate a compound or substance of interest {e.g., a therapeutic) for targeted intestinal delivery. 5.2 COMPOUNDS AND SUBSTANCES FOR ENCAPSULATION IN WHOLE SPORES

[00119] Any compound or substance of interest may be encapsulated in whole spores. As used herein, the term "encapsulate" and cognates thereof in the context of the whole spore means to take up a compound or substance by sorption, adhesion or bond, whether or not chemical or physical in nature, within the inner core of the whole spore. The term "encapsulate" is used interchangeably used with the terms "load" or "take up" and cognates thereof. As used herein, the term "sorption" and cognates thereof refer to absorption and adsorption. See, e.g., Section 5.5 and the Example Section, infra, for methods for encapsulating a compound and/or substance of interest in a whole spore, methods for co-encapsulating a compound and/or substance of interest in a whole spore and an agent that controls the rate of release of the compound and/or substance from the whole spore, and methods for coating a whole spore with an agent that controls the rate of release of the compound and/or substance from the whole spore.

[00120] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a therapeutic(s), a cosmetic product(s) or a component thereof, a personal care product(s) or a component thereof, a processed food(s) or a component thereof, a processed drink(s) or a component thereof, an agricultural product(s) or a component thereof, a household product(s) or a component thereof, toiletry product(s) or a component thereof, or a probe(s). In specific embodiments, the compound or substance of interest is a therapeutic. Examples of therapeutics include, but are not limited to small organic molecules, biologies, medicinal preparation of proteins, herbal medicines, inorganic and organometallic compounds (such as, lithium, platinum-based agents, gallium, and heavy metals), wound or burn healing agents, antiinflammatory agents, anti-irritants, antimicrobial agents (which can include antifungal and antibacterial agents), vitamins, vasodilators, topically effective antibiotics and antiseptics, or any other medicine. In some embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is hormone, antibody, cytokine, chemotherapeutic agent, or other agent useful or beneficial for treating a disease. In a specific embodiment, the therapeutic is 5 -fluorouracil. In other embodiments, the therapeutic is not 5-fluorouracil. In one embodiment, a compound(s) and/or substance(s) for encapsulation in the whole spores is a therapeutic other than 5- fluorouracil. In another embodiment, a compound(s) and/or substance(s) for encapsulation in the whole spores is a therapeutic agent other than a chemotherapeutic agent. [00121] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is an ink. In some embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is diagnostic agent. In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is an antimicrobial substance. In some embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a small molecule. In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is biomolecule. In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a macromolecule.

[00122] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a cosmetic product or a component thereof. In some embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a personal care product or a component thereof. Different spores can be selected for use in cosmetic and person care products, as e.g. , microbeeads, depending, inter alia, on the type of product, because different whole spores have different surface roughness. Examples of cosmetic and personal care products include, but are not limited to, makeup products (for example, foundations, powders, blushers, eye shadows, eye and lip liners, lipsticks, other skin colourings and skin paints), skin care products (for example, cleansers, moisturisers, emollients, skin tonics and fresheners, exfoliating agents and rough skin removers), fragrances, perfume products, essential oils, sunscreens, UV protective agents other than sunscreens, self tanning agents, after-sun agents, anti-ageing agents, anti- wrinkle agents, skin lightening agents, topical insect repellants, hair removing agents, hair restoring agents, or nail care products (such as nail polishes or polish removers). A perfume product may comprise more than one fragrance. In some embodiments, a cosmetic and personal care substance includes a high quality bioactive ingredients or compounds having cosmetic and personal care properties. In some embodiments, a cosmetic and personal care substance encapsulated in a whole spore is a compound that protects a subject from oxidation or UV light. In certain embodiments, a cosmetic and personal care substance encapsulated in a whole spore is flavor, aroma, nutrient, fragrance, phytochemical, or therapeutic.

[00123] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a processed food or a component thereof, or a processed drink or a component thereof. Processed foods or drinks include, for example, food additives, health food and supplements, flavours, aromas, nutrients, bioactives, or phytochemicals. In some embodiments, health food and supplements include nutrients or dietary supplements (such as vitamins, minerals, folic acid, omega-3 oils, fish oils, fibres, and so-called "probiotics" or "prebiotics"). Accordingly, in certain embodiments, one, two or more of the following compounds or substances are encapsulated in a whole spore: a food additive(s), a health food, a food

supplement(s), a flavor(s), an aroma(s), a nutrient(s), a bioactive molecule(s), and/or a phytochemical(s).

[00124] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a food additive. In some embodiments, food additives include acids, acidity regulators, anticaking agents, antifoaming agents, antioxidants, bulking agents, food coloring, color retention agents, emulsifiers, flavors, flavor enhancers, flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, thickeners, or thickening agents. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore.

[00125] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a flavour. In some embodiments, flavours include natural flavoring substances, nature-identical flavoring substances, or artificial flavoring substances. In some embodiments, flavours are selected from a group of flavorings consisting of diacetyl, acetylpropionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, and methyl salicylate. In some embodiments, flavours include salts, sugars or artificial sweeteners. In some embodiments, flavours include savory flavorants, for example, amino acids and nucleotides, in the form of sodium or calcium salts. In some embodiments, flavours are sour additives, such as organic and inorganic acids.

[00126] In a specific embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is caffeine. In other embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is not caffeine.

[00127] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is an aroma(s). In some embodiments, aromas include esters, linear-terpenes, cyclic-terpenes, aromatic, amines, alcohols, aldehydes, ketones, lactones, or thiols. [00128] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a nutrient. In some embodiments, nutrients include carbohydrates, proteins, fats, dietary minerals, vitamins, or dietary fiber. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore

[00129] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a bioactive compound or substance. In some embodiments, bioactives include fatty acids, flavonoids, caffeine, carotenoids, carnitine, choline, coenzyme Q, creatine, dithiolthiones, phytosterols, polysaccharides, phytoestrogens, glucosinolates, polyphenols, lipids, anthocyanins, prebiotics, or taurine. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore

[00130] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a phytochemical. Examples of phytochemicals include, but are not limited to, terpenoids (for example carotenoids, triterpenoid, monoterpenes, steroids), phenolic compounds (for example natural monophenols, polyphenols, aromatic acids, phenylethanoids),

glucosinolates (for example, isothiocyanate precursors, aglycone derivatives, organosulfides, organosulfur compounds, and indoles), betalains (for example betacyanins, betaxanthins), chlorophylls, organic acids, amines, carbohydrates (for example, monosaccharides, and polysaccharides) and protease inhibitors. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore

[00131] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a fuel. In some embodiments, a compound(s) and/or substance(s) for

encapsulation in whole spores is a disinfectant or cleaning agent.

[00132] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a lipid, proteinaceous agent (e.g., protein, polypeptide or peptide), fatty acid or carbohydrate.

[00133] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is an agricultural product or a component thereof. In some embodiments, the agricultural product is a pesticide or a fertilizer.

[00134] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a pesticide. In some embodiments, pesticides include chemically-related pesticides or pest specific formulations. Chemically-related pesticides include, for example, organophosphate pesticides, carbamate pesticides, organochlorine insecticides, pyrethroid pesticides, or sulfonylurea herbicides. Pest specific formulations include, for example, algicides, antifouling agents, antimicrobials, attractants, biopesticides, biocides, disinfectants, sanitizers, fungicides, fumigants, herbicides, insecticides, miticides, microbial pesticides, molluscicides, nematicides, ovicides, pheromones, repellents, or rodenticides. Accordingly, in certain embodiments, one, two or more of these pesticides are encapsulated in a whole spore.

[00135] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a fertilizer. In some embodiments, fertilizers include nitrogen fertilizers, phosphate fertilizers, potassium fertilizers, compound fertilizers, organic fertilizers or elemental compounds, for example, calcium, magnesium, and sulfur.

[00136] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a compound and/or substance found in a household product. Examples of household products (whether for internal or external use) include surface cleaners, disinfectants and other antimicrobial agents, fragrances, perfume products, air fresheners, insect and other pest repellants, laundry products (e.g. , washing and conditioning agents), fabric treatment agents (including dyes), cleaning agents, UV protective agents, dishwashing products, paints, varnishes, inks, dyes and other colouring products, and adhesive products.

[00137] In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a compound and/or substance found in a toiletry product. Examples of toiletry products include soaps; detergents and other surfactants; deodorants and antiperspirants;

lubricants; fragrances; perfume products; dusting powders and talcum powders; hair care products such as shampoos, conditioners and hair dyes; and oral and dental care products such as toothpastes, mouth washes and breath fresheners.

[00138] In certain embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a probe. In some embodiments, probes include fluorescence-tagged molecules. In some embodiments, probes include bovine serum albumin (BSA), calcein, or fluorescein isothiocyanate (FITC)-conjugated.

[00139] In certain embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a plant extract. In some embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a lipophilic compound(s) (e.g. , an oil(s)). In certain

embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a traditional herbal medicine. In some embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a modern pharmaceutical(s).

[00140] In a specific embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is an oil. In some embodiment, a compound(s) and/or substance(s) for

encapsulation in whole spores is Camellia japonica oil (Japanese tea oil), Camellia sinensis oil (tea seed oil) or Camellia oleifera oil (tea oil).

[00141] In a specific embodiment, a compound(s) and/or substance(s) for encapsulation in whole spores is a compound or substance disclosed in the Example Section, infra.

[00142] In other embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is not found in nature to be associated with the whole spore.

[00143] In certain embodiments, a whole spore is loaded with any suitable amount of the compound or substance of interest. The suitable amount of a compound or substance will depend on, inter alia, the intended use of a formulation comprising the whole spore and the compound or substance encapsulated in the whole spore. In some embodiment, the formulation includes the whole spore and the compound or substance at a weight ratio of from 100: 1 to 1: 1.

A larger whole spore may be needed to encapsulate a larger amount of the compound or substance.

[00144] In some embodiments, only one compound or substance of interest is encapsulated in the whole spore. In other embodiments, two or more compounds or substances of interest are encapsulated in the whole spore.

[00145] In certain embodiments, the encapsulated compound or substance is retained within cavities of the whole spore. In some embodiments, the encapsulated compound or substance is preferably retained within a central cavity of the whole spore. In some embodiments, a percentage of the encapsulated compound or substance is attached to a surface of the whole spore. In some embodiments, the percentage of encapsulated compound or substance attached to the surface of the whole spore is less than 5% by weight of the entire encapsulated amount of the compound or substance.

5.3 AGENTS FOR CONTROLLING THE RATE OF RELEASE OF COMPOUNDS

AND SUBSTANCES ENCAPSULATED IN WHOLE SPORES

[00146] In another aspect, provided herein are agents for controlling the rate of release of a substance or compound of interest encapsulated in a whole spore. [00147] In certain embodiments, agents for controlling the rate of release of a substance or compound of interest encapsulated in a whole spore include coating agents or co-loading agents. In some embodiments, coating agents include waxes, butters, starches, rosins, resins, hydrogels, alginate and polysaccharides. In another embodiment, agents include hydroxypropyl methyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, xanthan gum, Eudragit, Carbomers, oils and waxes and methacrylate copolymers. The coating may be a natural coating, such as starches, waxes, resins, rosins, etc. In one embodiment, a synthetic polymer coating is used for controlled release and improved product stability. In some embodiments, co-loading agents include glycerol, hydrogels, glucose and oils. In some embodiments, the co-loading agent is a viscous loading solution having a viscosity that is greater than the viscosity of water.

[00148] In certain embodiments, hydrogels for controlling the rate of release of a substance or compound of interest include water- swelling polymers. For example, ionic hydrogel polymers as well as non-ionic hydrogel polymers (e.g., non-ionic hydrophilic hydrogel polymers) can be used. As one example, a pharmaceutical-suitable homo-polymer hydrogel (such as a polymer polymerized from the same type of monomers without cross-linking to two or more different kinds of monomers, a polymer with the same kind of side chains, a non-copolymer) can be used. In some embodiments, a formulation includes the whole spore, a substance or compound of interest, and about 4% to 80% by weight of the non-cross-linked, water-swelling homo-polymer.

[00149] Examples of the non-cross-linked, water-swelling homo-polymer include, but are not limited to, hydroxypropyl methylcellulose (HPMC, e.g., METHOCEL™, etc.), alginate, sodium alginate, cellulose hydrogel, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; e.g., KLUCEL™, etc.), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentylcellulose, methyl cellulose, hydroxyethyl cellulose, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, poly-hydroxyalkyl methacrylate, polymethacrylic acid, polymethylmethacrylate, poly vinyl alcohol, sodium polyacrylic acid, calcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers, carboxypolymethylene, carboxyvinyl polymers, carboxymethylamide, polyoxyethyleneglycols, polyethylene oxide, and derivatives, their pharmaceutically equivalent salts, and mixtures thereof. [00150] In a specific embodiment, the agent for controlling the release rate of a compound or substance of interest from a whole spore is an agent described in the Example Section, infra. In a particular embodiment, the agent for controlling the release rate of a compound from a whole spore is alignate.

[00151] In a specific embodiment, the agent for controlling the release rate of a compound or substance of interest from a whole spore decreases the rate of release of the compound or substance from the spore. In specific embodiments, the presence of the agent reduces the rate of release of the compound by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the rate of release of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions. In specific embodiments, the presence of the agent results in a release rate within 1 hour that is reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the rate of release of the compound or substance within 1 hour from a whole spore not encapsulated or coated with the agent under identical conditions. In specific embodiments, the presence of the agent reduces the cumulative release of the compound by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the cumulative release of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions. In specific embodiments, the presence of the agent results in cumulative release within 1 hour that is reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the cumulative release of the compound or substance within 1 hour from a whole spore not encapsulated or coated with the agent under identical conditions.

[00152] In a specific embodiment, the agent for controlling the release rate of a compound or substance of interest from a whole spore prolongs the release time of the compound or substance from the spore. In specific embodiments, the presence of the agent prolongs the release time of the compound by 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h , 25 h, or 30 h relative to the release time of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions. In some embodiments, the total release time of the compound or substance from the spore encapsulated or coated with the agent is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h , 25 h, or 30 h. "Total release time" in this context refers to the time period of continuous release until at least 95% of the encapsulated compound or substance are released from the spore. In a specific embodiment, the total release time of the compound or substance from the spore encapsulated or coated with the agent is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h, 25 h, or 30 h without a burst effect. "Burst effect" in this context refers to a release of at least 90% of the encapsulated compound or substance within the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min from the onset of the release.

[00153] In certain embodiments, the coating agent, co-loading agent, hydrogel or other agent for controlling the release rate of a compound or substance of interest is engineered to co- encapsulate with the substance or compound in the whole spore. In some embodiments, a formulation includes a whole spore, an compound or substance of interest encapsulated within the whole spore, and a co-encapsulated agent for controlling the rate of release of the substance or compound from the whole spore. In some embodiments, the formulation includes one or more co-encapsulated release controlling agents.

[00154] In certain embodiments, the co-encapsulated agent is retained within cavities of the whole spore. In some embodiments, the co-encapsulated agent is preferably retained within a central cavity of the whole spore. In some embodiments, a percentage of the encapsulated agent is attached to a surface of the whole spore. In some embodiments, the percentage of co- encapsulated agent attached to the surface of the whole spore is less than 5% by weight of the entire encapsulated amount of the compound or substance.

[00155] In certain embodiments, the step of co-encapsulating the agent is performed as a concurrent or separate step to encapsulating the compound or substance in the whole spore. In some embodiments, co-encapsulating includes one or more distinct processing steps.

[00156] In certain embodiments, the agent for controlling the rate of release of a compound or substance of interest from a whole spore coated on the whole spore. In some embodiments, a formulation includes the whole spore, a compound or substance of interest encapsulated in the whole spore, and an agent for controlling the rate of release of the substance or compound from the whole spore, wherein the whole spore is coated with the agent. In some embodiments, the coat of a whole spore includes a microbead coat. In some embodiments, the microbead coat includes alginate microbeads.

5.4 METHODS OF MODIFYING THE PROPERTIES OF WHOLE SPORES

[00157] In another aspect, provided herein are methods of modifying the properties of a whole spore. In certain embodiments, modifying the properties of a whole spore includes modifying the structural features of the spore. Structural features of the spore include, for example, the size, shape or composition of the spore.

[00158] In some embodiments, modifying structural features of the spore includes modifying the surface of spore, for example, the surface roughness, altering the size or shape of the spore, or modifying the spore's structural robustness, for example by strengthening or weakening the spore's resistance to mechanical pressure. In some embodiments, modifying the mechanical robustness of the spore comprises using chemical processing. In some embodiments, chemical processes that structurally modify spores includes controlled application of acids, alkalis, oxidative processes, and solvents.

[00159] For example, chemical processing that exposes the spore to acid or alkali compounds that alters the exine polymer structure, causing the exine shell to fracture more easily may be used. If the mechanical robustness of the spores is decreased it may allow for more rapid spore breakdown, and more rapid release of the loaded compounds. In some embodiments, the chemical processing alters outer exine shell polymer structure of the spore, while maintaining the structural integrity of the spore. For example, oxidative processes degrade the exine polymer and cause the exine to fracture more easily. Other processing examples are treatments with fused potassium hydroxide, and in oxidizing mixtures such as hypochlorite/hydrochloric acid, potassium dichromate/sulphuric acid, hydrogen peroxide/sulphuric acid, and ozone. Other examples includes solvents (e.g. , 2-aminoethanol, 3-animopropanol, 2,2'2"-nitriltriethanol, and 4-methylmorpholine-N-oxide) that soften and eventually dissolve the exine polymer shell of the spores.

[00160] In some embodiments, modifying the properties of a whole spore includes exposing the whole spore to UV light to increase hydrophilicity of the whole spores. For example, exposure to UV light may alter the spore's hydrophilicity by changing its surface chemistry by converting hydrophobic surface proteins into their hydrophilic counterparts. See, e.g., the Example Section, infra, regarding UV light exposure. In some embodiments, hydrophilic and/or hydrophobic properties of a whole spore are controlled and modified by coatings in order to support water filtration and prevent clogging of the spore.

5.5 METHODS FOR ENCAPSULATING COMPOUNDS AND SUBSTANCES IN

WHOLE SPORES AND COATING SUCH WHOLE SPORES [00161] In another aspect, provided herein are methods for encapsulating a compound and/or substance of interest in a whole spore as well as co-encapsulating a compound and/or substance of interest and an agent that controls the release rate of the compound and/or substance from the whole spore. Any technique known to one of skill in the art may be used to encapsulate a compound and/or substance of interest in whole spore or co-encapsulate a compound and/or substance of interest and an agent that controls the release rate of the compound and/or substance from the whole spore. In a specific embodiment, a technique described in the Example Section, infra, is used to encapsulate a compound and/or substance of interest in a whole spore, or co- encapsulate a compound and/or substance of interest and an agent that controls the release of the compound and/or substance from the whole spore.

[00162] In addition, provided herein are methods for coating a whole spore with an agent that controls the release of an encapsulated compound and/or substance from the whole spore. In a specific embodiment, a technique described in the Example Section, infra, for a method for coating a whole spore with an agent that controls the release rate of an encapsulated compound and/or substance from the whole spore.

5.5.1 Passive Loading Method

[00163] In certain embodiments, a method of encapsulating a compound or substance of interest in a whole spore comprises contacting the compound or substance with the whole spore. In some embodiments, the step of contacting the compound or substance with the whole spore comprises dissolving the compound or substance in a solvent, suspending the whole spore in the solution, and allowing the whole spore to encapsulate the compound or substance for a specific duration. In some embodiment, the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution. In some embodiments, the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.

[00164] In some embodiments, the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature. In some embodiments, the cooling temperature is about 4° Celsius. In some embodiments, the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours. In some embodiments, the removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, centrifuging the solution is performed at 12000 rpm for a duration of 4 minutes.

5.5.2 Compression Loading Method

[00165] In certain embodiments, a method for encapsulating a compound or substance of interest in a whole spore comprises compressing the whole spore into a tablet and contacting the tablet with the compound or substance. In some embodiments, the step of contacting the tablet with the compound or substance comprises dissolving the compound or substance in a solvent, soaking the tablet of the whole spore in the solution, and allowing the whole spore to encapsulate the compound or substance for a specific duration. In some embodiments, the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution. In some embodiments, the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.

[00166] In some embodiments, the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature. In some embodiments, the cooling temperature is about 4° Celsius. In some embodiments, the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours. In some embodiments, the step of removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, the step of centrifuging the solution comprises the solution being centrifuged at 12000 rpm for a duration of 4 min.

[00167] In some embodiments, the step of compressing the whole spore into a table comprises applying a compression pressure of 5 ton or at least 1 ton for a duration of at least 10 sec or 20 sec. In some embodiments, the step of compressing the whole spore into a table further comprises filling the whole spore into die and applying the compression pressure to the die.

5.5.3 Vacuum Loading Method

[00168] In certain embodiments, a method for encapsulating a compound or substance of interest in a whole spore comprises contacting the compound or substance with the whole spore under vacuum pressure. In some embodiments, the step of contacting of the compound or substance with the whole spore under vacuum pressure comprises dissolving the compound or substance in a solvent, suspending the whole spore in the solution, applying a vacuum to the suspension, and allowing the whole spore to encapsulate the compound or substance for a specific duration. In some embodiments, the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution. In some embodiments, the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.

[00169] In some embodiments, the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature. In some embodiments, the cooling temperature is about 4° Celsius. In some embodiments, the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours. In some embodiments, the step of removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, centrifuging the solution is performed at 12000 rpm for a duration of 4 min.

[00170] In some embodiments, the step of applying a vacuum to the suspension comprises using a freeze-drier. In some embodiments, the vacuum includes a pressure of 2 mbar or at least less than 5 mbar.

5.5.4 Coating Methods

[00171] A coating is generally used in the context of applying an agent to the whole spore's surface, while co-encapsulation includes filling at least some of spore's cavities with the agent. Any technique known to one of skill in the art can be used to coat a whole spore with an agent that controls the release rate of a compound or substance of interest from the whole spore.

[00172] In certain embodiments, a method for coating a whole spore with an agent that controls release of a compound or substance of interest from the whole spore comprises using individual particle coating or agglomerate particle coating to coat the agent on the whole spore. In some embodiments, individual particle coating includes spray coating, sputtering, or applying vapor deposition. In some embodiments, agglomerate particle coating includes pressing spore pellets and dip coating, spray coating, sputtering, or applying vapor deposition. In some embodiments, agglomerate particle coating comprises mixing the whole spores with a co- encapsulating compound or substance and solidifying the mixture, by various techniques, to form agglomerates that contain the whole spores and the compound or substance.

5.5.5 Methods for Assessing Compound or Substance Encapsulation [00173] Uniformity in size, shape and morphology of the whole spore encapsulating a compound or substance is important for various uses of a product containing said spore.

Compound release and formulation of the product largely depend on these uniform micromeritic properties of the spore. A more uniform spore allows for encapsulating a larger amount of compound or substance in the whole spore. In addition, higher uniformity among the

encapsulating whole spores guaranties greater consistency in preparing and using formulations comprising whole spores encapsulating a compound or substance.

[00174] Any technique known to one of skill in the art or described herein may be used to assess the encapsulation of a compound and/or substance of interest in a whole spore (see, e.g., the Example Section, infra). In certain embodiments, a method of assessing the encapsulation of a compound or substance in a whole spore comprises using a dynamic image particle analyzer to assess structural characteristics of the whole spore. In some embodiments, the assessed structural characteristics include uniformity, size, shape and micromeritic properties of the whole cell.

[00175] For example, a dynamic image particle analyzer (DIPA) uses a high-resolution digital camera and objective lens to capture images of the particles, i.e. , the whole spore encapsulated with the compound or substance, flowing through a thin transparent flow cell. Particle size uniformity data is then generated based on digital signal processing of the images. Besides size determination, the digital particle images allows obtaining additional information including edge gradient, circularity, and the shape of whole spores. In some embodiments, size, edge gradient and circularity analysis by the DIPA is performed with an initial particle count of 10,000 whole spores for all the batch formulations and images are processed using software to obtain 1000 well focused whole spores. In some embodiments, representative data is plotted as a histogram and fitted with a Gaussian curve and values are reported with standard deviations. In a specific embodiment, DIPA is used as described in the Example Section, infra.

[00176] In certain embodiments, a method of assessing the encapsulation of a compound or substance in a whole spore comprises using a confocal laser scanning microscope to visualize the whole spore. In some embodiments, the method comprises mounting whole spores encapsulating the substance or compound on a sticky slide. In some embodiments, the method comprises measuring the fluorescence from the compound or substance encapsulated in the whole spore. In some embodiments, the compound or substance is a fluorescence probe or a fluorescence probe- tagged molecule. In certain embodiments, the compound or substance is a fluorescently labeled version of a compound described in Section 5.2, supra. In some embodiments, the compound or substance is FITC-conjugated BSA, fluorescein, 5 -fluorouracil, or calcein. In other

embodiments, the compound or substance is not is FITC-conjugated BSA, fluorescein, 5- fluorouracil, or calcein. In a specific embodiment, confocal laser scanning microscopy is used as described in the Example Section, infra.

[00177] Any technique known to one of skill in the art or described herein can be used to determine the amount of compound or substance encapsulated in a whole spore {see, e.g., the Example Section, infra). In certain embodiments, a method for determining the amount of compound or substance encapsulated in a whole spore comprises: (1) rupturing the compound or substance loaded whole spores; (2) incubating the ruptured whole spores in a solution to allow for maximum compound release into the solution; (3) separating the mass of the whole spore from the solution containing the compound by filtration; (4) using spectrographic analysis of the solution containing the compound (for example, UV spectroscopy) to determine light absorption properties of the solution containing the compound; and (5) comparing the determined light absorption properties against a standard absorption curve to determine the amount of compound or substance, wherein standard absorption curve is obtained from light absorption data collected from a series of solutions with a known amount of the compound.

[00178] In some embodiments, the method for determining the amount of compound or substance encapsulated in a whole spore further comprises repeating steps (l)-(5) using a placebo and subtracting the determined light absorption properties of the placebo from the determined light absorption properties of the whole spore prior to comparing the determined light absorption properties against a standard absorption curve to determine the amount of compound or substance. The additional step ensures an increased accuracy in determining the amount of compound or substance. In some embodiments, the amount of compound or substance in the whole spore, the percentage of compound or substance loading, and the percentage of encapsulation efficiency are determined by:

Absorbance x dilution factor

Amount of compound (mg) = (1),

Slope (standard curve) x 1000

Amount of compound

% compound loading = X 100 (2),

Weight of compound loaded spores

Practical compound loading

% Encapsulation efficiency = (3).

Theoretical compound loading [00179] In certain embodiments, a method for determining a weight ratio of a whole spore to a compound or substance encapsulated in the whole spore comprises using the method for determining the amount of compound or substance encapsulated in the whole spore to determine an amount of compound or substance and an amount of the whole spore, wherein the whole spore amount is measured from the separated mass of the whole spore in the above step (3). In some embodiments, the ratio is given by the amount of compound or substance : the amount of the whole spore. For example, 4 mg of compound and 6 mg of whole spore gives a ratio of 1 : 1.5.

[00180] Any technique known to one of skill in the art or described herein can be used to determine the rate of release of a compound or substance encapsulated in a whole spore (see, e.g., the Example Section, infra). In certain embodiments, a method for assessing the controlled release rate of a compound or substance encapsulated in a whole spore comprises incubating a formulation of the whole spore in a solution, allowing release of the encapsulated compound or substance into the solution, and determining the amount of released compound using standard analytical chemistry techniques. In some embodiments, standard analytical chemistry techniques include, for example, UV spectrometry.

[00181] In certain embodiments, a method for assessing the controlled release rate of a compound or substance encapsulated in a whole spore comprises performing steps (2)-(5) of the method for determining the amount of compound or substance encapsulated in a whole spore, wherein the incubation of the whole spores is stopped at a fixed time point.

[00182] Any technique known to one of skill in the art or described herein can be used to assess the allergies of a subject to a whole spore or a compound or substance-encapsulated whole spore. In certain embodiments, a method for assessing allergies in subjects comprises performing allergy blood testing or skin prick testing. In some embodiments, the method further comprises exposing the subject to a whole spore or a compound or substance-encapsulated whole spore. Exposure, for example, includes skin contact, inhalation or ingestion. Most pollen allergies are typically related to inhalation exposure. In some embodiments, the method comprises determining the response of a subject's skin upon contacting the compound or substance with the skin. 5.6 FORMULATIONS COMPRISING WHOLE SPORES OR WHOLE SPORES ENCAPSULATING A COMPOUND(S) OR SUBSTANCE(S). AND USES THEREOF

[00183] In another aspect, provided herein are formulations comprising whole spores, whole spores encapsulating a compound or substance of interest, or whole spores co-capsulating a compound or substance of interest and an agent that facilitates controlled release of the compound or substance, and uses thereof. In a specific embodiment, a formulation comprises whole spores encapsulating a compound or substance of interest. In certain embodiments, a formulation comprises whole spores co-encapsulating a compound or substance and an agent to facilitate controlled release of the compound or substance from the whole spore. In some embodiments, a formulation comprises whole spores encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore.

[00184] In certain embodiments, a formulation described herein further comprises one or more additional agents, such as a fluid vehicle(s), an excipient(s), a diluents(s), a carrier(s), a stabilizer(s), a surfactant(s), a penetration enhancer(s) or other agents for targeting delivery of the whole spore and/or the compound or substance of interest to the intended site of

administration. In one embodiment, a formulation comprises a whole spore(s) and a diluent or carrier. In another embodiment, a formulation comprises a whole spore(s) and a diluents(s) or pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to non-toxic carrier.

[00185] In a specific embodiment, a formulation comprises whole spores encapsulating a compound or substance of interest and a diluents or pharmaceutically acceptable carrier. In certain embodiments, a formulation comprises whole spores co-encapsulating a compound or substance and an agent to facilitate controlled release of the compound or substance from the whole spore and a diluents(s) or pharmaceutically acceptable carrier. In some embodiments, a formulation comprises whole spores encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore and a diluents(s) or pharmaceutically acceptable carrier.

[00186] A formulation described herein may comprise a weight ratio of compound or substance of interest to whole spore of from 0.0001 : 1 to 50: 1, such as from 0.001: 1 to 5: 1, 0.01: 1 to 5: 1, 0.1: 1 to 5: 1, or 0.5: 1 to 50: 1. [00187] In certain embodiments, suitable formulations are prepared by methods commonly employed using conventional, organic or inorganic additives or carriers, such as an excipient (e.g., sucrose, glucose, lactose, cellulose, sorbitol, talc, mannitol, calcium phosphate, starch, or calcium carbonate), a binder (e.g., cellulose, hydroxymethylcellulose, methylcellulose, polyvinylpyrrolidone, polypropylpyrrolidone, gum arabic, gelatin, polyethyleneglycol, starch, or sucrose), a disintegrator (e.g., starch, hydroxypropylstarch, carboxymethylcellulose, low substituted hydroxypropylcellulose, calcium phosphate, sodium bicarbonate, or calcium citrate), a lubricant (e.g., magnesium stearate, talc, light anhydrous silicic acid, or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, glycine, menthol, or orange powder), a preservative (e.g., sodium benzoate, methylparaben, sodium bisulfite, or propylparaben), a stabilizer (e.g., citric acid, acetic acid, or sodium citrate), a suspending agent (e.g., methylcellulose, aluminum stearate, or polyvinyl pyrroliclone), a dispersing agent (e.g., hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax (e.g., cocoa butter, polyethylene glycol, or white petrolatum).

[00188] The formulation of a whole spore, a whole spore encapsulating a compound or substance, a whole spore co-encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore, or a whole spore encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore will vary depending on the intended use. In addition, a formulation for administration to a subject may vary depending upon the route of administration to a subject. The formulations described herein can be administered by any route known to one of skill in the art. For example, a formulation described herein can be orally, parenterally, intradermally, intramuscularly, intraperitoneally, percutaneously, intravenously, subcutaneously, intranasally, epidurally, sublingually, intracerebrally, intravaginally,

transdermally, rectally, mucosally, by inhalation, or topically to the ears, nose, eyes, or skin. In a specific embodiment, a formulation described herein is administered to a subject orally. In another specific embodiment, a formulation described herein is administered to a subject parenterally (e.g., subcutaneously, intramuscularly or intravenously). The mode of

administration is left to the discretion of the health-care practitioner, and can depend in-part upon the site of the medical condition or the type of whole spore or the compound or substance.

[00189] A formulation may for example take the form of a lotion, cream, ointment, paste, gel, foam, a hydrogel lotion, a skin patch or any other physical form known for topical administration, including for instance a formulation which is, or may be, applied to a carrier such as a sponge, swab, brush, tissue, skin patch, dressing or dental fibre or tape to facilitate its topical administration. It may take the form of a viscous or semi-viscous fluid, or of a less viscous fluid such as might be used in sprays (for example nasal sprays), drops (e.g. eye or ear drops), aerosols or mouthwashes. In some embodiments, a topical formulation is a cosmetic or therapeutic lotion. In some embodiments, the whole spore described herein may be formulated as a composite powder-like material. This composite powder-like material is incorporated into a wide range of foods or drinks, processed foods, food supplements, etc.

[00190] In another aspect, provided herein are products comprising a formulation described herein. In yet another aspect, provided herein are products comprising the whole spore.

[00191] In some embodiments, provided herein are formulations for certain types of products, such as, e.g. , pharmaceutical products; herbal or nutraceutical products; personal healthcare products; cosmetics and personal care products (e.g. bath products, soaps, hair care products; nail care products, and dental products such as toothpastes, dentifrices, mouthwashes and dental flosses); food and drink products (including food and beverage additives and ingredients); and pesticides, herbicides and fertilizers; household products (whether for internal or external use, including surface cleaners, disinfectants and other antimicrobial agents, fragrances, perfume products, air fresheners, insect and other pest repellants, pesticides, laundry products (e.g. , washing and conditioning agents), fabric treatment agents (including dyes), cleaning agents, UV protective agents, dishwashing products, paints, varnishes, inks, dyes and other colouring products, and adhesive products); agricultural and horticultural products

(including pesticides, herbicides and fertilizers); toiletry products (including soaps; detergents and other surfactants; deodorants and antiperspirants; lubricants; fragrances; perfume products; dusting powders and talcum powders; hair care products such as shampoos, conditioners and hair dyes; and oral and dental care products such as toothpastes, mouth washes and breath

fresheners); fuels; explosives; propellants; and photographic materials. A whole spore, a whole spore encapsulating a compound or substance, a whole spore co-encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore, or a whole spore encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore may be added or included in any formulation known to one of skill in the art, including those described herein.

[00192] In certain embodiments, formulations of a whole spore encapsulating a compound or substance of interest are used in shower gels, toothpastes, mouthwash and face cleansers, to cure skin complaints, aging and stretch marks, to treat cuts and burns, and/or as an insect and lice repellent.

[00193] In certain embodiments, a formulation of whole spores encapsulating a compound or substance of interest comprises a plurality of Camellia pollen and oils for cosmetic and dermatology applications. Specifically, in some embodiments, the formulation comprises an effective quantity of Camellia japonica pollen grains or equivalent with one or more types of Camellia oil in order to reduce skin irritation and optimize therapeutic properties. The natural microsphere carrier for the pollen carries the added benefit of providing a therapeutic effect due to the components found in its natural composition

[00194] In certain embodiments, formulations for personal care products as described herein, comprise a whole spore or a whole spore encapsulating a compound or substance of interest.

[00195] In another aspect, provided herein is a method of treating a disease or condition in a subject, comprising administering to the subject a formulation comprising a whole spore encapsulating a compound or substance beneficial to treating the disease or condition (e.g., a therapeutic, herbal medicine or nutraceutical). In some embodiment, the therapeutic

encapsulated in the whole spore is beneficial for treating the disease or condition.

[00196] In certain embodiments, the formulations described herein are for treating treating skin or skin structure conditions (for example, acne, psoriasis or eczema), wound or burn healing, treating anti-inflammatory diseases or conditions, and/or use as anti-irritants or antimicrobial agents (including antifungal and antibacterial agents).

[00197] The term "subject" as used herein, refers to a patient, such as an animal, a mammal or a human, who has been the object of treatment, observation or experiment and is at risk of (or susceptible to) developing a disease or condition.

[00198] In another aspect, provided herein is a method for protecting a compound or substance of interest from heat, light (including UV light), water, oxygen, oxidizing agents or conditions, and other environmental hazards. Examples of protection and other benefits provided by the whole spore include: (1) protection from atmospheric effects, in particular from light and/or oxygen, and therefore from premature degradation; (2) physical protection to help reduce loss of the compound or substance by for instance evaporation, diffusion or leaching; (3) good uniformity in size, shape and surface properties, unlike typical synthetic encapsulating entities; (4) significant variation in spore size and shape between different species, allowing a formulation to be tailored dependent on the nature and desired concentration of the compound or substance, the site and manner of its intended application, the desired release rate, the likely storage conditions prior to use; (5) granularity providing an exfoliating effect; (6) protection against toxic or adverse effect of compound or substance by physically shielding the compound or substance from contact until release commences; (7) antioxidant for encapsulated compound or substance; and (8) tastelessness allowing taste masking of the compound or substance encapsulated in the whole spore.

[00199] In certain embodiment, the use of a whole spore encapsulating a compound or substance of interest in a formulation modifies the hydrophobicity, nitrogen/oxgen plasma, etc. of the compound or substance. In some embodiments, the use of a whole spore encapsulating a compound or substance of interest in a formulation improves the dispersion characteristics of the compound or substance.

[00200] In another aspect, provided herein, are methods of improving the stability of a compound or substance of interest, comprising encapsulating the compound or substance in a whole spore. Stability of a compound or substance can be assessed by technology known in the art.

[00201] In another aspect, provided herein are methods for preventing oxidation or providing protection against degradation of a compound or substance, comprising encapsulating the compound or substance in a whole spore. In some embodiments, the oxidation includes aerial oxidation.

[00202] In some embodiments, oxidative stability may be measured by measuring the rate of change in a parameter such as peroxide value. Additionally or alternatively, oxidative stability may be measured by measuring the rate of change of redox potential, thiobarbituric acid value, iodine value, anisidine value, TOTOX value (defined as two times the peroxide value added to the anisidine value) and/or free fatty acid content, and/or by the RANCIMAT, active oxygen or Schaal oven test methods, or by any other suitable test method. Other methods for determining oxidative stability includes using an oxidative stability instrument (OSI) or an oxidograph, which are automated versions of the more complicated AOM (active oxygen method). The

RANCIMAT method has become the most established and accepted into a number of national and international standards.

[00203] In another aspect, provided herein are methods of reducing the toxicity of a compound or substance, comprising encapsulating the compound or substance in a naturally occurring whole spore. In certain embodiments, the method allows for targeting a location of a subject's body for release of the compound or substance. In some embodiments, the methods allows for lowering the required amount of compound or substance to be administered to a subject. In some embodiments, the whole spore is co-encapsulated with an agent that controls the rate of release of the compound or substance spore. In certain embodiments, the whole spore is coated with an agent that controls the rate of release of the compound or substance from the spore. Any technique known to one of skill in the art can be used to assess the ability of the compound or substance-encapsulated whole spore to reduce the toxicity of the compound or substance.

[00204] In certain embodiments, allergies to a formulation for administration is tested before use.

[00205] In another aspect, provided herein are methods for masking the taste of a compound or substance of interest (e.g., a nutrient, phytochemical or bioactive molecule), comprising encapsulating the compound or substance in a naturally occurring whole spore and formulating the whole spore in a drink or food. In some embodiments, the whole spore is co- encapsulated with an agent that controls the rate of release of the compound or substance spore. In certain embodiments, the whole spore is coated with an agent that controls the rate of release of the compound or substance from the spore. Any technique known to one of skill in the art (e.g., surveys) or described herein (see, e.g., the Example Section, infra) can be used to assess the ability of the compound or substance-encapsulated whole spore to mask the taste of the compound or substance.

[00206] In certain embodiments, the method of encapsulating hydrophobic materials

(including but not limited to oil into pollen grains) as described herein, comprises first converting the pollen grains into hydrophilic microcapsules and then loading the hydrophobic material. This method optimizes loading of the hydrophobic material while minimizing surface contamination of the hydrophobic material which is important for cosmetic and food applications (e.g., tastemasking).

[00207] In another aspect, provided herein are methods for exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore. In another aspect, provided herein are methods for exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore engineered to encapsulate a compound or substance that is beneficial or useful in a cosmetic or personal care product. Any technique known to one of skill in the art can be used to assess the ability of a whole spore or a compound or substance- encapsulated whole spore to exfoliate skin.

[00208] In another aspect, a whole spore is used as a microbead. Such a microbead may be used for any application in which plastic microbeads are used, e.g., cosmetics, toothpastes, hair products, etc. In a specific embodiment, the whole spore used as a microbead is engineered to encapsulate a compound or substance of interest (e.g., a compound or substance that is beneficial or useful in a cosmetic or personal care product). SEM images of examples of whole spore microbeads are illustrated in FIG. 44.

6. EXAMPLES

6.1 EXAMPLE 1: LYCOPODIUM SPORES: A NATURALLY MANUFACTURED,

SUPER-ROBUST BIOMATERIAL FOR DRUG DELIVERY

[00209] This example reports on the exploration of natural plant based "spores" as well- defined microstructure materials for the encapsulation of biomacromolecules as a drug delivery platform. Benefits of encapsulation with natural "spores" include higher uniform size distribution including natural microridges, significant biomacromolecules loading and retention of natural spore constituents along with biomacromolecules. In addition, these natural spores with multiple therapeutic activities can be used as advanced materials to encapsulate a wide variety of medicaments, chemicals, and cosmetics. Here, we have explored for the first time natural spores as advanced materials to encapsulate biomacromolecules by three different microencapsulation techniques including passive, compression, and vacuum loading. The natural spore formulations developed by these techniques were extensively characterized with respect to size uniformity, shape, encapsulation efficiency, and localization of

biomacromolecules in the spores by confocal laser scanning microscopy (CLSM). We have also studied in-vitro release profiles of developed spore formulations in simulated gastric, intestinal fluids and also tunable release profile is demonstrated using vacuum-loaded spores. These results using extraordinary stable spore particles, provides the basis for further exploration on wide range of encapsulation materials.

6.1.1 Abstract

[00210] Natural lycopodium spores possess a very unique microstructure with uniformity in size and morphology, and are considered to comprise one of the most robust materials in the organic world. In addition, this material finds widespread use as a therapeutic material in herbal preparations and Chinese medicine for several human diseases. These natural spores were explored for the first time to encapsulate a model biomacromolecules by three different encapsulation techniques and were extensively characterized with respect to size uniformity, circularity, and encapsulation efficiency. FlowCam and SEM results confirmed uniform spores with a unique structure, and that this microstructure was retained after encapsulation with all three different techniques. Confocal laser micrographs revealed encapsulation of

biomacromolecules, in addition, tunable release has been achieved by various alginate coatings of spores to achieve several release profiles. This study provides a unique approach to utilize natural spores with unique materials properties, such as size uniformity and well-defined microstructures, as an advanced material for biomacromolecules encapsulation for controlled and targeted release applications.

6.1.2 Introduction

[00211] Plant based spores represent one form of natural encapsulation, and a wide range of specific plant species which produce spores are commonly found in the natural world. Γ 1 ' 21 Such natural packaging means are effective in protecting sensitive biological materials from environmental extremes in the form of prolonged desiccation, UV exposure, and predatory organisms. ^[3] A range of plants produce spores as a form of seed, which contains all the genetic material necessary to produce a new plant. ^[4' ^5] Such spores provide a ready-made capsule scaffold with high structural uniformity and a large internal cavity which may be used to encapsulate a wide range of materials. ^[6' ^7] Lycopodium clavatum is one species of the genus Lycopodium which produces spores and which has been identified to contain a range of promising phytochemicals for therapeutic applications ranging from stomach ailments to Alzheimer's disease. ^{[8 10]} Lycopodium spores provide a robust capsule structure and are commercially available in large quantities across globe. ^[6' ^7] Lycopodium spores are often used in traditional herbal medicine with a wide range of therapeutic benefits including improved osteogenesis, improved cognitive function,^[12] treatment of gastrointestinal disorders, ^[8] hepatoprotective activity,^[13] and antioxidative properties. ^[14] Recent studies demonstrated the use of processed lycopodium shells for encapsulation,^115"191 however, the production of lycopodium sporopollenin capsules requires the prolonged processing of natural spores with extreme chemical treatments at elevated temperatures, such that these resulting capsules are

Γ90-941

devoid of all other biological materials. ^" In many applications, this extensive processing may be unnecessary and potential therapeutic benefits may be lost. For applications in medicine, cosmetics, and food, enhanced effects may be obtained through the encapsulation of synergistic compounds, ^[25] and overall, the use of natural unprocessed spores provides significant benefits in terms of processing complexity and costs for a wide range of applications.

[00212] A major challenge in producing microencapsulated products is ensuring size monodispersity, ^[26'^27] which can have a large effect on drug release characteristics with respect to

Γ28 291

an intended target organ. ' In addition to size monodispersity, having well-defined microstructures plays an important role in exploring widespread applications. ^{[30 32]} Most conventional materials processing techniques used for encapsulation such as emulsion solvent evaporation, spray drying, and chemical conjugation fail to reliably provide either size

Γ26 27 30 321

monodispersity or well-defined microstructures. ' ' Although several studies have reported the use of empty exine microcapsules for the encapsulation of drugs, vaccines, and MRI contrast agents, as well as for use in cosmetics and food supplements,¹⁷'^16"18' ^22] the use of the natural 'spores' as a microencapsulation material and delivery vehicle still remains unexplored. In this regard, we have directed our efforts towards exploring systems to produce

biomacromolecule-loaded spores using three different microencapsulation techniques. The techniques we have developed to utilize natural spores are simple, cost effective, and versatile and can be applied to the development of several encapsulation products to overcome limitations of current encapsulated products while providing well-defined micromeritic properties. The specific scientific rationalities of the present work are i). Encapsulation of macromolecules into natural spores as biomaterials and the retention of natural spores constituents, ii).

Characterization of natural biomaterials with respect to size uniformity, shape and structure before and after encapsulation so as to provide valuable information on their potential usage as a pharmaceutical excipient. iii). Encapsulating macromolecules into natural spores so as to obtain uniform sized particles with well-defined micromeritic properties as a pharmaceutical encapsulating material, iv) Provide a feasible way to achieve a tunable release profile from macromolecule-loaded spores based on coating optimization intended to release drug in gastrointestinal tract. Hence, this study demonstrates the use of natural spores as a novel encapsulating material and this research provides a new dimension in the use of spores, which strongly supported by the use of lycopodium spores as plant-based medicine ^{[8 10]} for various ailments due to the intrinsic therapeutic benefits of spore constituents. In addition, our studies demonstrate that these medicinal spores can be encapsulated with molecules of interest for tailored applications.

[00213] Here, we have explored for the first time natural spores as advanced materials to encapsulate biomacromolecules by three different simple encapsulation techniques including passive, compression, and vacuum loading. The natural spores formulations developed by these techniques were extensively characterized with respect to size uniformity, shape, encapsulation efficiency, and localisation of biomacromolecules in the spores by confocal laser scanning microscopy (CLSM). We have also studied in-vitro release profiles of developed spore formulations in simulated gastric and intestinal fluids, further vacuum loaded formulations were chosen to achieve tunable release profiles by the use of alginate, a natural biomaterial, as a secondary coating material. Our encapsulation techniques involve the use of bovine serum albumin (BSA) as a model biomacromolecule to load into natural lycopodium spores.

6.1.3 Materials & Methods

6.1.3.1 Materials and chemicals

[00214] Natural lycopodium spores, bovine serum albumin (BSA), FITC-conjugated BSA, sodium alginate and other chemicals were purchased from Sigma (Singapore). Vectashield (H- 1000) medium was procured from Vector labs (CA, USA) and Sticky-slides, D 263 M Schott glass, No.l.5H (170 μπι, 25 mm x 75 mm) unsterile were procured from Ibidi GmbH (Munich, Germany).

6.1.3.2 Microencapsulation techniques to load macromolecule in to natural lycopodium spores

[00215] Encapsulation of macromolecules into natural lycopodium spores: Dissolve 75 mg BSA into 0.6 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg of spores in the BSA solution. Mix the suspension by vortexing (VWR, Singapore) for 5 min and transfer the tube to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4°C and 500 rpm for passive loading. In the case of compression loading, prepare a compressed tablet by using a hydraulic press at 5 ton pressure for 20 sec, soak the spore tablet in a BSA solution and allow for BSA uptake by the spore particles (Dimensions of spore tablets are provided in supporting

information). For the vacuum loading technique, use the BSA and spore particles suspension, slowly apply a 2 mbar vacuum in a freeze dryer (Labconco, MO, USA). Maintain the quantity of BSA, spore particles, and incubation time (2 hour) constant for all batches, and after incubation collect the BSA-loaded spore particles by centrifugation at 12000 rpm for 4 min and wash quickly using 0.5 ml water, then centrifuge to remove surface adhered BSA. Freeze the spores in a freezer at -70°C for 30 min and freeze dry for 24 hours, the final BSA-loaded lycopodium spores were stored at -20°C for further characterization. Prepare the placebo spores with the same procedure without BSA and preserve at -20°C.

[00216] Passive loading technique: Dissolve 75 mg BSA in to 0.6 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg natural spores into BSA solution. Mix the suspension by using vortex mixer (VWR, Singapore) for 5 min and transfer the tube to thermoshaker (Hangzhou Allsheng Inst. Singapore) set at 4°C, 500 rpm for 2 h incubation. Stop the process and collect the BSA-loaded spores by centrifugation at 12000 rpm for 4 min. Wash the spores quickly using 0.5 ml water and centrifuge to remove surface adhered BSA. Freeze the spores in freezer at -70°C for 30 min and freeze dry for 24 h. The resultant macromolecule loaded spores are stored in -20°C until further in-vitro characterizations. Prepare the placebo spores using similar procedure without BSA and preserve in -20°C.

[00217] Compression loading technique: Fill 150 mg natural spores into 12 mm die and compressed to form a tablet of around 10 - 12 mm dia. under hydraulic press to provide 5 ton load for 20 sec. using FTIR pellet maker. The dimensions of the spores tablet are mentioned in Table 2 and these tablets are soaked in 0.6 mL of 75 mg BSA containing aqueous solution in a 20 mL flat glass bottle for 2 h at 4°C to allow uptake of BSA molecules. Stop the process and collect BSA-loaded spores by centrifugation at 12000 rpm for 4 min. Wash quickly using 0.5 ml water and centrifuge to remove surface bound BSA. Freeze the spores in freezer at -70°C for 30 min and freeze dry for 24 h. The resultant spores are stored in -20°C until further

characterizations. Prepare the placebo spores with same procedure without BSA and preserve in -20°C. [00218] Table 2. Details of compressed lycopodium spore tablet^(a) used for compression technique

Weight Diameter of Thickness

of tablet of tablet

tablet⁰⁵' _(mm)(0 _(mm) (o

(mg)

126.77±6.44 13.29±0.02 1.47±0.02

^(a)Tablets used in BSA-loading by compression technique and results are mean of three batches (n=3) with standard deviation; ^<b)Weight determined in the Boeco BBX 22 (Germany) analytical balance;^(c) Diameter and thickness measured using digital vernier caliper.

[00219] Vacuum loading technique: Dissolve 75 mg BSA in to 0.6 mL purified water in a

1.5 mL centrifuge tube and suspend 150 mg spores, mix by using vortex mixer for 5 min. Apply vacuum at 2 mbar for 2 h using freeze drier. Stop the process and collect BSA-loaded lycopodium spores by centrifugation at 12000 rpm for 4 min. Wash quickly using 0.5 ml water and centrifuge to remove surface bound BSA freeze the spores in freezer at -70°C for 30 min and freeze dry for 24 h. The resultant particles are stored in -20°C until further characterization.

Prepare the placebo spores with same procedure without BSA and preserve in -20°C. In order to predict localization of BSA into natural lycopodium spores, FITC-conjugated BSA was encapsulated by three different techniques as mentioned in Section 6.1.3.2.

6.1.3.3 Characterizations of natural and macromolecule-loaded natural lycopodium spores

[00220] Dynamic image particle analysis by FlowCam (VS, Fluid Imaging Technologies, Maine, USA): Natural lycopodium spores and biomacromolecules-loaded spores with a pre -run volume of 0.5 mL (2 mg/ml) were primed manually into the flow cell. The focused spores were analyzed with a flow rate of 0.1 ml/min and a camera rate of 10 frames/sec leading to a sampling efficiency of about 9 %, and 1000 well focused spores were segregated by edge gradient ordering and manual processing. Representative data is plotted as a histogram and fitted with a Gaussian curve, and values are reported with standard deviations. (For a detailed description and calibration refer to the description in the next paragraph).

[00221] Dynamic image particle analysis by FlowCam®: Bench top system (FlowCamVS, Fluid Imaging Technologies, Maine, USA) was equipped with a 200 μπι flow cell (FC-200), a 20X magnification lens (Olympus®, Japan) and controlled by the visual spreadsheet software version 3.4.11. The system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/min and flow cell cleanliness was monitored visually before each sample run. Natural lycopodium spores and macromolecule-loaded spores (2 mg/ml) with a pre-run volume of 0.5 mL were primed manually into the flow cell and were analyzed with a flow rate of 0.1 ml/min, camera rate of 10 frames/s leading to a sampling efficiency of about 9 %. A minimum of 10,000 particles were fixed as count for each measurement and three separate measurements were performed and data analysis was carried out using highly focused 1000 spores segregated by edge gradient. Instrument was calibrated using polystyrene microspheres (50 ± 1 μπι,

Thermoscientific, USA) and representative data was plotted as histogram to fit Gaussian curve and values are reported with standard deviations (FIG. 2 and Table 3).

Table 3. Equivalent spherical diameter of natural spores and BSA-loaded spores

6.1.3.1 Lycopodium Spores Equivalent spherical diameter

(ESD^m ± SD) ^(a)

Natural spores before BSA loading 30.31 ± 1.87

Spores after passive technique 30.63 ± 1.92

Spores after compression technique 30.61 ± 1.92

Spores after vacuum technique 30.56 ± 1.88

[00222] Surface morphology evaluation by scanning electron microscopy (SEM): SEM imaging was performed using a FESEM 7600F (JEOL, Japan). Samples were coated with platinum at a thickness of 10 nm by using a JFC-1600 (JEOL, Japan) (20mA, 60 sec) and images were recorded by employing FESEM with an acceleration voltage of 5.00 kV at different magnifications to provide morphological information/to predict morphological observations.

[00223] Confocal laser scanning microscopy analysis: Confocal laser scanning

micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-Neofluar lOOx 1.3 oil objective M27 lens were used. Fluorescence from natural and macromolecule loaded lycopodium spores were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620. The laser scan speed was set at 67 sec per each phase (1024x1024:84.94 μπι sizes) and plane mode scanning with a 3.15 pixel dwell was used, and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany). See the next paragraph below for more details regarding the confocal laser scanning microscopy analysis.

[00224] Confocal laser scanning microscopy analysis of natural and macromolecule loaded natural spores: Confocal laser scanning micrographic analysis were done using a Carl Zeiss LSM700 (Germany) confocal microscopy equipped with three spectral reflected/fluorescence detection channels, six laser lines (405/458/488/514/543/633 nm) and connected to Zl inverted microscope (Carl Zeiss, Germany). Natural and macromolecule-loaded spores were mounted on sticky slides (Ibidi, Germany), a drop of mounting medium (Vectashield®) was added and spore particles were covered with another sticky slide. Images were collected immediately under the following conditions: laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-Neofluar 100X 1.3 oil objective M27 lens. Fluorescence from natural and macromolecule loaded spores were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620. The laser scan speed was set at 67 sec per each phase (1024x1024:84.94 μιη2 sizes) and plane mode scanning with 3.15 of pixel dwell. The iris was set as optimal for the sample conditions and all images were captured at mid of the particle (optical section) and other settings fixed same for all samples and at least three images were captured for each different sample and all images were processed at same conditions using ZESS 2008 software (ZEISS, Germany).

6.1.3.2 Encapsulation efficiency

[00225] Encapsulation efficiency: Suspend 5 mg BSA-loaded lycopodium spores in 1.4 mL PBS, vortex for 5 min and probe sonicate for 10 sec (3 cycles, 40 % amplitude). Filter the solution to collect extracted BSA using 0.45 μπι PES syringe filters (Agilent, USA). Measure the absorbance at 280 nm (Boeco-S220, Germany) using a placebo extract as a blank to compute the amount of BSA in the spore particles. In particular, measure the absorbance at 280 nm using placebo extract as blank to compute amount of BSA in the natural spores as below:

Amount of BSA (mg) = Absorbance x dilution factor

Slope (standard curve) x 1000

% Loading = Amount of BSA x 100

Weight of spores % Encapsulation efficiency = Practical Loading x 100

Theoretical loading

6.1.3.3 In-vitro drug release evaluation

[00226] In-vitro drug release studies: Suspend 5 mg of BSA-loaded lycopodium spores in pH 1.2 (0.1 M HCl), and phosphate buffer saline, pH 7.4, media. Incubate at 37°C at 50 rpm in an orbital shaker incubator (LM-400-D-220, Yihder, Taiwan), collect the release samples at specified time intervals by centrifugation at 14000 rpm for 30 sec, and replenish with fresh release media. Filter the release sample using PES syringe filters and measure absorbance at 280 nm. Compute the amount of BSA released using a BSA standard curve.

[00227] In-vitro drug release evaluation in simulated gastric fluid (0.1 M HCl, pH 1.2): Suspend 5 mg BSA-loaded spores and placebo in 1.4 ml media and incubate at 37°C, 50 rpm. Collect 1 ml release samples at specified time intervals by centrifugation at 14000 rpm 30 sec and replenish with fresh 1 ml release media. Filter the release sample using PES membrane filters (Agilent, USA) and measure absorbance at 280 nm using placebo as blank. Compute amount of BSA released using BSA standard curve.

[00228] In-vitro drug release evaluation in simulated intestinal fluid (PBS pH 7.4): Suspend 5 mg BSA-loaded spores and placebo in 1.4 ml media and incubate at 37°C, 50 rpm. Collect 1 ml release samples at specified time intervals by centrifugation at 14000 rpm 30 sec and replenish with fresh 1 ml release media. Filter the release sample using PES membrane filters (Agilent, USA) and measure absorbance at 280 nm using placebo as blank. Compute amount of BSA released using BSA standard curve.

6.1.3.4 Formulation of biomacromolecules-loaded spores for tunable release

[00229] 150 mg of BSA-loaded (vacuum loading) spores were mixed homogeneously with 1.5 ml of alginate (0.5 %, 1 %, 2 %) in water. The resulting suspension was slowly added to a 10 ml calcium chloride (8 %) solution using a blunt stain less steel 18 G needle under magnetic stirring, continue stirring for 5 minutes, and remove calcium chloride by centrifugation (1500 rpm, 2 minutes). Wash the beads twice using 1.5 ml water and freeze dry for 24 h. Store all the formulations in -20°C until further studies and use 10 mg of alginate coated lycopodium spores for in-vitro studies. 6.1.3.5 Statistical analysis

[00230] Statistical analysis was performed using two-tailed t -tests and P < 0.05 was considered as statistically significant. Encapsulations with natural spores and release experiments were repeated at least three times and all data are expressed as mean ± standard deviation (SD). 6.1.4 Results & Discussion

6.1.4.1 Macromolecule Encapsulation and Characterization of Natural Spores

[00231] FIGS. 1A-1E shows a schematic of the different encapsulation techniques developed to utilize natural spores as advanced encapsulating materials. Attractive features of our techniques include both versatility and simplicity with the potential to allow for application to a variety of small or large biomolecules under ambient processing conditions. FIG. 1A shows the origin of natural spores from the vascular plant lycopodium, these spores exhibit both well-defined size and microstructures. When these spores are suspended in a biomacromolecule solution (FIG. IB), the biomacromolecules enter the internal spore cavities through natural nanochannels in the spore wall of approximately 40 nm size.^[7] FIG. 1C depicts a biomacromolecule-loaded spore along with the spores natural cytoplasmic constituents. FIGS. ID, IE, and IF represent the three different microencapsulation techniques passive, compression, and vacuum loading, respectively. For the aforementioned three techniques, the spores are incubated in a biomacromolecule solution, with additional external forces being applied in the compression and vacuum processes for the encapsulation of biomacromolecules.

[00232] We have investigated natural spores with respect to uniformity of size, surface morphology, and physical changes arising during the course of the encapsulation process. In order to characterize natural spores, we employed dynamic imaging particle analysis (DIPA, FlowCam ) based on images captured using a high resolution digital camera (see materials & methods section). The size and morphology of natural spores are obtained based on digital signal processing of the highly focused images. In addition to size analysis, the spores images allow us to obtain additional information including edge gradient, circularity, and the shape of spore particles. Size, edge gradient, and circularity analysis by FlowCam was performed with an initial particle count of ~ 10,000 spore particles on all the batches of spores formulations and images were processed using FlowCam software to obtain 1000 highly focused spores. FIGS. 3A-3E show representative histogram data with Gausian curve fitting of equivalent spherical diameter (ESD) vs. Frequency, with an average ESD of 30.31 ± 1.87 μπι for (FIG. 3A) natural spores and an ESD of 30.63 ± 1.92 μπι, 30.61 ± 1.92 μπι, and 30.56 ± 1.88 μπι respectively for (FIG. 3B) passive, (FIG. 3C) compression, and (FIG. 3D) vacuum loaded spores.

[00233] The size uniformity and circularity of natural spores was supported by ESD data before and after biomacromolecule loading. The data is represented by curve fitting to histograms of circularity vs. frequency as shown in FIGS. 3A-3E. The shape of spores before and after BSA- loading are considered non-circular due to the characteristic microridges on spore surfaces with the resulting circularity value < 1 (ideal circle=l). The quality of the images used for data analysis is evident from the edge gradient vs. frequency data which indicates that well focused spores formulations were used during FlowCam analysis. In addition to these micromeritic data, FIGS. 3E, 3F, 3G, and 3H suggest the structural similarity of spores before loading, as well as after passive, compression, and vacuum loading techniques, respectively.

[00234] In the present study, natural spores are explored as novel encapsulating materials due to major benefits in utilizing these natural biomaterials for encapsulation research as well as cost- effective raw materials for large-scale industrial production. These spores provide distinct benefits compared to conventional materials due to: (i). Uniform size and monodispersity with well-defined micromeritic properties with no need for the formation of particles; (ii). Large scale raw material availability with consistent uniformity in an international market at a cost effective price of 25 USD per kg; (iii). Proven track record of human consumption of lycopodium spore capsules and their constituents for various therapeutic benefits; (iv). Robust capsule structure which has been shown to be stable under elevated temperatures, pressure and solvents, allowing for diverse encapsulation strategies of different molecules; and (v). Ready to use particles for encapsulation without the need for use of toxic organic solvents and which can be tailored for different bioactive release applications. In the past, encapsulation research has gained widespread importance due to the commercial potential of therapeutic molecules using various encapsulation materials such as natural polymer particles based mainly on chitosan, gelatin, starch, and sodium hyaluronate, and synthetic polymeric particles based mainly on poly(D,L- lactide-co-glycolide), polycaprolactone, polyanhydrides, and polyacrylates.^[26] In natural based polymers the use of solvents and toxic cross-linking agents is inevitable and may lead to toxic effects due to residual hazardous components. Natural based polymers are also predominantly of animal origin which leads to relatively higher processing costs to obtain raw materials. In addition, these conventional polymeric materials typically lack uniformity as well as defined microstructures. In the case of synthetic polymers, the use of solvents such as dichloromethane is a main prerequisite to develop particulate formulations and there is also a very high raw materials cost of 3000 to 10000 USD per kg in the international market. A further concern is that the acidic degradation products of PLGA based microspheres could potentially lead to deterioration of proteins, including denaturation, aggregation, and even chemical degradation. In addition, in the gastrointestinal tract a decrease in the pH value of the adjacent microenvironment may induce side effects such as inflammation. ^{[33 35]} Hence, it is essential to explore new biomaterials with versatile encapsulation strategies to meet the ever increasing demand for encapsulation biomaterials.

[00235] Further to characterize representative batches of spores, we utilized scanning electron microscopy (SEM) to examine any structural and morphological variations, and these images are displayed as FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D, respectively, for spores before loading, as well as after passive, compression, and vacuum loading techniques. Structural and morphological observations show that natural spores formulations have maintained their structural integrity without any denaturation, and exhibit size uniformity after biomacromolecule encapsulation using the three different microencapsulation techniques. Of note, as an encapsulation material, it is of the utmost importance to retain structural integrity after material processing at ambient temperatures. [36, 37]

[00236] The results from the quantitative determination of spore formulation yield after batch preparation in terms of loading and encapsulation efficiencies in spores are presented in Table 4, and a relatively higher batch yield was observed in the passive loading process due to one step passive loading as compared with multi-step compression and vacuum techniques. However, the encapsulation efficiency (EE) of passive and compression loadings are relatively similar, 42.7 ± 3.7% and 42.8 ± 1.1 %, whereas with the vacuum loading process a statistically significant (p < 0.05) higher EE of 59.2 ± 2.2 % was achieved.

Table 4. BSA-loaded lycopodium spores: formulation parameters^(a)

Lycopodium spores Theoretical BSA Weight of formulation BSA BSA

loading^(b) after lyophilization loading (%) Encapsulation

(%) (mg) efficiency^(c)

(%)

Passive filling 50 160.04 ± 7.04 21.3 ± 1.8 42.7 ± 3.7 Compression filling 50 137.31 ± 3.52 21.4 ± 0.5 42.8 ± 1.1

Vacuum filling 50 102.56 ± 4 23 29.6 ± 1.1 59.2 ± 2.2

^(a)Results are the mean of three batches (n=3) with standard deviation; ^{( )}Theoretical loading is based on 50% weight of natural lycopodium spores: ^<c) BSA encapsulation efficiency is determined using 5 mg of BS A-loaded natural spore particles.

[00237] Follow by the morphological characterization, we investigated the natural spores formulation with the aim to observe the localization of biomacromolecules within the natural spores. We encapsulated FITC-conjugated BSA into natural spores by the three aforementioned techniques and performed analysis using confocal laser scanning microscopy (CLSM). All the images were captured focusing on the middle section of spores mounted between thin glass slides and embedded within vectashield at lOOx with fixed settings (see experimental section). Confocal microscopic images of natural lycopodium spores before macromolecule loading are presented in FIG. 5A. It is evident from the row in FIG. 4A that no green color resulting from FITC-BSA was observed with spores before loading, whereas the blue and red channels show strong autofluorescence from the spore constituents which is even more evident from their overlay image. This autofluorescence can be attributed to the presence of sporoplasm

constituents. ^[38' ^39]

[00238] In the case of FITC-BSA loading by the passive technique (FIG. 5B), a strong green fluorescence was observed, confirming biomacromolecule loading into the natural spores, and is clearly evident from the overlay of all channels indicating FITC-BSA along with the natural spore constituents. In the case of the compression loading of biomacromolecules (FIG. 5C), we also observed a bright green fluorescence. However, it is important to note that the structural integrity and spores constituents are retained after compression at 5 ton for 20 sec. Relatively higher fluorescence is observed in the case of vacuum loading (FIG. 5D) and this supports our assertion that higher encapsulation efficiency may be obtained with the application of an external vacuum force in addition to incubation in a biomacromolecule solution. Although there appears to be a small amount of surface adhered FITC-BSA in the passive loading green channel image, and a thin layer of FITC-BASA may be adhered to the microridge structures of all loaded spores, all together, these data confirm that the significant majority of macromolecules are loaded inside the natural spore cavities and not in the reticulum chambers walled by the muri. To further support FITC-BSA localization inside the spore cavity, Z-stack images were provided in FIGS. 6 A and 6B. [00239] Plant based medicines have played a remarkable role as the main treatment option from ancient times and until now, these traditional plant-derived medicines play a crucial role as the primary form of healthcare in many developing countries. In addition, this has also led to the discovery of novel drug candidates for a variety of diseases that affect humankind. Lycopodium clavatum, known as club moss, is the most widespread species in the genus Lycopodium of the family Lycopodiaceae . Extractions of crushed spores from lycopodium are being used as a plant based medicine mainly for liver dysfunction, as well as urinary and digestive disorders. The medicinal effect of lycopodium is also reported to improve memory function and cerebral blood flow in memory impaired rat model. Specifically, in homeopathic Materia Medica,

Lycopodium is stated to have therapeutic effects on biliary stones and liver failure, and due to its diverse importance, application of these spores is emerging as a potential new treatment modality in health care. These proven therapeutic benefits have led to the commercialization of lycopodium based oral herbal formulations for the treatment of diverse health conditions such as anxiety, albuminuria, constipation, dysentery, gallstones, heartburn, hemorrhoids, impotence, indigestion, irritability, prostatitis, renal colic, and rheumatism. The therapeutic activity of these spore constituents are supported by bioassay-guided studies on Lycopodium clavatum indicating that a major component, a lycopodine alkaloid is responsible for the anti-inflammatory activity of the extract for wound-healing applications. ^[40] As other industrial applications, these spores have also been used as a dry powder in latex gloves and condom manufacturing. From this, it should be noted that there were several reports indicating cases of occupational allergy associated with a risk of sensitization to lycopodium spores. However, it has been proposed that this was probably due to daily prolonged exposure to a place where spores were highly abundant and that the spores may have also been carriers of allergenic latex proteins. ^[41] Previous reports indicate that lycopodium allergies are very rare and in clinical cases are never diagnosed due to a lack of allergenic symptoms and these often involve local treatment. ^[42] In particular, a review of 61 clinical cases of rhinitis or skin reactions associated with lycopodium extracts indicates that the majority of cases were weakly positive and without clinical significance. ^[42] In our study the lycopodium spores based formulations involve quantities of spores as low as 5 mg for bioactive release as safe oral formulations.

6.1.4.2 Modulating Macromolecules Release from Natural Spores [00240] We have demonstrated natural spores as fascinating materials for the encapsulation of various molecules with BSA as a model protein. We further studied in-vitro release profiles of BSA from these natural spores separately in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.4) conditions. The in-vitro release of BSA in SGF

(FIG. 7A) indicates 90% biomacromolecules release in the first 5 minutes and complete release was observed in 30 to 60 minutes. There was no significant difference among the release from BSA-loaded spores prepared using different techniques (p > 0.05). In case of intestinal conditions (FIG. 7B), a similar burst release was observed with spore formulations prepared by three different loading techniques suggesting no significant release differences in simulated gastric and intestinal conditions. The observed release trend indicates fast release in both simulated conditions and is evident due to the high aqueous solubility of BSA resulting in rapid release from nanodomains of natural spores. This supports our findings on natural spores which indicate a lack of a barrier to retard biomacromolecules release in SGF and SIF for a longer period of time.¹⁷' ¹⁶' ^18] Similar drug release profiles were reported with the use of processed sporopollenin microcapsules of lycopodium clavatum spores. ^[16' ^18] To explore the structural changes after FITC-conjugated BSA release in PBS, we performed CLSM after complete release and the results indicate the absence of any green colour in the spores confirming the complete release of FITC-BSA from natural spores (see FIGS. 8A-8C). However, a low intensity green colour is observed surrounding exine shell of the spores with external FITC-BSA binding and interestingly the autofluorescence of these natural spores was retained after processing for encapsulation and release. These observations clearly indicate the intact sporoplasm in the natural spores.

[00241] Further, in order to achieve sustained tunable release, we have optimized the process to incorporate biomacromolecules-loaded natural spores into alginate microbeads. These microbeads were prepared by using vacuum loaded spores in different concentrations of alginate solutions viz., 0.5%, 1%, and 2% (w/v). We have investigated the release profiles from natural spores with varying amount of alginates in SGF and SIF and results are presented in FIG. 7C and FIG. 7D, respectively. In the case of SGF, the release profiles from natural spores indicate a systematic release trend based on the amount of alginate used in the formulations. There was a higher burst release with 0.5% alginate formulations compared to 1% and 2% alginate, also sustained release up to 8 h without any burst effect was achieved using natural spores in 2% alginate beads. In SIF, the tunable release was more prominent with varying amount of alginate up to 8 h, representing the gastrointestinal transit time. Statistical analysis of biomacromolecules release from natural spores was performed separately in SGF and SIF and results indicate significant differences (p < 0.05) among the various formulations. In addition, a significant difference (p < 0.05) in release profiles was achieved after alginate coating using 1% and 2% alginate. It is crucial to study in-vitro release profile in both simulated gastric and intestinal conditions and in particular the observed macromolecule release from spores indicates pH independent release profiles. With further development such spores based formulations could be highly beneficial in oral dosage formulations for drugs targeting gastrointestinal diseases which involve varied absorption windows and require drug release in different parts of the GI tract.^[43' ^44] In addition, the large internal cavity of these natural spores provides high therapeutic loading and release in the gastrointestinal tract.

[00242] In order to evaluate the structure of the microbeads after alginate coating, SEM analysis was performed on 0.5%, 1%, and 2% alginate coated spores, and the results are depicted in FIGS. 9A-9C. Interestingly, spores were fused inside alginate microbeads and surface analysis reveals spores were intact with variable layers of coating with 0.5%, 1%, and 2% alginate. These morphological observations further support the concentration-dependent release profiles, which indicate that alginate acts as barrier to retard macromolecule release from spores. Hence, our results provide a way to tune the biomacromolecules release from natural lycopodium spores by varying the amount of alginate. Such release profiles are crucial in targeting the drug absorption window in the human gastrointestinal track^[43' ^45] to aid developments in new oral therapeutics for new disease targets.

6.1.5 Conclusion

[00243] In conclusion, we have successfully demonstrated the encapsulation of

biomacromolecules into natural spores by three different microencapsulation techniques viz., passive, compression and vacuum loading. Significant encapsulation was achieved using vacuum loading techniques and encapsulation of macromolecules was confirmed by laser scanning microscopy by localization of FITC-BSA in the natural spores. The surface characterization and dynamic image particle analysis (DIPA) revealed higher uniformity in size of all the developed formulations along with well-defined microridges. The demonstrated potential of natural spores for encapsulation applications provides motivation for their further exploration as delivery vehicles for various agents, including small molecules, peptides, and cosmetics. Indeed, different natural spores have unique architectures that are formed by natural bio-templating processes and their precise uniformity and robust stability make these ideal biomaterials for encapsulation purposes, especially in light of our environmentally friendly encapsulation strategies which bypass the need for harsh chemical treatments. Importantly, we have also demonstrated that the release of biomacromolecules from within Lycopodium spores can be controlled by forming a biocompatible alginate coating as a tunable profile. In the future, the integration of natural and synthetic biomaterials offers rich opportunities to develop multifunctional delivery platforms for both medical and biotechnology applications.

6.1.6 References Cited in Example 1

1. Stanley, R.G. and H.F. Linskens, Pollen: biology, biochemistry, management. 1974:

Berlin: Springer- Verlag 307p.. Illustrations. Palynology (KR, 197506433).

2. Aya, K., et al., The Gibberellin perception system evolved to regulate a pre-existing GAMYB-mediated system during land plant evolution. Nature communications, 2011. 2: p. 544.

3. Ariizumi, T. and K. Toriyama, Genetic regulation of sporopollenin synthesis and pollen exine development. Annual review of plant biology, 2011. 62: p. 437-460.

4. Wang, A., et al., The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proceedings of the National Academy of Sciences, 2003. 100(24): p. 14487-14492.

5. Knox, R. and J. HESLOP-HARRISON, Pollen-wall proteins: localization and enzymic activity. Journal of cell science, 1970. 6(1): p. 1-27.

6. Gaonkar, A.G., et al., Microencapsulation in the Food Industry: A Practical Implementation Guide. 2014: Elsevier.

7. Diego-Taboada, A., et al., Hollow Pollen Shells to Enhance Drug Delivery.

Pharmaceutics, 2014. 6(1): p. 80-96.

8. Banerjee, J., et al., A better understanding of pharmacological activities and uses of phytochemicals of Lycopodium clavatum: A. Journal of Pharmacognosy and Phytochemistry, 2014. 3(1): p. 207-210.

9. Bai, F., et al., Free energy landscape for the binding process of Huperzine A to acetylcholinesterase. Proceedings of the National Academy of Sciences, 2013. 110(11): p. 4273-4278.

10. Yu, D., et al., Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A. Proceedings of the National Academy of Sciences, 2013. 110(8): p. E746-E755.

11. Wang, C, et al., Effect of a novel compound from Lycopodium obscurum L. on osteogenic activity of osteoblasts in vitro. 2013.

12. Hanif, K., et al., Effect of homeopathic Lycopodium clavatum on memory functions and cerebral blood flow in memory-impaired rats. Homeopathy, 2015. 104(1): p. 24-28.

13. da Silva, G.H., et al., Hepatoprotective effect of Lycopodium clavatum 30CH on experimental model of paracetamol-induced liver damage in rats. Homeopathy, 2014. Durdun, C, et al., Antioxidant potential of Lycopodium clavatum and Cnicus benedictus hydroethanolic extracts on stressed mice. Scientific Works-University of Agronomical Sciences and Veterinary Medicine, Bucharest Series C, Veterinary Medicine, 2011. 57(3): p. 61-68.

Archibald, S.J., et al., How does iron interact with sporopollenin exine capsules? An X- ray absorption study including microfocus XANES and XRF imaging. Journal of Materials Chemistry B, 2014.2(8): p. 945-959.

Atwe, S.U., Y. Ma, and H.S. Gill, Pollen grains for oral vaccination. Journal of Controlled Release, 2014.194: p. 45-52.

Ma, H., et al., Preparation of a novel rape pollen shell microencapsulation and its use for protein adsorption and pH-controlled release. Journal of microencapsulation, 2014. 31(7): p. 667-673.

Diego-Taboada, A., et al., Protein free microcapsules obtained from plant spores as a model for drug delivery: Ibuprofen encapsulation, release and taste masking. Journal of Materials Chemistry B, 2013.1(5): p. 707-713.

Barrier, S., et al., Sporopollenin exines: A novel natural taste masking material. LWT- Food Science and Technology, 2010.43(1): p. 73-76.

Diego-Taboada, A., et al., Sequestration of edible oil from emulsions using new single and double layered microcapsules from plant spores. Journal of Materials Chemistry, 2012.22(19): p. 9767-9773.

Wakil, A., et al., Enhanced bioavailability of eicosapentaenoic acid from fish oil after encapsulation within plant spore exines as microcapsules. Lipids, 2010.45(7): p. 645- 649.

Lorch, M., et al., MRI contrast agent delivery using spore capsules: controlled release in blood plasma. Chem. Commun., 2009(42): p. 6442-6444.

Paunov, V.N., G. Mackenzie, and S.D. Stoyanov, Sporopollenin micro-reactors for in- situ preparation, encapsulation and targeted delivery of active components. Journal of Materials Chemistry, 2007.17(7): p. 609-612.

Barrier, S., et al., Viability of plant spore exine capsules for microencapsulation. Journal of Materials Chemistry, 201 1.21(4): p. 975-981.

Gertsch, J., Botanical drugs, synergy, and network pharmacology: forth and back to intelligent mixtures. Planta Medica-Natural Products and MedicinalPlant Research, 2011. 77(1 1): p. 1086.

Mundargi, R.C., et al., Nano/micro technologies for delivering macromolecular therapeutics using poly (D, L-lactide-co-glycolide) and its derivatives. Journal of Controlled Release, 2008.125(3): p. 193-209.

Zhang, H., et al., Fabrication of a Multifunctional Nano - in - micro Drug Delivery Platform by Microfluidic Templated Encapsulation of Porous Silicon in Polymer Matrix. Advanced Materials, 2014.26(26): p. 4497-4503.

Jamil, F., et al., Review on stomach specific drug delivery systems: development and evaluation. IJRPBS, 201 1.2(4): p. 1427-1433.

Satwara Rohan S, P.S.C., Jivani Rishad R, Patel Parul K, Pancholi S S, Formulation Approaches to Enhance the Bioavailability of Narrow Absorption Window Drugs. Inventi Rapid: Pharm Tech, 201 1(3). Lee, W., et al., The reliable targeting of specific drug release profiles by integrating arrays of different albumin-encapsulated microsphere types. Biomaterials, 2009. 30(34): p. 6648-6654.

Fattahi, P., A. Borhan, and M.R. Abidian, Microencapsulation of chemotherapeutics into monodisperse and tunable biodegradable polymers via electrified liquid jets: Control of size, shape, and drug release. Advanced Materials, 2013. 25(33): p. 4555-4560.

Duncanson, W.J., et al., Microfluidic synthesis of advanced microparticles for encapsulation and controlled release. Lab on a Chip, 2012. 12(12): p. 2135-2145.

Wang, M., et al., A spheres -in- sphere structure for improving protein-loading poly (lactide-co-glycolide) microspheres. Polymer Degradation and Stability, 2010. 95(1): p. 6-13.

Lee, E.S., et al., Protein complexed with chondroitin sulfate in poly (lactide-co-glycolide) microspheres. Biomaterials, 2007. 28(17): p. 2754-2762.

Chen, X., et al., Preparation and properties of BSA-loaded microspheres based on multi- (amino acid) copolymer for protein delivery. International journal of nanomedicine, 2014. 9: p. 1957.

Ma, G., Microencapsulation of protein drugs for drug delivery: Strategy, preparation, and applications. Journal of Controlled Release, 2014. 193: p. 324-340.

Anal, A.K. and H. Singh, Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science & Technology, 2007. 18(5): p. 240-251.

Mitsumoto, K., K. Yabusaki, and H. Aoyagi, Classification of pollen species using autofluorescence image analysis. Journal of bioscience and bioengineering, 2009. 107(1): p. 90-94.

oshchina, V. and V. Karnaukhov, Changes in pollen autofluorescence induced by ozone. Biologia plantarum, 1999. 42(2): p. 273-278.

Orhan, I., et al., Appraisal of anti-inflammatory potential of the clubmoss, Lycopodium clavatum L. Journal of ethnopharmacology, 2007. 109(1): p. 146-150.

Rask - Andersen, A., et al., Asthma, skin symptoms, and allergy in a condom factory. Allergy, 2000. 55(9): p. 836-841.

SALEN, E.B., LYCOPODIUM ALLERGY1. Allergy, 1951. 4(4): p. 308-319.

Ensign, L.M., R. Cone, and J. Hanes, Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Advanced drug delivery reviews, 2012. 64(6): p. 557-570.

MacAdam, A., The effect of gastro-intestinal mucus on drug absorption. Advanced drug delivery reviews, 1993. 11(3): p. 201-220.

McEntee, M.K.E., et al., Tunable transport of glucose through ionically - crosslinked alginate gels: Effect of alginate and calcium concentration. Journal of applied polymer science, 2008. 107(5): p. 2956-2962. EXAMPLE 2: NATURAL SUNFLOWER POLLEN AS A DRUG DELIVERY

VEHICLE [00244] In this example bovine serum albumin (BSA) was loaded into natural Helianthus annuus (sunflower) pollen employing the same three encapsulation techniques (passive, compression, and vacuum loading) .

6.2.1 Introduction

[00245] The fabrication of uniform micron-scale capsules with complex architectures is a long-standing goal towards successful materials encapsulation strategies. Γ 1 ' 21 While various fabrication approaches have been devised in the past few decades, the biosynthetic capabilities of nature have evolved over much longer time scales to produce large quantities of exquisitely complex microcapsules with high fidelity. One such example includes micron-scale pollen grains, which encapsulate sensitive biological materials for long durations under harsh conditions. ^[3'^4] In nature, pollen grains ensure the reproductive capabilities of plants by protecting nucleic acids and other genetic materials from unfavorable environmental conditions such as prolonged desiccation, high temperatures, ultraviolet light, and microbial damage. ^[5] The genetic material is stored within the cytoplasmic core of the pollen grain and surrounded by a double layer shell consisting of an in tine and an exine layer. ^[6'^7] The outermost exine layer contains the sporopoUenin biopolymer, which is considered to be one of nature's most resilient materials. ^[8'^9] With growing recognition of pollen's unique material properties along with its organic production and simple collection, there has been renewed interest in exploring pollen and its components as promising biomaterials.

[00246] In recent years, the robust exine capsule, isolated from pollen grains and plant spores, has demonstrated strong potential as a delivery vehicle for the encapsulation of various materials including pharmaceutical drugs, vaccines, contrast agents, and oils.^{[10 21]} In the materials encapsulation strategies explored thus far, high-purity exine capsules are required and obtained through vigorous and prolonged processing of pollen grains or spores under harsh conditions using organic solvents, acids at elevated temperatures, or systematic exposure to numerous enzymes. ^[22] Indeed, it is commonly assumed that "It is possible to attach drugs to the outside of complete [pollen] particles, but the loading is restricted and the drug receives little protection and its release is relatively uncontrolled. "^[12] At the same time, as part of the natural pollen cycle, both the sporopoUenin exine and cellulosic intine layers are permeable and undergo dehydration and hydration which facilitates materials loading as the surrounding fluid is drawn into the internal pollen cavity. ^{[23 26]} This suggests that materials encapsulation inside unprocessed pollen grains should be possible provided a suitable encapsulation route is identified. The potential advantages of natural sunflower pollen grains as a drug delivery vehicle are enormous; (1) Proven track record as safe for human oral consumption due to use as a biosupplement and in herbal medicine. (2) Common constituent of 'bee pollen' for human consumption for nutritional and therapeutic benefits. (3) Economical raw material for materials encapsulation without the need for the use of organic solvents and harsh ultrasonication or homogenisation conditions. (4) Unique microstructure with uniform size distribution and inner cavity. The primary disadvantage of natural sunflower pollen grains is the presence of pores which result in rapid drug release, however this can be addressed with suitable polymer coating techniques to modulate the release depending on the need to target different regions of the gastrointestinal track.

[00247] Motivated by the biological function of pollen grains, we hypothesize that encapsulation strategies can be devised to load biomacromolecules into natural pollen grains. This approach would drastically reduce processing requirements for materials encapsulation in exine-reinforced microcapsules while also taking advantage of the innate therapeutic benefits of natural pollen, which is often used as a dietary supplement and in traditional herbal

medicine. Γ27 ' 281 In particular, multifloral bee collected pollen, of which Helianthus annuus (sunflower) is a common constituent, ^[29'^30] has been associated with a wide range of nutritional and therapeutic effects. Γ27 ' 281

[00248] As proof-of-concept to test our hypothesis, we investigated natural, unprocessed sunflower pollen grains as microscale materials for the efficient encapsulation of

biomacromolecules. Through the comparison of three different encapsulation strategies (passive hydration, hydraulic compression, and vacuum-assisted), we demonstrate multiple routes to achieve high-efficiency protein loading with bovine serum albumin (BSA) as a model biomacromolecule. Importantly, the methods used are environmentally friendly and preserve the complex architecture of natural pollen grains, including size, uniformity, and surface features. Furthermore, we demonstrate that a controlled release profile is achievable by encapsulating pollen grains inside alginate hydrogel beads. Taken together, our findings offer compelling evidence that natural pollen grains are excellent drug delivery vehicles.

6.2.2 Materials & Methods

6.2.2.1 Materials and chemicals [00249] Natural sunflower pollen grains (defatted) were procured from Greer Labs (NC,

USA), bovine serum albumin (BSA), FITC-conjugated BSA and other reagents were purchased from Sigma (Singapore). Vectashield (H-1000) medium was procured from Vector labs (CA,

USA) and Sticky-slides, D 263 M Schott glass, No.l.5H (170 μτη, 25 mm x 75 mm) unsterile were procured from Ibidi GmbH (Munich, Germany).

6.2.2.2 Microencapsulation techniques to load macromolecule (BSA) into natural sunflower pollen grains

[00250] Encapsulation of macromolecules into natural pollen grains: Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg of natural pollen grains in the BSA solution. Mix the suspension by vortexing (VWR, Singapore) for 5 min and transfer the tube to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4°C and 500 rpm for passive loading. In the case of compression loading, prepare compressed tablet by using a hydraulic press at 5 ton pressure for 20 sec, soak the tablet in BSA solution and allow for BSA uptake by the pollen grains (Dimensions of compressed tablets are provided in supporting information). The 5 ton compression pressure employed is able to retain intact sunflower pollen structure with some portion of pollen cytoplasmic constituents, as indicated by red and blue channel CLSM autofluorescence. For the vacuum loading technique, use a BSA and pollen grains suspension, and slowly apply a 2 mbar vacuum in a freeze dryer (Labconco, MO, USA). Maintain the quantity of BSA, pollen grains and incubation time (2 hour) constant for all the batches, and after incubation collect the BSA-loaded pollen grains by centrifugation at 12000 rpm for 4 min and wash using 0.5 ml water, then centrifuge to remove surface adhered BSA. Freeze the pollen grains in a freezer at -70°C for 30 min and freeze dry for 24 hours, store the final BSA-loaded pollen grains at -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve at -20°C. See the following paragraphs for additional details regarding the methods.

[00251] Passive filling: Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg natural pollen grains into BSA solution. Mix the suspension by vortex (VWR, Singapore) for 5 min and transfer the tube to thermoshaker

(Hangzhou Allsheng Inst. Singapore) at 4°C, 500 rpm for 2 h. Stop the process and collect the BSA-loaded particles by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface adhered BSA. Freeze the pollen grains in a freezer at -70°C for 30 min and freeze dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C.

[00252] Compression filling: Fill 150 mg natural sunflower pollen grains into 12 mm die and compress to form a tablet of around 10 - 12 mm dia. under hydraulic press to provide 5 ton load for 20 sec. using FTIR pellet maker. The pellet formed in this method is soaked in 0.5 mL of 75 mg BSA containing aqueous solution in 20 mL flat glass bottle for 2 h at 4°C to allow swelling of pollen grains thereby BSA is entrapped in the pollen grains. Stop the process and collect the BSA-loaded particles by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA. Freeze the pollen grains in a freezer at - 70°C for 30 min and freeze-dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C.

Table 5. Details of compressed natural sunflower pollen tablet ^<a)

Weight of Diameter of Thickness of

tablet (mg) ^(b) tablet^(c) (mm) tablet^(c) (mm)

127.73 ± 2.20 12.99 ± 0.02 0.98 ± 0.02

^(a)Tablets used in BSA-loading by compression technique and results are mean of three batches (n=3) with standard deviation; ^<b)Weight determined in the Boeco (Germany) balance with accuracy of 1 mg; ^<c)Diameter and thickness measured using digital vernier caliper.

[00253] Vacuum filling: Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL centrifuge tube and suspend 150 mg pollen grains and vortex for 5 min to homogenize. Apply a vacuum at 2 mbar for 2 h using a freeze-drier. Stop the process and collect BSA-loaded pollen grains by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA, then freeze the pollen grains in a freezer at -70°C for 30 min and freeze dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C.

[00254] In order to predict localization of BSA into natural pollen grains, FITC-conjugated BSA was encapsulated by three different techniques as mentioned in section 1.1. with a batch size of 22.5 mg containing 7.5 mg FITC-BSA per batch of natural pollen grains. 6.2.2.3 Characterization of natural and macromolecule-loaded natural pollen grains

[00255] Dynamic image particle analysis by FlowCam : FlowCam VS benchtop system (FlowCam^® VS, Fluid Imaging Technologies, Maine, USA). 0.5 mL (2 mg/ml) of natural sunflower pollen grains and macromolecule-loaded grains with a pre -run volume of 0.5 mL (primed manually into the flow cell) were analyzed with a flow rate of 0.1 ml/min and a camera rate of 10 frames/s leading to a sampling efficiency of about 9 %, and 1000 highly focused pollen grains were segregated by edge gradient ordering. Representative data is plotted as a histogram and fitted with a Gaussian curve and values are reported with standard deviations. (For a detailed description refer to the next paragraph).

[00256] Dynamic image particle analysis by FlowCam®: FlowCam VS benchtop system (FlowCam® VS, Fluid Imaging Technologies, Maine, USA) was equipped with a 200 μπι flow cell (FC-200), a 20X magnification lens (Olympus®, Japan) and controlled by the visual spreadsheet software version 3.4.11. The system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/min and flow cell cleanliness was monitored visually before each sample run. Natural sunflower pollen grains and macromolecule-loaded grains of 0.5 mL (2 mg/ml) with a pre -run volume of 0.5 mL (primed manually into the flow cell) were analyzed with a flow rate of 0.1 ml/min and a camera rate of 10 frames/s leading to a sampling efficiency of about 9 %. A minimum of 10,000 particles were fixed as the count for each measurement and three separate measurements were performed and data analysis was carried out using 1000 well focused pollen grains segregated by edge gradient. The instrument was calibrated using polystyrene microspheres (50 ± 1 μπι, Thermoscientific, USA)

Representative data was plotted as a histogram and fitted with a Gaussian curve and values are reported with standard deviations. The DIPA uses a high-resolution digital camera and objective lens to capture images of the particles flowing through a thin transparent flow cell. Particle size uniformity data is then generated based on digital signal processing of the images. Besides size determination, the digital particle images allow us to obtain additional information including edge gradient, circularity, and the shape of pollen grains. Size, edge gradient and circularity analysis by FlowCam was performed with an initial particle count of 10,000 pollen grains for all the batches of pollen formulations and images were processed using the FlowCam software to obtain 1000 well focused pollen grains. Table 6. Equivalent spherical diameter of natural sunflower pollen and BSA-loaded pollen grains

Sunflower pollen grains Equivalent spherical

diameter (ESD, μπι ± SD)

Natural pollen before BSA-loading 37.93 ± 1.41

BSA-loaded pollen by passive 36.54 ± 1.45

BSA-loaded pollen by vacuum 36.17 ± 1.36

BSA-loaded pollen by compression 36.95 ± 1.35

[00257] Surface morphology evaluation by scanning electron microscopy (SEM): SEM imaging was performed using a FESEM 7600F (JEOL, Japan). Samples were coated with platinum at a thickness of 10 nm by using a JFC-1600 (JEOL, Japan) (20mA, 60 sec) and images were recorded by employing FESEM with an acceleration voltage of 5.00 kV at different magnifications to provide morphological information/ observe morphological characteristics.

[00258] Confocal laser scanning microscopy analysis: Confocal laser scanning

micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-NeofluarlOOxl.3 oil objective M27 lens were used. Fluorescence from natural and macromolecule loaded pollen grains were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620. The laser scan speed was set at 67 sec per each phase (1024x1024:84.94 μπι sizes) and plane mode scanning with a 3.15 pixel dwell was used and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany). See the next paragraph for additional information regarding the confocal laser scanning microscopy analysis.

[00259] Confocal laser scanning microscopy analysis of natural and macromolecule loaded natural pollen grains: Confocal laser scanning micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope equipped with three spectral

reflected/fluorescence detection channels, six laser lines (405/458/488/514/543/633 nm) and connected to Zl inverted microscope (Carl Zeiss, Germany). Natural and macromolecule-loaded pollen grains were mounted on sticky slides (Ibidi, Germany), a drop of mounting medium (Vectashield®) was added and pollen grains were covered with another sticky slide. Images were collected immediately under the following conditions: laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-Neofluar lOOx 1.3 oil objective M27 lens. Fluorescence from natural and macromolecule loaded pollen grains were collected in

photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620. The laser scan speed was set at 67 sec per each phase (1024x1024: 84.94 μιη2 sizes) and plane mode scanning with 3.15 of pixel dwell. The iris was set as optimal for the sample conditions and all images were captured at the mid region of the particle (optical sections) and other settings were fixed the same for all samples and at least three images were captured for each different sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany).

6.2.2.4 Encapsulation efficiency:

[00260] Suspend 5 mg BSA-loaded pollen grains in 1.4 mL PBS, vortex for 5 min and probe sonicate for 10 sec (3 cycles, 40 % amplitude). Filter the solution to collect the extracted BSA using 0.45 μπι PES syringe filters (Agilent, USA). Measure the absorbance at 280 nm (Boeco- S220, Germany) using a placebo extract as a blank to compute the amount of BSA in the pollen grains.

[00261] Suspend 5 mg BSA-loaded SP in 1.6 mL PBS vortex for 5 min and probe sonication for 10 sec (3 cycles, 40 % amplitude). Filter the solution to collect extracted BSA using 0.45 μπι PES syringe filters. Measure the absorbance at 280 nm using a placebo extract as a blank to compute the amount of BSA in the pollen grains as below:

Amount of BSA (mg) = Absorbance x dilution factor

Slope (standard curve) x 1000

% Loading = Amount of BSA x 100

Weight of pollen grains

% Encapsulation efficiency = Practical loading x 100 Theoretical loading

6.2.2.5 In vitro Drug Release Studies

[00262] In-vitro drug release studies: Suspend 5 mg of BSA-loaded pollen grains and placebo in pH 1.2 (0.1 M HC1), and phosphate buffer saline, pH 7.4, media. Incubate at 37°C at 50 rpm in an orbital shaker, collect the release samples at specified time intervals by

centnfugation at 14000 rpm for 30 s, and replenish with fresh release media. Filter the release sample using PES filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.

[00263] In-vitro drug release evaluation in simulated gastric fluid (0.1 M HCl, pH 1.2): In- vitro drug release evaluation in simulated intestinal fluid (pH 1.2): Suspend 5 mg BSA-loaded pollen grains and placebo in pH 1.2 media. Incubate at 37°C at 50 rpm and collect the release samples at 5 min, 15 min and 30 min by centrifugation at 14000 rpm for 30 sec, replenish with fresh release media and continue the release study. Filter the release sample using PES membrane filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.

[00264] In-vitro drug release evaluation in simulated intestinal fluid (PBS pH 7.4):

Suspend 5 mg BSA-loaded pollen grains and placebo in pH 7.4 media. Incubate at 37°C at 50 rpm and collect the release samples at 5 min, 15 min and 30 min by centrifugation at 14000 rpm for 30 sec, replenish with fresh release media and continue the release study. Filter the release sample using PES membrane filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.

6.2.2.6 Formulation of macromolecule-loaded natural pollen grains into alginate beads:

[00265] 150 mg of BSA-loaded (vacuum loading) pollen grains were mixed homogeneously with 1.5 ml of alginate (2 %) in water. The resulting suspension was slowly added to 10 ml calcium chloride (8 %) using a blunt 18 G needle under magnetic stirring, continue stirring for 5 minutes, and remove calcium chloride by centrifugation (1500 rpm, 2 minutes). Wash the beads twice using 1.5 ml water and freeze dry for 24 h. Store all the formulations in -20°C for further studies and use 10 mg of alginate coated pollen grains for in-vitro studies. Please see the paragraph below for additional information.

[00266] In order to control the macromolecule release from natural pollen grains, these are incorporated into alginate beads. 150 mg of BSA-loaded (vacuum loading) pollen grains were mixed homogeneously with 1.5 ml alginate (2 %) in water. The resulting suspension was slowly added using blunt 18 G needle to a 10 ml calcium chloride (8 %) solution under magnetic stirring, the stirring was continued for 5 minutes and the calcium chloride was removed by centrifugation (1500 rpm, 2 minutes) followed by washing twice with 1.5 ml water and freeze- drying for 24 h. All the formulations were stored in -20°C for further studies.

6.2.2.7 Statistical analysis

[00267] Statistical analysis was performed using two-tailed t -tests and <0.05 was considered as statistically significant. Encapsulations with natural pollen and release

experiments were repeated at least three times and all data are expressed as mean ± standard deviation (SD).

6.2.3 Results and Discussion

[00268] FIGS. 10A-10D outline the basic hydration and encapsulation process, then subsequent release. Hydration begins with taking dried sunflower pollen grains with the cytoplasmic material intact (FIG. 10A) and combining with a BSA solution (FIG. 10B). The BSA solution is absorbed into the pollen grain as the pollen grain swells and the BSA solution fills the additional volume created by the swelling process (FIG. IOC). This process may be natural, as in the passive loading method, or assisted, as in the compression or vacuum loading methods. After the BSA is released in the simulated intestinal or gastric fluid, the pollen grains remain swollen although all BSA is released (FIG. 10D).

[00269] To demonstrate a platform technology to utilize natural pollen grains as a drug delivery vehicle, the grains have been characterized methodically to assess uniformity in size, morphology, and physical changes arising during the course of the encapsulation process. In order to characterize natural pollen grains we employed dynamic imaging particle analysis

(DIPA) Γ31 ' 321 (see supporting experimental section). FIG. 11 A shows representative data by curve fitting histograms of equivalent spherical diameter (ESD) vs. frequency with average an ESD of 37.93 ± 1.41 μπι for natural pollen grains and an ESD of 36.54 ± 1.45 μπι, 36.95 ± 1.35 μπι, and 36.17 ± 1.36 μπι respectively for passive, compression, and vacuum loaded pollen grains. The natural pollen grains uniformity with reference to size was evident from ESD data before and after macromolecule loading, which is an important prerequisite of particulate drug delivery systems. ^[33] In addition to pollen size uniformity, the pollen circularity was measured before and after BSA loading and the data is represented by curve fitting to histograms of circularity vs. frequency as shown in FIG. 1 IB. The shape of pollen capsules before and after BSA-loading are considered non-circular due to the characteristic distribution of spikes on the pollen surface with the resulting circularity value < 1 (ideal circle=l). The quality of the image focus of the images used for data analysis is evident from FIG. 11C, and the edge gradient vs. frequency data which is represented indicates that highly focused pollen grains were used during DIPA analysis.

[00270] In addition to quantitative data, images, FIGS. 12A, 12B, 12C, and 12D indicate the structural similarity of pollen before loading as well as passive, compression, and vacuum loading techniques, respectively. In addition to DIPA, we characterized each representative batch of pollen grains using scanning electron microscopy (SEM) to examine any morphological changes^[13] and these images are displayed as FIG. 13A, 13B, 13C, 13D, respectively, for pollen before loading and after loading by passive, compression, vacuum loading techniques. These structural and morphological observations indicate that our pollen grain formulations have maintained their structural integrity without any denaturation, and exhibit size uniformity after macromolecule encapsulation using different techniques. This is important, as any proposed drug delivery vehicle for controlled and targeted delivery demands uniformity in size and shape for efficient quality control and performance and it can be a major challenge in encapsulation processess to control the product quality. ^[34] It is clearly evident from the morphological observations that pollen apetures exist and that each spike on these pollen grain surfaces is surrounded by pores which both allow for the possible uptake of macromolecules and facilitate the pollen hydration process. ^{[23 26]} The results from quantitative determination after

macromolecule encapsulation in terms of loading and encapsulation efficiencies in pollen grains are depicted in Table 7. The encapsulation efficiency (EE) of passive and compression loading is similar 37.2 ± 4.4 % and 37.8 ± 3.2 %, whereas with the vacuum loading process a statistically significant (p<0.05) higher EE of 65.7 ± 1.8 % was achieved. The possible reasons for higher macromolecule encapsulation are evident from applying a vacuum as an external force in addition to macromolecule uptake during the natural pollen rehydration process. ^{[23 26]} Although some existing reports discuss the encapsulation of drugs into extracted exine capsules, ^[15] we have focused on encapsulation processes involving natural pollen grains through the encapsulation of a model macromolecule.

[00271] Table 7: BSA-loaded Sunflower pollen: formulation parameters'^

Natural sunflower Theoretical BSA BSA

pollen BSA loading loading Encapsulation

_(%) ^{( )}

(%) efficiency (%)^(c)

Passive loading 50 18.6 ± 2.2 37.2 ± 4.4

Compression loading 50 18.8 ± 1.5 37.8 ± 3.2

Vacuum loading 50 32.8 ± 0.9 65.7 ± 1.8

' Results are means of three batches (n=3) with standard deviation; ' Theoretical loading is based on 50% weight of natural pollen: ^(c) BSA encapsulation efficiency is determined using 5 mg BSA-loaded natural pollen grains.

[00272] In order to directly visualize qualitatively and understand the intrapollen presence of loaded macromolecules in the presence of pollen cytoplasmic constituents, we encapsulated FITC-conjugated BSA into natural pollen grains by the three aforementioned techniques and performed analysis using confocal laser scanning microscopy. All images were captured focusing on the middle section of pollen grains. Confocal microscopic images of natural sunflower pollen grains before macromolecule loading are presented in FIG. 14A. It is evident from row (A) that no green color contributing to FITC-BSA was observed with pollen grains before loading, where as blue and red channels show strong autoflorescence from pollen constituents which is even more evident from their overlay image.

[00273] As a pollen grain, a male gametophyte exhibits autofluorescence based on the presence of compounds like carotenoids, phenolics, and terpenoids and this supports the observed autofluorescence in natural sunflower pollen. ^[35] In the case of FITC-BSA loading by the passive technique (FIG. 14B), a strong green fluorescence was observed confirming macromolecular loading into the natural pollen grains and is clearly evident from the overlay of all channels indicating FITC-BSA along with the natural pollen constituents. In the case of the compression loading of macromolecules (FIG. 14C), we also observed a bright green fluorescence. However, in addition, the pollen grains appeared as compressed although still retaining their structural integrity. Relatively higher overall fluorescence is observed in the case of vacuum loading (FIG. 14D) and this supports our assertion that higher encapsulation efficiency may be obtained with the application of an external vacuum force in addition to the natural pollen rehydration process. ^{[23 26]} CLSM Z-stack imaging was performed for FITC-BSA- loaded pollen grains loaded by vacuum loading techniques (FIGS. 15A-15B in supporting information) to provide further confirmation of the encapsulation of FITC-BSA into natural pollen grains.

[00274] The encapsulation of macromolecules into natural pollen grains was evident and we further studied in-vitro release profiles of BSA from these natural pollen grains separately in simulated intestinal (PBS, pH 7.4) and gastric conditions (pH 1.2). See FIGS. 16A-16C. The in- vitro release of BSA in PBS (FIG. 16A) indicates 80 % release in the first 5 minutes and complete release was observed in 30 to 60 minutes. There was no significant difference among the release from BSA-loaded pollen grains prepared using different techniques (p> 0.05). The high percent of release is expected to be due to the high number of pores and apertures on pollen grains resulting in rapid release which was also similar to the rate of drug release reported earlier with lycopodium exine capsules. ^[15] In simulated gastric conditions (FIG. 16B) a similar burst release was observed with all three different loading techniques suggesting no significant release differences in simulated gastric and intestinal conditions.

[00275] Further, in order to control the macromolecule release, we selected vacuum loaded- BSA pollen grains to be incorporated into natural biopolymer alginate beads by ionic crosslinking using calcium chloride. Interestingly, after macromolecule loading, the pores of the pollen grains are predominantly not covered, however, after alginate coating substantial closure of pollen pores is clearly indicated, thus acting as barrier for macromolecule release. Our initial pollen coating optimization using 0.1 % and 0.5 % alginate provided a thin alginate coating but was not suitable for modulating the release of macromolecules. Further optimization using a 2 % alginate solution (FIGS. 17A-17D and FIGS. 18A-18D) was found to provide an adequate barrier for delayed macromolecule release and was also necessary to form a viscous enough hydrogel so as to prevent BSA leaching during the coating process. By using 2 % alginate as the hydrogel medium, the release was retarded and extended up to 20 h and there was a significant difference (p< 0.05) with release after coating compared to release before coating in both simulated conditions. Among the BSA release from coated pollen grains in simulated gastric and intestinal conditions, a more retarded release is observed in gastric conditions. A similar release profile was reported in a previous study also using alginate gel microspheres and alternatively, controlled release of drugs was also reported by constructing a self-assembly of human serum albumin and L-a-dimyristoylphosphatidic acid (DMPA) to form layer-by-layer assembly on drug crystals. ^{[37 39]} The DMPA based multilayer approach controlled the drug release based on capsule wall thickness^[37] and this provides further evidence that greater control over protein release may be obtained by modulating the permeability of the coating layer.

[00276] To observe the condition of the pollen grains after FITC-BSA release, we used pollen grains after in-vitro release in simulated intestinal media and performed confocal microscopy analysis. FIGS. 19A, 19B, and 19C clearly indicate the release of FITC-BSA from pollen grains prepared using three different techniques and the pollen structure was found to remain intact. It is also evident from the CLSM images that a low amount of BSA binding to the exine has occurred and is clearly visible by the resulting 'green ring'.

[00277] Taken together, for the first time, we have demonstrated the use of natural pollen grains as a drug delivery vehicle by encapsulating BSA as a model macromolecule using encapsulation techniques based on passive, compression and vacuum loading. Encapsulation of up to 65 % was achieved using the vacuum loading technique suggesting a simple means of encapsulating therapeutic compounds into natural pollen grains. Using these methods, different natural pollen grains can be explored for encapsulation of small molecules, proteins, peptides, growth factors, and biosimilars in a simple process without the need for the use of harsh encapsulation conditions where the stability of therapeutic molecules may be compromised. In addition, we have demonstrated a way to retard the release of macromolecules with pollen grains by the use of a natural biopolymer, through crosslinking alginate with calcium ions to achieve a controlled release up to 20 hours. This is of particular interest in the field of controlled delivery of therapeutic molecules, where different drug release profiles are needed to improve the therapeutic benefit of active ingredients. Our group is currently pursuing ongoing studies into the use of natural pollen grains and their exine capsules as efficient drug delivery carriers of natural origin.

6.2.4 References Cited in Example 2

1. G. Ma, . Control. Release 2014, 193, 324.

2. A. K. Anal, H. Singh, Trend. Food Sci. Tech. 2007, 18, 240.

3. R. G. Stanley, H. F. Linskens, Pollen: Biology, Biochemistry, Management, Springer- Verlag, Berlin, Germany 1974, p. 307. K. Aya, Y. Hiwatashi, M. Kojima, H. Sakakibara, M. Ueguchi-Tanaka, M. Hasebe, M. Matsuoka, Nature Communications 2011, 2, 544.

T. Ariizumi, K. Toriyama, Annu. Rev. Plant Biol. 2011, 62, 437.

A. Wang, Q. Xia, W. Xie, R. Datla, G. Selvaraj, Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14487.

R. B. Knox, J. Heslop-Harrison, . Cell Sci. 1970, 6, 1.

W. T. Fraser, A. C. Scott, A. E. S. Forbes, I. J. Glasspool, R. E. Plotnick, F. Kenig, B. H. Lomax, New Phytologist 2012, 196, 397.

S. Barrier, A. Lobbert, A. J. Boasman, A. N. Boa, M. Lorch, S. L. Atkin, G. Mackenzie, Green Chem. 2010, 12, 234.

G. Mackenzie, S. Beckett, S. Atkin, A. Diego-Taboada, in Microencapsulation in the Food Industry: A Practical Implementation Guide, (Eds: A. G. Gaonkar, N. Vasisht, A. R. Khare, R. Sobel), Elsevier, San Diego, USA 2014, Ch. 24.

S. J. Archibald, S. L. Atkin, W. Bras, A. Diego-Taboada, G. Mackenzie, J. F. W. Mosselmans, S. Nikitenko, P. D. Quinn, M. F. Thomas, N. A. Young, . Mater. Chem. B 2014, 2, 945.

A. Diego-Taboada, S. T. Beckett, S. L. Atkin, G. Mackenzie, Pharmaceutics 2014, 6, 80. S. U. Atwe, Y. Ma, H. S. Gill, . Control. Release 2014, 194, 45.

H. Ma, P. Zhang, J. Wang, X. Xu, H. Zhang, Z. Zhang, Y. Ning, . Microencapsul. 2014. 31, 667.

A. Diego-Taboada, L. Maillet, J. H. Banoub, M. Lorch, A. S. Rigby, A. N. Boa, G. Mackenzie, . Mater. Chem. B 2013, 1, 707.

S. Barrier, A. S. Rigby, A. Diego-Taboada, M. J. Thomasson, G. Mackenzie, S. L. Atkin, LWT-Food Sci. Technol. 2010, 43, 73.

A. Diego-Taboada, P. Cousson, E. Raynaud, Y. Huang, M. Lorch, B. P. Binks, Y. Queneau, A. N. Boa, S. L. Atkin, S. T. Beckett, G. Mackenzie, . Mater. Chem. 2012, 22, 9767.

A. Wakil, G. Mackenzie, A. Diego-Taboada, J. G. Bell, S. L. Atkin, Lipids 2010, 45, 645. M. Lorch, M. J. Thomasson, A. Diego-Taboada, S. Barrier, S. L. Atkin, G. Mackenzie, S. J. Archibald, Chem. Commun. 2009, 42, 6442.

V. N. Paunov, G. Mackenzie, S. D. Stoyanov, . Mater. Chem. 2007, 17, 609.

S. Barrier, A. Diego-Taboada, M. J. Thomasson, L. Madden, J. C. Pointon, J. D. Wadhawan, S. T. Beckett, S. L. Atkin, G. Mackenzie, . Mater. Chem. 2011, 21, 975. B. P. Binks, A. N. Boa, M. A. Kibble, G. Mackenzie, A. Rocher, Soft Matter 2011, 7, 4017.

N. Firon, M. Nepi, E. Pacini. Ann Bot 2012, 109, 1201.

G. Bohne, E. Richter, H. Woehlecke, R. Ehwald, Ann. Bot. 2003, 92, 289.

J. Dumais, . Exp. Bot. 2013, 64, 4681.

E. Katifori, S. Alben, E. Cerda, D. R. Nelson, J. Dumais, Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7635.

M. G. R. Campos, C. Frigerio, J. Lopes, S. Bogdanov, Journal of ApiProduct and ApiMedical Science 2010, 2, 131.

M. G. Campos, S. Bogdanov, L. B. D. Almeida- Muradian, T. Szczesna, Y. Mancebo, C. Frigerio, F. Ferreira, . Apicult. Res. 2008, 47, 154.

I. Keller, P. Fluri, A. Imdorf, Bee World 2005, 86, 3.

S. S. Greenleaf, C. Kremen, Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13890. 31. S. Zolls, D. Weinbuch, M. Wiggenhorn, G. Winter, W. Friess, W. Jiskoot, A. Hawe, AAPS J. 2013, 15, 4.

32. N. R. Goss, N. Mladenov, C. M. Seibold, K. Chowanski, L. Seitz, T. B. Wellemeyer, M.

W. Williams. Aim. Envir. 2013, 80, 549.

33. J. A. Champion, Y. K. Katare, S. Mitragotri, . Control. Release 2007, 16, 3.

34. P. L. Lam, R. Gambari, . Control. Release 2014, 178, 25.

35. A. J. Castro, J. D. Rejon, M. Fendri, M. J. Jimenez-Quesada, A. Zafra, J. C. Jimenez- Lopez, M. I. Rodriguez-Garcia, J. D. Alche, in Microscopy: Science, Technology, Applications and Education (Eds: A. Mendez-Vilas, J. Diaz), Formatex Research Center, Spain 2010. pp. 607-613.

36. D. M. Hariyadi, Y. Wang, S. C-Yu Lin, T. Bostrom, B. Bhandari, A. G. A. Coombes, Microencapsul. 2012, 29, 250.

37. Z. An, G. Lu, H. Mohwald, J. Li, Chemistry 2004, 10, 5848.

38. Z. An, G. Lu, H. Mohwald, J. Li, Biomacromol. 2006, 7, 580.

39. Q. He, Y. Cui, J. Li, Chem Soc Rev. 2009, 38, 2292.

6.3 EXAMPLE 3: ENCAPSULATION AND CONTROLLED RELEASE

FORMULATIONS OF 5-FLUORURACIL FROM NATURAL LYCOPODIUM CLAY AT M SPORES

[00278] This example demonstrates a cost-effective, simple approach to produce oral- controlled release formulations of 5-fluorouracil (5-FU) based on natural L. clavatum spores. The data provided in this example demonstrates that the vacuum loading technique provides the highest encapsulation efficiency of 49% compared to the passive and compression loading techniques. Micrometric properties of the 5-FU loaded spores confirmed a uniform size distribution, and surface characterization of 5-FU spores verified no evidence of residual 5-FU, indicating encapsulation of 5-FU inside spores. Uniform Eudragit RS100 coatings (ERS) on 5- FU loaded spores provide a controlled release of 5-FU for up to 30 hours. The demonstrated features of 5-FU loaded spores indicate a potential oral drug delivery system for gastrointestinal cancer treatment and other maladies.

6.3.1 Materials & Methods

6.3.1.1 Materials

[00279] The following materials were used: whole L. clavatum spores, 5-fluorouracil (5- FU), ammonium hydroxide and ethanol were purchased from Sigma (Singapore). Polystyrene microspheres (50 ± 1 mm) were purchased from Thermoscientific (CA, USA). Eudragit RSI 00 (ERS) was procured from Evonik Industries (Essen, Germany) and perfluoroalkoxy polymer (PFA) flasks were procured from Vitlab (Grossostheim, Germany). Stainless steel casted pellet press die (13 mm) was procured from Specac (Kent, UK).

6.3.1.2 Encapsulation of 5-FU into whole L. clavatum spores by passive loading technique

[00280] 5-Fluorouracil solution was prepared by dissolving 75 mg of drug in a 1.8 mL mixture of ethanol and 1 N ammonium hydroxide (1: 1) solution. Whole L. clavatum spores (150 mg) were suspended in the prepared solution. The suspension was vortexed for 5 min and the tube was transferred to a thermoshaker (Hangzhou Allsheng Inst. Singapore) set at 500 rpm for 2 hours incubation at room temperature. The 5-FU loaded spores were collected by centrifugation at 4500 rpm for 3 min. The spores were washed using 4 mL deionized water and centrifuged to remove surface adhered 5-FU. The 5-FU loaded spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours. The resulting 5-FU loaded spores were stored in a dry cabinet at room temperature until further characterization. The placebo passive-loaded spores without 5-FU were prepared by using the same procedure as described above.

6.3.1.3 Encapsulation of 5-FU into whole L. clavatum spores by compression loading technique

[00281] 150 mg of L. clavatum spores were filled in a 13 mm pellet press die and compressed to form a tablet under a hydraulic press with a 5 ton load for 20 sec (die diameter 13 mm; area 132.75 mm ; 370 MPa). The dimensions of the spore tablet are listed in the Table 8. The tablet was soaked in a 1.8 mL 5-FU solution in a 20 mL flat glass bottle for 2 hours to allow for the uptake of 5-FU. The 5-FU loaded spores were collected by centrifugation at 4500 rpm for 3 min. The spores were washed using 4 mL deionized water and centrifuged to remove surface bound 5-FU. The spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours. The resulting spores were stored in a dry cabinet until further characterization. The placebo compression-loaded spores without 5-FU were prepared by using the same procedure as described above.

[00282] Table 8. Compression loading technique: L. clavatum spore tablet dimensions'"'

^(a)Tablets used in 5-FU-loading by compression technique and results are mean of three batches (n=3) with standard deviation; ^<b)Weight determined in the Boeco BBX 22 (Germany) analytical balance; ^<c)Diameter and thickness measured using digital vernier caliper 6.3.1.4 Encapsulation of 5-FU into whole L. clavatum spores by vacuum loading technique

[00283] Vacuum-assisted 5-FU loading was performed by suspending 150 mg of L.

clavatum spores in 1.8 mL of 5-FU solution. The suspension was vortexed for 5 min. The sample was placed in a freeze-drier (Lanconco, USA) and a 1 mbar vacuum was applied for 2 hours. The process was stopped and the 5-FU loaded L. clavatum spores were washed using 4 mL water and centrifuged to remove surface bound drug. The spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours. The resulting spore particles were stored in a dry cabinet until further characterization. The placebo vacuum-loaded spores without 5-FU were prepared by using the same procedure without 5-FU as described above.

6.3.1.5 Surface morphology evaluation by scanning electron microscopy (SEM)

[00284] The evaluation of the surface morphology of the 5-FU loaded spores was conducted using a FESEM 7600F (JEOL, Japan). A platinum coating of 10 nm thickness was deposited on each sample by using an auto fine coater JFC-1600 (JEOL, Japan) at 20 mA for 60 sec. Images were taken with an acceleration voltage of 5 kV at various magnifications.

6.3.1.6 Dynamic image particle analysis (DIPA)

[00285] The benchtop system (FlowCamVS, Fluid Imaging Technologies, Maine, USA) was installed with a visual spreadsheet software version 3.4.11., 200 μπι flow cell (FC-200) and a 20x magnification lens (Olympus , Japan). The flow cell was cleaned by flushing the system with 1 mL of deionized water at a flow rate of 0.5 mL/min. The instrument was calibrated using polystyrene microspheres (50 ± 1 μπι) and a pre -run volume of 0.5 mL of whole L. clavatum spores and 5-FU loaded spores were primed and transferred into the flow cell. Analysis was carried out at a flow rate of 0.1 mL/min and a frame rate of 10 FPS leading to a sampling efficiency of about 9%. The count for each analysis was fixed at the minimum of 10,000 particles and highly focused particles were selected by edge gradient for data analysis. The representative data reported in this work is an average of triplicate measurements with standard deviation (n = 3).

6.3.1.7 Preparation of Eudragit RSlOO-coated spore formulations

[00286] 5-FU loaded L. clavatum spores were coated using Eudragit RS100 (ERS) at two different ERS concentrations (2.50% w/v and 10.0% w/v). The coating solutions were prepared by slowly dissolving Eudragit RS100 in acetone. For the coating process, 150 mg of 5-FU loaded (vacuum method) spores were added to 1.2 mL of Eudragit RS100 solution in a PFA round bottom flask and the solvent was evaporated in a vacuum desiccator for 1 hour. Further, spores were dried in vacuum oven (Memmert GmbH, Germany) at 1 mbar for 1 hour. The dried spore formulation was then gently powdered using an agate pestle and mortar and stored in a dry cabinet until further characterization.

6.3.1.8 Encapsulation efficiency

[00287] 10 mg of 5-FU loaded L. clavatum spores were suspended in 10 mL of pH 7.4 phosphate -buffered saline (PBS), mixed using a vortex mixer (IKA, Staufen, Germany) for 5 min, and subjected to probe sonication (Qsonica, Newtown, USA) at room temperature for 15 sec at 40% amplitude (3 cycles). The supernatant was collected after centrifugation at 4500 rpm for 3 min and the absorbance at 266 nm was measured using a UV spectrometer (Boeco-S220,

Germany) with placebo spores as the blank. The amount of 5-FU present in the whole L.

clavatum spores was calculated using the following equations:

Absorbance at 266 nm x dilution factor

Amount of BSA (mg) = -— - — (7),

⁶ Slope (standard curve) x 1000

Amount of 5-FU , _

% 5-FU loading = . X 100 (8),

Weight of 5-FU loaded spores

Practical 5-FU loading

% Encapsulation efficiency = X 100 (9).

Theoretical 5-FU loading

6.3.1.9 In-vitro drug release in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)

[00288] In-vitro 5-FU release was performed initially for 2 hours in SGF followed by SIF to simulate gastrointestinal conditions. 0.1 M hydrochloric acid solution with a pH value of 1.2 was used as SGF and a PBS buffer with a pH of 7.4 was used as SIF. 10 mg of 5-FU loaded L.

clavatum spores were suspended in 10 mL of release media and incubated at 37° Celsius while stirring at 50 rpm in an orbital shaker incubator (LM-450D, Yihder, Taiwan). At predetermined time points, 1 mL of release media was collected and replenished with fresh release media. The absorbance in the release sample was measured using a UV spectrometer (Boeco-S220,

Germany) at 266 nm. In-vitro drug release from Eudragit RSlOO-coated 5-FU spore formulations was performed using 30 mg of sample in 5 mL of release media.

6.3.1.10 Statistical Analysis [00289] Statistical analysis was performed using two-tailed t-tests and p < 0.05 was considered statistically significant. 5-FU encapsulation with natural spores and in-vitro release experiments were repeated at least three times and all data are expressed as mean ± standard deviation (SD).

6.3.2 Results and Discussion

6.3.2.1 Microencapsulation of 5-FU into natural L. clavatum spores

[00290] In order to encapsulate 5-FU into spores, the solubility of 5-FU was first increased to 50 mg/mL by dissolving the drug in a mixture of ethanol and 1 N ammonium hydroxide (1: 1). The higher solubility of 5-FU facilitated higher drug loading into the spores as loading into the spores would be limited by aqueous solubility of the drug (Garea et al., Int. J. Pharm. 491 (2015) 299-309). By suspending L. clavatum spores in a 5-FU solution, the drug was able to enter the internal cavity of the spores through nanoscale channels on the spore wall (Diego-Taboada et al. , Pharmaceutics 6 (2014) 80-96). Three different encapsulation techniques were employed to load 5-FU into whole L. clavatum spores with the data presented in Table 9.

[00291] Table 9. Formulation parameters of 5-Fluorouracil loaded L. clavatum spores^(a)

w Theoretical loading is based on total initial weig ht of batch (225 mg); ^{( )} Results are the mean of three

independent batches (n=3) with standard deviation; ^<c) 5-FU encapsulation efficiency is determined using 10 mg of 5-FU loaded whole L. clavatum spore.

[00292] With a theoretical loading capacity of 33% 5-FU, the vacuum-assisted loading resulted in a significantly higher encapsulation efficiency (EE) of 49% (p < 0.05) compared to the passive loading technique. In the case of the compression loading technique, a relatively lower EE was observed in comparison to the vacuum loading technique albeit with no significant difference. These observations are consistent with the fact that the loading of drug molecules was influenced by the external energy supplied during the encapsulation process. In the case of passive loading, no external forces were involved and loading might be limited by drug passage into the internal cavity by nanoscale channels located on the spore wall (Diego-Taboada et al. , Pharmaceutics 6 (2014) 80-96; Barrier et al., J. Mater. Chem. 21 (2011) 975-981). [00293] The compressed tablet enabled spores to incorporate higher drug concentrations into the internal cavity by virtue of the elastic exine wall (Barrier et al., J. Mater. Chem. 21 (2011) 975-981). It is notable that compression of spores at 5 ton was not detrimental, indicating the robust structure of L. clavatum spores. The vacuum-assisted loading of 5-FU into whole L. clavatum spores at 1 mbar facilitated forced passage of drug molecules into the internal cavity of spores. Barrier et al. (Diego-Taboada et al., J. Mater. Chem. B. 1 (2013) 707-713; Barrier et al., J. Mater. Chem. 21 (2011) 975-981) reported similar encapsulation data with drugs and proteins encapsulated in sporopollenin exine capsules (SECs) produced from L. clavatum spores, supporting the higher EE of 5-FU into L. clavatum spores by vacuum-assisted loading. In the case of 5-FU encapsulation, attempts to load 5-FU into crosslinked natural polymers resulted in EE in the range of 8% to 53% based on the drug-to-polymer ratio. These results therefore provided insight into how to load low aqueous soluble drugs in a way that optimizes

encapsulation efficiency by employing different loading techniques.

6.3.2.2 Micromeritic properties of 5-fluorouracil loaded spores

[00294] To understand the micromeritic properties of 5-FU loaded L. clavatum whole spores, dynamic imaging particle analysis (DIP A) was performed on the loaded spores as described in Section 6.3. FIGS. 20A-20D illustrate the results from the DIP A. It is evident from diameter measurements (FIG. 20A) that the whole spores with a native diameter of 30 ± 0.45 mm remain unchanged after 5-FU encapsulation by all three encapsulation techniques. The diameter of spores before and after 5-FU loading is listed in Table 10 . The 5-FU loaded spores retained the intact microstructure with uniform size distribution. In order to investigate the uniform shape of 5-FU loaded spores, the circularity and aspect ratio were measured and the data are illustrated in FIGS. 20B and 20C. It is evident from FIG. 20B that the circularity of 5-FU loaded spores was near to circular shape and that encapsulation of 5-FU into spores by the three different techniques was favorable in retaining the native shape of the spore. To support the shape uniformity of spores, the data for aspect ratio of 5-FU loaded spores also indicates that there was no change in spore microstructure. Edge gradient as illustrated in FIG. 20D indicates that all the micromeritic properties based on image analysis were obtained using well-focused particles.

[00295] Table 10 . Diameter of L. clavatum spores before and after 5-FU loading

L. clavatum spores | Diameter (μπι + SD) Before 5-FU loading 30.0 ± 0.45

5-FU passive 30.0 ± 0.88

5-FU compression 32.2 ± 0.23

5-FU vacuum 32.6 ± 1.73

^(a)FlowCam measurements performed in triplicate and reported are one of representative values with standard deviation (SD)

[00296] The images captured during DIPA are presented in FIGS. 21A-21D for spores before 5-FU loading, as well as after loading by passive, compression and vacuum loading techniques, respectively. The DIPA images indicate that all spores after 5-FU loading retained well-defined microstructures supporting the DIPA data for the uniform size distributions.

[00297] To further evaluate the structure and morphology of 5-FU loaded spores, L.

clavatum spores before and after 5-FU loading were analyzed by SEM as described in Section 6.3.1.5. The SEM images after 5-FU loading by passive, compression and vacuum are presented in FIGS. 22A-22D, respectively. The structural and morphological data for spores before 5-FU loading showed characteristic well-defined ornamentation with reticulate structure and uniform size distribution. In the case of 5-FU loaded L. clavatum spores achieved by the three different encapsulation techniques, the spore's native microstructure and ornamentation were retained. The 5-FU encapsulated spores showed no detrimental effect to the spore microstructure by drug loading even after the use of external factors such as compression at 5 ton and with 1 mbar vacuum. The surface of the 5-FU loaded spore was clean without any evidence of residual drug aggregation suggesting the encapsulated drug was principally inside the spore's internal cavity. Hence, the data for 5-FU loaded spores supports that the disclosed methods to encapsulate 5-FU in whole L. clavatum spores offers excellent potential as a multiparticulate oral delivery system with uniform size distribution and well-defined surface morphology.

6.3.2.3 ERS coating

[00298] To investigate effects of ERS coating, SEM analysis was performed on ERS-coated spores prepared as described in Section 6.3.1.7. FIGS. 23A and 23B illustrated the SEM images of ERS-coated spores using 2.5% and 10% ERS concentrations, respectively. The surface morphology of whole spores after coating indicates that spores were coated with ERS, and that the ERS coating was higher in the case of 10% ERS-coated spores. The muri located on the spores was filled with the coating material which acts as a barrier for 5-FU release.

6.3.3 In-vitro release studies [00299] In-vitro release studies of 5-FU loaded spores were performed in simulated gastrointestinal conditions. FIGS. 24A and 24B illustrate the 5-FU release profiles in SGF (pH 1.2) and SIF (pH 7.4), respectively. High release rates of up to 90% were observed in the initial 10 min and complete 5-FU release was observed within 60 min due to exit via the nanochannels in the exine wall (Diego-Taboada et al., Pharmaceutics 6 (2014) 80-96). Similarly higher 5-FU release in SGF for stomach-targeted release was reported by Bhardwaj et al. (Bhardwaj et al., Scientific World Journal (2014) 705259) using floating micro-spheres, and recently Diego- Taboada et al. (Diego-Taboada et al., J. Mater. Chem. B. 1 (2013) 707-713) have reported higher ibuprofen release from L. clavatum sporopollenin exine capsules (SECs) within 1 hour in SGF. Hence, the 5-FU release from natural spores indicates that a suitable polymeric coating to retard the drug release in simulated gastrointestinal conditions would be beneficial.

[00300] To modulate 5-FU release from spores a polymethacrylate (Eudragit RS 100) coating was employed. Eudragit RS 100 is a copolymer of ethyl acrylate, methyl methacrylate and is widely used as a coating material to develop controlled release formulations (Alai et al. , J. Microencapsul. 30 (2013) 519-529; Piao et al, AAPS PharmSciTech. 11 (2010) 630-636). The initial coating and in-vitro release studies in simulated gastrointestinal conditions using different concentrations of Eudragit RS 100 indicates that coatings with 2.5% w/v and 10% w/v ERS provided a suitable coating on whole L. clavatum spores. FIG. 24C illustrates the data for in-vitro release profiles using ERS coated spores, indicating that the ERS coating significantly (p < 0.05) retarded 5-FU release under simulated gastrointestinal conditions. The inset (FIG. 24C) indicates around 70% of 5-FU was released in the initial 2 hours and by increasing the ERS concentration to 10% the 5-FU release was reduced to 50%. Further, in-vitro 5-FU release was extended up to 30 hours and a significant (p < 0.05) difference in 5-FU release was observed with 10% ERS- coated spores in comparison to 2.5% coating, suggesting that 10% ERS coating is beneficial to achieve controlled 5-FU release from whole spores.

[00301] In the case of 2.5% and 10% ERS coated whole spores, the enteric coating covered the spore microstructure, there by closing the nanochannels on the exine wall (Diego-Taboada et al., Pharmaceutics 6 (2014) 80-96). The 5-FU release from spores was controlled by the enteric coating on 5-FU loaded spore. There was no lag time observed in 5-FU release from enteric coated spores due to pH independent release behavior of ERS. Further, 5-FU release from ERS coated spores was gradually decreased in controlled fashion based on the ERS concentration used in coating the spores. The in-vitro release data indicates that 5-FU release from the ERS coated spores was a result of polymer erosion from the surface of spores, as the enteric coating was higher the 5-FU release was lowered during 30 hours. Hence, the possible mechanism of 5- FU release from enteric coated spores was a combination of dissolution, diffusion erosion and is consistent with previous finding (Piao et al., AAPS PharmSciTech. 11 (2010) 630-636).

[00302] Thus, in-vitro release of 5-FU from L. clavatum spores can be controlled in gastrointestinal conditions by ERS coating. Similar 5-FU release profiles from modified sodium alginate microspheres were reported by Sanli et al. (Sanli et al., Drug Deliv. 21 (2014) 213-220) with controlled release up to 12 hours under simulated gastrointestinal conditions. The controlled gastrointestinal release of 5-FU is highly beneficial in the treatment of breast, stomach and colon cancer, possibly avoiding furthermore repeated doses. Hence, the disclosed results for 5-FU loaded L. clavatum whole spores indicate that whole spores could encapsulate and control the release of 5-FU under gastrointestinal conditions.

6.4 EXAMPLE 4: ENCAPSULATION OF BOVINE SERUM IN PINE POLLEN

[00303] In this example bovine serum albumin (BSA) was loaded into whole pine pollen grains employing three different encapsulation techniques (passive, compression, and vacuum loading).

6.4.1 Materials & Methods

[00304] Encapsulation of BSA into natural, unprocessed pine pollen grains: 75 mg BSA (50 wt% based on pollen grains weight) was dissolved into 0.5 mL purified water in a 1.5 mL polypropylene tube and 150 mg of whole pollen grains was suspended in the BSA solution. The suspension was mixed by vortexing (VWR, Singapore) for 5 min and the tube was transferred to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4° Celsius and 500 rpm for passive loading. For the vacuum loading technique, the BSA and pollen grains suspension was used, and a 2 mbar vacuum was slowly applied in a freeze dryer (Labconco, MO, USA). The quantity of BSA, pollen grains, and incubation time (2 hours) were maintained constant for all batches, and after incubation the BSA-loaded pollen grains were collected by centrifugation at 12000 rpm for 4 minutes, washed using 0.5 mL water, and then centrifuged to remove surface adhered BSA. The pollen grains were frozen in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hour. The final BSA-loaded pine pollen grains were stored at -20° Celsius until further characterization. Scanning electron microscopy analysis and calculation of loading efficiency (LE) and encapsulation efficiency (EE) was performed as described in the above Examples. 6.4.2 Results

[00305] Initially BSA was loaded into whole unprocessed pine pollen grains by utilizing the vacuum loading technique as described above. After encapsulation of BSA, surface cleanliness was observed in relation to the number of washings. One water wash was determined to be adequate to remove residual surface adhered BSA (see FIG. 25 A, which shows the surface cleanliness of BSA-loaded pine pollen grains after zero, one, two or three washing steps).

Loading efficiency (LE) and encapsulation efficiency (EE) data was also measured for the BSA- loaded pine pollen formulations after each washing step. As shown in FIG. 25B, at zero washings, immediately after encapsulation, approximately 80% of the BSA was still present in the formulation, and that approximately 27 wt.% of the formulation comprised BSA, resulting in a loading ratio of about 1:3 (BSA:pollen grain). After one wash step approximately 40% of the BSA remained in the formulation, and approximately 13 wt.% of the formulation comprised BSA, resulting in a loading ratio of about 1 :7 (FIG. 28B). Subsequent washing resulted in further reductions in both LE and EE (FIG. 28B). The use of FITC-conjugated BSA (FITC-BSA) allowed visualizing the BSA within pollen grains through confocal laser scanning microscopy (CLSM). As shown in FIG. 25C, the majority of the FITC-BSA was present near the surface of the pine pollen wing cavities, as observed by confocal laser scanning microscopy.

[00306] Next, the process of prolonged passive-loading of BSA in pine pollen was explored. It was observed that over the period of one hour there was minimal loading occurring for pine pollen incubated in BSA solution concentrations of 10 mg/ml and 150 mg/ml (FIG. 26). When prolonged passive loading of pine pollen for four BSA solution concentrations (5, 25, 125, 250 mg/ml) over the duration of 1, 2, 3, 7 days was performed, it was observed that loading into the central cavity increased for days 1, 2, 3 and reached a maximum at 3 days, as 3 day and 7 day loading appeared the same (FIG. 27). Thus, these results demonstrate that prolonged passive loading was more effective for central cavity loading, whereas vacuum loading was more effective for wing cavity loading.

6.5 EXAMPLE 5: ENCAPSULATION OF BOVINE SERUM IN CAMELLIA

POLLEN [00307] In this example bovine serum albumin (BSA) was load into whole camellia pollen grains employing a passive loading technique.

6.5.1 Materials & Methods

[00308] Encapsulation of Macromolecules into natural pollen grains: 75 mg BSA (50 wt% based on pollen grains weight) was dissolved into 0.5 mL purified water in a 1.5 mL

polypropylene tube and 150 mg of whole camellia pollen grains was suspended in the BSA solution. The suspension was mixed by vortexing (VWR, Singapore) for 5 min and the tube was transferred to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4° Celsius and 500 rpm (without washing) for passive loading. Scanning electron microscopy analysis was performed as described in the above Examples.

6.5.2 Results

[00309] The process of prolonged passive-loading of BSA in pine pollen was explored. Over a period of 1 hour significant loading was observed for camellia pollen incubated in a BSA solution concentration of 150 mg/ml (FIG. 28). The results indicate that the onset of loading for one of the camellia pollen grains (dashed square) occurred between 9 and 20 min of incubation time. Most camellia pollen grains showed significant BSA-loading after about 20 min of incubation time.

6.6 EXAMPLE 6: ENCAPSULATION OF CALCEIN INTO LYCOPODIUM

CLAY AT M SPORES

[00310] In this example calcein was load into whole L. clavatum spores employing the passive loading technique.

6.6.1 Materials & Methods

[00311] Calcein (pharma grade), whole L. clavatum spores, and other solvents were purchased from Sigma-Aldrich (Singapore). Polystyrene microspheres (50 ± 1 μηι) were purchased from Thermoscientific (CA, USA).

[00312] Encapsulation of calcein into L. clavatum spores. Calcein loading into spores was performed using a modified passive loading technique. A calcein mixture was loaded by dissolving 22 mg calcein in 2.2 mL DMSO, and spores (1 g) were suspended in the calcein solution in 50 mL polypropylene tubes. The suspension was mixed for 10 minutes using a vortex mixer (IKA, Staufen, Germany) to form a homogeneous suspension. The calcein suspension was incubated at room temperature overnight with intermittent stirring at 200 rpm for 5 hours. The suspension was filtered using vacuum filtration. Calcein-loaded formulations were transferred to a 250 ml beaker washed using 40 ml hot water (45° Celsius), and collected. The collected formulations were frozen at -20° Celsius for 1 hour, and freeze-dried (Labconco, MO, USA) for 24 hours. The spore formulations were collected, weighed and stored in a dry cabinet until further characterizations.

[00313] Confocal laser scanning micro graphic ( CLSM) analysis. Confocal laser scanning micrographic (CLSM) analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines at 405 nm (6.5 %), 488 nm (6 %), and 633 nm (6 %) with differential inference contrast (DIC) in an EC Plan-NeofluarlOOxl.3 oil objective M27 lens were used. Fluorescence from calcein-loaded spores were collected in photomultiplier tubes equipped with the following emission filters: 416-477 nm, 498-550 nm, and 572-620 nm. The laser scan speed was set at 67 sec per each phase (1024x1024:84.94 μπι sizes) and a plane mode scanning with a 3.15 pixel dwell was used. At least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany).

6.6.2 Results

[00314] The process of passive-loading of calcein in L. clavatum spores was explored.

Through the use of confocal scanning laser microscopy (CLSM), significant calcein was observed to present in the spores after loading (compare FIG. 29B (images after loading) to FIG. 29 A (images prior to loading).

6.7 EXAMPLE 7: ENCAPSULATION OF CAMELLIA OIL IN CAMELLIA

POLLEN

[00315] In this example camellia oil was loaded into whole camellia pollen grains employing the vacuum loading technique.

6.7.1 Materials & Methods

[00316] Encapsulation of Oil into Natural Pollen Grains. 1 mg nile red was dissolved into 0.5 ml camellia oil in a 1.5 mL polypropylene tube and 150 mg of whole camellia pollen grains was suspended in the oil solution. The suspension was mixed by vortexing for 5 min, and then, without washing, a 2 mbar vacuum was slowly applied in a freeze dryer (Labconco, MO, USA). [00317] The process of vacuum-loading of oil in camellia pollen was explored. Through the use of confocal scanning laser microscopy (CLSM), it was observed that significant camellia oil was present in the camellia pollen formulation after vacuum loading (FIG. 30).

[00318] FIGS. 31A and 3 IB illustrate that the size and morphology of whole Camellia pollen grains significantly differ from the size and morphology of Camellia pollen grain's isolated sporopollenin exine capsules (SEC) as measured by DIPA. The DIPA was performed as described in above Examples. The average diameter of the whole pollen grain was on average

37 nm compared to SEC's average diameter of 31 nm. In addition, the whole pollen grains displayed more characteristic surface features as compared to the SEC's smoother surface.

6.8 EXAMPLE 8: ENCAPSULATION OF CAFFEINE IN LYCOPODIUM

CLAY AT M SPORES AND CONTROLLED RELEASE FORMULATIONS

[00319] In this example caffeine (CF) was load into whole L. clavatum spores employing a modified passive loading technique.

6.8.1 Materials & Methods

[00320] Caffeine (pharma grade), L. clavatum spores and other solvents were purchased from Sigma- Aldrich (Singapore). Polystyrene microspheres (50 ± 1 μηι) were purchased from Thermoscientific (CA, USA).

[00321] Encapsulation of caffeine ( CF) into L. clavatum spores: Caffeine loading into spores was performed using a modified passive loading technique. CF equivalent to 50 % theoretical loading was dissolved in 11 mL dichloromethane with or without co-encapsulant (1.8 % w/v, Eudragit RS 100). Spores (1 g) were suspended in CF solution in 50 mL polypropylene tubes. The suspension was mixed for 10 min using a vortex mixer (IKA, Staufen, Germany) to form a homogeneous suspension. The CF suspension was incubated at room temperature overnight with intermittent stirring at 200 rpm for 5 hours. The suspension was filtered by using vacuum filtration. The CF-loaded formulations were then transferred to a 250 ml beaker and washed using 40 ml hot water (45° Celsius). After collecting, formulations were frozen at -20° Celsius for 1 hour and freeze-dried (Labconco, MO, USA) for 24 hours. The spore formulations were collected, weighed and stored in a dry cabinet until further characterizations. Placebo spores were prepared with the same procedure, except CF, and also stored in the dry cabinet at room temperature. [00322] To investigate CF encapsulation by CLSM, a fluorescent calcein-CF mixture was loaded according to the same procedure by dissolving 22 mg calcein in 2.2 mL DMSO and uniformly mixing with above CF solution. For some of the CF-loaded spores, an additional co- encapsulant, Eudragit RS 100 (ERS) was applied together with CF according to the process described in the previous paragraph with co-encapsulant (1.8 % w/v, Eudragit RS 100).

[00323] Surface morphology evaluation by scanning electron microscopy (SEM): FESEM 7600F (JEOL, Japan) was used for SEM image processing. Spores before and after CF loading were coated with platinum at a thickness of 10 nm using JFC-1600 (JEOL, Japan) (20mA, 60 sec). Images were recorded with an acceleration voltage of 5.00 kV at different magnifications to observe morphological changes before and after CF encapsulation into the spores.

[00324] Dynamic image particle analysis (DIP A) The micromeritic properties of the spores before and after CF loading were analyzed by the benchtop system (FlowCamVS, Fluid Imaging Technologies, Maine, USA) equipped with a 200 μπι flow cell (FC-200), and a 20X

magnification lens (Olympus^®, Japan). The system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/rnin and flow cell cleanliness was visually inspected before each sample run. Spores before and after CF loading with a concentration of 2 mg/ml were primed manually into the flow cell (a pre-run volume of 0.5 mL) and were analyzed with a flow rate of 0.1 ml/min and a camera rate of 14 frames/s leading to a sampling efficiency of approximately 12.2 %. A minimum of 10,000 spores were fixed as the particle count for each measurement and three independent measurements were performed. Data analysis was carried out using 1000 well-focused spores obtained from the raw data by segregating based on edge gradient. The instrument was calibrated using polystyrene microspheres (50 ± 1 μπι). The IB- spline curves for diameter, circularity, aspect ratio and edge gradient were reported using mean values ± standard deviation of three independent measurements.

[00325] Confocal Scanning Microscopy. Confocal laser scanning micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with differential inference contrast (DIC) in an EC Plan-NeofluarlOOxl.3 oil objective M27 lens were used. Fluorescence from CF- calcein-loaded spores were collected in photomultiplier tubes equipped with the following emission filters: 416-477 nm, 498-550 nm, and 572-620 nm. The laser scan speed was set at 67 sec per each phase (1024x1024:84.94 μπι sizes) and plane mode scanning with a 3.15 pixel dwell was used and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS,

Germany).

[00326] Determination of CF encapsulation efficiency: 10 mg of CF-loaded spores were suspended in 10 mL of PBS, then vortexed for 10 min before collecting the supernatant by centrifugation for 5 min at 4500 rpm and filtering by using a 0.45 μπι PES syringe filter (Agilent, CA, USA). The CF extraction was repeated twice and all the extracted CF solution was pooled to measure the absorbance. In case of CF formulations with coencapsulant, 10 mg CF-loaded spores were suspended in 1 ml DCM, vortexed for 5 min to dissolve the coencapsulant polymer. 10 ml PBS vortex was added for 10 min and centrifuged at 4500 rpm for 5 min. The aqueous layer was collected and the solution filtered using Advantech filter paper. The CF extraction was repeated according to the same procedure. The absorbance values were measured at 275 nm (Boeco-S220, Germany) using a placebo extract as a blank and the amount of CF in the spores was calculated using a CF standard curve and following equations:

Absorbance x dilution factor

Amount of CF (mg) = -_f — r — (10),

⁶ Slope (standard curve) x 1000

Amount of CF , _

% CF loading = . X 100 (11),

Weight of 5-FU loaded spores

Practical CF loading

% CF Encapsulation efficiency = X 100 (12).

Theoretical CF loading

[00327] In-vitro release studies of CF-loaded spores: In order to predict in-vitro release profiles of CF-loaded spores formulations, the release studies were performed in simulated saliva fluid pH 6.8 (SSF) for up to 5 min. 10 mg CF-loaded spores were suspended in 20 mL SSF and incubated at 37° Celsius, 50 rpm in a orbital shaker incubator LM-450D (Yihder, Taiwan). At predetermined time points 1 ml of release sample was collected and replenished with fresh release fluids. The absorbance of release sample was measured using UV spectrometer (Boeco- S220, Germany) at 275 nm with placebo as blank. [00328] Statistical Analysis: Statistical analysis was performed using two-tailed t-tests and p < 0.05 was considered as statistically significant. Encapsulation efficiency and in vitro release data are reported as mean values ± standard deviation of three independent experiments.

6.8.2 Results

[00329] FIGS. 32A-32B shows the L. clavatum spores before CF loading (FIG. 32A) and after CF-loading with co-encapsulant Eudragit RS 100 (ERS) by SEM. Micromeritic properties of CF-loaded spores confirmed a uniform size distribution, indicating monodisperse

multiparticulate taste masked formulation. Scanning electron microscopy and confocal laser scanning microscopic analysis confirmed encapsulation of caffeine into spores without residual CF on the surface. FIGS. 33A-33B show CLSM images of spores with sporoplasm before CF- Calcein loading (FIG. 33 A) and after CF-loading into spores with coencapsulant ERS. The CF encapsulated with Eudragit RS 100 (ERS) as coencapsulant provide highest encapsulation efficiency, 12 %.

[00330] In-vitro release profiles in simulated saliva fluid confirmed lower release profiles compared to physical mixture of CF with spores. The controlled release of CF from CF-loaded spores with ERS as coencapsulant confirmed extended CF release for up to 24 hours indicating

CF taste masked formulations suitability for oral controlled release applications (FIG. 34).

[00331] Table 11 provides the percentages of caffeine loading into L. clavatum spores using the modified passive loading technique described above.

Table 11 . Caffeine-loaded L. clavatum spores loaded by a modified passive loading technique

Theoretical CF CF loading CF Encapsulation

Formulations

loading (%) _(%) ^{( )} efficiency (%)^(c)

Spores-CF 50 1.6 ± 0.1 3.2 ± 0.2

Spores-CF-E 50 6.0 ± 0.1 12 ± 0.2

Theoretical loading is based on CF in total initial weight of batch (2 g). Results are the mean of three independent batches (n=3) with standard deviation. ^(c) CF encapsulation efficiency is determined using 10 mg of CF loaded L. clavatum spores.

6.9 EXAMPLE 9: TASTE MASKING BY ENCAPSULATION OF CAFFEINE IN

LYCOPODIUM CLAVATUM SPORES [00332] In this example the CF-loaded L. clavatum spores with ERS as coencapsulent of Example 8 were investigated for their taste-masking properties using passive loading as described in the above Examples.

[00333] Human taste-masking studies were performed in healthy human volunteers with informed consent and institutional ethical committee approval. Human subjects of either sex with age range between 18 and 47 years old were selected. Subjects suffering from fever, cold, smokers, mouth sores, wounds were excluded from the study with the inclusion criteria being healthy volunteer who meet basic perception criteria. The selected healthy volunteers were assessed to establish their basic perception level towards caffeine bitterness. The surface temperature of the tongue was recorded using an Infrared (IR) thermometer and the screening of pure CF was performed to determine an individual's threshold and perception of bitterness recognition with respect to CF. Ten human volunteers who reported a bitter taste for CF standard solutions were recruited for the taste trials.

[00334] Firstly, the human volunteers were administered orally 2 mL of pure caffeine solution starting with water (blank) and different CF dose (0.5, 1, 5, 10 mg). The volunteers were requested to score the bitterness on a scale of 0 to 5 for each solution, where 0 indicates none and 5 indicates strong bitterness. In this step, the bitterness recognition threshold for all the human volunteers was assessed. Based on the qualified volunteer selection and a washout period of 24 hours, the volunteers were requested to place test products (physical mixture of CF with spores and CF loaded spores) on their tongue for duration of 30 sec. Both the products were

administered randomly (blinded) and volunteers were instructed to score the products on a scale of 0 to 5. The scores provided by all human volunteers were averaged and expressed as mean ± SD. The mean scores between the physical mixture of CF with spores and CF loaded spores formulation were compared using t-test at 95 % confidence level. P < 0.05 was considered as statistically significant.

[00335] Table 12 . Evaluation of caffeine (CF) test solution and taste masking formulations in human volunteers'^

4 5 3 3 3 4 3 2 4 4 3 4

5 10 5 4 5 5 5 4 5 5 4 5

Natural pollen spores for taste masking: Eq uivalent to 10 mg of CF

1 Physical 4 5 5 5 5 4 4 3 4 5

mixture:

spores and CF

(negative

control)

2 Encapsulated 0 0 0 0 0 0 0 1 0 2

CF in spores

with

coencapsulant

(lead

formulation)

Evaluation score is based on scale 0 to 5: 0 is no bitterness and 5 is highest bitterness.

Taste threshold of the human volunteers reached with 1 mg CF.

[00336] Table 12 lists the individual bitterness evaluation scores of all human volunteers for formulations including: water, CF at 0.5 mg, 1 mg, 5mg, and 10 mg in 2 ml water, a physical mixture of CF and L. clavatum spores (negative control), and CF-loaded/ERS-coencapsulated L. clavatum spores (lead formulation). FIGS. 35A and 35B show the corresponding bitterness score histograms for these formulations.

[00337] Thus, the bitter taste of caffeine can be effectively masked by encapsulating CF into L. clavatum spores by the modified passive loading technique or by coencapsulating with ERS. The results from the human trials confirmed taste masking of CF from CF encapsulated spores formulations, making them suitable for masking the bitter taste of commercial food supplements and pharmaceuticals.

6.10 EXAMPLE 10: HYDROPHOBICITY SURFACE MODIFICATION BY

UV/OZONE EXPOSURE

[00338] In this example surface modification to the natural Camellia pollen grains were investigated.

6.10.1 Materials & Methods

[00339] Surface Modification Using Ultraviolet (UV) Ozone Cleaner. The surface of the pollen grains were modified by exposure to UV-Ozone cleaner. A thin layer of whole Camellia pollen grains (approximately 50 mg) was spread evenly on a 90 x 15 mm petri dish and UV- Ozone treated using a benchtop PSD Series UV-Ozone cleaner (Novascan, United States). The UV-Ozone treatment of the pollen grains ranged from 30 sec to 120 min. [00340] Contact Angle Measurements: A thin layer of the UV-Ozone treated pollen was spread out on self-adhesive carbon tape on a glass slide. A 2 μL· bead of water was slowly lowered onto the pollen layer. The contact angle was measured using Attension Theta Optical Tensiometer (Biolin Scientific Holding AB, Sweden) with OneAttension 1.0 software.

Measurements were taken at 0.7 X magnification and for 10 sec at 12 frames per sec (fps).

6.10.2 Results

[00341] FIG. 36 illustrates the contact angle data for UV-Ozone treated Camellia pollen grains showing a decrease in contact angle with increasing UV-Ozone treatment duration.

Treatment of Camellia pollen with UV-Ozone produced a decrease in contact angle with increasing UV-Ozone treatment duration. The observed decrease in contact angle indicates a decrease in hydrophilicity of the pollen grains upon modifying the pollen surface through exposure to UV-Ozone.

[00342] As further illustrated in FIGS. 37-42, UV-Ozone treatment improved the hydrophilicity of pollen grains and sporopollenin exine capsules (SECs) in order to aid aqueous dispersion of the pollen grains and encapsulated materials therein. FIG. 37 shows SEM images of the UV-Ozone treated and untreated pollen grains, indicating improved surface roughness in the case of the UV-Ozone treated pollen grains. FIG. 39 shows SEM images of the UV-Ozone treated and untreated Camellia SECs. FIG. 38 shows aqueous solutions of the UV-Ozone treated and untreated Camellia pollen grains. FIG. 40. shows aqueous solutions of the UV-Ozone treated and untreated Camellia SECs. FIG. 41 A shows an aqueous solution of Camellia seed oil. FIG. 41B shows an aqueous solution of unloaded and untreated Camellia SECs. FIG. 41C shows an aqueous solution of Camellia SECs that were oil loaded, ethanol washed and UV-Ozone treated. FIG. 42 shows CLSM images illustrating macromolecular encapsulation in Camellia pollen grains and SECs.

[00343] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A whole spore engineered to encapsulate a compound or substance of interest.

2. A whole spore engineered to encapsulate a compound or substance of interest and coated with an agent to facilitate controlled release of the compound or substance of interest from the whole spore.

3. A whole spore engineered to encapsulate a compound or substance of interest and an agent to facilitate controlled release of the compound or substance of interest from the whole spore.

4. The whole spore of claim 1, 2 or 3, wherein the whole spore is a Abies spore, a Agrocybe spore, a Aspergillus niger spore, a Bacillus subtilis spore, a Cantharellus minor spore, a Epicoccum spore, a Cuburbita spore, a Cucurbitapapo spore, a Ganomerma spore, a

Lycopodium clavatum spore, a Myosotis spore, a Penicillium spore, a Periconia spore, a ryegrass spore, a Timothy grass spore, a maize spore, a hemp spore, a rape hemp spore, a wheat spore, a Urtica dioica spore, a sunflower spore, a corn spore, a pine spore, a cattail spore, a rape spore, a dandelion spore, a rye spore, a Baccharis spore, a Chorella, a Camellia spore, a ragweed spore, a mulberry spore, or a pecan spore.

5. The whole spore of claim 1, 2 or 3, wherein the whole spore has a size in the range of 0.5 μπι to 300 μπι.

6. The whole spore of claim 5, wherein the whole spore has a size in the range of 40 μπι ιο 100 μπι.

7. The whole spore of claim 6, wherein the whole spore has a size in the range of 1 μπι to 40 μπι.

8. The whole spore of claim 5, wherein the whole spore has a size in the range of 1 μπι ιο 100 μπι.

9. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a therapeutic agent.

10. The whole spore of claim 9, wherein the therapeutic agent is a small organic molecule, a peptide, a nucleic acid, a protein, a polymer, a biologic, a medicinal preparation of proteins, a herbal medicine, an inorganic compound, an organometallic compound, lithium, a platinum-based agent, or gallium.

11. The whole spore of any one of claims 1 to 8, wherein the compound or substance is an oil.

12. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a fragrance.

13. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a cleaning agent.

14. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a disinfectant agent.

15. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a pesticide.

16. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a herb.

17. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a food ingredient.

18. The whole spore of claim 17, wherein the food ingredient is caffeine.

19. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a herbicide.

20. The whole spore of any one of claims 1 to 8, wherein the compound or substance is a fuel.

21. A formulation comprising the whole spore of any one of claims 1 to 20 and a diluent or carrier.

22. A formulation comprising the whole spore of any one of claims 1 to 9 and a diluent or pharmaceutically acceptable carrier.

23. The formulation of claim 22 which is for topical administration.

24. The formulation of claim 22 which is for parenteral administration.

25. A method of treating a disease or condition in the subject, comprising administering the formulation of claim 22, 23, or 24, wherein the therapeutic agent encapsulated in the whole spore is beneficial for treating the disease or condition.

26. A cosmetic product or personal care product comprising the whole spore of any one of claims 1 to 9.

27. A food or drink product comprising the whole spore of any one of claims 1 to 8, 16, 17 or 18.

28. An herbal product comprising the whole spore of any one of claims 1 to 8 or 16.

29. A pesticide comprising the whole spore of any one of claims 1 to 8 or 15.

30. A herbicide comprising the whole spore of any one of claims 1 to 8 or 19.

31. A method for masking the taste of a compound or substance, comprising encapsulating the compound or substance in a whole spore and formulating that in a drink or food product.

32. The method of claim 31, wherein the encapsulation comprises contacting the compound or substance with the whole spore.

33. The method of claim 31, wherein the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure.

34. The method of claim 31, 32 or 33 further comprising coating the whole spore with agent for controlling the release of the compound or substance from the spore.

35. A method of improving the stability of a compound or substance, comprising encapsulating the compound or substance in a whole spore.

36. The method of claim 35, wherein the encapsulation comprises contacting the compound or substance with the whole spore.

37. The method of claim 35, wherein the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure.

38. The method of claim 35, 36 or 37 further comprising coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore.

39. A method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore.

40. A method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore engineered to encapsulate a compound or substance that is beneficial or useful in a cosmetic or personal care product.

41. A method of reducing the toxicity of a compound or substance, comprising encapsulating the compound or substance in a whole spore.

42. The method of claim 41, wherein the encapsulation comprises contacting the compound or substance with the whole spore.

43. The method of claim 41, wherein the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure.

44. The method of claim 41 , 42 or 43 further comprising coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore.

45. A method of preparing a formulation comprising a compound or substance of interest and the whole spore of any one of the claims 1 -20, comprising encapsulating the compound or substance of interest in the whole spore.