WO2020180311A1 - Plant-based exosome compositions and use thereof for rejuvenating skin - Google Patents

Abstract

A topical composition for regulating skin condition is described. The composition comprises an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier. The isolated exosomes are obtained from a plant's leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours. Uses of the exosome-containing compositions for regulating human skin include inducing increased skin integrity by cell renewal, enhancing water content or moisture of skin, reducing trans epidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, and improving the appearance of scars and skin abrasions.

Description

PLANT-BASED EXOSOME COMPOSITIONS AND USE THEREOF FOR

REJUVENATING SKIN

TECHNICAL FIELD

[0001] The present disclosure relates to plant exosome compositions, and preparation thereof, for uses including rejuvenating, regulating and conditioning skin.

BACKGROUND

[0002] Existing treatments for aging and wrinkled skin are temporary and many treatments are ineffective or have unwanted side effects. During the aging process, skin loses thickness and resiliency due to a loss of collagen and other elastic proteins in the dermal layers. These losses can result in fine lines and wrinkles. Common non-invasive methods for treating fine lines and wrinkles include application of formulations topically to the skin. The formulations commonly include alpha and beta hydroxyl acids, retinoic acids, argirelines, and vitamins. None of these formulations completely eliminate wrinkles and many are expensive. In addition, while some formulations irritate the skin to elicit a wound healing response, this does not result in replenishment of the thinning skin to sufficiently treat and/or prevent age-related defects. Thus, an unmet need remains for more effective topical formulations for regulating skin condition such as improving skin damage, wrinkles, and other defects including scars, keloids, skin discolorations, and skin abrasions.

[0003] Skin aging is characterized by a decrease in collagen synthesis and an increase in collagen breakdown. It is generally accepted that the breakdown of collagen is mediated by metalloproteinases [1]. The loss in dermal collagen is believed to contribute to the appearance of fine lines and wrinkles. It is believed that biological factors that stimulate collagen production in wound healing might provide a benefit for aging skin. As a result, formulations for regulating skin condition and/or rejuvenating skin such as those for treating and/or reducing the appearance of fine lines and wrinkles can include growth factors, peptide fragments, and other biologically active molecules. [0004] Plant-based skincare products have been used for centuries with varying degrees of effectiveness. Naturally derived botanical components, including biologically active molecules such as polyphenols, polysaccharides, glycoconjugates and glycoproteins are isolated from plants and incorporated into skincare products to improve skin tone and quality; however, single components are largely ineffective by themselves. This result necessitates choosing several individual components to combine for efficacy.

[0005] Mammalian cells are known to secrete extracellular vesicles known as microvesicles or exosomes. Exosomes were once thought of as contaminating debris in cell culture, however, it has become recognized that the secreted microvesicles are packed with protein and RNA cargos and play a role in cellular signaling and communication. Exosomes contain functional mRNA, miRNA, DNA, and protein molecules that can be taken up by target cells and affect target cell biology. Proteomic and genomic analysis of exosome cargo has revealed a broad range of signaling factors that are both cell type-specific as well as differentially regulated based on the secreting cells’ environment [2]. HSP70 has been previously shown to be a cargo constituent of exosomes [3, 4, 5]. The genetic information contained in exosomes may influence or even direct the fate of the target cell, for example by triggering target cell activation, migration, growth, differentiation or de-differentiation, or by promoting apoptosis or necrosis. As such, exosomes may provide additional cell factors for assistance in wound healing and epithelial remodeling.

[0006] Additionally, exosomes have been identified from other organisms, such as certain bacteria and Drosophila, suggesting that they are an evolutionarily conserved method of secretion and intercellular communication [6, 7]. This has led to questioning whether plants also secrete exosome or exosome-like vesicles (ELVs) [8]. The recent identification of apoplastic vesicles and ELVs from fruit juices strongly supports the presence of exosome secretion in plants [9-12]. As such, plant-derived exosome and ELV cargo may be regulated by the plant’ s environment. Plants respond to environmental stressors in a similar manner as mammalian cells as exemplified by exposure to heat stress increasing heat shock protein gene expression (HSP70 and HSP100) in Aloe barbadensis Miller (Aloe vera) [13]. Herein, we provide evidence that these changes are translated into exosome or ELV cargo content.

SUMMARY

[0007] In a first aspect of the invention, a topical composition for regulating skin condition includes an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier. The isolated exosomes are isolated from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours. In a feature of this aspect, the level of heat shock stress-response molecule in the heat-shocked plant exosome is from about 10 times to about 20 times higher than the level of heat shock stress-response molecule in the exosomes from non-heat shocked plants. In another feature of this aspect, the heat shock stress-response molecule comprises HSP70.

[0008] In yet another feature of this aspect, the composition further includes from about 0.1 to about 20% of a moisturizing agent. With regard to this feature, the moisturizing agent comprises one or more of panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof. The composition may also include one or more of a vitamin B3 compound, tocopherol or esters of tocopherol, and/or an anti-oxidant. The composition may comprise tocopherol nicotinate.

[0009] In an additional feature of this aspect, the plant is from the Asphodelaceae family. In another feature of this aspect, the plant is of the Aloe genus, including Aloe vera, Aloe barbadensis Miller, Aloe aborenscens or Aloe vera L. In yet another feature, the composition is in the form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.

[00010] In a second aspect of the invention, a method for regulating skin condition comprises applying to human skin at least once a day over at least seven days a topical composition according to the first aspect, wherein regulating skin condition includes one or more of inducing increased skin integrity by cell renewal, enhancing water content or moisture of skin, reducing trans epidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, or improving the appearance of scars and skin abrasions.

[00011] In a third aspect of the invention, a glove for conditioning the skin has a coating composition on the inside thereof, wherein the coating composition includes isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and a powder carrier. The plant-derived exosomes are isolated from a plant’s leaf flesh

conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of from about 33 °C to about 45 °C for about 1 hour to about 3 hours.

[00012] In a fourth aspect of the invention, a method of stimulating a skin rejuvenating response in human primary fibroblasts includes treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules. The exosomes are obtained from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours.

[00013] In a feature of the fourth aspect, the heat shock temperature is from about

33°C to about 37 °C. In another feature of the fourth aspect, the heat shock temperature is from about 40°C to about 45 °C.

[00014] In an additional feature, the skin rejuvenating response includes increasing production of collagen I in human primary fibroblasts by about 10% to about 80% relative to control. With regard to this feature, the skin rejuvenating response may include increasing production of collagen I in human primary fibroblasts by at least 20% relative to control, by at least 40% relative to control and/or by at least 60% relative to control. For example, collagen I production in human primary fibroblasts may be increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, and/or 80% relative to control.

[00015] In another feature of the fourth aspect, the skin rejuvenating response may comprise increasing production of hyaluronic acid in human primary fibroblasts by about 10% to about 50% relative to control. With regard to this feature, the skin rejuvenating response may include increasing production of hyaluronic acid in human primary fibroblasts by at least 20%, by at least 30%, and/or by at least 40% relative to control. For example, hyaluronic acid production may increase by about 10%, 15%, 20%, 25%, 30%, 35%, and/or 40%, relative to control.

[00016] In another feature of the fourth aspect, the skin rejuvenating response may comprise increasing production of elastin protein in human primary fibroblasts by about 100% to about 400% relative to control. With regard to this feature, the skin rejuvenating response may include increasing production of elastin protein in human primary fibroblasts by at least 120%, by at least 150%, by at least 200%, and/or by at least 300% relative to control.

[00017] In a fifth aspect of the invention, a method of regulating an inflammatory response in mammalian cells includes treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules. The exosomes are obtained from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45 °C for about 1 hour to about 3 hours.

[00018] In a feature of the fifth aspect, the heat shock temperature is from about 33°C to about 37 °C. In another feature of the fifth aspect, the heat shock temperature is from about 40 °C to about 45 °C.

[00019] In an additional feature of this aspect, regulating the inflammatory response comprises reducing the amount of inflammatory response molecules in mammalian cells upon exposure of the cells to immune response inducing bacteria. With regard to this feature, the amount of inflammatory response molecules is reduced by about 20% to about 50%. For example, the amount of inflammatory response molecules may be reduced by at least 30% or by at least 40%. In further features of the fifth aspect, the inflammatory response molecule may comprise IL6 or TNFa.

BRIEF DESCRIPTION OF THE DRAWINGS [00020] FIGS. 1A and IB are graphs showing the size distribution of a representative sample of A) non-heat shocked (mean 156nm, mode 115nm) and B) 45°C for 3 hours heat-shocked (mean 156nm, mode 117nm) aloe vera-derived isolated exosomes according to one or more embodiments of the present disclosure.

[00021] FIG. 2 is a graph showing the relative changes in HSP70 mRNA transcript levels in exosomes isolated from aloe vera according to one or more embodiments of the present disclosure before and after exposure to heat shock. Values shown are Relative Quantification values determined by the AACt method relative to the non-heat shocked exosome values.

[00022] FIG. 3 is a bar graph showing the amount of cell proliferation in human primary fibroblasts after 3 day incubation with serum free medium, medium with 10% fetal bovine serum, or varying concentrations of aloe vera-derived exosomes isolated according to one or more embodiments of the present disclosure. Values shown are percent increase in proliferation relative to serum free medium.

[00023] FIG. 4 is a graph showing the amount of collagen I production in human primary fibroblasts after a 48 hour incubation with medium control, transforming growth factor b (TGF beta), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of collagen.

[00024] FIG. 5 is a graph showing the amount of hyaluronic acid produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of hyaluronic acid.

[00025] FIG. 6 is a graph showing the amount of elastin protein produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of elastin.

[00026] FIG. 7 is a graph showing quantified interleukin 6 (IL-6) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM dexamethasone and without exosomes (Dex 100nM+LPS), with lpg/ml LPS in combination with exemplary aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). Values shown are pg/ml of IL-6.

[00027] FIG. 8 is a graph showing quantified tumor necrosis factor alpha (TNFa) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM

dexamethasone and without exosomes (Dex 100nM+LPS), with lpg/ml LPS in combination with exemplary aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). Values shown are pg/ml of TNFa.

DETAILED DESCRIPTION

[00028] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[00029] There is an unmet need for more effective topical formulations for regulating skin conditions such as, for example, the treatment and prevention of skin damage, wrinkles, effects of aging, and defects including scars, keloids, skin discolorations, and skin abrasions. Described herein are plant-derived exosome compositions and methods for their preparation and use, to regulate skin conditions.

[00030] Exosomes represent a compelling therapeutic for a range of indications, especially those requiring delivery to tissues with reduced vasculature or prominent necrosis. Exosomes, unlike stem cells, do not require an oxygenated blood supply to exert their impact. And, because exosomes fuse with cell membranes directly, there is no requirement for receptor mediated uptake of their pro-healing cargos. Accordingly, the isolated plant-derived exosomes produced according to the methods provided herein can have advantages over existing systemic pharmaceuticals or direct application of plant extracts for regulating skin conditions.

[00031] The plant-derived exosome-containing compositions described herein are based on the context-dependency of the loading of exosomes. More specifically, as described herein, exosome loading can be engineered to result in exosomes having enhanced skin conditioning activities, such as and including, increased proliferative and anti-inflammatory activities. In exemplary embodiments, isolated exosomes can be prepared from aloe vera leaf flesh in a controlled environment, wherein the plant is exposed to various stimuli to manipulate the exosomal cargo. In an example of providing exosomes engineered for improved skin conditioning activity, aloe vera leaves can be subjected to relatively high temperature (otherwise known as“heat shock”) to produce exosomes having increased levels of heat shock stress-response molecules, including stress-response proteins in the HSP70 protein family. HSP70 proteins are a family of proteins expressed in response to heat stress or heat shock. HSP70 proteins have three major functional domains: N-terminal ATPase domain, substrate binding domain, and C-terminal domain.

[00032] As used herein, the term“increased levels” of heat shock stress-response molecules means that the amount of stress-response molecules present in exosomes of a plant that has been subjected to a relatively high temperature (or heat shock) is higher than the amount of stress-response molecules present in exosomes of a plant subjected to conventional plant exposure temperatures (for example, room temperature, which is generally around 25 °C). For example, increased levels may include increases of 5% to 200% relative to plants having no heat shock treatment. For example, the level of heat shock stress-response molecules in exosomes of a heat shocked plant may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175% or 200% greater than the level of heat shock stress-response molecules in exosomes of non-heat shocked plants. Additionally, the level of heat shock stress-response molecules in exosomes of a heat shocked plant may be 2, 3, 4, 5, 10, 15, 20, 25, or 30 times greater than the level of heat shock stress-response molecules in exosomes of non-heat shocked plants.

[00033] It is demonstrated herein that the isolated exosomes having increased heat shock stress-response proteins have increased proliferative, anti-inflammatory, collagen production, hyaluronic acid production and elastin production activity in cell cultures.

[00034] The terms“exosomes”,“exosome-like vesicles”,“microvesicles”,“secreted microvesicles”,“extracellular vesicles", and“secreted vesicles” are used interchangeably herein for the purposes of the specification and claims.

[00035] The terms“freeze drying” and“lyophilization” are used interchangeably herein for the purposes of the specification and claims.

[00036] The terms“stress-response molecules” and“heat shock stress-response molecules” are used interchangeably herein for the purposes of the specification and claims. These terms are meant to include molecules present in exosomes that are secreted by plant cells subjected to high temperature (otherwise known as“heat shock”). Often, heat shock stress-response molecules are proteins. Similarly, the terms“exosomes” and“heat shock exosomes” and“heat shocked exosomes” are used interchangeably herein for the purposes of the specification and claims to represent exosomes that are secreted by plant cells subjected to high temperature (otherwise known as“heat shock”).

[00037] The terms“a,”“an,” and“the” refer to“one or more” when used in this application, including the claims.

[00038] Throughout this specification and the claims, the terms“comprise,”

“comprises,” and“comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term“include” and its grammatical variants are intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[00039] For the purposes of this specification and claims, the term“about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[00040] Described herein is a plant-derived, exosome-containing composition comprising isolated plant-based exosomes containing heat shock stress-response molecules and a carrier. The heat shock stress-response molecules can be any molecules present in the plant exosomes that are secreted by plant cells in response to being subjected to a growing temperature that is relatively higher than the growing temperature to which the plant was exposed previously. Heat shock stress-response molecules are typically proteins produced by cells in response to exposure to stressful conditions, such as heat shock. Heat-shock proteins are named according to their molecular weight. For example, Hsp60, Hsp70 and Hsp90 refer to families of heat shock proteins on the order of 60, 70, and 90 kilodaltons in size, respectively.

[00041] A plant may be accustomed to being grown at a temperature of about room temperature, which is about 25°C. Thus, any growing temperature higher than 25°C could be a relatively higher temperature. A plant may also be accustomed to a growing temperature that is higher or lower than room temperature.

[00042] A relatively higher growing temperature may include a temperature at least

10°C greater than the growing temperature to which the plant was previously exposed. Using room temperature as an example, a relatively higher growing temperature may include 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C. Increasing the growing temperature of the plant may include increasing the temperature to a range of about 30°C to about 45 °C, about 30°C to about 40°C, about 32°C to about 38°C, about 33°C to about 37°C, and/or about

40 °C to about 45 °C.

[00043] A plant can be subjected to a relatively higher growing temperature for various periods of time. For example, a plant can be subjected to a relatively higher temperature for a period of time of about 30 minutes to about 4 hours, including, for example, 30 minutes, 60 minutes, 90 minutes, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours. After the plant is subjected to a relatively higher growing temperature for a period of time (i.e., heat shocked), the plant may then be exposed to a relatively lower growing temperature for a period of time. For example, the plant may be exposed to a temperature of about 25 °C to about 27 °C for about 24 hours to about 72 hours subsequent to heat shocking.

[00044] The carrier may be an acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more exosomes to a subject. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrates (e.g. starch, sodium starch glycolate, etc); or wetting agents (e.g. sodium lauryl sulphate, etc).

[00045] An exemplary embodiment of a plant-derived exosome-containing composition includes a composition comprising: i) isolated aloe vera plant exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier. The aloe vera exosomes can be produced by a process comprising: (a) growing the plant, wherein the growing includes a step of heat shocking the plant by increasing the growth temperature for a set period of time; and (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the leaf flesh.

[00046] The composition can be in any number of physical forms, including, but not limited to a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.

[00047] Isolation of the exosomes can be performed using various processes and/or combination of processes. For example, isolating the exosomes can be carried out by centrifugation, including one or more centrifugation steps. For example, the one or more centrifugation steps can include centrifugation at relatively lower speeds of 400 to 500 x g, at relatively moderate speeds of 10,000 to 20,000 x g, and relatively higher speeds of 80,000 to 100,000 x g or greater. Moreover, isolating the exosomes can further include freeze drying isolated exosomes. Freeze dried exosomes can be stored at room temperature.

[00048] The plant may be any plant believed to secrete heat shock response molecules in response to a heat shock process such as that described above. For example, the plant may be of the Asphodelaceae family. The plant can further be of the genus Aloe, including, but not limited to, Aloe vera and Aloe barbadensis. The heat shock stress-response molecules can include heat shock proteins, such as proteins from the following protein families: HSP60, HSP70, and HSP90.

[00049] In one embodiment, a method is provided for making plant-derived exosomes having increased levels of heat shock stress-response molecules, the method including:

growing the plant, wherein the growing condition includes a step of heat shocking the plant by increasing the growth temperature from a range of about 20°C to about 30°C, to about 33°C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’s leaf flesh contains the exosomes having the increased levels of heat shock stress-response molecules.

[00050] As will be described in greater detail below in Example 2, heat shock stress does not affect the size distribution of plant-derived exosomes. For many applications, it is desirable for exosomes to be in a size range of about 30 nm to about 150 mn. Advantageously, the results of the testing described in Example 2 indicate that exosomes produced after heat shock treatment are within the size range typically used for most applications.

[00051] The results of testing in Example 2 are shown in FIGs. 1A and IB. FIG. 1A shows the size distribution of a representative sample of isolated exosomes from non-heat shocked aloe vera with mean of 156 nm and a mode of 115 nm. FIG. IB shows the size distribution of a representative sample of isolated exosomes from the leaf flesh of an aloe vera plant 6 hours after a 3 hour 45 °C heat shock with a mean of 156nm and a mode of 116nm. As shown, there is no significant effect on the size distribution of exosomes following a heat shock stress.

[00052] As described below in Example 3, heat shock increases the production of heat shock stress-response molecules in plant cells, thereby resulting in an increased amount of stress-response molecules in exosomes derived from the heat-shocked plant cells. In this regard, as shown in FIG. 2, exposure to heat shock for 2 to 3 hours significantly upregulated the transcript levels of an exemplary heat shock stress-related molecule, HSP70, in the contents of the exosomes, and the upregulation persisted for 24 hours after removal of the heat shock stress.

[00053] The effects of isolated exosomes derived from heat-shocked aloe vera plants on cultured human primary fibroblasts are described in Example 4. In this regard, FIG. 3 shows that treatment with isolated exosomes from the plant leaf flesh of heat shocked plants significantly increased proliferation of human primary fibroblasts. In addition, the level of proliferation of the human primary fibroblasts induced by the isolated exosomes approached that induced by fetal bovine serum.

[00054] Degradation of collagen fiber and the extracellular matrix, as well as, significantly reduced elasticity and barrier functions are associated with skin aging.

Experimental results shown in Example 4 demonstrate that isolated exosomes prepared from aloe vera leaf flesh of heat shocked plants can induce collagen I, hyaluronic acid and elastin synthesis in human primary fibroblasts.

[00055] For example, FIG. 4 shows that treatment with isolated exosomes from heat shocked plants increased collagen I production of human primary fibroblasts. Specifically, exposure of the plant to heat shock increased the beneficial effects of the isolated exosomes on human primary fibroblast collagen I production by about 40% to about 80% and this exosome activity persisted up to 24 hours after heat shock exposure. In addition, the increase in collagen I production of the human primary fibroblasts induced by the isolated exosomes approached that of the individual growth factor, TGF-b.

[00056] FIG. 5 shows that treatment with the isolated exosomes from heat shocked plants increased hyaluronic acid production of human primary fibroblasts. Specifically, exposure of the plant to heat shock increased the effects of the isolated exosomes on human primary fibroblast hyaluronic acid production by about 31% to about 45% and this exosome activity persisted up to 24 hours after heat shock exposure. In addition, the increase in hyaluronic acid production of the human primary fibroblasts induced by the isolated exosomes approached that of the individual growth factor, IL-1.

[00057] FIG. 6 shows that treatment with the isolated exosomes from heat shocked plants increased elastin protein production of human primary fibroblasts by 140 - 375%. In addition, the increase in elastin protein production of the human primary fibroblasts induced by the isolated exosomes surpassed that of the individual growth factor, IL-1 (35%). These data indicate that the isolated exosomes can have a role in regulating skin condition and improving skin elasticity, barrier function and tensile properties.

[00058] Lypopolysaccharide (LPS) is a major component of the outer membrane of

Gram-negative bacteria and is known to induce a strong immune response in mammalian cells contributing to inflammation and related pathogenesis lethal to skin and soft tissue cells. Previous reports indicate the induction of inflammatory cascades in PBMCs in response to LPS, including the inflammatory molecules IL6 and TNFa [14-16]. In an experiment described in Example 5, PBMCs were concomitantly exposed to LPS (lpg/ml) and isolated exosomes from aloe vera leaf flesh from heat shocked plants. The results in FIGs 7 and 8 indicate a significant elevation in IL-6 (87-fold) and TNFa (62-fold) protein secretion in PBMCs induced by LPS at lpg /ml. The elevation is reduced by 25-50% by isolated exosomes from plants exposed to heat shock. These data indicate that isolated exosomes from heat shocked plants can inhibit the production of inflammatory cytokines including IL6 and TNFa that act locally to recruit monocytes to the site of inflammation.

[00059] In one embodiment, a method is provided for treating a skin condition, the method including one or more of putting on, embedding into, or filling an area on the skin of a living body a composition of the present disclosure including isolated plant-derived exosomes having increased levels of heat shock stress-response molecules, wherein the condition of the skin is treated.

[00060] The skin condition can include, for example, one or more of a wound, a bum, a bum resulting from radiation treatment, a discoloration, a scar, and a keloid.

[00061] In one embodiment, a topical composition is provided for regulating skin condition, the composition comprising an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier.

[00062] In one embodiment a topical composition is provided for regulating skin condition, the composition including: i) an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the isolated exosomes are isolated from a plant’s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of about 33°C to about 37°C for about 1 hour to about 3 hours, and/or about 40°C to about 45°C for about 1 hour to about 3 hours.

[00063] In one embodiment, a topical composition for regulating skin condition is provided, the composition comprising: i) an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the isolated exosomes are produced by a process comprising: (a) growing a plant, wherein the growth condition includes a step of heat shocking plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33 °C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’ s leaf flesh contains the exosomes having the increased levels of heat shock

stress-response molecules; and (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the leaf flesh.

[00064] In one embodiment, a method is provided for making a topical composition for regulating skin condition, the method including: combining isolated exosomes having increased levels of heat shock stress-response molecules with a carrier, wherein the exosomes are isolated from the flesh of a plant’ s leaf conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of about 33°C to about 37°C for about 1 hour to about 3 hours, and/or of about 40°C to about 45°C for about 1 hour to about 3 hours.

[00065] In the compositions provided for regulating skin conditions, regulating skin conditions can include one or more of inducing increased skin integrity by cell renewal; enhancing water content or moisture of skin; reducing trans epidermal water loss, skin flaking, and scaling; improving skin thickness; enhancing skin tensile properties; reducing the appearance of dermal fine lines and wrinkles; improving skin texture; reducing skin pores size; enhancing skin smoothness; improving skin age spots; improving skin tone; or improving the appearance of scars and skin abrasions.

[00066] In the compositions provided for regulating skin condition, the composition can further include from about 0.1 to about 20% of a moisturizing agent. The moisturizing agent can include one or more of panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof.

[00067] In the compositions provided for regulating skin condition, the composition can further include a vitamin B3 compound. Exemplary, vitamin B3 compounds can include tocopherol nicotinate, niacinamide, and inositol hexanicotinate

[00068] In the compositions provided for regulating skin condition, the composition can further include an anti-oxidant. The anti-oxidant can include one or a combination of tocopherol or esters of tocopherol.

[00069] In the compositions provided for regulating skin condition, the isolated exosomes can be freeze dried.

[00070] In one embodiment, a method is provided for regulating a human skin condition which includes applying to human skin at least once a day over at least seven days a topical composition as described herein comprising isolated exosomes having increased levels of heat shock stress-response molecules. The method can further include applying the topical to human skin at least twice a day over at least fourteen days.

[00071] In one embodiment, a coating composition is provided for conditioning skin or hair, the coating composition including: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the plant-derived exosomes are produced by a process including: (a) growing the plant, wherein the growth condition includes a step of heat shocking the plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33 °C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’ s leaf flesh contains the exosomes having the increased levels of heat shock

stress-response molecules; and (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the plant’s leaf flesh.

[00072] In the coating compositions for conditioning skin or hair, the process for producing the isolated plant-derived exosomes can further include freeze drying the isolated exosomes.

[00073] In the coating compositions for conditioning skin or hair of the present disclosure, the process for producing the isolated plant-derived exosomes can further include freeze drying the isolated exosomes and the carrier can be a dry powder.

[00074] The coating compositions for conditioning skin or hair of the present disclosure can be a dry powder coating composition applied to the inside of a glove.

[00075] The coating compositions for conditioning skin or hair of the present disclosure can be in the form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.

[00076] In one embodiment, a glove is provided for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition including: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a powder carrier, wherein the isolated plant-derived exosomes are produced by a process including: (a) growing the plant, wherein the growth condition includes a step of heat shocking the plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33°C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45 °C for about 1 hour to about 3 hours, and wherein the plant’s leaf flesh contains the exosomes having the increased levels of heat shock stress-response molecules; (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the plant’s leaf flesh; and (c) freeze drying the isolated exosomes.

[00077] In one embodiment, a glove is provided for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition comprising: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a powder carrier, wherein the plant-derived exosomes are isolated from the plant’ s leaf flesh conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of about 33°C to about 37 °C for about 1 hour to about 3 hours, and/or of about 40°C to about 45 °C for about 1 hour to about 3 hours.

[00078] In one embodiment, a glove is provided for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition comprising isolated plant-derived exosomes having increased levels of heat shock stress-response molecules and a powder carrier.

[00079] In one embodiment, a method is provided for making a glove for conditioning the skin, the glove having a coating composition on the inside thereof, the method comprising applying to the inside of the glove, a coating composition according to the present disclosure comprising isolated exosomes having increased levels of heat shock stress-response molecules and a powder carrier.

EXAMPLES

[00080] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Example 1 Preparation of Exosomes with Increased Levels of Heat Shock Stress-Response

Molecules using Heat Shock

[00081] The following examples describe the production and isolation of exosomes having increased levels of heat shock stress-response molecules. Heat shock stress-response molecules have been shown to provide enhanced proliferative, extracellular matrix production, and anti-inflammatory activities.

[00082] Aloe vera plants were grown and sampled under the following conditions.

Initially, the plants were maintained at room temperature, approximately 25 ^°C with ample water and light. Leaf samples from plants grown in the room temperature conditions were taken by removing a 3 inch leaf section for further preparation. Leaf section preparation is described below. The plant was then heat shocked by warming it to 35^°C in an incubator for 2 hours. After the 2 hour incubation, 3 inch leaf sections from the heated plant were dissected for further preparation.

[00083] For leaf section preparation, the inner leaf pulp containing parenchymal mesophyll cells was carefully removed to avoid collecting any outer leaf skin material and then homogenized. Homogenization was performed using a Precellys tissue homogenizer, ceramic beads and sterile PBS to disrupt the plant pulp tissue. The homogenate was transferred to a larger tube and the beads were washed 4 times with sterile PBS to collect all of the homogenate, which was also transferred to the larger tube for storage at -80^°C until used for exosome isolation.

[00084] In separate preparations, the same procedure as described above was followed except that the temperature used in the heat shock step was about 42 ^°C in some preparations and about 45^°C in other preparations, and the time period of heat shock ranged from as short as about 1 hour to as long as about 3 hours.

[00085] For all samples, the exosomes produced by the plants were isolated from the inner leaf pulp homogenate using the following procedure. The homogenate was diluted in phosphate-buffered-saline (PBS) at a ratio of about 16g of homogenate per 50 mL PBS. The diluted homogenate mixture was put through a freeze thaw cycle by freezing at -80^°C and then allowing the mixture to thaw at room temperature, whereupon the volume was increased to 300 ml. by adding PBS.

[00086] The diluted homogenate mixture was then fractionated by differential centrifugation to isolate the exosomes produced by the plants. In particular, the diluted homogenate mixture was subjected to centrifugation at 4,000 x g for 20 min at room temperature to pellet cell debris (in 50, 250, or 500 mL screw cap vessels). The clarified supernatant was collected and subjected to centrifugation at 12,000 x g (Avg. RCF) for 30 minutes at room temperature to remove any intact chloroplasts. The subsequent clarified supernatant was brought up to 360 mL with PBS and subjected to centrifugation at 100,000 x g (Avg. RCF) for 2 hrs at 4^°C to pellet exosomes. The supernatant was aspirated and the pellet(s) suspended in minimum volume of DPBS (300-1000 pL).

[00087] Manufacturer’s instructions were followed to estimate protein and RNA concentration using a NANODROP (THERMO FISHER, Corp) spectrophotometer. Particle diameter and concentration were assessed by tunable resistive pulse sensing (TRPS; (qNano, Izon Science Ltd) using a NP150 nanopore membrane at a 47 mm stretch. The concentration of particles was standardized using multi-pressure calibration with 110 nm carboxylated polystyrene beads at a concentration of 1.2x 10E13 particles/mL. The isolated exosomes were aliquoted into appropriate volumes into 1.5 mL screw cap tubes.

[00088] It was determined that the isolated exosomes described above could be stored at

-80^°C and then thawed at a later date for use without a detectable decrease in activity.

Example 2

Size Characterization of Isolated Heat Shock Exosomes

[00089] Size characterization was performed on isolated exosomes produced according to the method described in Example 1. The results were compared to isolated exosomes that were not heat shocked..

[00090] The isolated exosomes were analyzed using the QNANO (IZON SCIENCE,

Ltd), following manufacturer’s instructions. The graphs in FIGs. 1A and IB show the resulting size distribution of representative samples from non-heat shocked and heat shocked aloe vera plants. FIG. 1A shows the particle size distribution of exosomes derived from non-heat shocked aloe vera plants. As can be seen, the exosomes had a mean diameter of 156 nm and a mode diameter of 115 nm. FIG. IB shows the particle size distribution of exosomes derived from an aloe vera plant 6 hours after it was heat shocked at 45 ^°C for 3 hours. The heat shocked exosomes had a mean diameter of 156 nm and a mode diameter of 117 nm. As shown, exposure to heat shock stress did not significantly change the mean particle size of the isolated exosomes. Thus, heat shock treatment does not detrimentally alter or impact the applications in which heat shocked exosomes can be used.

Example 3

Heat Shock Induces Stress Response Molecules in Plant-Derived Exosomes

[00091] Isolated exosomes produced according to the method described in Example 1 were characterized using the following procedures.

[00092] To determine and compare changes in heat shock stress response molecules in the isolated plant exosomes, total RNA was isolated from exosomes derived from the leaf pulp of aloe vera plants exposed to no heat shock, 35^°C heat shock for 2 hours, 45 ^°C heat shock for 3 hours, and 24 hour room temperature recovery after 45^°C heat shock for 3 hours. Specifically, 2 x 10^L10 exosomes from each treatment were used to isolate total RNA using a micro RNeasy kit from Qiagen and the manufacturer’s protocol. Total RNA was quantified spectrophometrically using manufacturer’s instructions and a NANODROP (THERMO FISHER, Corp) spectrophotometer. cDNA was prepared using 300ng total RNA per reaction in 20 microliter final volume and qPCR performed using lOng of cDNA per triplicate, primers for HSP70 and 18s genes, and SYBR Green master mix in a final volume of 15 microliters. Relative gene expression was determined by the AACt method using 18s gene expression as the internal housekeeping gene and comparisons made to the no heat shock control exosomes. HSP70 is a heat stress response molecule. In this testing, HSP70 was used as a marker to show that heat stress responses to the plant were transferred to the plant exosomes. One of skill in the art will understand that other stress response molecules can be transferred to the plant exosomes.

[00093] The results are shown in FIG. 2, which shows the relative changes in HSP70 mRNA transcript levels in exosomes isolated from aloe vera plants for each of the treatments set forth above. Values shown are Relative Quantification values determined by the AACt method (Fold Change) relative to the non-heat shocked exosome values. Statistical significance compared to the non-heat shocked sample was determined using student’s t-test and a p-value < 0.05, significance is denoted by a As can be seen in FIG. 2, induction of a heat shock stress at both 35^°C and 45^°C increased the transcript level of the stress response molecule, HSP70, relative to that for non-heat treated plants. The transcript level at a heat shock temperature of 35 °C was less than the transcript level at a heat shock temperature of 45 °C. The data shows a 10X increase in transcript level of the stress response molecule HSP70 after a heat shock of 35 °C in comparison to the same for non-heat shocked exosomes. Transcript level of HSP70 after a heat shock of 45 °C was about 17X that for non-heat shocked exosomes, and transcript level of HSP70 after heat shock of 45 °C and 24 hour waiting period was about 22X that for non-heat shocked exosomes. As shown, the increase in stress response molecules was maintained for at least 24 hours after removing the heat shock stress. The data indicate that stress response molecules are significantly increased in isolated exosomes by subjecting the aloe vera plant to a heat shock for 2-3 hours, and that these effects persist for at least 24 hours after the heat shock. Temperatures higher than 45 °C were not tested because of concerns that higher temperatures would detrimentally affect the health of the plant.

Example 4

Effects of Isolated Exosomes on Cultured Human Fibroblasts

[00094] The effects of the isolated exosomes produced according to the method described in Example 1 on cultured human fibroblasts were determined as described below.

[00095] Isolated exosomes were added to low density human primary fibroblasts

(3,000 cells/well) in 96-well culture plates in serum free medium and incubated for 3 days. To compare the proliferative effects of the isolated exosomes, the cells were also treated with another inducer, 10% Fetal Bovine Seram (FBS). After 3 days, the cells were treated with CELL TITER BLUE REAGENT (PROMEGA) for 2 hours to assess proliferation.

[00096] FIG. 3 graphically shows the results of the cell proliferation in the human primary fibroblasts. The values shown are percent increase in proliferation compared to media only treatment. The data in FIG. 3 shows that treatment with the heat shocked isolated exosomes increased proliferation of human primary fibroblasts. For example, heat shocked exosomes produced up to 25% more fibroblasts than a media only control. While aloe vera-derived exosomes with no heat shock demonstrated a dose dependent induction of proliferation, many of the heat-shocked exosomes provided a greater increase in proliferation than the non-heat shocked exosomes. Moreover, the data shows that different concentrations of isolated exosomes have differing effects on the proliferation of human primary fibroblasts. Unexpectedly, relatively lower concentrations of isolated exosomes (e.g., 1.11 x 10⁷ particles/mL and 3.33 xlO⁷ particles/mL) led to higher fibroblast proliferation levels than some relatively higher concentrations of isolated exosomes (e.g., 1.00 x 10⁸ particles/mL) from heat-shocked aloe vera plants. Thus, the data suggests that the concentration of isolated exosomes from heat-shocked aloe vera plants can be selected or tuned to provide optimum results and that relatively lower amounts of exosomes can be used to provide results approaching that of much higher amounts of exosomes. In addition, the level of proliferation of the fibroblasts induced by the isolated exosomes approached that induced by 10% FBS.

[00097] Aging and various skin conditions are associated with degradation of the extracellular matrix, loss of elasticity, decreased barrier function and collagen fiber degeneration. The following experiments were performed to determine if isolated exosomes prepared from aloe vera plants using the procedures described in Example 1 could induce collagen I synthesis, hyaluronic acid synthesis, and elastin synthesis in human dermal fibroblasts.

[00098] For the experiments, isolated exosomes produced by aloe vera plants were tested along with serum-free conditioned medium from vehicle and growth factors using a similar procedure. For the assessment of collagen synthesis, human dermal fibroblast cells (10,000 cells/well) were treated for 48 hours with one of media only, 20ng/ml TGF -l, or isolated exosomes (1c10^L8 particles/mL), some from non-heat-shocked aloe vera plants and some from heat-shocked aloe vera plants. After the 48 hours, the conditioned medium was removed, clarified by centrifugation, and diluted into a procollagen I C-peptide HTRF ELISA (Cisbio Bioassays) assay.

[00099] The resulting data are shown in FIG. 4, which is a graph showing the amount of collagen I production (in ng/mL) in human primary fibroblasts after a 48 hour incubation with medium control, transforming growth factor b (TGF beta), or exosomes isolated from aloe vera plants. Values shown are ng/ml of collagen. As shown in FIG. 4, treatment with the isolated exosomes only from heat shocked aloe vera plants increased collagen I production of human fibroblasts. Exosome samples from heat-shocked plants were obtained at the following times: 1) after heat shock of 35°C for 2 hours, 2) after 3 hours of 45 °C heat shock, and 3) after 24 hours post 45°C heat shock. Collagen I production was increased by 42 - 78% for all three heat-shocked aloe vera plant exosome types relative to media alone. Exosomes from aloe vera plants not receiving a heat shock stress did not increase collagen I production. The highest collagen I production was measured for exosomes obtained immediately after heat shock of 35°C for 2 hours. TGF -l is a known activator for collagen I synthesis;

however, it is also known to cause scarring and excess production of collagen. TGF -l was included in the testing for comparison with exosomes from heat shocked aloe vera plants.

The results indicate that exosomes from heat-shocked aloe vera plants provide a favorable alternative for collagen I synthesis.

[000100] For the assessment of isolated exosomes on dermal fibroblast hyaluronic acid synthesis, human dermal fibroblast cells were treated for 48 hours with one of media only, lng/ml IL-1, or the isolated exosomes (1c10^L7 particles/mL). After the 48 hours, the conditioned medium was removed, clarified by centrifugation, and diluted into a hyaluronic acid ELISA assay (Corgenix Medical Corp.). The resulting data are shown in FIG. 5, which is a graph showing the amount of hyaluronic acid produced (in ng/mL) by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera plants (including non-heat- shocked plants and heat-shocked plants). The exosomes were isolated from heat-shocked plants at varying times: 1) immediately after heat shock of 35°C for 2 hours, 2) 6 hours post 45°C heat shock, and 3) 24 hours post 45°C heat shock. Values shown are ng/ml of hyaluronic acid.

[000101] As shown in FIG. 5, treatment with the isolated exosomes from heat-shocked aloe vera plants increased hyaluronic acid production of human fibroblasts and approached that induced by IL-1. The highest hyaluronic acid production was measured for exosomes obtained immediately after heat shock of 35°C for 2 hours, showing a 45% increase in production in comparison to media only hyaluronic acid production (control). Hyaluronic acid production for exosomes obtained 6 hours post heat shock (38% increase) was slightly higher than that for exosomes obtained 24 hours post heat shock (31% increase). The increase in production of hyaluronic acid after treatment with exosomes from heat shocked aloe vera plants was greater (31 - 45%) than the increase in production after treatment with exosomes from non-heat shocked aloe vera plants (19%).

[000102] The effects of isolated exosomes (1c10^L7 particles/mL) from aloe vera plants on elastin synthesis were determined using a similar method to that for hyaluronic acid synthesis. The 48 hour clarified conditioned medium was diluted into a human elastin ELISA assay (Wuhan USCN Business Co.). The resulting data are shown in FIG. 6, which is a graph showing the amount of elastin protein (in ng/mL) produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera plants. The exosomes were isolated from heat-shocked plants at varying times: 1) immediately after heat shock of 35°C for 2 hours, 2) 6 hours post 45°C heat shock, and 3) 24 hours post 45 °C heat shock. Values shown are ng/ml of elastin. FIG. 6 shows that isolated exosomes from heat shocked aloe vera plants greatly increased elastin synthesis, by 139 - 377% relative to media only samples (control), including above that induced by IL-1 (36%), and that this activity was maintained out to 24 hour following heat shock. Exosomes from non-heat shocked aloe vera plants increased elastin protein production as well. The limit for clinically relevant elastin induction is currently unknown. Thus, it is beneficial to understand the effect on elastin production after treatment with exosomes from heat-shocked as well as non-heat shocked plants. Example 5

Plant-Derived Exosomes Inhibit Production of Inflammatory Cytokines

[000103] The potential of isolated exosomes from heat-shocked aloe vera plants to inhibit IL6 and TNFa protein secretion in human peripheral blood mononuclear cells (PBMCs) was examined as described below.

[000104] Lypopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is known to induce a strong immune response in mammalian cells promoting the secretion of inflammatory cytokines. Previous reports indicate the induction of inflammatory cascades in PBMCs in response to LPS, including the

inflammatory molecules IL6 and TNFa [14-16]. To evaluate the anti-inflammatory impact of treatment with isolated exosomes prepared from heat shocked aloe vera plants, human PBMCs were concomitantly exposed to LPS (lpg/ml) and exemplary isolated exosomes from heat shocked aloe vera plants. Dexamethasone, a synthetic glucocorticoid known to reduce inflammation in response to LPS, was included as a control comparator.

[000105] To measure inflammatory response, multiplexed quantification of secreted cytokine protein levels for the inflammatory cytokines IL6 and TNFa was performed using MagPix and Milliplex assay kits. PBMCs were seeded in 96-well plates and incubated overnight prior to treatments. Cells were treated as follows: 1) without LPS and without exosomes (Media Only), 2) with lpg/ml LPS and without exosomes (lpg/ml LPS), 3) with lpg/ml LPS and lOOnM dexamethasone (Dex lOOnM + LPS), and 4) with lpg/ml LPS in combination with heat-shocked aloe vera-derived isolated exosomes (lpg/ml LPS + Aloe Exosomes).

[000106] The quantified cytokine data are shown in the graph in FIGs. 7 and 8. More specifically, FIG. 7 is a graph showing quantified interleukin 6 (IL-6) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and lOOnM dexamethasone (Dex IOOhM+LPS) only, with lpg/ml LPS (lpg/ml LPS) only, and a series of exemplary samples with lpg/ml LPS in combination with aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). The exemplary samples of aloe vera derived exosomes all used a concentration of 1 x 10^L8 particles/mL of exosomes isolated from plants at varying times: 1) no heat shock, 2) immediately after heat shock of 35 °C for 2 hours, 3) immediately after heat shock of 45°C for 3 hours, 4) 6 hours post 45°C heat shock, and 5) 24 hours post 45 °C heat shock were used. Y axis values shown are pg/ml of IL-6. Treatment with aloe exosomes isolated immediately after heat shock of 45 °C for 3 hours and 6 hours post 45 °C heat shock provided decreased production of IL-6. Aloe exosomes isolated 6 hours post 45°C heat shock provided the biggest decrease in production of IL-6, which most closely approached IL-6 production decrease achieved by treatment with lOOnM dexamethasone.

[000107] FIG. 8 is a graph showing quantified tumor necrosis factor alpha (TNFa) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM

dexamethasone and without exosomes (Dex 100nM+LPS), and a series of exemplary samples with lpg/ml LPS in combination with aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). The exemplary samples of aloe vera derived exosomes all used a concentration of 1 x 10^L8 particles/mL of exosomes isolated from plants at varying times: 1) no heat shock, 2) immediately after heat sho

[000108] ck of 35°C for 2 hours, 3) immediately after heat shock of 45°C for 3 hours, 4) 6 hours post 45°C heat shock, and 5) 24 hours post 45°C heat shock were used. Y-axis values shown are pg/ml of TNFa. Treatment with aloe exosomes isolated immediately after heat shock of 45 °C for 3 hours and 6 hours post 45 °C heat shock provided decreased production of TNFa relative to no treatment. The biggest decrease in production of TNFa was provided by treatment with exosomes isolated 6 hours post 45 °C heat shock. [000109] The results indicate a significant elevation in IL-6 (87-fold) and TNFa (62-fold) protein secretion in PBMCs induced by LPS at lpg/ml. The elevation is reduced by the isolated aloe vera exosomes from heat-shocked plants by about 40% for TNFa and about 50% for IL-6. These data indicate that the isolated exosomes described herein can inhibit the production of inflammatory cytokines including IL6 and TNFa that act locally to recruit monocytes to the site of inflammation·

References

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9. Regente, M., et ak, Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins. FEBS Lett, 2009. 583(20): p. 3363-66. PMID 19796642.

10. Regente, M., et ak, Apoplastic exosome-like vesicles: a new way of protein secretion in plants? Plant Signal Behav, 2012. 7(5): p. 544-6. PMID 22516827.

1 1 . Ju, S., et ak, Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol Ther, 2013. 21(7): p. 1345-57. PMID 23752315.

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[000110] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. These patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[000111] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present Examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

Claims

THAT WHICH IS CLAIMED:

1. A topical composition for regulating skin condition, the composition comprising: i. an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules, wherein the isolated exosomes are obtained from a plant’s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45 °C for about 1 hour to about 3 hours; and

ii. a carrier.

2. The composition of claim 1, wherein the level of heat shock stress-response molecule in the heat- shocked plant exosome is from about 10 times to about 20 times higher than the level of heat shock stress-response molecule in the exosomes from non-heat shocked plants.

3. The composition of claim 1, wherein the heat shock stress-response molecule

comprises HSP70.

4. The composition of claim 1, further comprising from about 0.1 to about 20% of a moisturizing agent.

5. The composition of 4, wherein the moisturizing agent comprises one or more of

panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof.

6. The composition of claim 1, further comprising one or more of a vitamin

B3 compound tocopherol or esters of tocopherol, and an anti-oxidant.

7. The composition of claim 1, further comprising tocopherol nicotinate.

8. The composition of claim 1, wherein the plant is from the Asphodelaceae family.

9. The composition of claim 1, wherein the plant is of the Aloe genus, including Aloe vera, Aloe barbadensis Miller, Aloe aborenscens or Aloe vera L.

10. The composition of claim 1, wherein the composition is in the form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.

11. A method for regulating skin condition which comprises applying to human skin at least once a day over at least seven days a topical composition according to claim 1, wherein regulating skin condition includes one or more of inducing increased skin integrity by cell renewal, enhancing water content or moisture of skin, reducing trans epidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, or improving the appearance of scars and skin abrasions.

12. A glove for conditioning the skin having a coating composition on the inside thereof, the coating composition comprising: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a powder carrier, wherein the plant-derived exosomes are isolated from a plant’s leaf flesh conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of from about 33 °C to about 45 °C for about 1 hour to about 3 hours.

13. A method of stimulating a skin rejuvenating response in human primary fibroblasts, the method comprising treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules, wherein the exosomes are obtained from a plant’s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45 °C for about 1 hour to about 3 hours.

14. The method of claim 13, wherein the heat shock temperature is from about 33°C to about 37°C.

15. The method of claim 13, wherein the heat shock temperature is from about 40°C to about 45°C.

16. The method of claim 13, wherein the skin rejuvenating response comprises increasing production of collagen I in human primary fibroblasts by about 20% to about 80% relative to control.

17. The method of claim 16, wherein the skin rejuvenating response comprises increasing production of collagen I in human primary fibroblasts by at least 20% relative to control.

18. The method of claim 16, wherein the skin rejuvenating response comprises increasing production of collagen I in human primary fibroblasts by at least 40% relative to control.

19. The method of claim 16, wherein the skin rejuvenating response comprises increasing production of collagen I in human primary fibroblasts by at least 60% relative to control.

20. The method of claim 13, wherein the skin rejuvenating response comprises increasing production of hyaluronic acid in human primary fibroblasts by about 20% to about 50% relative to control.

21. The method of claim 20, wherein the skin rejuvenating response comprises increasing production of hyaluronic acid in human primary fibroblasts by at least 20% relative to control.

22. The method of claim 20, wherein the skin rejuvenating response comprises increasing production of hyaluronic acid in human primary fibroblasts by at least 30% relative to control.

23. The method of claim 20, wherein the skin rejuvenating response comprises increasing production of hyaluronic acid in human primary fibroblasts by at least 40% relative to control.

24. The method of claim 13, wherein the skin rejuvenating response comprises increasing production of elastin protein in human primary fibroblasts by about 100% to about 400% relative to control.

25. The method of claim 24, wherein the skin rejuvenating response comprises increasing production of elastin protein in human primary fibroblasts by at least 120% relative to control.

26. The method of claim 24, wherein the skin rejuvenating response comprises increasing production of elastin protein in human primary fibroblasts by at least 150% relative to control.

27. The method of claim 24, wherein the skin rejuvenating response comprises increasing production of elastin protein in human primary fibroblasts by at least 200% relative to control.

28. The method of claim 24, wherein the skin rejuvenating response comprises increasing production of elastin protein in human primary fibroblasts by at least 300% relative to control.

29. A method of regulating an inflammatory response in mammalian cells, the method comprising treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules, wherein the exosomes are obtained from a plant’s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45 °C for about 1 hour to about 3 hours.

30. The method of claim 29, wherein the heat shock temperature is from about 33°C to about 37°C.

31. The method of claim 29, wherein the heat shock temperature is from about 40°C to about 45°C.

32. The method of claim 29, wherein the regulating the inflammatory response comprises reducing the amount of inflammatory response molecules in mammalian cells upon exposure of the cells to immune response inducing bacteria.

33. The method of claim 32, wherein the amount of inflammatory response molecules is reduced by about 20% to about 50%.

34. The method of claim 33, wherein the amount of inflammatory response molecules is reduced by at least 30%.

35. The method of claim 33, wherein the amount of inflammatory response molecules is reduced by at least 40%.

36. The method of claim 32, wherein the inflammatory response molecule comprises IL6.

37. The method of claim 32, wherein the inflammatory response molecule comprises TNFa.