WO2010009146A1 - Nanoagrégats pour l’administration de nanoparticules médicamenteuses faiblement hydrosolubles - Google Patents

Nanoagrégats pour l’administration de nanoparticules médicamenteuses faiblement hydrosolubles Download PDF

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Publication number
WO2010009146A1
WO2010009146A1 PCT/US2009/050565 US2009050565W WO2010009146A1 WO 2010009146 A1 WO2010009146 A1 WO 2010009146A1 US 2009050565 W US2009050565 W US 2009050565W WO 2010009146 A1 WO2010009146 A1 WO 2010009146A1
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Prior art keywords
nanoparticles
nanocluster
nanoparticle
drug substance
insulin
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PCT/US2009/050565
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English (en)
Inventor
Cory J. Berkland
Mark Bailey
Nashwa El Gendy
Carl Plumley
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University Of Kansas
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Publication of WO2010009146A1 publication Critical patent/WO2010009146A1/fr
Priority to US12/954,509 priority Critical patent/US8906392B2/en
Priority to US14/535,177 priority patent/US9278069B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1688—Processes resulting in pure drug agglomerate optionally containing up to 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)

Definitions

  • the present invention relates generally to delivery vehicles that can be used to transport active ingredients to a subject.
  • the delivery vehicles can be nanoclusters that can be used in preventative or therapeutic applications.
  • Nanoparticles as drug delivery vehicles has been employed for a variety of indications (John 2003). Nanoparticles, for example, have been shown to improve the dissolution of poorly water-soluble drugs and enhance the transport of drugs both intra- and paracellularly.
  • literature indicates that plasmid DNA can be effectively delivered by polycantionic polymers that form nanoparticles when mixed with DNA resulting in enhanced gene expression (Kumar 2003).
  • Research efforts on nanoparticle-mediated gene therapy also address treating genetic disorders such as Cystic Fibrosis (Griesenbach 2004).
  • nanoparticle formulations are designed for action at the cellular level. This assumes the efficient delivery of the nanoparticle to the appropriate cellular target.
  • current nanoparticle treatment options are limited in the ability to access the cellular target.
  • two research groups are currently investigating microencapsulated nanoparticles as a mode of nanoparticle delivery to the pulmonary epithelium (Sham 2004, Grenha 2005). These efforts are hindered by the common inability to control microparticle size, distribution, and difficulty in delivering a large payload of therapeutic nanoparticles.
  • the present invention overcomes the deficiencies in the art by providing effective drug delivery systems that can: (1) formulate nanoparticles as a nanocluster to facilitate handling, administering, or targeting, for example; and (2) maintain the cluster or disperse the nanoparticles at the targeted site.
  • a nanocluster comprising a plurality of nanoparticles.
  • the nanocluster is maintained at the targeted site (e.g., the nanocluster does not disperse into separate nanoparticles).
  • the nanoparticles disperse in response to an environmental cue.
  • the nanocluster in certain non-limiting embodiments, can have a size of about 1 to about 200 microns.
  • the nanocluster size is l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 microns.
  • the size of the nanocluster can be greater than 200 microns (e.g., 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 600, 700, or more microns in size.)
  • the nanocluster of the present invention can also have a variety of shapes (e.g., spherical and non-spherical shapes).
  • the nanocluster can be solid or hollow. A person of ordinary skill in the art will recognize that a solid nanocluster can be completely solid throughout or can have spaces, such as pores or a hollow core, that are created by the packing of the nanoparticles within the nanocluster.
  • the size of these packing spaces can be from about 1 nm to about 1000 nm, in non-limiting aspects. In certain aspects, the size of the packing spaces can be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nanometers, in non-limiting aspects. Hollow nanoclusters can have an empty space or cavity. The size of the cavity can vary, for example, from about 50 m to about 20 ⁇ m, in non limiting aspects.
  • the size of the cavity for example, can be 50, 100, 150, 200, 250, 300, 3500, 400, 450, 500, 550, 600, 650, 700, 750, 800 . . . 20 ⁇ m, and any range derivable therein.
  • the nanoparticles that are included in the nanocluster are not held (e.g., adhered or chemically bound (e.g., covalent bond, non-covalent bond, van der waals forces)) together by a functional group on the nanoparticles.
  • the nanoparticles can be in direct contact with one another in some aspects. In other aspects, the nanoparticles are not in direct contact with one another. In certain embodiments of the present invention, the nanoparticles are not encapsulated. In other embodiments, the nanoparticles do not include a functional group. In other aspects, however, the nanoparticles can include a functional group such as, for example, a carboxyl, sulhydryl, hydroxyl, or amino group. All types of functional groups that can be used to bind other nanoparticles together, active ingredients to the surface of nanoparticles, or other compounds are contemplated as being useful with the present invention.
  • the nanocluster can include an active ingredient.
  • active ingredients that are contemplated as being useful in the context of the present invention include those known to a person of ordinary skill and those described throughout this specification.
  • active ingredients can include medical pharmaceuticals and specialties such as preventive agents, for example vaccines, diagnostic agents, for example tracers of various types and imaging enhancers, therapeutic agents, for example small molecules (e.g., nucleic acids, proteins, peptides, polypeptides, etc.), drugs, peptides, and radiation, immuno-modulators, vaccine and virus vectors, and combinations of these classes.
  • the nanoparticles can include particular embodiments, respirable non-medical specialties such as physiochemical agents, for example gas antidotes, biophysical modulators, for example paramagnetics, emitters, for example electromagnetic wave emitters, and imaging enhancers.
  • the active ingredients in certain embodiments, can be associated with the nanoparticles.
  • the active ingredients can be entangled, embedded, incorporated, encapsulated, bound to the surface (e.g., covalently or non-covalently bonded), or otherwise associated with the nanoparticle.
  • the active ingredient is the nanoparticle.
  • the nanoparticles can include a polymer material (including, for example, biodegradable and non-biodegradable polymers). Non-limiting examples of polymer materials that can be used include those known to a person of ordinary skill and those described throughout this specification.
  • the nanoparticles can include a mixture of a polymer and an active ingredient.
  • the nanocluster or nanoparticles, or both can include at least one, two, three, four, five, six, seven, or more different active ingredients.
  • the nanocluster or nanoparticles include a first drug on its surface, and a second active ingredient encapsulated within the nanocluster or nanoparticles or other incorporated into the nanocluster or nanoparticle material. It is contemplated that a nanocluster can release the active ingredients in a given environment, or after a given period of time in a controlled manner. For example, a nanocluster having at least one active ingredient can be released in response to an environmental cue or after a pre-determined amount of time.
  • a nanocluster having at least two different active ingredients can be released in response to different environmental cues or after pre-determined periods of time.
  • active ingredient 1 can be released first and then active ingredient 2 can be released second.
  • the release of the first active ingredient can improve the performance of the second active ingredient.
  • the nanoclusters of the present invention can include a dispersing material that holds the plurality of nanoparticles together and/or disperses the nanoparticles in response to an environmental cue.
  • the dispersing materials that can be used with the present invention include those materials that are known to a person of skill in the art and those that are disclosed throughout this specification.
  • Non-limiting examples of dispersing material include liquid sensitive materials (e.g., water-soluble materials (e.g., polymers)), biodegradable polymers, polyelectrolytes, metals, surfactants, polymeric cross-linkers, small molecule cross-linkers, pH sensitive materials, pressure sensitive materials, enzymatic sensitive materials, and temperature sensitive materials.
  • Non-limiting examples of environmental cues that can be used with the present invention include liquid (e.g., water, blood, mucous, solvent, etc.), a selected pH range, a selected temperature range, an electric current, a selected ionic strength, pressure, the presence of a selected enzyme, protein, chemical, electromagnetic wavelength range (e.g., visible light, UV light, infrared, ultraviolet light, microwaves, X-rays, and gamma-rays), or the presence of an external force (e.g., vibration, shearing, shaking, etc.).
  • the dispersing material can be coated onto the surface of the nanoparticles before or after nanocluster formation.
  • the dispersing material can be between the nanoparticles or link the nanoparticles together (e.g., covalently or non-covalently couple a first nanoparticle to a second nanoparticle).
  • the dispersing material can be adhered to or covalently or non-covalently coupled to the nanoparticles.
  • the nanociuster can include from about 1% to about 99% by weight or volume of the nanoparticles or dispersing materials.
  • the nanocluster can also be completely made up of nanoparticles (i.e., 100%).
  • the nanocluster includes from about 10% to about 90%, 15% to about 80%, 20% to about 70%, 30% to about 60%, and about 40% to about 50% of nanoparticles or dispersing materials.
  • the nanocluster includes at least 50% of the nanoparticles or dispersing material.
  • compositions comprising a nanocluster of the present invention.
  • the composition in certain non-limiting aspects can have a plurality (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 200, 300, 400, 500, or more nanoclusters.
  • the composition can further include an active ingredient.
  • the composition can be formulated into a dry powder, an aerosol, a spray, a tablet, or a liquid.
  • compositions of the present invention can include at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nanoclusters of the present invention.
  • the compositions of the present invention can include a plurality of identical or similar nanoclusters.
  • the compositions of the present invention can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nanoclusters that have different characteristics (e.g., different active ingredients attached, different shapes, hollow or solid, etc.).
  • the compositions of the present invention can be formulated into a pharmaceutically acceptable carrier.
  • a method of preventing or treating a disease or condition in a subject comprising administering a therapeutically effective amount of a composition comprising a nanocluster of the present invention to a subject (e.g., human, pigs, horses, cows, dogs, cats, mouse, rat, rabbit, or any other mammal and non-mammals) in need of the composition.
  • a subject e.g., human, pigs, horses, cows, dogs, cats, mouse, rat, rabbit, or any other mammal and non-mammals
  • the method can further include a method for determining whether a subject is in need of the prevention or treatment.
  • the disease or condition can include all types of diseases or conditions known to a person of skill in the art and discussed throughout this specification.
  • the disease or condition can be a pulmonary associated disease or condition (e.g., common cold, flu, cystic fibrosis, emphysema, asthma, tuberculosis, severe acute respiratory syndrome, pneumonia, lung cancer, etc.), a circulatory disease or condition, a muscular disease or condition, a bone disease or condition, an infection, a cancer, etc.
  • the method can include the administration of a second therapy used to treat or prevent the disease (e.g., combination therapy).
  • the compositions of the present invention are administered nasally. Other modes of administration known to those of skill in the art or discussed in this specification are also contemplated.
  • the nanoclusters within the composition are delivered to the deep lung (e.g., bronchiole or alveolar regions of the lung).
  • the nanoclusters of the present invention can be used to deliver vaccines or components of vaccines.
  • cells of the immune system especially macrophages and dendrocytes, are targets for immunization.
  • APCs antigen-presenting cells
  • APCs are typically capable of phagocytosis of particles in the range of 1 to 10 ⁇ m.
  • the nanoclusters of the present invention can also have a particular mass density.
  • the mass density can be greater than, equal to, or less than 0.1 g/cm J .
  • the mass density of the nanoclusters of the present invention can be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 g/cm 3 , or greater.
  • Also disclosed is a method of preparing a nanocluster comprising: (i) obtaining a plurality of nanoparticles; (ii) obtaining a dispersion material (when desired); and (iii) admixing (i) and (ii), wherein the admixture is formulated into a nanocluster.
  • obtaining a plurality of nanoparticles comprises: (i) obtaining an aqueous suspension of nanoparticles; (ii) emulsifying the suspension into a non-aqueous phase; (iii) allowing water in the aqueous suspension to absorb into the non-aqueous phase; (iv) allowing the nanoparticles to aggregate together; and (v) retrieving the aggregated nanoparticles.
  • obtaining a plurality of nanoparticles includes: (i) obtaining a non-aqueous suspension of nanoparticles; (ii) emulsifying the suspension into an aqueous phase; (iii) allowing liquid in the non-aqueous suspension to absorb into the aqueous phase; (iv) allowing the nanoparticles to aggregate together; and (v) retrieving the aggregated nano particles.
  • the disclosed method represents a non-limiting method with other methods being evident by one skilled in the art (e.g. Emulsion/solvent evaporation, extraction, spray-drying, spray freeze-drying, self-assembly in solution, etc.).
  • the nanoclusters can be prepared in a solution without using spray and/or freeze dry techniques. It is also contemplated that the nanoclusters can be recovered from the solution by using freeze dry or spray dry techniques that are known to those of skill in the art. As noted throughout this specification, the nanocluster can be included within a composition.
  • the composition can be formulated into a liquid, a spray, an aerosol, or a dry powder in non-limiting embodiments.
  • a method of delivering an active ingredient to a subject in need comprising obtaining composition comprising a nanocluster of the present invention and an active ingredient and administering the composition to the subject.
  • the active ingredient is encapsulated in the nanoparticle, incorporated within the nanoparticle material, conjugated to the nanoparticle, absorbed or coupled to the nanoparticle.
  • a method of preparing a nanocluster comprising: (i) obtaining a first nanoparticle and a second nanoparticle; and (ii) admixing the first and second nanoparticles, wherein the nanoparticles self assemble to form a nanocluster.
  • the first and second nanoparticles can have hydrophobic properties, hydrophilic properties, or a mixture of both.
  • the first or second nanoparticles can have an electrostatic charge.
  • the first nanoparticle can be positively charged and the second nanoparticle negatively charged, and vice versa.
  • the self-assembly in particular embodiments can be based on an electrostatic interaction between the first and second nanoparticles.
  • the self-assembly can be based on a hydrophobic or hydrophilic interaction between the first and second nanoparticles.
  • the first and second nanoparticles can self assemble in solution to form the nanocluster in certain embodiments.
  • preparation of the nanoclusters does not require the use of spray and/or freeze dry techniques; rather nanocluster formation can occur in solution.
  • the nanoclusters can be recovered from the solution by using freeze dry or spray dry techniques that are known to those of skill in the art.
  • the method of preparing the nanocluster can further comprise obtaining a dispersion material and admixing the dispersion material with the first and second nanoparticles.
  • a method of storing nanoparticles comprising forming the nanoparticles into a nanocluster.
  • the nanoparticles for instance, can be stored as a liquid, a spray, and aerosol, or a dry powder.
  • the method of storing the nanoparticles can further comprise returning the nanocluster to nanoparticles.
  • returning the nanocluster to nanoparticles can include subjecting the nanocluster to an environmental cue.
  • environmental cues include water, a selected pH, a selected temperature, a selected enzyme, a selected chemical, a selected electromagnetic wavelength range, vibration, or shearing.
  • the nanocluster can include a dispersing material that holds the nanoparticles together and/or disperses the nanoparticles in response to an environmental cue.
  • dispersing materials include a water soluble polymer, a biodegradable polymer, a polyelectrolyte, a metal, a polymeric cross-linker, a small molecule cross-linker, a pH sensitive material, a surfactant, or a temperature sensitive material.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1 Therapeutic nanoparticles are organized into a nanocluster having a defined (and tunable) diameter. Upon contact with an environmental cue, the dispersive material triggers dispersion of the nanoparticles.
  • FIG. 2 Electron micrographs of (A) —100 nm silica particles that compose the (B) ⁇ 6 ⁇ m nanocluster. (C) Represents typical nanocluster distribution. Scale bar in (C) represents 10 ⁇ m.
  • Nanoclusters can be fabricated with a broad or narrow size distribution (left top and bottom). Adjusting fabrication conditions and/or dispersing material used allows for the formation of a solid (top right) or hollow (bottom right) clusters.
  • FIG. 4 Uniform ( ⁇ 75 ⁇ m) nanoclusters composed of polystyrene nanoparticles.
  • FIG. 5 Electron micrographs of (A) 225 nm silica nanoparticles coated with a dispersion material (light gray corona) and (B) a 9 ⁇ m nanocluster of the silica nanoparticles coated with dispersion material.
  • FIG. 6. The dispersion of nanoclusters overtime composed of nanoparticles coated with a hydrolysable polymer was a function of pH as determined by (A) absorption of light at 480 nm and (B) visual inspection.
  • FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F Laser scanning confocal micrographs of PLGA nanoparticle nanoclusters.
  • FITC-labeled PVAm-coated nanoparticles (FIG. 8A and FIG. 8D) and rhodamine-labeled PEMA-coated nanoparticles (FIG. 8B and FIG. 8E) are both identified within the nanocluster structure.
  • FIG. 9. Scanning electron microscope (SEM) image of a population of nifedipine nanoparticles.
  • FIG. 10 SEM image of nifedipine nanoparticle clusters.
  • FIG. 1 Illustration of the geometric diameter of the nanoclusters comprising DOTAP/PLGA nanoparticles and ovalbumin.
  • FIG. 12 SEM images of the nanoclusters comprising DOTAP/PLGA nanoparticles and ovalbumin.
  • FIG. 13 The particle size distributions of paclitaxel nanoparticle agglomerates in suspension after flocculation and resuspended after lyophilization.
  • FIG. 14 Aerodynamic size distributions of paclitaxel nanoparticle agglomerates after lyophilization.
  • FIG. 15 The distribution of Paclitaxel powder as received and nanoparticle agglomerate formulations deposited on the stages of a cascade impactor at a flow rate of -30 L/min.
  • FIG. 16 In-vitro dissolution profiles of paclitaxel in PBS (pH 7.4) from pure paclitaxel powder and two different nanoparticle (NP) and nanoparticle agglomerate formulations (NA).
  • FIG. Viability of A549 cells in the presence of formulation components as determined by an MTS assay.
  • FIG. 18 The particle size distributions of budesonide nanoparticle agglomerates in suspension after flocculation and resuspended after lyophilization.
  • FIG. 19 Aerodynamic size distributions of budesonide nanoparticle agglomerates after lyophilization.
  • FIG. 20 The distribution of budesonide nanoparticle agglomerate formulations (A) Fl, (B) F2, and (C) F3 deposited on the stages of a cascade impactor. (D) Formulations were compared with stock budesonide at a flow rate of ⁇ 30 L/min.
  • FIG. 21 Transmission electron micrographs of A) Fl nanoparticles and B) Fl nanoparticle agglomerates.
  • FIG. 22 13C CP/MAS spectra of budesonide, excipients, and budesonide formulations.
  • the nanoparticle agglomerates spectrum was expanded 8 times vertically to produce the nanoparticle agglomerates x8 spectrum to aid the interpretation of the budesonide peaks.
  • FIG. 24 13C CP/MAS spectra from spectral editing experiment.
  • FIG. 25 In-vitro dissolution profiles of budesonide in PBS (pH 7.4) from budesonide stock and three different nanoparticle (NP) and nanoparticle agglomerate formulations (NA).
  • FIG. 26 Viability of A549 cells in the presence of formulation components as determined by an MTS assay.
  • FIG. 27 Percent volume as a function of particle diameter for a flocculated solution of NIF/SA nanoparticles in water (421.7 +/- 26.2 ran, -32.16 +/- 3.75 mV) after addition of NaCl to 0.1 M. Also shown is the same solution after homogenization at 25000 RPM for 30 seconds.
  • FIG. 28 Aerodynamic Diameter size distribution for the sample of nanoparticle flocculates shown in Figure 1.
  • FIG. 29 A collection of SEM images for nanoparticles directly after sonication (A), newly prepared flocculates (B), flocculate powders after residing under room conditions and devoid of light for 1 month (C), and pure nifedipine crystals as received (D).
  • FIG. 30 DSC outputs for the optimal formulation of nanoparticles, pure nifedipine, and flocculated nanoparticles.
  • FIG. 31 Percent drug dissolution vs. Time as deduced via HPLC UV spectroscopy for the nifedipine/stearic acid nanoparticles, flocculates, and the drug in pure crystalline form.
  • FIG. 32 Cascade impactor readings for nifedipine/stearic acid nanoparticles, flocculates, and drug as received in pure form.
  • FIG. 33 Particle size distributions for a flocculate sample and portions of the sample after three homogenization regimes.
  • a nanoparticle solution (Before) was allowed to flocculate to completion without homogenization for 4 hours. Portions of the sample were then subject to increasingly powerful homogenization regimes (Low, Mid, High) from 5, 15, and 25 kRPM for 30 seconds, respectively.
  • FIG. 34 Particle size distributions for the flocculation of a nanoparticle suspension ( 336.1 +/- 5.9 nm, -34.42 +/- .73 mV) under a range of NaCl molarities (0.01 , 0.1, 1, and 10 Molar) marked from lowest to highest. Salt was added with homogenization at 15 kRPM for 30 seconds.
  • FIG. 35 Particle size distributions for a nanoparticie solution and portions of the solution with MgSO4 added to vary molarities (0.1, 0.25, 0.5) marked as low, mid, high, respectively. Salt was added with homogenization at 15 kRPM for 30 seconds.
  • FIG. 36 The effects of sonication and homogenization on a flocculated suspension of nanoparticles.
  • a solution of nanoparticles was allowed to flocculate to completion under 0.1 M CaC12. Portions of the solution were then subject to vary shear stresses.
  • Horn refers to 2 minutes of homogenization at 15 kRPM, and Son refers to subsequent sonication at 60% amplitude for 10 seconds.
  • FIG. 37 Schematic of a typical Anderson cascade impactor. Adapted from Reference: Pharacotherapy, copyright 2003 Pharmacotherapy Publications.
  • FIG. 38 Outline of insulin processing method.
  • FIG. 39 Mass fraction of insulin in pellet vs. PH. Each value represents mean ⁇ S.D of three experiments.
  • FIG. 40 Microparticle size vs. Nanoparticle size. Each value represents mean ⁇ S.D of three experiments.
  • FIG. 41 SEM micrographs of insulin particles; (A) and (B) are unprocessed insulin particles (scale bars 30 pm and 10 pm, respectively); (C) and (D) are insulin microparticles after processing (scale bars 10 pm and 2 pm, respectively).
  • FIG. 42 Tap density of insulin powders. Each bar shows mean ⁇ S.D. of three experiments.
  • FIG.43 Circular dichroism of dissolved insulin powders.
  • the top panel shows isothermal spectra, and the bottom panel shows variable temperature scan at a wavelength of 210 nm.
  • Each value of the variable temperature scan represents mean ⁇ S.D. of three experiments.
  • FIG. 44 13C CP/MAS NMR spectra for insulin powders; (A) Unprocessed; (B) Insulin microparticles; (C) Lyophilized insulin nanoparticles; (D) Centrifuged and dried insulin nanoparticles.
  • FIG. 45 Percent crystallinity of insulin particles, as determined by the HPLC dissolution method described in the U.S. Pharmacopeia and National Formulary. Each bar shows mean ⁇ S.D. of three experiments.
  • FIG. 46 Dissolution of insulin powders over time. Each value represents mean ⁇ S.D. of three experiments.
  • Nanoparticles offer several advantages for delivering drugs (e.g. Improved dissolution of low solubility API, intracellular and transcellular transport, etc.), the use of nanoparticles, for example, can be hindered by the inability to deliver nanoparticles to the site of drug action (e.g. Dried nanoparticles are too small to deposit efficiently in the lungs, can avoid detection by APCs, etc.).
  • nanoparticles are often difficult to handle at an industrial scale and a controlled clustering process may ease handling and allow facile reconstitution and formulation of nanoparticles or nanoclusters for delivering drugs.
  • the nanoclusters of the present invention can be used to deliver active ingredients to a targeted site.
  • the size and distribution of the disclosed nanoclusters and nanoparticles can be designed for a desired route of administration and/or for the treatment of a particular disease or condition.
  • the nanoclusters provide an effective and efficient drug delivery system that can carry nanoparticles to a targeted site via the nanocluster.
  • the nanocluster is maintained at the targeted site.
  • the nanocluster can disperse the nanoparticles at the targeted site.
  • the nanoclusters can be formulated with the appropriate physicochemical properties to carry and controllably release therapeutic nanoparticles or active ingredients to a targeted site.
  • nanoclusters can be prepared in a solution without using standard spray and/or freeze dry techniques known to those of ordinary skill in the art.
  • a nanocluster of the present invention can include a plurality of nanoparticles with or without a dispersing material that holds the plurality of nanoparticles together.
  • the dispersing material can also be used to disperse the nanoparticles in response to an environmental cue.
  • An active ingredient can also be incorporated into the nanocluster.
  • the nanoparticle can be the active ingredient.
  • This delivery system provides the advantage of particle clusters appropriately sized for delivery (e.g., lung, nasal passage, M-cells in the digestive tract, uptake by antigen presenting cells, etc.) with the benefits of nanoparticles, such as improvements in drug solubility, bioavailability, transport through biological barriers, intracellular delivery, etc.
  • delivery e.g., lung, nasal passage, M-cells in the digestive tract, uptake by antigen presenting cells, etc.
  • nanoparticles such as improvements in drug solubility, bioavailability, transport through biological barriers, intracellular delivery, etc.
  • changing the nature of the dispersing material allows for the development of an environmentally responsive nanoparticle delivery system and/or biosensors.
  • the special arrangement of nanoparticles within the cluster can allow discrete control over the duration and concentration of an active ingredient, a concept that can also be facilitated by the independent formulation of each nanoparticle type before cluster formation.
  • the inventors have successfully formulated nanoclusters from a variety of nanoparticulate materials and have controlled the dispersion of clusters into constituent nanoparticles in aqueous solution (see Examples 1-3 below).
  • the inventors obtained a colloidal suspension of nanoparticles in deionized water which is subsequently emulsified into octanol. Water in the dispersed droplets then absorbs into the octanol phase Nanoparticles can pack together as water is extracted from individual droplets until an aggregate of nanoparticles remains (FIG. 2).
  • the size of the droplet in certain non-limiting embodiments, can serve as a template for controlling the size of the resulting nanoclusters depending on the concentration of nanoparticles within the droplet.
  • the clustered nanoparticles in FlG 2 can be held together by hydrophobic, coulombic, and/or Van der Waals forces and can resist dispersion into aqueous media.
  • Nanoparticles are described in further detail in the following subsections.
  • a nanoparticle is a microscopic particle whose size is measured in nanometers.
  • the nanoparticles of the present invention have a size of from about 1 to about 3000 nanometers.
  • the nanoparticle has a size of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, or more nanometers, or any iange derivable
  • the material is the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) PLGA is a well-studied polymer for drug delivery and is FDA-approved for a number of in vivo applications.
  • PLGA biodegradable polymer poly(lactic-co-glycolic acid)
  • polyesters such as poly(lactic acid), poly(L-lysine), poly(glycolic acid) and poly(lactic-co-glycolic acid)
  • poly(lactic acid-co-lysine) poly(lactic acid-graft-lysine)
  • polyanhydrides such as poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), copolymers of these monomers and the like
  • poly(anhydride-co-imides such as poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), copolymers of these monomers and the like
  • poly(anhydride-co-imides such as poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexan
  • the nanoparticles include hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan, chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc. And their derivatives, co-polymers, and mixtures thereof.
  • HPC hydroxypropyl cellulose
  • NIPA N-isopropylacrylamide
  • PVA polyethylene glycol
  • PVA polyvinyl alcohol
  • polyethylenimine chitosan
  • chitin dextran sulfate
  • heparin chondroitin sulfate
  • gelatin etc.
  • a non-limiting method for making nanoparticles is described in U.S. Publication 2003/0138490, which is incorporated by reference.
  • the nanoparticles can be associated with an active ingredient (e.g., entangled, embedded, incorporated, encapsulated, bound to the surface, or otherwise associated with the nanoparticle).
  • the active ingredient is the nanoparticle.
  • the active ingredient is a drug such as a pure drug (e.g., drugs processed by crystallization or supercritical fluids, an encapsulated drug (e.g., polymers), a surface associated drug (e.g., drugs that are absorbed or bound to the nanoparticle surface), a complexed drugs (e.g., drugs that are associated with the material used to form the nanoparticle).
  • the nanoparticles of the present invention do not include a functional group.
  • the nanoparticles can include a functional group such as, for example, a carboxyl, sulhydryl, hydroxyl, or amino group. All types of functional groups that can be used to bind other nanoparticles together, active ingredients to the surface of nanoparticles, or other compounds are contemplated as being useful with the present invention.
  • the functional groups can be available for drug binding (covalent or electrostatic).
  • the dispersing material can serve several functions. For example, it can be used to hold (e.g., adhere or chemical bind (e.g., covalent bond, no-covalent bond, van der wall forces) the nanoparticles to one another via the dispersing material.
  • the dispersing material can disperse the nanoparticles at a targeted site in response to an environmental cue. This dispersing can occur, for example, when the dispersing material breaks-down, disintegrates, or other changes in such a way that it is no longer capable of holding the nanoparticles together.
  • Non-limiting examples of dispersing materials that are contemplated as being useful with the present invention include liquid sensitive materials (e.g., water-soluble materials) such as polyoxyethylene sorbitan fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene derivatives, and analogues thereof, sugar esters, sugar ethers, sucroglycerides, (e.g.
  • Sucrose, xylitol and sorbitol) etc. biodegradable polymers (see list of polymers for nanoparticle preparation), polyelectrolytes such as dextran sulfate, polyethylenimine, chitosan, chondroitin sulfate, heparin, heparin sulfate, poly(L-lysine), etc., metals (calcium, zinc, etc.), polymeric cross-linkers (polymethacrylate or similar derivatives with this functionality, poly(glutamic acid), poly(phosphorothioates), poly(propylene fumarate)-diacrylate, etc.
  • biodegradable polymers see list of polymers for nanoparticle preparation
  • polyelectrolytes such as dextran sulfate, polyethylenimine, chitosan, chondroitin sulfate, heparin, heparin sulfate, poly(L-lysine), etc.,
  • polymers with appropriate terminal or side chain reactive groups small molecule cross-linkers (di-expoxies, di-acids, di-amines, etc.) such as 2-methylene-l,3-dioxepane, gluteraldehyde, dithiobis succinimidyl propionate, pH sensitive materials such as poly( ⁇ -glutamic acid), enzymatic sensitive materials such as poly(amino acids) (peptides, proteins, etc.) like poly(N-substituted alpha/beta-asparagine)s, polysaccharides, lipids, oils, etc., and temperature sensitive material such as (2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(2-ethylacrylic acid-co-N-[4-(phenylazo)phenyl]methacrylamide), polymers of acrylic acid or acrylamide and related polymers including and co-polymers or blends of these in addition to those previously mentioned
  • Non-limiting examples of surfactants include esters of glycerin, esters of propylene glycol, fatty acid esters of polyethylene glycol, fatty acid esters of polypropylene glycol, esters of sorbitol, esters of sorbitan anhydrides, carboxylic acid copolymers, esters and ethers of glucose, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether phosphates, fatty acid amides, acyl lactylates, soaps, TEA stearate, DEA oleth-3 phosphate, polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soya sterol, steareth-2, steareth-20, steareth-21, ceteareth-20
  • Non-limiting examples of environmental cues that can cause the dispersing material to no longer be capable of holding the nanoparticles together include liquid (e.g., water, blood, mucous, solvent, etc.), a selected pH range, a selected temperature range, an electric current, a selected ionic strength, pressure, the presence of a selected enzyme, protein, DNA, chemical, electromagnetic wavelength range (e.g., visible light, UV light, infrared, ultraviolet light, microwaves, X-rays, and gamma-rays), or the presence of an external force (e.g., vibration, shearing, shaking, etc.).
  • the nanoclusters of the present invention can include an active ingredient.
  • Active ingredients include, but are not limited to, any component, compound, or small molecule that can be used to bring about a desired effect.
  • desired effects of the present invention include diagnostic and therapeutic effects.
  • a desired effect can include the diagnosis, cure, mitigation, treatment, or prevention of a disease or condition.
  • An active ingredient can also affect the structure or function of body part or organ in a subject.
  • Active ingredients which can be used by the present invention include but are not limited to nucleic acids, proteins and peptides, hormones and steroids, chemotherapeutics, NSAIDs, vaccine components, analgesics, antibiotics, anti-depressants, etc.
  • Non-limiting examples of nucleic acids that can be used include DNA, cDNA, RNA, iRNA, siRNA, anti-sense nucleic acid, peptide-nucleic acids, oligonucleotides, or nucleic acids that are modified to improve stability (e.g., phosphorothioates, aminophosphonates or methylphosphonates).
  • Proteins and peptides that can be used with the present invention include but are not limited to human growth hormone, bovine growth hormone, vascular endothelial growth factor, fibroblast growth factors, bone morphogenic protein, tumor necrosis factors, erythropoietin, thrombopoietin, tissue plasminogen activator and derivatives, insulin, monoclonal antibodies (e.g., anti-human epidermal growth factor receptor2 (Herceptin), anti-CD20 (Rituximab), anti-CD 18, anti-vascular endothelial growth factor, anti-lgE, anti-CD 1 Ia) and their derivatives, single-chain antibody fragments, human deoxyribonuclease 1 (domase alfa, Pulmozyme), type- 1 interferon, granulocyte colony-stimulating factor, luteinizing hormone releasing hormone inhibitor peptides, leuprolide acetate, endostatin, angiostatin, porcine factor VIII clotting factor
  • Non-limiting examples of hormones and steroids that can be used include norethindrone acetate, ethinyl estradiol, progesterone, estrogen, testosterone, prednisone and the like.
  • Chemotherapeutics that can be used include but are not limited to taxol (Paclitaxel), vinblastine, cisplatin, carboplatin, tamoxifen and the like.
  • Non-limiting examples of NSAIDs include piroxicam, aspirin, salsalate (Amigesic), diflunisal (Dolobid), ibuprofen (Motrin), ketoprofen (Orudis), nabumetone (Relafen), piroxicam (Feldene), naproxen (Aleve, Naprosyn), diclofenac (Voltaren), indomethacin (Indocin), sulindac (Clinoril), tolmetin (Tolectin), etodolac (Lodine), ketorolac (Toradol), oxaprozin (Daypro), and celecoxib (Celebrex).
  • Vaccine components that can be used include but are not limited to Hepatitis B, polio, measles, mumps, rubella, HIV, hepatitis A (e.g., Havrix), tuberculosis, etc.
  • Non-limiting examples of analgesics include but are not limited to aspirin, acetaminophen, ibuprofen, naproxen sodium and the like.
  • Antibiotics include but are not limited to amoxicillin, penicillin, sulfa drugs, erythromycin, streptomycin, tetracycline, clarithromycin, tobramycin, ciprofloxacin, terconazole, azithromycin and the like.
  • Anti-depressants include but are not limited to Zoloft, fluoxetine (Prozac), paroxetine (Paxil), citalopram, venlafaxine, fluvoxamine maleate, imipramine hydrochloride, lithium, nefazodone and the like.
  • Varying nanoparticle type or size, dispersion properties, dispersing materials, and processing conditions can be used to tune the nanocluster to a targeted size, density, and/or dispersability.
  • FIGS. 3 and 4 illustrate that varying processing conditions can be used to create nanoclusters with a broad or narrow size range and also allows for the formation of solid or hollow nanoclusters. Controlling the droplet size in an emulsion or sprayed from a nozzle can facilitate the formation of uniform nanoclusters. Varying the solvent and extraction phase, temperature, humidity, etc. As well as the properties of the nanoparticles can control the morphology of the nanocluster.
  • nanoparticle-carrying solution may result in a core/shell structure while slow remove of this phase allows time for nanoparticles to diffuse from the interface and form a more dense nanocluster structure.
  • controlling nanoparticle physicochemical properties can provide a driving force for the nanoparticle towards or away from the droplet interface, thus, leading to a core/shell structure or solid matrix, respectively.
  • the inventors can produced a wide range of monodisperse nano clusters (FIG. 4).
  • a variety of techniques can be used to characterize nanoclusters that have been created by varying nanoparticle type or size, dispersion properties, dispersing materials, and processing conditions. These techniques can be used to mechanistically determine how processing parameters affect particle physicochemical properties.
  • the aerodynamic diameter of a dried nanocluster powder can be determined by an Aerosizer LD (available at the Center for Drug Delivery Research, KU), which will also provide supportive data on dry particle geometric diameter, size distribution, aggregation and density.
  • Aerosizer LD available at the Center for Drug Delivery Research, KU
  • a helium pycnometer (Micromeritics AccuPyc 1330 helium gas pycnometer) located in Dr.
  • Eric Munson's lab can be used to more accurately determine the density of different nanocluster formulations. For example, a sample of nanocluster powder is measured into a 1 cm sample and weighed. The density of the sample is determined by helium displacement of the sample compared to a secondary empty chamber. Measurements are conducted in triplicate for each of three samples and the average and standard deviation calculated. Particle exterior and interior morphology (interior viewed via cryo-fracturing (Berkland 2004)) can be investigated via scanning electron microscopy (LEO 1550).
  • One embodiment of this invention includes methods of treating, preventing, or diagnosing a particular disease or condition by administering the disclosed nanoclusters to a subject.
  • the nanoclusters are administered alone or can be included within a pharmaceutical composition.
  • An effective amount of a pharmaceutical composition generally, is defined as that amount sufficient to ameliorate, reduce, minimize or limit the extent of the disease or condition. More rigorous definitions may apply, including elimination, eradication or cure of the disease or condition.
  • phrases "pharmaceutical or pharmacologically acceptable” can include but is not limited to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, such as, for example, a human.
  • the preparation of a pharmaceutical composition is generally known to those of skill in the art. Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
  • animal (e.g., human) administration it is preferred that the preparations meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • “Therapeutically effective amounts” are those amounts effective to produce beneficial results in the recipient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's, 1990).
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's, 1990
  • the actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active ingredient or a nanocluster, for example.
  • the an active ingredient or nanocluster may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the composition may also include various antioxidants to retard oxidation of one or more active ingredient or nanocluster.
  • various antioxidants to retard oxidation of one or more active ingredient or nanocluster.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • compositions of the present invention may include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the compositions may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • isotonic agents such as, for example, sugars, sodium chloride or combinations thereof.
  • nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained.
  • the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation.
  • various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.
  • the compositions are prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof.
  • the oral composition may be prepared as a syrup or elixir.
  • a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition should be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • compositions of the present invention can include any number of combinations of nanoparticles, dispersion materials, active ingredients, and other components. It is also contemplated that that the concentrations of these ingredients can vary.
  • a composition of the present invention can include at least about 0.0001% to about 0.001%, 0.001% to about 0.01%, 0.01% to about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.
  • the percentage can be calculated by weight or volume of the total composition.
  • concentrations can vary depending on the addition, substitution, and/or subtraction of nanoparticles, dispersion materials, active ingredients, and other components.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheal Iy, intranasally, intravitreally, intravaginally, intrauterinely, intrarectally, intrathecally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington's, 1990).
  • inhalation
  • compositions of the present invention can precede or follow the other agent treatment by intervals ranging from minutes to weeks. It is contemplated that one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6- 12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, where several days (2, 3, 4, 5, 6 or 7), several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) or even several months ( 1 , 2, 3, 4, 5, 6, or more) lapse between the respective administrations.
  • compositions including a nanocluster is "A” and the secondary agent, is "B”: A/B/A B/ AJB B/B/A AJAJB AJBIB BIAJA A/B/B/B BIAJBIB B/B/B/A B/B/A/B AIAJBIB AJBIAIB AJBIBIA BIBIAJA B/A/B/A B/A/A/B AJAIAIB B/A/A/A A/B/A/A A/A/B/A.
  • nanoparticles, dispersion materials, active ingredients, and other components described in the claims and specification can be obtained by any means known to a person of ordinary skill in the art.
  • these ingredients can be isolated by obtaining the source of such nanoparticles, dispersion materials, active ingredients, and other components.
  • the ingredients can be purified by any number of techniques known to a person of ordinary skill in the art. Non-limiting examples of purification techniques include Polyacrylamide Gel Electrophoresis, filtration, centrifugation, dialysis, High Performance Liquid Chromatography (HPLC), Gel chromatography or Molecular Sieve Chromatography, and Affinity Chromatography.
  • kits can include, in non-limiting aspects, the nanoparticles, dispersion materials, active ingredients, and other components described in the claims and the specification.
  • the kit can include a composition that includes a nanocluster.
  • the nanocluster can include, for example, a plurality of nanoparticles and a dispersing material that holds the plurality of nanoparticles together and/or disperses the nanoparticles in response to an environmental cue.
  • Containers of the kits can include a bottle, dispenser, package, compartment, or other types of containers, into which a component may be placed.
  • the container can include indicia on its surface.
  • the indicia for example, can be a word, a phrase, an abbreviation, a picture, or a symbol.
  • the containers can dispense a pre-determined amount of the component (e.g. Compositions of the present invention).
  • the composition can be dispensed in a spray, an aerosol, or in a liquid form or semi-solid form.
  • the containers can have spray, pump, or squeeze mechanisms.
  • the kit can include a syringe for administering the compositions of the present invention.
  • kits of the present invention also can include a container housing the components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired bottles, dispensers, or packages are retained.
  • a kit can also include instructions for employing the kit components as well the use of any other compositions, compounds, agents, active ingredients, or objects not included in the kit. Instructions may include variations that can be implemented. The instructions can include an explanation of how to apply, use, and maintain the products or compositions, for example.
  • Nanoclusters of the present invention can be prepared by the following procedure: Two syringe pumps (Harvard Apparatus 4400 and Isco) are connected to the inner and outer ports of a coaxial nozzle to pass a colloidal suspension of nanoparticles (see above formulation) in aqueous solution and 1-octanol (Fisher Scientific) as droplet carrying liquid, respectively. The two immiscible liquids are injected at appropriate flows to produce monodisperse aqueous droplets, which contain the colloidal suspension of nanoparticles, in the octanol phase.
  • Two syringe pumps Hard Apparatus 4400 and Isco
  • the two immiscible liquids are injected at appropriate flows to produce monodisperse aqueous droplets, which contain the colloidal suspension of nanoparticles, in the octanol phase.
  • nanoclusters are formed after water in the droplets dissolves into 1 -octanol resulting in packing of the nanoparticles into a spherical structure (FIG. 4). Nanoclusters are then washed with ethanol to remove residual 1-octanol and can be freeze dried for analysis. Similar results were achieved by simply adding the nanoparticle suspension to the octanol phase and stirring to form a primary emulsion.
  • the inventors coated silica nanoparticles with poly(N-vinylformamide) and cross-linked this polymer with a hydrolyzable cross-linker (2-bis[2,2'-di(N-vinylformamido)ethoxy]propane) to form nanoclusters that dispersed in response to a decrease in pH (FIG. 5).
  • This set-up was used to determine the ability to disperse nanoclusters in response to environmental cues.
  • the clustered nanoparticles were slightly different in appearance due to the presence of the polymer, but the size distribution remained consistent with previous experiments.
  • the nanoclusters were dispersed into aqueous solution as a function of time and pH (FIG. 6).
  • a turbidity assay was used to measure optical density at 480 nm over time, the opacity of the solution indicating the relative dispersion of the clusters into constituent nanoparticles.
  • the dispersion of the nanoclusters could also be visually tracked over time (FIG. 6B).
  • Size analysis of the solution phase of dispersed nanoclusters via laser light scattering indicated that polydisperse agglomerates of nanoparticles were liberated. These agglomerates further dispersed into individual nanoparticles overtime (FIG. 6C).
  • PLGA nanoparticles were prepared by a modified emulsion/solvent extraction method using different polyelectrolyte coating materials to control surface charge (Table 1 ).
  • Polyvinylamine (PV Am) was used as a cationic coating material and was synthesized in house (see Experimental).
  • Polyethylene-alt-maleic acid (PEMA) was synthesized by hydrolysis of the anhydride from of this polymer as adapted from methods reported previously.
  • the resulting polyelectrolyte-coated PLGA nanoparticles possessed excellent uniformity and high surface charge (Table 1 ).
  • Nanoparticles were analyzed for size and zeta potential using dynamic light scattering and conductivity measurements (Brookhaven ZetaPALS), respectively, in the appropriate media (water or organic). Studies confirmed the maintenance of particle surface charge upon lyophilization and after more than one week of incubation at 37° C, pH 7.4 (data not shown). PV Am-coated nanoparticles were notably larger than PEMA-coated nanoparticles for this experiment; however, this size is readily controlled. Nanoparticles can be made by using reported techniques, for example; emulsion polymerization, emulsion solvent extraction, reverse emulsions of the same, precipitation, crystallization, freeze drying, spray freeze drying, salting out, etc. (Wittaya-Areekul et al. 2002).
  • Nanocluster Formation Nanoparticle clusters were produced by slow addition of 3 mL of PVAm-coated nanoparticles into 10 mL of PEMA-coated nanoparticles under gentle stirring. Nanocluster formation was induced by electrostatic self-assembly of the oppositely charged nanoparticles. Increasing the concentration of mixed nanoparticles resulted in a corresponding increase in the cluster diameter (FIG. 7). The geometric size distribution of nanoclusters was determined in aqueous solution (Isoton) using a Coulter Multisizer III. Geometric size distributions were relatively broad exhibiting standard deviations that were 60-70% of the average geometric diameter.
  • the aerodynamic size distributions were determined from freeze dried nanoclusters using time of flight measurements obtained by an Aerosizer LD. Nanocluster aerodynamic size distributions were narrower than the geometric size distributions as indicated by the increased volume percent (FIG. 7B) and decreased standard deviations (35-60% of the mean; Table 2). Free PEMA-coated nanoparticles were detected as a rising tail in the geometric size distributions, but were not detected in the aerodynamic size distributions. In addition, few free nanoparticles were observed in scanning electron micrographs (FIG. 7C) indicating that nanoparticles that were not associated with nanoclusters in solution bound to nanoclusters during lyophilization.
  • Fine Particle Fraction defined as the fraction of diy particles with d aer o ( ⁇ m) ⁇ 5 ⁇ m.
  • PVAm-coated nanoparticles were labeled with a green fluoroisothiocyanate (FITC) dye and PEMA-coated nanoparticles were labeled with a red rhodamine dye.
  • FITC green fluoroisothiocyanate
  • PEMA-coated nanoparticles were labeled with a red rhodamine dye.
  • the following includes examples of preparing various non-limiting nifedipine and loratadine nanoparticles.
  • Nifredipine nanoparticle 1020 nm Nifredipine nanoparticle— Nifedipine (50 mg) was dissolved in 3 ml of methylene chloride. Dumped nifedipine solution into 0.125% Cetyltrimethylammonium bromide (CTAB) solution (30 mL) and sonicated for 60 s. The particle suspension was placed into a hood for two hours to evaporate the methylene chloride. The resulting nanoparticle had a particle size of 1020 nm and a polydispersity of 0.24.
  • CTAB Cetyltrimethylammonium bromide
  • Nifedipine nanoparticle Nifedipine (50 mg) was dissolved in 3 ml of methylene chloride. Dumped nifedipine solution into 0.5% CTAB solution (30 mL) and sonicated for 60 s. The particle suspension was placed into a hood for two hours to evaporate the methylene chloride. The resulting nanoparticle had a particle size of 660 nm and a polydispersity of 0.17.
  • Nifedipine nanoparticle— Nifedipine (50.2 mg) was dissolved in 3 ml of ethanol. Dumped nifedipine solution into 0.5% CTAB solution (30 mL) and sonicated for 60 s. The particle suspension was placed into a hood for two hours to evaporate the ethanol. The resulting nanoparticle had a particle size of 480 nm and a polydispersity of 0.12. 2373 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 ml of ethanol. Dumped nifedipine solution into 0.3% PIuronic F-68 solution (30 mL) and homogenized at 15,000 rpm for 60 s.
  • the particle suspension was placed into a hood for two hours to evaporate the ethanol.
  • the resulting nanoparticle had a particle size of 2373 nm and a polydispersity of 0.09. 897 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 ml of ethanol.
  • nifedipine solution into 0.6% PIuronic F-68 solution (30 mL) and homogenized at 15,000 rpm for 60 s.
  • the particle suspension was placed into a hood for two hours to evaporate the ethanol.
  • the resulting nanoparticle had a particle size of 897 nm and a polydispersity: 0.07.
  • Nifedipine nanoparticle 639 nm Nifedipine nanoparticle—Nifedipine (30 mg) was dissolved in 2 ml of ethanol. Dumped nifedipine solution into 0.9% PIuronic F-68 solution (30 mL) and homogenized at 15,000 rpm for 60 s. The particle suspension was placed into a hood for two hours to evaporate the ethanol. Resulting nanoparticle had a particle size of 639 nm and a polydispersity of 0.005.
  • Loratadine nanoparticle-Loratadine (10 mg) was dissolved in 1 ml of ethanol. Dumped loratadine solution into 0.9% PIuronic F-68 solution (10 mL) and homogenized at 15,000 rpm for 60 s. The particle suspension was placed into a hood for two hours to evaporate the ethanol. The resulting nanoparticle had a particle size of 391 nm and a polydispersity of 0.005.
  • the nanoparticle is pure nifedipine (a calcium channel blocker that treats high blood pressure).
  • the nanoparticle is coated with a cationic surfactant (CTAB).
  • CAB cationic surfactant
  • a polyanion (sodium alginate) couples with the CTAB which induces nanocluster formation.
  • nifedipine nanoparticles Preparation of nifedipine nanoparticles: Nifedipine (50 mg) was dissolved in methylene chloride (3 ml). The solution was poured completely into a CTAB concentration-known aqueous solution (Table 3). The solution was sonicated for 60 s. Subsequently, the particle suspension was placed into a hood for two hours to evaporate the methylene chloride The suspension was diluted to 1 mg/ml.
  • FIG. 9 is a scanning electron microscope (SEM) image of a population of nifedipine nanoparticles.
  • nifedipine nanoparticle clusters Preparation of nifedipine nanoparticle clusters: Algenic acid aqueous solution (10 ml, 1 mg/ml) was poured into nifedipine nanoparticle aqueous suspension (10 ml, 1 mg/ml) and the mixture was homogenized with a homogenizer (about 2000 rpm) for 2 min. Dry Nifedipine nanoparticle clusters were obtained by freeze-drying.
  • FIG. 10 is a SEM image of nifedipine nanoparticle clusters.
  • nanoparticle is a biodegradable polymer (PLGA) coated with a cationic lipid (DOTAP).
  • DOTAP cationic lipid
  • Ovalbumin couples to the surface of the coated nanoparticle which induces nanocluster formation.
  • PLGA nanoparticles were prepared using a modified emulsion-solvent evaporation technique (Kazzaz et al., 2000; Mainardes et al., 2005, both of which are incorporated by reference).
  • a cationic surface charge was incorporated using the lipid ],2-dioleoy]-3-trirnethylammoniurn-propane (DOTAP; Avanti Polar Lipids, Inc.; Alabaster, Ala.) as the coating material.
  • DOTAP lipid ],2-dioleoy]-3-trirnethylammoniurn-propane
  • 3 mL PLGA (0.41 dL/g inherent viscosity; Lactel; Pelham, AIa.) dissolved in an acetone/methanol mixture (5/1) at 1.67% (w/v) was added to 25 mL DOTAP (50 ⁇ M) and sonicated at 50% power using a sonic dismembrator (Fisher Scientific; Pittsburgh, Pa.) for 60 s on ice. This was repeated for a total of 6 batches.
  • the batches were combined and stirred at moderate speed in the hood overnight to evaporate the solvent.
  • the washed nanoparticles were sonicated in a water bath for 15 min and again filtered through a Kim Wipe to remove any large agglomerates.
  • the resulting particles were then characterized using a Zeta Potential Analyzer (Brookhaven Instruments; Holtsville, N.Y.) to measure particle size and surface charge (.zeta.): the nanoparticles had an average size of 343.0 ⁇ 8.6 (ran), a polydispersity of 0.232 ⁇ 0.022, and a zeta potential of 36.44 ⁇ 0.56 (mV).
  • the nanoparticle suspension was diluted in 1 mM sodium nitrate solution for surface charge measurements.
  • Ovalbumin was used as a model protein.
  • Three solutions containing approximately 0.4, 1.5 and 2.5 mg/mL ovalbumin were prepared in phosphate buffered saline (PBS), and the exact concentration of each solution was determined using UV absorbance spectroscopy (Table 4).
  • Three labeled, 15 mL centrifuge tubes, 6 mL DOTAP nanoparticles and 1 mL ovalbumin solution were added. The samples were tumbled gently on an end-over-end tube rotator for 45 min at 4° C.
  • the resulting nanoclusters were analyzed using a Multisizer 3 Coulter Counter (Beckman Coulter, Inc.; Fullerton, Calif.) to measure their geometric diameter.
  • the nanoclusters were lyophilized using a Labconco bench-top lyophilizer (Kansas City, Mo.) and further characterized to determine the aerodynamic diameter (Aerosizer; Amherst Process Instruments Inc.) and morphology (SEM) (Table 5).
  • FIG. 11 illustrates the geometric diameter of the DOTAP/PLGA nanoparticles with ovalbumin.
  • FIG. 12 includes SEM images of the nanoclusters comprising DOTAP/PLGA nanoparticles and ovalbumin.
  • a multi-stage liquid impactor (MSLI) fitted with a mouthpiece and throat assembly can be used to evaluate the deposition performance of various particle formulations administered from a dry powder inhaler.
  • DPI dry powder inhaler
  • Rotahaler® particles are first encapsulated in a large, two-piece gelatin capsule. The capsule is placed into a small compartment in the DPI, which is then twisted to either separate or rupture the capsule immediately prior to breath actuation. Since no propellants or compressed gases are used for these DPIs, the breathing force of the patient, or in our case the volumetric flow rate through the MSLI, disperses the powder.
  • Nanoclusters can be formulated for controlled release of ciprofloxacin for ⁇ 1 week.
  • a complete analysis of nanocluster physicochemical properties, dispersion and release of the drug can be prepared by the methods described throughout this specification.
  • the nanoclusters in one embodiment, can be made with nanoparticles of pure ciprofloxacin or ciprofloxacin encapsulated in PLGA nanoparticles.
  • Ciprofloxacin is a broad spectrum antibiotic, especially effective against gram negative bacteria (Geller 2002, Geller 2003, Marier 2003) having the following formula: Nanocluster dispersability and ciprofloxacin release kinetics: Nanocluster formulations can be reformulated to determine controlled release of ciprofloxacin, taking care to maintain the same fabrication procedure and resulting structure designed for deep lung deposition. Ciprofloxacin (Sigma, Inc.) can be encapsulated by co-dissolving with the polymer phase and will be partially suspended in the polymer phase or dissolved in a co-solvent if low solubility in the polymer phase is an issue. Dissolution studies ascertain the release kinetics of ciprofloxacin.
  • the release of ciprofloxacin from the various nanocluster formulations will be conducted in triplicate and the average and standard deviation is calculated.
  • the initial loading of ciprofloxacin in nanocluster formulations is determined by dissolving -10 mg of each formulation in triplicate in dimethylsulfoxide and measuring the absorbance at -350 nm. Absorbance values for formulations of nanoclusters without ciprofloxacin are used as blanks.
  • the calculated amount of ciprofloxacin per mass of polymer is termed the drug loading. This number can be divided by the mass of ciprofloxacin per mass of polymer entered into the experiment to calculate the drug encapsulation efficiency.
  • the summed mass of ciprofloxacin released over time is then divided by the drug loading to arrive at the cumulative percent released.
  • Analogous samples of nanoclusters can be prepared to determine the dispersion kinetics based on measuring the turbidity of the sample solution at 480 nm (see preliminary data above). Reformulation and optimization of controlled release: Generating a near constant release of ciprofloxacin for -1 week may include reformulation of nanoclusters. If drug "bursting" (rapid initial release) occurs or increased duration of release is desired, higher molecular weight PLGA or PLGA with a higher lactide content will be used as each of these prolong degradation of the polymer phase.
  • Paclitaxel nano-agglomerates as dry powder for pulmonary delivery
  • Paclitaxel (PX), L-a-phosphatidylcholine (lecithin; Lee), cetyl alcohol (CA), L-leucine (Leu), polyvinylpyrrolidone (PVP K90, Mw -36,000) and sodium chloride were purchased from Sigma Chemicals Co, USA.
  • Pluronic F- 127 (PL, Mw -12,220) was purchased from BASF, The Chemical Company, USA.
  • Polyvinyl alcohol (PVA; Mw 22,000, 88% hydrolyzed) was purchased from Acros Organics, New Jersey, USA. Potassium dihydrogen phosphate, disodium hydrogen phosphate, acetone, ethanol and acetonitrile were purchased through Fisher Scientific.
  • FBS Fetal bovine serum
  • Penicillin-streptomycin was purchased from MB Biomedical, LLC. Trypsin- EDTA was purchased through Gibco.
  • MTS reagent [tetrazolium compound; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] was purchased from Promega, USA. Double-distilled water was used throughout the study, provided by an EASYpure® RODI (Barnstead International, Model # Dl 3321).
  • Nanosuspensions were prepared using a precipitation technique. The drug was precipitated by direct injection of acetone solution of paclitaxel, 0.1% w/v, in waters at a rate of 1 mL/min under sonication (Fisher Scientic, Sonic Dismembrator) with an amplitude of 46%.
  • the chosen surfactants for the study included hydrophobic (cetyl alcohol), hydrophilic (PL, PVA and PVP K90) and amphoteric (lecithin).
  • hydrophobic and amphoteric surfactants were added to the drug organic solvent solution and the contents were allowed to stand at room temperature for 30 to 45 minutes with occasional vortexing to allow complete solubilization of the drug and the surfactants.
  • Hydrophilic surfactants were added to the aqueous phase. Surfactants were used individually or in combination as reported.
  • the particle size and zeta potential of the nanosuspensions were determined by dynamic light scattering (Brookhaven, ZetaPALS). Zeta potential measurements were performed using 1 mM KCl solution. All measurements were performed in triplicate.
  • paclitaxel nano-agglomerates were prepared by addition of L-leucine powder to agglomerate nanoparticle suspensions followed by homogenization at 25,000 rpm for 30 sec. The amount of L-leucine added was adjusted to a drug:leucine ratio equal to 1 :1. The size of paclitaxel nanoparticle agglomerates was measured in Isoton diluent using a Coulter Multisizer 3 (Beckman Coulter Inc.) equipped with a 100 mm aperture after three hours of incubation with the flocculating agent.
  • the particle size of the dispersed nanoparticle agglomerates as well as the resuspended lyophilized powder was measured using a Coulter Multisizer 3. The particle size distributions are shown in FIG. 13. 2.
  • the fixed- height cone method was used. A glass funnel with cut stem surface of 5 mm internal diameter was fixed at 2.5 cm height over a flat surface. The powders were allowed to flow gently through the funnel until a cone was formed and reached the funnel orifice. The flow of powder was then stopped and the average diameter of the formed cone (D) was measured. The area of the base of the cone was taken as a measure of the internal friction between the particles.
  • the aerodynamic size distributions of the agglomerate powders were measured directly from lyophilized powder by time-of-flight measurement using an Aerosizer LD (Amherst Instruments) equipped with a 700 mm aperture operating at 6 psi.
  • d gco geometric diameter
  • p particle bulk density
  • pa water mass density ( 1 g/cm ). Aerodynamic size distributions of paclitaxel nanoparticle agglomerates is shown in FIG. 14. Tapped density measurements underestimate particle bulk densities since the volume of particles measured includes the interstitial space between the particles. The true particle density, and therefore the aerodynamic diameter of a given powder, is expected to be slightly larger than reported.
  • Aerodynamic characteristics of selected nanoparticle agglomerates were studied in vitro using a Tisch Ambient Cascade Impactor (Tisch Environmental, Inc., Ohio). The study was carried out by applying ⁇ 20 mg powder manually into the orifice of the instrument at an air flow rate of -30 L/min. Cut-off particle aerodynamic diameters for each stage of the impactor were: pre-separator (10.00 mm), stage 0 (9.00 mm), stage 1 (5.8 mm), stage 2 (4.7 mm), stage 3 (3.3 mm), stage 4 (2.1 mm), stage 5 (1.1 mm), stage 6 (0.7 mm), stage 7 (0.4 mm) and filter (0 mm).
  • Nanoparticle agglomerates deposited on each stage of the impactor were determined by measuring the difference in weight of filters placed on the stages.
  • the mass median aerodynamic diameter, MMAD, and geometric standard deviation, GSD were obtained by a linear fit of the cumulative percent less-than the particle size range by weight plotted on a probability scale as a function of the logarithm of the effective cut-off diameter.
  • FIG. 15 shows the distribution of Paclitaxel powder as received and nanoparticle agglomerate formulations deposited on the stages of a cascade impactor at a flow rate of ⁇ 30 L/min.
  • D. Imaging of particles by Transmission Electron Microscopy Image data was used to corroborate the size of nanoparticles and nanoparticle agglomerates and to observe their morphological aspects.
  • TEM Transmission electron micrographs
  • the lyophilized powder for the prepared nanoparticle agglomerates was weighed and the yield was calculated using the following expression:
  • Paclitaxel loading efficiency was assessed by dispersing one mg of the lyophilized powder in 10 mL ethanol. The dispersion was sonicated in a bath-type sonicator (Branson 3510) for 30 min. Then the solution was centrifuged (Beckman, Avanti TM) at -15,000 rpm for 30 min to remove insoluble ingredients and the amount of drug in the supernatant was determined spectrophotometrically (Agilent C) at 228 nm. Drug loading was defined as follows:
  • the dissolution of the prepared nanoparticles and nanoparticle agglomerates was determined and compared with the dissolution characteristics of the drug powder as received.
  • the dissolution of paclitaxel was carried out at 37 ⁇ 0.5° C in a 1 liter beaker.
  • the solution was stirred at a constant speed (100 rpm) using a magnetic stirrer (Barnstead, Thermolyne MIRAKTM).
  • serial samples (1 mL) of the medium were withdrawn from the dialysis bag and centrifuged for 30 minutes at ⁇ 13,000 rpm.
  • the nanoparticles-free supernatant was removed and extracted with 3 mL of ethanol.
  • the ethanol extract was analyzed for paclitaxel concentration using a reverse-phase HPLC method. Studies were conducted in triplicate.
  • a Shimadzu HPLC system including a solvent delivery pump (Shimadzu LC-IOAT), a controller (Shimadzu SCL- 1 OA), an autoinjector (Shimadzu SlL- 1 OAxL), and a UV detector (Shimadzu SPD- 1 OA) was used in this study.
  • FIG. 16 shows the in-vitro dissolution profiles of paclitaxel in PBS (pH 7.4) from pure paclitaxel powder and two different nanoparticle (NP) and nanoparticle agglomerate formulations (NA).
  • NP nanoparticle
  • NA nanoparticle agglomerate formulations
  • cytotoxicity of selected nanoparticles and nanoparticle agglomerates was assessed using the CellTiter 96® Aqueous Cell Proliferation Assay (Promega) and compared with paclitaxel powder as received, lecithin, PVP K90, L- leucine, physical mixtures of these ingredients and blank nanoparticle agglomerates.
  • 8 x 104 A549 cells / well were seeded in 96-well microtiter plates. At the end of the incubation period (12 h), 20 ml of MTS reagent solution was added to each well and incubated for 3 h at 37 0 C.
  • FIG. 17 shows the viability of A549 cells in the presence of formulation components as determined by an MTS assay.
  • MMAD Mass median aerodynamic diameter obtained from Aerosizer.
  • d % EF Percent emitted fraction.
  • e RF Percent respirable fraction.
  • fMMAD Mass median aerodynamic diameter.
  • Nanoparticle technology represents an attractive approach for formulating poorly water soluble pulmonary medicines.
  • nanoparticle suspensions used in nebulizers or metered dose inhalers often suffer from physical instability in the form of uncontrolled agglomeration or Ostwald ripening.
  • processing such suspensions into dry powders can yield broad particle size distributions.
  • a controlled nanoparticle flocculation process has been developed. Nanosuspensions of the poorly water soluble drug budesonide were prepared by dissolving the drug in organic solvent containing surfactants followed by rapid solvent extraction in water. Different surfactants were employed to control the size and surface charge of the precipitated nanoparticles.
  • Selected budesonide nanoparticle suspensions exhibited an average particle size ranging from -160-230 nm, high yield and high drug content.
  • Nanosuspensions were flocculated using leucine, which produced micron-sized agglomerates. Freeze-drying the nanoparticle agglomerates yielded dry powders with desirable aerodynamic properties for inhalation therapy.
  • the dissolution rates of dried nanoparticle agglomerate formulations were significantly faster than that of stock budesonide.
  • the results of this study suggest that nanoparticle agglomerates possess the microstructure desired for lung deposition and the nanostructure to facilitate rapid dissolution of poorly water soluble drugs. Pulmonary dosage forms have established an important role in the local treatment of lung diseases.
  • Nanoparticles whether amorphous or crystalline, offer an interesting way of formulating drugs having poor water solubility. By presenting drugs at the nanoscale, dissolution can be rapid and as a result the bioavailability of poorly soluble drugs can be significantly improved.
  • Nanoparticles have been disregarded to some extent in dry powder dosage forms because particles ⁇ 1 mm have a high probability of being exhaled before deposition, are prone to particle growth due to Ostwald ripening and can suffer from uncontrolled agglomeration. Conversely, particles exhibiting an aerodynamic diameter from 1 to 5 mm are more likely to bypass the mouth and throat, resulting in augmented deposition in the lung periphery.
  • Budesonide is a potent nonhalogenated corticosteroid with high glucocorticoid receptor affinity, airway selectivity and prolonged tissue retention. It inhibits inflammatory symptoms, such as edema and vascular hyperpermeability.
  • Budesonide is already applied through dry powder inhalers (DPI, Pulmicort), metered dose inhalers (pMDI, Rhinocort) or ileal-release capsules (Entocort).
  • DPI dry powder inhalers
  • pMDI metered dose inhalers
  • Entocort ileal-release capsules
  • Nanoparticle suspensions were evaluated by measuring particle size, polydispersity and zeta potential. Nanosuspensions were then flocculated and lyophilized to obtain dry powders composed of micron-sized agglomerates. Nanoparticle agglomerates were characterized by the determination of particle size, aerolization efficiencies, flowability characteristics, process yield and loading efficiency.
  • A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
  • the cell culture medium (Ham's F-12 Nutrient Mixture, Kaighn's modified with L-glutamine) was purchased through Fisher Scientic.
  • Fetal bovine serum (FBS) was purchased from Hyclone.
  • Penicillin-streptomycin was purchased from MB Biomedical, LLC. Trypsin- EDTA was purchased through Gibco.
  • MTS reagent [tetrazolium compound;
  • Nanosuspensions were prepared using a precipitation technique. Briefly, solutions of budcsonide in acetone were prepared at concentrations of 0.1 and 0.2% w/v and water was used as nonsolvent. To precipitate the drug, the solution of budesonide was directly injected into the non-solvent at a rate of 1 mL/min under sonication (Fisher Scientic, Sonic Dismembrator) with an amplitude of 46% in an ice bath.
  • the selected surfactants for the study included hydrophobic (cetyl alcohol and Span 85), hydrophilic (PL, PVA and PVP) and amphoteric (lecithin).
  • hydrophobic and amphoteric surfactants were added to the drug organic solvent solution and the contents were allowed to stand at room temperature for 30 to 45 minutes with occasional vortexing to allow complete solubilization of the drug and the surfactants.
  • Hydrophilic surfactants were added to the aqueous phase. Surfactants were used individually or in combination as reported. 2. Flocculation of budesonide nanoparticles
  • the budesonide nanoparticle agglomerates were prepared by slow addition of L-leucine solution (1 % w/v) in water to flocculate nanoparticle suspensions during homogenization at 25,000 rpm for 30 sec. The amount of L-leucine added was adjusted to a drug:leucine ratio equal to 1 : 1. The size of budesonide nanoparticle agglomerates was measured in Isoton diluent using a Coulter Multisizer 3 (Beckman Coulter Inc.) equipped with a 100 mm aperture after three hours of incubation with the flocculating agent.
  • the flocculated suspensions were kept overnight at room temperature to allow evaporation of acetone and then frozen at -80° C and transferred to a freeze dryer (Labconco, FreeZone 1 ). Drying lasted for 36 hours to remove all appreciable water content. Lyophilized powder was stored at room temperature for further characterization.
  • the size and zeta potential of the nanosuspensions were determined by dynamic light scattering (Brookhaven, ZetaPALS). Zeta potential measurements were performed using 1 mM KCl solution. All measurements were performed in triplicate.
  • the particle size of the dispersed nanoparticle agglomerates as well as the resuspended lyophilized powder was measured using a Coulter Multisizer 3.
  • the aerodynamic size distributions of the agglomerate powders were measured directly from lyophilized powder by time-of-flight measurement using an Aerosizer LD (Amherst Instruments) equipped with a 700 mm aperture operating at 6 psi.
  • Aerodynamic characteristics of selected nanoparticle agglomerates were studied in vitro using a Tisch Ambient Cascade Impactor (Tisch Environmental, Inc., Ohio). The study was carried out by applying -20 mg powder manually into the orifice of the instrument at three air flow rates; ⁇ 15 L/min, ⁇ 30 L/min and -60 L/min. Cut-off particle aerodynamic diameters at 30 L/min for each stage of the impactor were: pre-separator (10.00 mm), stage 0 (9.00 mm), stage 1 (5.8 mm), stage 2 (4.7 mm), stage 3 (3.3 mm), stage 4 (2.1 mm), stage 5 (1.1 mm), stage 6 (0.7 mm), stage 7 (0.4 mm) and filter (0 mm).
  • Nanoparticle agglomerates deposited on each stage of the impactor were determined by measuring the difference in weight of filters placed on the stages.
  • the mass median aerodynamic diameter, MMAD, and geometric standard deviation, GSD were obtained by a linear fit of the cumulative percent less-than the particle size range by weight plotted on a probability scale as a function of the logarithm of the effective cut-off diameter. 4.
  • TEM Transmission electron microscopy
  • Image data was used to corroborate the size of nanoparticles and nanoparticle agglomerates and to observe their morphological aspects.
  • Transmission electron micrographs (TEM) were obtained for budesonide nanoparticles and nanoparticle agglomerates using a JEOL 1200 EXII transmission electron microscope.
  • carbon-coated grids Electro Microscopy Sciences
  • a filter paper After this, the grid was blotted with a filter paper and air dried for 1 hr. 5.
  • SSNMR analysis SSNMR analysis.
  • Budesonide loading efficiency was assessed by dispersing one mg of the lyophilized powder in 10 mL ethanol. The dispersion was sonicated in a bath-type sonicator (Branson 3510) for 2 hours and then kept overnight at room temperature to allow complete dissolution of the drug by ethanol. Then the solution was centrifuged (Beckman, Avanti TM) at ⁇ 15,000 rpm for 30 min to remove insoluble surfactants and L-leucine and the amount of drug in the supernatant was determined spectrophotometrically (Agilent C) at 243 nm. Drug loading was defined as follows:
  • the fixed- height cone method was used. A glass funnel with cut stem surface of 5 mm internal diameter was fixed at 2.5 cm height over a flat surface. The powders were allowed to flow gently through the funnel until a cone was formed and reached the funnel orifice. The flow of powder was then stopped and the average diameter of the formed cone (D) was measured. The area of the base of the cone was taken as a measure of the internal friction between the particles.
  • the dissolution of the prepared nanoparticles and nanoparticle agglomerates was determined and compared with the dissolution characteristics of the stock drug.
  • the dissolution of budesonide was carried out at 37 ⁇ 0.5oC in a 400 mL beaker.
  • the solution was stirred at a constant speed (100 rpm) using a magnetic stirrer (Barnstead, Thermolyne MIRAKTM).
  • aliquots (5 ml) of the medium were removed and fresh medium was immediately added to continue the dissolution study. Studies were conducted in triplicate.
  • the budesonide concentration was analyzed using a reverse-phase HPLC method.
  • a Shimadzu HPLC system including a solvent delivery pump (Shimadzu LC-IOAT), a controller (Shimadzu SCL-IOA), an autoinjector (Shimadzu SIL-IOAxL), and a UV detector (Shimadzu SPD-IOA) was used in this study.
  • the peak areas were integrated using Shimadzo Class VP (Version 4.3).
  • cytotoxicity of selected nanoparticles and nanoparticle agglomerates was assessed using the CellTiter 96® Aqueous Cell Proliferation Assay (Promega) and compared with stock budesonide, lecithin, leucine, physical mixtures of these ingredients and blank nanoparticle agglomerates.
  • 8 x 104 A549 cells /well were seeded in 96-well microtiter plates.
  • 20 ml of MTS reagent solution was added to each well and incubated for 3 h at 37 0 C.
  • the absorbance was measured at 490 nm using a microtiter plate reader (SpectraMax, M25, Molecular Devices Corp., CA). The percentage of viable cells with all tested concentrations was calculated relative to untreated cells.
  • the mechanism to control nanoparticle agglomeration is mainly driven by leveraging the competitive processes of attraction (van der Waals force) and repulsion (electrostatic repulsive force or steric hindrance barrier or both). If particles are mainly stabilized electrostatically, disruption of the electrostatic double layer surrounding the particles will result in the agglomeration of nanoparticles.
  • the addition of flocculating agents has also been speculated to decreases the cohesion between particles. It is thought that these agents may interfere with weak bonding forces between small particles, such as Van der Waals and Coulomb forces. These agents may act as weak links or "chain breakers" between the particles which are susceptible to disruption in the turbulent airstream created during inhalation.
  • the amino acid, L-leucine, used as a flocculating agent in these studies may also act as an anti-adherent material to yield a high respirable fraction of the agglomerated budesonide nanoparticles.
  • Nanoparticle agglomerates were prepared through the slow incorporation of a flocculating agent (L-leucine) during homogenization (25,000 rpm) for 30 sec.
  • the geometric size distribution of the prepared nanoparticle agglomerates was measured in Isoton diluent using a Coulter Multisizer 3.
  • the size average of the three selected nanoparticle agglomerate formulations ranged from -2-4 mm (Table 15).
  • the size distributions of resuspended lyophilized powders were slightly broader and the average particle size was slightly increased, when compared to the nanoparticle agglomerates prior to lyophilization (Table 15 and FlG. 18). This may be due to the deposition of nanoparticles on agglomerates during lyophilization or to cohesion between agglomerates as a result of drying.
  • the key physical parameter that predicts the site of aerosol deposition within the lungs for particles larger than several hundred nanometers is the aerodynamic diameter (d aer o)-
  • the aerodynamic diameter of the flocculated nanoparticles, measured by an Aerosizer LD was smaller than the geometric diameter and the aerodynamic size distribution was narrower than the geometric size distribution (Table 15 and FIG. 19). When compared to the geometric diameter, the lower aerodynamic diameter was likely due to the low density of nanoparticle agglomerates.
  • d geo geometric diameter
  • p particle bulk density
  • pa water mass density ( 1 g/cm J ). Tapped density measurements underestimate particle bulk densities since the volume of particles measured includes the interstitial space between the particles. The true particle density, and therefore the aerodynamic diameter of a given powder, is expected to be slightly larger than reported.
  • Particles with a daero between 1 and 5 mm that are inhaled via the mouth are capable of efficient alveolar deposition, whereas daero between 4 and 10 mm are more likely to deposit primarily in the tracheobronchial region of the lungs. Therefore, the budesonide nanoparticle agglomerates with daero in the 2-2.5 mm range are expected to deposit primarily in the alveolar region of the lungs.
  • Aerosizer results and theoretical MMAD calculations were corroborated by cascade impaction studies at air flow rates of ⁇ 15 L/min, -30 L/min and ⁇ 60 L/min (FIG. 20). At these flow rates, most nanoparticle agglomerates were deposited in stages 6 and 7 of the cascade impactor which was suggestive of efficient aerosolization and a high fine particle fraction.
  • the aerosolization efficiency of nanoparticle agglomerates was represented by the percent emitted fraction (%EF), percent respirable fraction (RF), mass-median aerodynamic diameter (MMAD) and geometrical standard deviation (GSD) The percent emitted fraction was determined from the following equation:
  • d n is the diameter at the nth percentile of the cumulative distribution.
  • the mass— mean geometric size of nanoparticle agglomerates ranged between 3 and 4 mm with a GSD of -2.3 mm (Table 16). Typical GSD values for aerosol particles are between 1.3-3 0.17
  • the mass-mean aerodynamic diameter (MM AD) of the selected nanoparticle agglomerates, as calculated from the cascade impaction results (Table 16) was close to that obtained from the Aerosizer (Table 15) although it was slightly smaller than the theoietical density values calculated from the tapped density indicating the suitability of the prepared nanoparticle agglomerate powders for peripheral lung deposition (i e , ⁇ 3 mm)
  • MMAD Mass median aerodynamic diameter.
  • hGSD Geometric standard deviation.
  • Electron microscopy was used to study the morphology of budesobide nanoparticle and nanoparticle agglomerate formulations.
  • FIG. 23 shows the 13C spectra of budesonide by itself and in formulations.
  • Both the budesonide as received and the leucine exhibit relatively narrow lines (several tens of hertz), indicating that these samples are crystalline.
  • Lecithin also had narrow lines, which is consistent with it being a crystalline form of phosphatidylcholine; however, it is a semi-solid and therefore cannot be crystalline.
  • the budesonide that was melt quenched had significantly broader lines (several hundreds of hertz) indicating that the budesonide is consistent with it being amorphous.
  • the peaks for budesonide are similar to the peaks in the budesonide that was melt quenched, although the peak at ⁇ 180 ppm shows that there is a small amount of crystalline budesonide in the nanoparticles.
  • the tall, sharp peaks in the spectrum of the nanoparticle agglomerates align with the peaks in the leucine spectrum and showed that the leucine in the formulation has undergone phase separation and has crystallized to some extent.
  • the peak at 180 ppm showed that the amount of crystalline budesonide had increased in the formulation of the nanoparticle agglomerates. This was consistent with the shape of several other budesonide peaks in the spectrum.
  • Budesonide consists of 25 carbons (FIG. 23); however, the spectrum of the budesonide as received had at least 27 resolved peaks and several peaks that may be the result of several overlapping peaks. The extra peaks did not seem to be due to splitting, as would be expected if there were more than one molecule in the asymmetric unit cell.
  • the budesonide is a racemic mixture of both epimers that have been shown to pack differently in the crystal lattice. Therefore, spectral editing was used in an attempt to assign the peaks in the spectrum to determine if the differences in the two epimers could be used to explain the "extra" peaks.
  • the spectral editing experiment allowed the assignment of carbon type (C, CH, CH2, or CH3) to a peak, these assignments could then be combined with predictions to assign the peaks to specific carbons within the molecule.
  • the carbon type of most of the peaks could be assigned from these experiments (FIG. 24) with the exception of a few of the aliphatic peaks (particularly ⁇ 30 ⁇ ⁇ 40 ppm).
  • Budesonide nanoparticle and nanoparticle agglomerates showed improved dissolution rates
  • a dissolution study of budesonide was conducted for the prepared nanoparticles and nanoparticle agglomerates and compared to the unprocessed drug.
  • the cumulative percentage of drug dissolved after 8 hours (Q8h) was found to be slower than that of the nanoparticles and faster than that of the stock budesonide (Table 15). This finding was the expected result of increasing the surface area by decreasing the particle size.
  • F2 and F3 nanoparticle and nanoparticle agglomerate formulations showed faster drug dissolution than Fl which may be due to the incoiporation of the hydrophilic surfactant, PL (FIG. 25).
  • the cytotoxicity of the different budesonide formulations were compared to stock budesonide, lecithin, leucine, physical mixtures of Fl components and blank Fl nanoparticle agglomerates (FIG. 26).
  • Stock budesonide, excipients and physical mixtures of Fl components up to 5 mg/mL did not show any significant cytotoxicity in A549 cells at the end of 12 hours.
  • Blank Fl nanoparticle agglomerates did induce a very low level of cytotoxicity where the IC50 was found to be 0.97 mg/mL.
  • Fl nanoparticles and nanoparticle agglomerates also induced very low level of cytotoxicity with 1C50 values equal to 1.67 mg/mL and 1.91 mg/mL, respectively.
  • the 1C50 values occurred at higher concentrations than the maximum daily dose of inhaled budesonide currently prescribed.
  • Nanosuspensions were successfully prepared yielding nanoparticles in the range of ⁇ 160-230 nm. This was accomplished by using surfactants proven to be safe for human use such as lecithin. Nanosuspensions were flocculated using L-leucine and the resulting nanoparticle agglomerates were analyzed. Nanoparticle agglomerates were efficiently aerosolized and offered a high fine particle fraction suitable for accessing the peripheral lung.
  • Nanoparticle agglomerates also exhibited significantly faster budesonide dissolution when compared to the stock powder.
  • budesonide nanoparticle agglomerates demonstrated a desirable microstructure for efficient lung deposition and nanostructure for rapid dissolution of poorly water soluble drugs.
  • Pulmonary administration of poorly water soluble drugs represents a leading challenge in the drug delivery industry. These drugs are generally not suitable for delivery to the aqueous lumen of the GI tract, and are often susceptible to enzymatic degradation.
  • This thesis investigates a general method for producing micron sized dry powders of a general class of drugs, poorly water soluble small molecule drugs, for their use in pulmonary drug delivery. These drugs are stable at room conditions in crystalline form, and typically soluble in ethanol.
  • Our model drug in this body of work was Nifedipine, a well known calcium channel blocker used to treat various symptoms of hypertension. Formulation methods already exist for some drugs that fall into this category, but the resulting powders show limited performance.
  • Particulates for Pulmonary Drug Delivery Drug delivery is a rapidly growing field of research, and the pulmonary route has not been neglected throughout this process.
  • technologies become more adept at characterizing and manipulating the microscopic and submicron world, our ability to reliably produce entities on this scale continually improves, and drug delivery science has rapidly sought to put these improvements to application.
  • the ideal drug nanoparticle is equipped with a milieu of targets and sensors; everything it needs to seek and destroy the pathogen, or rebuild the damaged tissue. Indeed, we are many years from this idealized super particle, but more simple particles on this scale still yield enormous benefits.
  • nanoparticles and microparticles are a benchmark design strategy for drug delivery scientists, and possibly the only strategy for those interested in pulmonary delivery. Pulmonary Physiology and Molecular Transport
  • Hydrophilic drugs bear the opposite challenge. Even though they may readily disperse throughout the surfactant layers, they still may not be provided with a suitable pathway to the capillaries just beyond the tightly grouped epithelial barrier. The surface cells in this region of the lungs are characterized by their minute morphologies and lack of interstitial spacing between cells.
  • Pulmonary delivery stands out among the various delivery schemes for many reasons. Inhalers can be stored and used for isolated emergency events, such as those that may arise through such diseases as diabetes and chronic bronchitis. Drug entities delivered via the lungs are able to avoid first pass metabolism in the liver and this may increase overall bioavailability.
  • Nanoparticles (-700 nm, -3OmV) of the hypertension drug Nifedipine were synthesized via known solvent anti-solvent precipitation techniques.
  • the resulting colloids were destabilized via ionic charge interactions using common salts at different solution molarities to achieve a final particle size distribution suitable for delivery of particulates to the deep lung.
  • the flocculated solutions were freeze dried and collected as dry powders for further characterization. Synthesis and separation techniques were optimized for nanoparticle size, flocculate size, overall yield, and flowability. Performance of the final powders was found suitable for delivery of Nifedipine to the deep lung.
  • Nifedipine is one such drug that bears complicated pharmacodynamics in the traditional oral dosage form. Nifedipine shows limited systemic bioavailability via the oral route due to a combination of enzymatic effects in the stomach and small intestine, primarily from P450 reductase and CYP3A mediated drug metabolism. Though it is effective in easing symptoms of sever hypertension, it sometimes can not be used for this reason due to elevated vasodilation and extreme hypotension.
  • Nifedipine Pulmonary administration of Nifedipine is one such strategy that might help alleviate the aforementioned difficulties.
  • Nifedipine is a dihydropyridine and resides in a class of calcium antagonists known as calcium channel blockers. It is a small molecule poorly water soluble drug with the site of action at the calcium channels residing on the surface of all cells and primarily acts upon smooth muscle cells and heart muscle cells. Many different drugs bear similar physical and chemical properties to nifedipine, such as its hydrophobicity and cyclic structure, so the methods contained in this paper may be applicable to formulating a host of drugs for pulmonary delivery.
  • Nifedipine (NIF), stearic acid (SA), arachidonic acid (AA), sodium chloride and calcium chloride were purchased from Sigma Chemicals Co, USA and used as received in solid form.
  • Acetone, methanol, ethanol, and phosphate buffered salts were purchased from Fisher Scientific and used as received.
  • Nanoparticles were prepared by the rapid mixing of ethanol with dissolved nifedipine and stearic acid into a larger aqueous volume, known as a solvent anti-solvent precipitation technique. Briefly, 10 mg of Nifedipine and 1 mg of stearic acid were completely dissolved in 1 ml of ethanol and allowed to stir overnight. Upon complete dissolution this solution was added to 29 ml of cold deionized water under probe sonication at 60% amplitude for 20 seconds. The resulting colloid was then immediately frozen and lyophilized, or stored in a 4°C refrigerator until further processing into flocculations. At this time, a small sample was taken from the solution for sizing and imaging. All solution vials and reaction vessels were kept covered from any light sources, as Nifedipine exhibits considerable photosensitivity from UV and visible light spectra. Preparation of nanoparticle flocculations
  • Nanoparticle colloids were destabilized via a largely understood combination of ionic and thermodynamic force interactions to produce stable flocculations of nanoparticles. Briefly, 30 ml of the nanoparticle suspension was taken from refrigeration and solid salt crystals were added in various amounts. Directly after addition, the suspensions would be subject to vigorous mixing via a homogenization probe operating at 20,000 RPM. Three different salt species were tested for their ability to form flocculations: sodium chloride, calcium chloride, and magnesium sulfate. Salts were commonly added in a 1 :1 ratio of salt to NIF. Colloid stability was also tested under a range of salt molarities and flocculation behaviors were observed under all conditions. Nanoparticle characterization
  • Nanoparticle size, polydispersity, and zeta potential were all measured in solution directly after synthesis and prior to flocculation using a zetaPALS dynamic light scattering device. Size and polydispersity were first measured. Briefly, ImI of the solution was added to a standard cuvette and the remaining volume was filled with deionized water. Data were collected in three runs and combined to arrive at a final size for each solution. Measurements were taken at 90 degrees to the incident light source while assuming a viscosity and refractive index of pure water. After arriving at a combined size, a second cuvette was filled with 1 ml of our colloid solution and the remaining volume was filled with KCl. A known voltage was then applied to this solution and data were analyzed via online software to determine the zeta potential of the particles in solution. Flocculate characterization
  • Flocculated nanoparticles were studied in solution and as a dry powder. After the flocculation event was complete, a small volume ( ⁇ 3ml) of the solution was analyzed using a
  • a cascade impactor was then used to collect data on powder performance in the lung. Briefly, eight filters were preweighed and set onto collection plates which were housed within eight airtight stages arranged serially and stacked on a level setting. Air was then pumped through the stages at 30 liters per minute via a vacuum pump and 10 mg of sample was introduced at the top of the impactor device. The powders were allowed to deposit amongst the stages for 20 seconds, after which time the air flow was stopped. Filters were then removed from the stages and weighed a second and final time.
  • the powders were characterized via two simple tests: a tap density test, and a test for angle of repose.
  • the tap (bulk) density was determined by demarcating a small cuvette with known volumes, and then inserting a small mass of powder into the cuvette and tapping it vertically against a padded bench top 50 times. The density was tested in triplicate.
  • the angle of repose was measured by placing a volume of powder on a glass slide and tilting the slide until the powder began to move down the slide, and recording the angle between the slide and the horizontal. This test was also performed in triplicate for each sample.
  • Nanoparticles, microparticles and pure drug crystals were imaged via a scanning electron microscope.
  • the samples were deposited onto mica slides in solution (or as received for the crystals) and allowed to evaporate over night.
  • the slides were then coated with a 2 nm gold surface using a voltage controlled gold sputtering device and subject to a vacuum chamber whereby image data were subsequently collected.
  • Size distributions for a sample of nanoparticle flocculations are shown in FIG. 28.
  • This data reveals the particle size distribution from a sample of nanoparticles (geometric diameter: 421.7 +/- 26.2 nm, zeta potential: -32.16 +/- 3.75) before and after homogenization at 25,000 RPM for 30 seconds.
  • the data were collected in solution, and as such it is not ideal data for studying the powder characteristics of the flocculates.
  • it is important at this stage in the synthesis to verify flocculation as it is well known that particles can agglomerate upon lyophilization and we wanted to verify that this is not the case in our experiments.
  • the samples reveal a fairly monodisperse distribution of sizes between about 2 and 20 microns, with an average diameter of about 10 microns. More so, the data reveals very stable microstructure in the flocculates. Their distributions are barely altered after intense homogenization, and they maintain their shape almost entirely.
  • FIG. 28 shows a typical aerodynamic size distribution as collected via this method.
  • the theoretical mass-mean aerodynamic diameter (d aero ) of the nanoparticles was determined from the geometric particle size and tapped density using the following relationship: d gco I ; aero ⁇ V a
  • d geo geometric diameter
  • p particle density
  • p a water mass density (1 g/cm J ).
  • the aerodynamic diameter will be some fraction of the geometric diameter. This is the case for our flocculate samples. Their geometric diameters are shown to be much larger, on average, than the aerodynamic diameters. For the samples shown in FIGS. 22 and 23, the average geometric diameter is about five times larger than the average aerodynamic diameter. They are shown to be about 10 and 2 microns, respectively.
  • FIG. 29 shows SEM micrographs of the nanoparticles, flocculates and pure drug. These images further validate the data collected, thus far.
  • the nanoparticles are shown to bear an elliptical morphology with an average diameter somewhere below one micron, but not as small as 100 nanometers.
  • the flocculate images reveal a highly textured morphology, with many small and similarly shaped lumps protruding from the surface. These features are indicative of the mechanism behind particle formation, as they are probably the result of nanoparticles grouped together during the flocculation step. Also, we can see a somewhat porous assembly in the imaged marked 'C.
  • the pure drug is shown to bear a highly crystalline structure, and is received in crystals larger than 100 micrometers in some cases. This crystalline morphology is not seen in any of the other images, thus indicating the potential change in overall crystallinity.
  • the particles have such a large surface area per mass, they will be much more susceptible to heating and thus will not require as much heat to induce a state transition. This difference would then show up as smaller troughs in the DSC endotherms. It is also worth noticing the consistently changed shape of the nifedipine peak for the nanoparticle samples. They reveal an exothermic peak indicative of a crystallization event, or some other energy producing phenomena. It may be surmised that the stearic acid and nifedipine interact upon melting of the nifedipine, whereby they arrange with respect to each other into a semi crystalline state of lower energy than just the randomly organized fluids initially present. No other theories for these exotherms have been presented at this point.
  • stage F is shown here as stage 8, simple to ensure a numerical ordering to the graph.
  • the outputs reveal different behaviors for each of the samples. 61
  • the pure drug mostly deposits in the earlier stages, 1-3. These stages prepresent the pharyx and primary bronchi and so it may be assumed that these powders would not enter the lungs whatsoever.
  • the nanoparticles show the bulk of their deposition between stages 4-6 and these represent the secondary, and terminal bronchiolar and alveolar regions.
  • the flocculates show similar deposition patterns. Indeed, these are suitable regions for delivery of particles to the lungs and so it may be shown here that both the nanoparticle samples and their corresponding flocculates are able to deposit efficiently to the lungs. The exact reasons for this similarity, given different processing steps, may be elusive for the time being.
  • the nanoparticles are able to agglomerate upon lyophilization and hence bear similar structural properties to the flocculates.
  • this similarity does not necessarily detract from the advantages of the flocculation since the flocculated particles are more likely able to be harvested directly from solution without the expensive processing step of lyophilization.
  • nifedipine Stearic acid stabilized pure drug nanoparticles of nifedipine were synthesized via ultrasonication in a pure aqueous solution. These colloids were destabilized under different salt molarities and/or salt:drug mass ratios to induce particle flocculation and subsequent microstructure formation. Nifedipine changes morphology upon processing with and without stearic acid. Nanoparticles revealed enhanced dissolution kinetics when compared to the pure drug and flocculated samples. The resulting dried powders exhibited suitable flowability characteristics and size distributions for pulmonary drug delivery. Throughout the course of preparing samples for the primary study, data were collected to help optimize the formulation and gain understanding of the processes at work. The results of these studies are shown here, with a brief discussion concerning their significance.
  • Diabetes is a set of diseases characterized by defects in insulin utilization, either through autoimmune destruction of insulin-producing cells (Type I) or insulin resistance (Type II) Treatment options can include regular injections of insulin, which can be painful and inconvenient, often leading to low patient compliance
  • novel formulations of insulin are being investigated, such as inhaled aerosols.
  • Sufficient deposition of powder in the peripheral lung to maximize systemic absorption requires precise control over particle size and density, with particles between 1 and 5 ⁇ m in aerodynamic diameter being within the respirable range.
  • Insulin nanoparticles were produced by titrating insulin dissolved at low pH up to the pi of the native protein, and were then further processed into microparticles using solvent displacement.
  • Diabetes mellitus is a set of diseases characterized by defects in insulin utilization, either from autoimmune destruction of insulin-producing cells (Type I) or insulin resistance (Type II).
  • Type I insulin-producing cells
  • Type II insulin resistance
  • Current treatment methods involve regular injections of insulin, which can be both painful and inconvenient, thus often leading to low patient compliance.
  • Inhaled aerosols have been shown to be an effective means to treat local diseases of the lung. Additionally, the large surface area of the lungs (-140 m 2 ) and their ready access to systemic circulation makes them a possible candidate for noninvasive, systemic drug delivery. This is particularly good for macromolecular drugs such as peptides, proteins, and DNA.
  • p re f is a reference density (for example 1 g/cm 3 for water) and ⁇ is the shape factor (equal to 1 for a sphere).
  • penetration enhancers such as polyoxyethylene 9 lauryl ether and sodium glycocholate, which have been shown to induce acute inflammation in the lung. It may therefore be desirable to create an inhalable form of insulin that does not contain excipients so as to avoid any potential complications that might arise.
  • Lyophilized insulin powder from bovine pancreas (0.5% zinc content) and phosphate buffered saline premix (PBS) were purchased from Sigma (St. Louis, MO). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA) and used without further purification.
  • zeta potential ( ⁇ ) of the nanoparticles in ImM potassium chloride solution Three runs of 15 cycles were acquired, and the mean zeta potential was recorded. Some samples were frozen at -8O 0 C and lyophilized using a Labconco bench top lyophilizer (Kansas City, MO) for further analysis. A range of pH values near the pi of the native protein were determined in which the nanoparticle colloid was preserved. Particle sizes and zeta potentials were measured for each sample. Nanoparticle samples within this pH range (from 4.92 to 5.09) were centrifuged at 13,000 rpm for 10 minutes and the supernatant concentration of insulin was analyzed using UV absorbance spectroscopy (Agilent 8453). All pH values were measured in triplicate. The measured concentration was used to calculate the mass of insulin in the pellet from the original insulin mass and total volume.
  • the aerodynamic diameters of the lyophilized powders were determined using an Aerosizer LD (Amherst Process Instruments Inc.)- Data were collected over -70 seconds under high shear force (-3.4 kPa) using a 700 ⁇ m nozzle.
  • Post-processing secondary structural changes in samples were analyzed by dissolving particles in 0.01 N HCI solution and analyzing using circular dichroism spectroscopy (CD; Jasco J-810, Easton, MD) to determine conformational differences between processed and unprocessed insulin, as well as thermal stability differences between groups.
  • CD spectra were acquired in three accumulations from 260-195 nm with a scanning speed of 50 nm/min and 1.0 nm resolution.
  • Thermal stability was determined at a wavelength of 210 nm from 10-80 0 C with a scanning speed of 15 °C/hr. Thermal stability spectra were acquired in triplicate. Insulin concentration in prepared solutions was determined by UV absorbance spectroscopy. 7. Crystallinity of processed insulin. NMR: Spectra were collected using a Tecmag Apollo spectrometer operating at 300 MHz using ramped amplitude cross-polarization (RAMP), magic-angle spinning (MAS), and SPINAL-64 decoupling. Samples were packed in 4 mm o.d.
  • HPLC The crystalline insulin content of the materials was determined using the method in the insulin zinc suspension monograph of the 2005 U.S. Pharmacopoeia National Formulary, with minor modifications.
  • Buffered acetone TS was produced by dissolving 8.15 g of sodium acetate and 42 g of sodium chloride in 100 mL of water, to which 68 mL of 0.1 N hydrochloric acid and 150 mL of acetone were added, the mixture was then diluted with water to make 500 mL. Approximately 0.5 mg of insulin was placed in a 1.5 mL microcentrifuge tube and 33.3 pL of a 1 :2 mixture of water and buffered acetone TS was added to the tube to extract any amorphous insulin.
  • the sample was immediately centrifuged at 13,000 rpm for one minute, the supernatant was decanted, and the extraction was repeated. Additionally, -0.5 mg of insulin was placed in another microcentrifuge tube to be used as a control. Both insulin samples were each dissolved in 33.3 pL of 0.01 N hydrochloric acid and analyzed by HPLC, with each sample being prepared in triplicate.
  • the HPLC was a Shimadzu system that consisted of an SCL-IOA system controller,
  • Aqueous mobile phase was prepared by dissolving 28.4 g of anhydrous sodium sulfate in 1000 mL of water, to which 2.7 mL of phosphoric acid was added and the pH was adjusted to 2.3 with ethanolamine. The aqueous mobile phase was then mixed 74:26 with acetonitrile. The separation was performed on a 4.6 x 250 mm Symmetry® Cl 8 column from Waters that was maintained at 4O 0 C.
  • each insulin particle sample was suspended in PBS (pH 7.4).
  • the solution was placed in a 100,000 Dalton biotech grade cellulose ester dialysis tube (Spectrum Labs, Collinso Dominiguez, CA) and placed in PBS solution to a final volume of 45 mL. All samples were incubated at 37°C and shaken at 50 rpm on a shaker table. 1 mL aliquots were taken at various time points up to 8 hours from the bulk solution and replaced with 1 mL of fresh PBS. The insulin concentration was measured using a Coomassie Plus colorimetric protein quantification assay (Thermo Fisher Scientific, Waltham, MA).
  • a calibration curve was used to correlate the insulin concentration with the measured absorbance, with insulin concentrations ranging between 1 and 25 ⁇ g/mL being used as the standard. Dissolved mass was calculated from the measured concentration, and was then normalized to the total loaded mass to determine the percent dissolved. All experiments were performed in triplicate. Analysis of variance (ANOVA) was used to determine statistically significant differences between groups (p ⁇ 0.05). Comparisons among groups were done using a Fisher's F-test. 9. Estimation of bulk powder density. The bulk density of the dry powder was estimated using a micro-tap test approach, as defined in the U.S. Pharmacopoeia National Formulary, with slight modifications.
  • Zn insulin nanoparticles were created by titrating dissolved insulin to the pi of the native protein, which resulted in a colloidal suspension of nanoparticles.
  • Particle sizes and zeta potentials were analyzed over a pH range of 4.92 to 5.09, and ranged from 292.5 nm to 592.1 nm (Table 24. Zeta potentials ranged from 10.86 mV to 18.89 mV. Neither particle sizes nor zeta potentials correlated strongly with the pH of the solution.
  • Insulin microparticles were produced from insulin nanoparticle suspensions through solvent displacement. This was achieved by adding aliquots of insulin nanoparticle suspension to ethanol and stirring overnight. The geometric diameter of the insulin microparticles was determined to be 3.408 ⁇ 1.35 gm. No correlation was determined to exist between insulin nanoparticle size and microparticle size (FIG. 40). SEM imaging revealed differences in the morphology of the unprocessed insulin and the insulin microparticles (FIG. 41). The unprocessed insulin agglomerates appear to have a more regular structure, while the microparticles have more of a leaf-like morphology. This leaf-like shape could aid in the aerosolizability of the insulin microparticles, and would suggest a shape factor, ⁇ , of less than 1. 3. Aerosol properties of insulin particles.
  • the aerodynamic diameters of the unprocessed insulin powder, lyophilized insulin nanoparticles, and lyophilized insulin microparticles were measured with an Aerosizer LD and are shown in Table 25.
  • the large aerodynamic diameter of the insulin nanoparticles is most likely due to uncontrolled agglomeration, which probably occurred during lyophilization.
  • the smaller aerodynamic diameter of the insulin microparticles compared to the geometric diameter of the microparticles was expected because of the lower density of the insulin microparticles (FIG. 42).
  • Circular dichroism was employed to analyze the secondary structure and thermal stability of processed insulin powders. Isothermal scans of dissolved, unprocessed insulin powder, dissolved nanoparticles, and dissolved microparticles reveal near- identical spectra with minima at 210 nm, suggesting that any changes in secondary structure that might occur during processing were reversible upon dissolution (FIG. 43). This overlap was also reflected in the thermal stability CD scans, which show a slight change in molar ellipticity from 10-80 0 C starting at about 5O 0 C for all samples. 5. Crystallinity of processed insulin.
  • the crystallinity of the insulin particles was examined using 13C CP/MAS NMR (FIG. 44).
  • the spectra display differences in the aliphatic region (-0 to 75 ppm), although these differences are difficult to correlate with the physical state of insulin. More obvious differences between the samples arise in the carbonyl (-175 ppm) and aromatic (-137 ppm) regions.
  • the peak at - 137 ppm in the unprocessed insulin seems to be more narrow and better resolved than peaks at -129 ppm. These same lines in the other samples are broader, to the point where peaks at -129 ppm cannot be resolved.
  • the peak at -175 ppm in the unprocessed insulin is more narrow, with two very clear shoulders at -180 ppm and -173 ppm. Other samples only show one broad peak at -175 ppm. Crystallinity of the insulin particles was also examined using the buffered acetone method described in the U.S. Pharmacopoeia National Formulary. The results suggest that the unprocessed insulin particles are between 80% and 88% crystalline, which is much greater than both the nanoparticles and microparticles, which were estimated to be between 2% and 8% crystalline, and between 17% and 24% crystalline, respectively (FIG. 45). 6. Dissolution of insulin particles.
  • the concentration of insulin was measured over time in PBS solution to determine the dissolution rate of the different powders (FIG. 46).
  • the unprocessed insulin follows Higuchi dissolution kinetics, and the nanoparticles and microparticles appear to show a burst dissolution phenomenon after 15 minutes.
  • the dissolved masses of neither the nanoparticles nor the microparticles were significantly different from the dissolved mass of the unprocessed insulin after 8 hours. 7.
  • Bulk powder density The tap test method was used to determine the bulk density of the insulin powders before and after processing.
  • Density of the unprocessed insulin powder was determined to be 0.48 ⁇ 0.08 mg/ ⁇ L (FIG. 42).
  • the nanoparticle bulk density was determined to be 0.28 ⁇ 0.04 mg/ ⁇ L, and the bulk density of the insulin microparticles was determined to be 0.063 ⁇ 0.004 mg/ ⁇ L.
  • Analysis of variance revealed a p-value of 0.00025 (p ⁇ 0.05), indicating a statistically significant difference between the bulk densities of each group.
  • Nanoparticles with sizes within the respirable range were produced from the solvent-induced agglomeration of insulin nanoparticles.
  • Nanoparticles were produced using titration and were shown to have a strong correlation between pH and particle size (FIG. 39).
  • Microparticles were then produced using ethanol to displace the aqueous solvent and induce nanoparticle agglomeration.
  • the proposed mechanism for this agglomeration is a combination of decreased electrostatic interactions between nanoparticles due to the addition of the organic phase, and the deposition of dissolved insulin onto the surface of the nanoparticles, forming microparticles with a leaf-like morphology.
  • the sizes of the microparticles were independent of the size of the nanoparticles used, and had a mean aerodynamic diameter that was roughly between 0.436 gm and 4.294 gm. This range of particle sizes is similar to other dry powder insulin formulations, such as Exubera (3.5 ⁇ m), and a formulation based on the Spiros technology (2-3 ⁇ m). Additionally, these particles were smaller than those produced using AIR technology (5-30 ⁇ m).
  • the crystallinity of the insulin particles was first examined using 13C CP/MAS NMR (FIG. 44). Due to their highly ordered nature, crystalline materials will have relatively narrow lines in a 13C CP/MAS spectrum, while disordered or amorphous materials have relatively broad lines. Insulin consists of 51 amino acids and therefore the spectrum will be quite complicated because every amino acid will have at least an amide and a carbon, each of which will have slightly different conformations and thus different chemical shifts. Because of this, even the 13C CP/MAS spectrum of a crystalline protein will appear to have broad lines even though it of many narrow lines with slightly different chemical shifts.
  • Crystallinity was also determined by dissolution testing, as defined by the 2005 U.S. Pharmacopoeia National Formulary, with modifications. Buffered acetone TS was used to dissolve the amorphous insulin from each sample, the concentration of which was then determined and used to estimate the crystallinity of the particles.
  • the unprocessed insulin was shown to be about 17 times more crystalline than the nanoparticles, and 4 times more crystalline than the microparticles (FIG. 45).
  • the dissolution rate of both the microparticles and the nanoparticles exhibited a burst effect over the first few minutes when compared to the unprocessed insulin (FIG. 46).
  • This burst may be due to the rapid dissolution of amorphous material deposited on the surface of the particles during processing or possibly during lyophilization.In the case of the nanoparticles, it is probable that the large total surface area of the particles also plays a significant role in increasing the dissolution rate. This may be beneficial in a pulmonary insulin formulation if the desired therapeutic effect is rapid control of spikes in glucose levels. This type of formulation may be adjusted for sustained control of glucose over long periods of time, or for postprandial glucose control.

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Abstract

La présente invention concerne des compositions et des procédés de préparation d’un nanoagrégat comprenant une pluralité de nanoparticules qui comprennent une substance médicamenteuse. L’invention concerne également des procédés permettant de prévenir ou de traiter une maladie chez un patient par l’administration d’une quantité thérapeutiquement efficace d’une composition comprenant les nanoagrégats de la présente invention.
PCT/US2009/050565 2005-12-16 2009-07-14 Nanoagrégats pour l’administration de nanoparticules médicamenteuses faiblement hydrosolubles WO2010009146A1 (fr)

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EP2526926A1 (fr) * 2011-05-25 2012-11-28 Justus-Liebig-Universität Gießen Nanoparticule de polymère biocompatible dotée de matières actives pour l'application pulmonaire
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US8815294B2 (en) 2010-09-03 2014-08-26 Bend Research, Inc. Pharmaceutical compositions of dextran polymer derivatives and a carrier material
US8968786B2 (en) 2007-06-22 2015-03-03 Board Of Regents, The University Of Texas System Formation of stable submicron peptide or protein particles by thin film freezing
US9084727B2 (en) 2011-05-10 2015-07-21 Bend Research, Inc. Methods and compositions for maintaining active agents in intra-articular spaces
US9192584B2 (en) 2010-04-12 2015-11-24 Alison J. Foster Relating to antiviral compositions
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US8685458B2 (en) 2009-03-05 2014-04-01 Bend Research, Inc. Pharmaceutical compositions of dextran polymer derivatives
US9757464B2 (en) 2009-03-05 2017-09-12 Bend Research, Inc. Pharmaceutical compositions of dextran polymer derivatives
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EP2683362A4 (fr) * 2011-03-10 2014-09-17 Univ Texas Dispersions de nanoparticules de protéines
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