HK1203052B - Nanoparticles with enhanced mucosal penetration or decreased inflammation - Google Patents

Description

Nanoparticles that enhance mucosal penetration or reduce inflammation

Technical Field

The present invention is in the field of nanoparticles, particularly nanoparticles that rapidly penetrate mucus, such as human mucus, and methods of making and using the same.

Background Art

Local delivery of therapeutic agents via biodegradable nanoparticles often offers advantages over systemic drug administration, including reduced systemic side effects and controlled drug levels at the target site. However, controlled drug delivery at mucosal surfaces is limited by the presence of the protective mucus layer.

Mucus is a viscoelastic gel that coats all exposed epithelial surfaces not covered by the skin (such as the respiratory tract, gastrointestinal tract, nasopharyngeal tract, female reproductive tract, and ocular surface). Mucus effectively traps conventional particulate drug delivery systems via spatial and/or adhesive interactions. Due to mucus turnover, most therapeutic agents delivered topically to mucosal surfaces have problems with retention and poor distribution, which limits their efficacy. Biodegradable nanoparticles that deeply penetrate into the mucus barrier can provide improved drug distribution, retention, and efficacy at the mucosal surface.

A dense coating of low molecular weight polyethylene glycol (PEG) allows nanoparticles to rapidly penetrate the highly viscoelastic human mucus secretions. The hydrophilic and bioinert PEG coating effectively minimizes adhesive interactions between the nanoparticles and mucus components. Biodegradable mucus-penetrating particles (MPPs) have been prepared by physical adsorption of certain PLURONICs (e.g., F127) onto prefabricated mucoadhesive nanoparticles. Additionally, MPPs have been prepared by nanoprecipitation using diblock copolymers of poly(sebacic acid) and PEG.

However, the range of biodegradable MPPs prepared by nanoprecipitation is limited because it requires dissolution of the drug and polymer in a water-miscible solvent. Many hydrophobic drugs can benefit from local delivery using MPPs due to severe systemic side effects, but their poor solubility in water-miscible organic solvents limits their effective encapsulation into MPPs by nanoprecipitation.

Other emerging classes of drugs, including nucleic acids, peptides, and proteins, have immense potential for treating diseases at mucosal sites. However, these hydrophilic drugs cannot be readily formulated into MPPs via nanoprecipitation. Emulsion solvent evaporation is widely used to effectively encapsulate both hydrophilic drugs (water-in-oil-in-water double emulsions) and hydrophobic drugs (oil-in-water single emulsions) into biodegradable nanoparticles. However, the resulting particles are mucoadhesive when using the conventional emulsifier, polyvinyl alcohol (PVA).

New methods for preparing mucus-penetrating particles are needed that can encapsulate a wide range of drugs into nanoparticles without compromising mucus penetration properties as described above. Similarly, formulations for administration via injection are needed. It has been determined that similar coatings that enhance mucosal permeation also reduce inflammation induced by drug particles.

It is therefore an object of the present invention to provide a method for preparing particles and the resulting particles, which can encapsulate a wide range of drugs into biodegradable nanoparticles without reducing mucus penetration properties or increasing inflammation induced by the particles as described above.

Another object of the present invention is to provide particles, such as nanoparticles and microparticles, having high drug loading and dense coatings of surface-altering materials to provide efficient drug delivery via various routes of administration.

Summary of the Invention

Nanoparticles formed from an emulsion of one or more core polymers, one or more surface-altering materials, and one or more low molecular weight emulsifiers have been developed. The particles are prepared by dissolving the one or more core polymers in an organic solvent, adding the solution of the one or more core polymers to an aqueous solution or suspension of the emulsifier to form an emulsion, and then adding the emulsion to a second solution or suspension of the emulsifier to effect formation of the nanoparticles. The core and emulsifier can be separate, combined, or in the form of a block copolymer containing one or more blocks of the surface-altering material. In a preferred embodiment, the surface-altering material is polyethylene glycol having a molecular weight of about 1 kD to about 10 kD, preferably about 1 kD to about 5 kD, and more preferably about 5 kD. In this embodiment, the polyethylene glycol has a density of about 1 to about 100 chains/ ^nm² , preferably about 1 to about 50 chains/nm², more preferably about 5 to about 50 chains/nm², and most preferably about 5 to about 25 chains/nm² when measured using 1 H NMR.

Critical points for the molecular weight of the emulsifier are illustrated using examples. In preferred embodiments, the molecular weight of the one or more emulsifiers is less than 1500, 1300, 1200, 1000, 800, 600, or 500 amu. Preferred emulsifiers include sodium cholate, dioctyl sodium sulfosuccinate, cetyltrimethylammonium bromide, saponin, 20, 80, and sugar esters. The surface-altering material is present in an amount effective to neutralize or substantially neutralize the surface charge of the particles when the one or more emulsifiers are added. The emulsifier has an emulsifying capacity of at least about 50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The nanoparticles are particularly suitable for delivering therapeutic agents, prophylactic agents, nutritional agents, or diagnostic agents. These nanoparticles can be administered enterally, parenterally, or topically, preferably to a mucosal surface. In a preferred embodiment, the particles are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, or subconjunctivally. In one embodiment, the particles are administered to the pulmonary airways, intranasally, intravaginally, rectally, or buccally.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1a and 1b are representative trajectories of PLA-PEG and PCL-PEG nanoparticles containing CHA and PVA prepared by the emulsification method. Figures 1c and 1d are graphs showing the overall mean geometric mean square displacement (<MSD>/μm ² ) as a function of time (time scale/s). Figures 1e and 1f are graphs showing the logarithmic distribution of the effective diffusivity (D _eff ) of individual particles at a time scale of 1 s. Figures 1g and 1h are graphs showing the estimated fraction of particles able to penetrate a physiological 30 μm thick mucus layer over time. Data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as SEM.

Figures 2a and 2b demonstrate the effect of PEG MW on the transport rate of MPPs in human cervicovaginal mucus: Figure 2a is a graph showing the overall mean geometric mean square displacement (MSD/ ^μm² ) as a function of the time scale (s). Figure 2b is a graph showing the logarithmic distribution of the effective diffusivity ( _Deff ) of individual particles at a time scale of 1 s. Particles were prepared using an emulsion method using PLGA-PEG (6 wt% PEG). Data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as SEM.

Figure 3a is a graph showing the ensemble mean geometric mean square displacement (MSD/μm ² ) as a function of the time scale /s. Figure 3b is a graph showing the logarithmic distribution of the effective diffusivity (D _eff ) of individual particles at a time scale of 1 s. Figure 3c is a graph showing the estimated fraction of particles predicted to be able to penetrate a 30 μm thick mucus layer over time. Data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as SEM.

Figures 4a-c are schematic diagrams illustrating the effect of surface PEG coverage ([Γ/Γ*]) on mucus penetration of nanoparticles. Figures 4a-c show the preparation of PLGA-PEG nanoparticles with surface PEG coatings at increasing coverage. As surface PEG coverage increases, the PEG state changes from a mushroom (adjacent PEG chains do not overlap, [Γ/Γ*] < 1, Figure 4a) to a brush (adjacent PEG chains overlap, 1 < [Γ/Γ*] < 3, Figure 4b) to a dense brush ([Γ/Γ*] > 3, Figure 4c). At low PEG coverage ([Γ/Γ*] < 1, Figure 4a), mucin fibers strongly adhere to the nanoparticle core. At intermediate PEG coverage (1 < [Γ/Γ*] < 3, Figure 4b), mucin fibers can still be partially absorbed into the nanoparticle core. At high PEG coverage ([Γ/Γ*] > 3, Figure 4c), the nanoparticle core is completely shielded by the bioinert PEG corona, resulting in no mucin absorption into the nanoparticle. Figure 4c shows that nanoparticles with low PEG coverage were immobilized in mucus, nanoparticles with medium PEG coverage were hindered or even immobilized in mucus, and nanoparticles with high and very high PEG coverage were able to rapidly penetrate mucus.

DETAILED DESCRIPTION

I. Definition

As used herein, "nanoparticles" generally refer to particles of any shape having a diameter of about 1 nm up to (but not including) about 1 micron, more preferably about 5 nm to about 500 nm, and most preferably about 5 nm to about 100 nm. Nanoparticles having a spherical shape are generally referred to as "nanospheres."

As used herein, "average particle size" generally refers to the statistical average particle size (diameter) of particles in a population of particles. The diameter of substantially spherical particles can be referred to as the physical or hydrodynamic diameter. The diameter of non-spherical particles can preferably refer to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle can refer to the maximum straight-line distance between two points on the particle surface. The average particle size can be measured using methods known in the art (e.g., dynamic light scattering).

As used herein, "mass median aerodynamic diameter" (MMAD) refers to the median aerodynamic diameter of a plurality of particles. "Aerodynamic diameter" is the diameter of a sphere of unit density that typically has the same settling velocity in air as the powder, and is therefore a useful way to characterize aerosolized powders or other dispersed particles or particle formulations in terms of settling behavior. Aerodynamic diameter encompasses particle or particle shape, density, and the physical size of the particle or particles. MMAD can be determined experimentally using methods known in the art, such as using cascade impaction.

As used herein, "tap density" refers to a measure of powder density. Tap density can be determined using the USP Bulk Density and Tapped Density method, United States Pharmacopia convention, Rockville, Md., 10th Edition Supplement, 4950-4951, 1999. Characteristics that may contribute to low tap density include irregular surface texture and porous structure.

"Monodisperse" and "homogeneous particle size distribution" are used interchangeably herein and describe a plurality of nanoparticles or microparticles in which the particles have the same or nearly the same diameter or aerodynamic diameter. As used herein, a monodisperse distribution refers to a distribution of particles in which 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or more of the distribution is within 5% of the mass median diameter or aerodynamic diameter.

As used herein, "pulmonary administration" refers to administration of a pharmaceutical formulation containing an active agent into the lungs by inhalation. As used herein, the term "inhalation" refers to the drawing of air into the alveoli of the lungs. Air inhalation can be performed through the mouth or nose. Air inhalation can be performed by self-administering the formulation while inhaling or by administration to a patient who is on a ventilator through a ventilator.

As used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, according to the guidelines of agencies such as the Food and Drug Administration.

As used herein, "biocompatible" and "biologically compatible" generally refer to materials that are generally non-toxic to the recipient, along with any metabolites or degradation products thereof, and that do not cause any significant side effects to the recipient. Generally speaking, a biocompatible material is one that does not induce a significant inflammatory or immune response when administered to a patient.

Unless otherwise specified, "molecular weight" as used herein generally refers to the relative average chain length of the bulk polymer. In practice, molecular weight can be estimated or characterized using various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weight is reported as weight average molecular weight (Mw), as opposed to number average molecular weight (Mn). Capillary viscometry provides an estimate of molecular weight, such as the intrinsic viscosity determined from a dilute polymer solution using a specific set of concentration, temperature, and solvent conditions.

As used herein, "hydrophilic" refers to the property of having an affinity for water. For example, a hydrophilic polymer (or hydrophilic polymer segment) is a polymer (or polymer segment) that is primarily soluble in aqueous solutions and/or has a tendency to absorb water. Generally speaking, the more hydrophilic a polymer is, the more it tends to dissolve in water, mix with water, or be wetted by water.

As used herein, "hydrophobicity" refers to the property of lacking affinity for water or even repelling water. For example, the more hydrophobic a polymer (or polymer fragment) is, the more it tends not to dissolve in water, mix with water, or be wetted by water.

As used herein, "mucus" refers to a viscoelastic, natural substance containing primarily mucin-type glycoproteins and other substances that protects the epithelial surfaces of various organs/tissues, including the respiratory, nasal, cervicovaginal, gastrointestinal, rectal, visual, and auditory systems. As used herein, "sputum" refers to a highly viscoelastic mucus secretion that, in addition to mucin-type glycoproteins, also consists of various macromolecules such as DNA, actin, and other cellular debris released by dead cells. "Sputum" is commonly found in the pathogenic airways of patients suffering from obstructive pulmonary diseases, including but not limited to asthma, COPD, and CF. As used herein, "CF mucus" and "CF sputum" refer to mucus and sputum, respectively, from patients suffering from cystic fibrosis.

As used herein, "mucus degrader" refers to a substance that increases the rate of mucus clearance when administered to a patient. Mucus degraders are known in the art. See, for example, Hanes, J. et al., Gene Delivery to the Lung, Pharmaceutical Inhalation Aerosol Technology , Marcel Dekker, Inc., New York: 489-539 (2003). Examples of mucus degraders include N-acetylcysteine (NAC), which cleaves disulfide and sulfhydryl bonds present in mucin. Other mucus degraders include mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, denufosol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, neltenexine, erdosteine, and various DNases (including rhDNase).

As used herein, "CF mucus-resistant/diffusible particles" refer to particles that exhibit reduced or low mucoadhesion in CF mucus and, therefore, pass through CF mucus at a higher rate than other particles. The particles may be characterized by high diffusivity within CF mucus. In certain embodiments, the CF mucus-resistant/diffusible particles have an effective diffusivity in CF mucus greater than about 0.01 μm ² /s, more preferably greater than about 0.5 μm ² /s, and most preferably greater than about 1 μm ² /s. In preferred embodiments, a particle population may be characterized as "CF mucus-resistant/diffusible" if at least 30%, more preferably at least 40%, and most preferably at least 50% of the particle population diffuses across a 10 μm thick layer of CF sputum within one hour.

As used herein, "cystic fibrosis" (CF) refers to an inherited genetic disease caused by one or more mutations in the gene encoding the cystic fibrosis transmembrane transport regulator (CFTR). In patients with cystic fibrosis, mutations in CFTR, which is endogenously expressed in the respiratory epithelium, lead to a decrease in apical anion secretion, causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to increased mucus accumulation in the lungs and concurrent microbial infections that ultimately cause death in CF patients. In addition to respiratory diseases, CF patients are often plagued by gastrointestinal problems and pancreatic insufficiency, which, if left untreated, can lead to death. Sequence analysis of the CFTR gene on the CF chromosome has revealed a variety of diseases that cause mutations. To date, more than 1,000 diseases that cause CF gene mutations have been identified (http://www.genet.sickkids.on.ca/cftr/). The most common mutation is the deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and it is commonly referred to as ΔF508-CFTR. This mutation occurs in about 70 percent of cystic fibrosis cases and is associated with severe disease. Cystic fibrosis affects about one in every 2,500 births in the United States.

As used herein, "cystic fibrosis transmembrane transport regulator" (CFTR) refers to a transmembrane protein that is essential for maintaining electrolyte transport throughout the body, including respiratory and digestive tissues. CFTR consists of approximately 1480 amino acids that encode a protein composed of tandemly repeated transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The gene encoding CFTR has been identified and sequenced. See Gregory, R.J., et al. Nature 347:382-386 (1990); Rich, D.P., et al. Nature 347:358-362 (1990) and Riordan, J.R., et al. Science 245:1066-1073 (1989).

As used herein, "nucleic acid" refers to DNA, RNA, and nucleic acid molecules that have been modified to enhance stability for various therapeutic purposes. An example is a gene encoding the human cystic fibrosis transmembrane transport regulator (CFTR) protein, its analogs, and variants, which can be expressed in CF individuals to at least partially correct some of the symptoms characteristic of CF. This also includes molecules such as DNA fragments, including regions for introducing corrections or modifications into genes, such as triple helix-forming DNA, which can be used to correct endogenous CF genes in at least some CF patient genes. It should be noted that this term is not limited to the CFTR gene, but is applied to every genetic material that can be used to treat, diagnose, or cure a disease.

The phrases "parenteral administration" and "parenterally administered" are art-recognized terms and include modes of administration other than enteral and topical administration, such as injection, and include, but are not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intraconjunctival, and intrasternal injection and infusion.

As used herein, the term "surfactant" refers to an agent that reduces the surface tension of a liquid.

The term "therapeutic agent" refers to an agent that can be administered to prevent or treat a disease or condition. The therapeutic agent can be a nucleic acid, a nucleic acid analog, a small molecule, a peptide mimetic, a protein, a peptide, a carbohydrate or sugar, a lipid, or a surfactant, or a combination thereof.

The term "treating" or "treating" refers to preventing a disease, disorder, or condition from occurring in an animal that may be susceptible to the disease, disorder, and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder, or condition, e.g., arresting its development; and alleviating the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and/or condition. Treating a disease or condition includes ameliorating at least one symptom of a particular etiology or condition, even if the underlying pathophysiology is not affected, such as treating pain in a subject by administering an analgesic, even if the agent does not treat the cause of the pain.

As used herein, the term "targeting moiety" is a moiety that is designated to be located at or away from a specific location. The moiety can be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The entity can be, for example, a therapeutic compound, such as a small molecule, or a diagnostic entity, such as a detectable marker. The location can be a tissue, a specific cell type, or a subcellular compartment. In one embodiment, the targeting moiety directs the localization of an active entity. The active entity can be a small molecule, a protein, a polymer, or a metal. The active entity can be suitable for therapeutic, prophylactic, or diagnostic purposes.

The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and/or onto the particles described herein, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. The effective amount may vary depending on factors such as the disease or condition being treated, the specific targeting construct being administered, the size of the subject, and the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation.

The terms "conjugated" and "encapsulated" refer to binding, formulating, or otherwise including an active agent in a composition that permits release (e.g., sustained release) of the agent in a desired application. The terms encompass any manner of incorporating a therapeutic agent or other substance into a polymer matrix, including, for example, attachment to monomers of the polymer (via covalent, ionic, or other binding interactions), physical incorporation, encapsulation of the agent in a polymer coating, incorporation into the polymer, distribution throughout the polymer matrix, attachment to the surface of the polymer matrix (via covalent or other binding interactions), encapsulation within the polymer matrix, and the like. The terms "co-conjugated" or "co-encapsulated" refer to a therapeutic agent or other substance being combined with at least one other therapeutic agent or other substance in a subject composition.

II. Mucus-penetrating nanoparticles (MPPs)

A. Core Polymer

Many biocompatible polymers can be used to prepare nanoparticles. In one embodiment, the biocompatible polymer is biodegradable. In another embodiment, the particles are non-degradable. In other embodiments, the particles are a mixture of degradable and non-degradable particles.

Exemplary polymers include, but are not limited to, cyclodextrin-containing polymers, particularly cationic cyclodextrin-containing polymers, such as those described in U.S. Pat. No. 6,509,323;

polymers prepared from lactones, such as poly(caprolactone) (PCL);

Polyhydroxy acids and copolymers thereof, such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and blends thereof;

Polyalkyl cyanoacrylate;

polyurethane;

polyamino acids, such as poly-L-lysine (PLL), poly(valeric acid), and poly-L-glutamic acid;

Hydroxypropyl methacrylate (HPMA);

Polyanhydrides;

Polyester;

Polyorthoesters;

poly(esteramide);

polyamide;

Poly(ester ether);

polycarbonate;

Polyalkylene groups, such as polyethylene and polypropylene;

Polyalkylene glycols, such as poly(ethylene glycol) (PEG); polyalkylene oxides (PEO);

Polyalkylene terephthalates, such as poly(ethylene terephthalate); ethylene vinyl acetate polymer (EVA);

Polyvinyl alcohol (PVA);

Polyvinyl ether;

Polyvinyl esters, such as poly(vinyl acetate);

Polyvinyl halides, such as poly(vinyl chloride) (PVC); polyvinyl pyrrolidone; polysiloxanes;

Polystyrene (PS);

Cellulose, including derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, and carboxymethyl cellulose;

Acrylic acid polymers, such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (collectively referred to herein as "polyacrylic acid");

Polydioxanone and its copolymers;

polyhydroxyalkanoates;

Polypropylene fumarate;

polyoxymethylene;

Poloxamer;

poly(butyric acid);

trimethylene carbonate; and

Polyphosphide nitride.

Copolymers of the above (such as random, block or graft copolymers) or blends of the above listed polymers may also be used.

Functional groups on polymers can be capped to modify the properties of the polymer and/or to modify (e.g., reduce or increase) the reactivity of the functional groups. For example, the carboxyl termini of formic acid-containing polymers (such as lactide-containing and glycolide-containing polymers) can be optionally capped, for example, by esterification, and the hydroxyl termini can be optionally capped, for example, by etherification or esterification.

Copolymers of PEG or its derivatives with any of the above-mentioned polymers can be used to make polymeric particles. In certain embodiments, PEG or its derivatives can be located at internal positions of the copolymer. Alternatively, PEG or its derivatives can be located near or at the terminal positions of the copolymer. For example, one or more of the above polymers can be end-capped with polyethylene glycol blocks. In some embodiments, the core polymer is a blend of a PEGylated polymer and a non-PEGylated polymer, wherein the base polymers are the same (e.g., PLGA and PLGA-PEG) or different (e.g., PLGA-PEG and PLA). In certain embodiments, microparticles or nanoparticles are formed under conditions that allow PEG domains to phase separate or otherwise localize to the particle surface. Only the PEG domains localized to the surface can exhibit or include the function of a surface modifier. In specific embodiments, the particles are prepared from one or more polymers end-capped with polyethylene glycol blocks as surface-modifying materials.

The weight average molecular weight of a given polymer can vary, but is typically about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Daltons, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, 5,000 Daltons to 75,000 Daltons, 5,000 Daltons to 50,000 Daltons, or 5,000 Daltons to 25,000 Daltons.

Examples of preferred natural polymers include proteins (such as albumin), collagen, gelatin and prolamins (eg zein) and polysaccharides (such as alginate).

In some embodiments, the particles can be used as nanoparticle gene carriers. In these embodiments, the particles can be formed from one or more polycationic polymers complexed with one or more negatively charged nucleic acids.

Cationic polymers can be any synthetic or natural polymer that carries at least two positive charges per molecule and has sufficient charge density and molecular size to bind to nucleic acids under physiological conditions (i.e., pH and salt conditions encountered in vivo or within cells). In certain embodiments, the polycationic polymer contains one or more amine residues.

Suitable cationic polymers include, for example, polyethyleneimine (PEI), polyallylamine, polyvinylamine, polyvinylpyridine, aminoacetalized poly(vinyl alcohol), acrylic or methacrylic polymers with one or more amine residues (e.g., poly(N,N-dimethylaminoethyl acrylate)), polyamino acids (e.g., polyornithine, polyarginine, and polylysine), protamine, cationic polysaccharides (e.g., chitosan), DEAE-cellulose and DEAE-dextran, and polyamidoamine dendrimers (cationic dendrimers), as well as copolymers and blends thereof. In a preferred embodiment, the polycationic polymer is PEI.

Cationic polymers can be linear or branched, can be homopolymers or copolymers, and when containing amino acids can have L or D configuration, and can have any mixture of these characteristics. Preferably, the cationic polymer molecule is sufficiently flexible to allow it to form a tight complex with one or more nucleic acid molecules.

In some embodiments, the polycationic polymer has a molecular weight between about 5,000 and about 100,000 Daltons, more preferably between about 5,000 and about 50,000 Daltons, and most preferably between about 10,000 and about 35,000 Daltons.

B. Materials that promote diffusion through mucus

The nanoparticles are preferably coated with or contain one or more surface-altering agents or materials. As used herein, "surface-altering agent" refers to an agent or material that modifies one or more properties of the particle with respect to the surface, including but not limited to hydrophilicity (e.g., making the particle more hydrophilic or less hydrophilic), surface charge (e.g., making the surface neutral or nearly neutral or more negative or positive), and/or enhancing transport in or through body fluids and/or tissues (e.g., mucus). In some embodiments, the surface-altering material provides a direct therapeutic effect, such as reducing inflammation.

Examples of surface-altering agents include, but are not limited to, proteins, including anionic proteins (e.g., albumin); surfactants; sugars or sugar derivatives (e.g., cyclodextrins); therapeutic agents; and polymers. Preferred polymers include heparin, polyethylene glycol ("PEG"), and poloxamers (polyethylene oxide block copolymers). The most preferred material is PEG.

Examples of surfactants include, but are not limited to, L-α-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidylcholine (DPPC), oleic acid, sorbitan trioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearoyl polyoxyethylene (2) ether, lauryl Polyoxyethylene (4) ether, block copolymer of ethylene oxide and propylene oxide, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetylpyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cottonseed oil and sunflower oil, lecithin, oleic acid and sorbitan trioleate.

In one embodiment, the particle coating is provided with or contains polyethylene glycol (PEG). PEG can be applied as a coating to the particle surface. Alternatively, PEG can be in the form of a block covalently bound (e.g., internally or at one or both ends) to the core polymer used to form the particle. In a specific embodiment, the particle is formed by a block copolymer containing PEG. In a more specific embodiment, the particle is prepared by a block copolymer containing PEG, wherein the PEG is covalently bound to the end of the base polymer.

Representative PEG molecular weights include 300 Da, 600 Da, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 6 kDa, 8 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 50 kDa, 100 kDa, 200 kDa, 500 kDa, and 1 MDa, as well as all values within the range of 300 daltons to 1 MDa. In a preferred embodiment, the molecular weight of PEG is about 5 kD. PEGs of any given molecular weight can differ in other characteristics such as length, density, and branching.

i. Evaluate surface density

The surface density of poly(ethylene glycol) (PEG) on nanoparticles is a key parameter in determining their successful in vivo administration. Controlled delivery of drugs to mucosal surfaces is challenging due to the presence of a protective mucus layer, and mucus-permeable particles show promise in terms of improved resistance profile, retention, and efficacy at mucosal surfaces. Dense coatings of PEG on biodegradable nanoparticles can allow rapid penetration through mucus due to greatly reduced adhesive interactions between mucus components and the nanoparticles. However, it remains unclear how to optimally control surface PEG density and how to prepare biodegradable, mucus-permeable nanoparticles for in vivo administration.

Different methods have been used to assess the surface PEG density on nanoparticles, including those that directly measure changes in the nanoparticle's physiochemical properties, such as surface charge and hydrodynamic diameter. However, these methods cannot provide quantitative information on the number of PEG chains per square meter of nanoparticle surface.

To directly quantify surface PEG density, various techniques have been applied. Thermogravimetric analysis (TGA) can be used to calculate PEG content, but is limited to inorganic materials and requires the use of relatively large amounts of sample.

The reaction of dyes and reagents (such as fluorescent dyes) with functional PEG is widely used for PEG quantification. In these methods, unreacted PEG molecules with functional groups (such as -SH, _-NH2 , etc.) are quantified by fluorescence analysis or colorimetric quantification after reaction with specific reagents, and the surface PEG density is obtained by subtracting the unreacted PEG portion in the supernatant. However, these methods are limited to surface PEGization and functional PEG. Similar methods for quantifying the surface PEG density on PRINT nanoparticles by measuring the signal of unreacted fluorescein-PEG in the supernatant are limited to surface modification of nanoparticles with PEG. Therefore, these quantitative analyses are not suitable for measuring the PEG density on biodegradable nanoparticles prepared from PEG-containing block copolymers (such as the widely used poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) and poly(lactic acid)-poly(ethylene glycol) (PLA-PEG)).

Nuclear magnetic resonance (NMR) can be used to assess the surface PEG density on the PEG-containing polymeric nanoparticles described herein both qualitatively and quantitatively (the PEG peak is typically observed at approximately 3.65 ppm). When the nanoparticles are dispersed in the NMR solvent D ₂ O, only surface PEG (and not PEG embedded in the core) can be directly detected by NMR. Therefore, NMR provides a means for directly measuring the surface density of PEG.

In some embodiments, the surface density of PEG can be controlled by preparing particles from a mixture of PEGylated and non-PEGylated particles. For example, the surface density of PEG on PLGA nanoparticles can be precisely controlled by preparing particles from a mixture of poly(lactic-co-glycolic acid) and poly(ethylene glycol) (PLGA-PEG). Quantitative ^1H nuclear magnetic resonance (NMR) can be used to measure the surface PEG density on nanoparticles. Multiple particle tracking in human mucus and studies of mucin binding and tissue distribution in the mouse vagina have revealed a threshold PEG density of approximately 10-16 PEG chains per 100 square nanometers for PLGA-PEG nanoparticles to be effective in penetrating mucus. This density threshold can vary depending on various factors, including the core polymer used to prepare the particles, particle size, and/or PEG molecular weight.

The density of the coating can vary based on various factors, including the surface-altering material and the particle components. In one embodiment, the density of the surface-altering material (e.g., PEG) is at least 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, 10, 15, 20, 25, 40, 50, 60, 75, 80, 90, or 100 chains/nm² as measured using ^1H NMR. The above ranges include all values from 0.1 to 100 units/nm².

In certain embodiments, the density of the surface-altering material (e.g., PEG) is about 1 to about 25 chains/nm², about 1 to about 20 chains/nm², about 5 to about 20 chains/nm², about 5 to about 18 chains/nm², about 5 to about 15 chains/nm², or about 10 to about 15 chains/nm². The concentration of the surface-altering material (e.g., PEG) can also vary. In some embodiments, the target concentration of the surface-altering material (e.g., PEG) is at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more. The above ranges include all values from 0.5% to 25%. In another embodiment, the concentration of the surface-altering material (e.g., PEG) in the particle is at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more. The above ranges include all values from 0.5% to 25%. In other embodiments, the content of the surface-altering material (e.g., PEG) on the surface of the particle is at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. The above range includes all values from 0.5% to 25%.

In certain embodiments, the density of the surface altering material (eg, PEG) is such that the surface altering material (eg, PEG) adopts an extended brush configuration.

In other embodiments, the mass of the surface-altering portion is at least 1/10,000, 1/7500, 1/5000, 1/4000, 1/3400, 1/2500, 1/2000, 1/1500, 1/1000, 1/750, 1/500, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/20, 1/5, 1/2, or 9/10 of the mass of the particle. The above ranges include all values from 1/10,000 to 9/10.

C. Emulsifier

The particles described herein contain an emulsifier, particularly a low molecular weight emulsifier. The emulsifier is incorporated into the particles during the particle formation process and thus becomes a component of the finished particles. The emulsifier can be encapsulated within the particles, fully or partially dispersed within the polymer matrix (e.g., a portion of the emulsifier protrudes from the polymer matrix), and/or associated with the particle surface (e.g., covalently or non-covalently).

As used herein, " low molecular weight " generally refers to an emulsifier having a molecular weight less than 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400 or 300 amu. In certain embodiments, the molecular weight is less than 1300 amu. In certain embodiments, the molecular weight is from about 300 amu to about 1200 amu.

Emulsifiers can be positively charged, negatively charged, or uncharged. Examples of negatively charged emulsifiers include, but are not limited to, cholic acid sodium salt (CHA, MW = 430) and dioctyl sodium sulfosuccinate (DSS, MW = 455). Examples of positively charged emulsifiers include, but are not limited to, cetyltrimethylammonium bromide (CTAB, MW = 364). Examples of uncharged emulsifiers include, but are not limited to, saponin (MW = 1191), TWEEN 20 (MW = 1,225), TWEEN 80 (MW = 1310), and sugar ester D1216 (sucrose laurate, SE, MW = 524).

In addition to having a low molecular weight, the emulsifier must be able to adequately stabilize the emulsion droplets during particle formation to prevent particle aggregation. The emulsifying capacity of a particular emulsifier can be calculated using the following equation and can be expressed as a percentage.

Emulsifying capacity = nanoparticle weight/(nanoparticle weight + aggregated particle weight) × 100%

In some embodiments, the emulsifying capacity is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. This range includes all values between 50 and 95.

In addition to properly stabilizing the emulsion droplets to prevent aggregate formation, the stabilizer must be small enough to be completely shielded by the surface-altering material corona (e.g., PEG) at the particle surface, thereby providing a neutral or near-neutral surface charge. The transport of charged particles can be hindered by interactions between charged particles and oppositely charged species in the body. For example, the ability of a particle to rapidly penetrate mucus depends, at least in part, on the particle's surface charge. Therefore, the emulsifier must be small enough so that if the emulsifier is charged (e.g., positively or negatively charged), the charge is shielded by the corona of the surface-altering material (e.g., PEG) so that the surface charge is zero or substantially zero, e.g., -10 to 10 eV, -5 to 5 eV, -3 to 3 eV, -2 to 2 eV, or -1 to 1 eV.

D. Therapeutic, prophylactic, nutritional and/or diagnostic agents

1. Therapeutic agents

In some embodiments, the particles have one or more therapeutic agents encapsulated therein, dispersed therein, and/or covalently or non-covalently associated with the surface. The therapeutic agent can be a small molecule, protein, polysaccharide or carbohydrate, nucleic acid molecule, and/or lipid.

i. Small molecule therapeutics

Exemplary classes of small molecule therapeutics include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, anti-epileptics, antipsychotics, neuroprotectants, antiproliferatives (e.g., anticancer agents), anti-infectives (e.g., antibacterial and antifungal agents), antihistamines, anti-migraine drugs, antimuscarinics, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergic agents, electrolytes, gastrointestinal drugs, muscle relaxants, nutrients, vitamins, parasympathomimetics, stimulants, anorexias, and anti-somniacs.

ii. Nucleic Acids

In some embodiments, the pharmaceutical agent is one or more nucleic acids. The nucleic acids can alter, correct, or replace endogenous nucleic acid sequences. The nucleic acids are used to treat cancer, correct defects in genes involved in other lung diseases and metabolic diseases affecting lung function, such as genes used to treat Parkinson's disease and ALS, where the genes are delivered to the brain via nasal delivery.

Gene therapy is a technique used to correct a defective gene that contributes to the development of a disease. Researchers can use one of several methods to correct the defective gene:

A normal gene can be inserted into an unspecified location within the genome to replace a non-functional gene. This approach is the most common.

Abnormal genes can be exchanged for normal genes through homologous recombination.

• Abnormal genes can be repaired by selective reversion mutations, which return the gene to its normal function.

Can change the regulation of specific genes (the extent to which a gene is turned on or off).

Nucleic acids can be DNA, RNA, chemically modified nucleic acids, or combinations thereof. For example, methods for enhancing the stability of nucleic acid half-life and resistance to enzymatic cleavage are known in the art and may include one or more modifications or substitutions to nucleobases, sugars, or polynucleotide linkages. Nucleic acids can be custom synthesized to contain properties suitable for the desired use. Common modifications include, but are not limited to, the use of locked nucleic acids (LNA), unlocked nucleic acids (UNA), morpholinos, peptide nucleic acids (PNA), phosphorothioate linkages, phosphoacetate linkages, propyne analogs, 2'-O-methyl RNA, 5-Me-dC, 2'-5' linked phosphodiester linkages, chimeric linkages (mixed phosphorothioate and phosphodiester linkages and modifications), conjugation to lipids and peptides, and combinations thereof.

In some embodiments, nucleic acids include internucleotide linkage modifications such as phosphate analogs with achiral and uncharged intersubunit linkages (e.g., Sterchak, E.P. et al., Organic Chem., 52:4202, (1987)) or uncharged morpholino-based polymers with achiral intersubunit linkages (see, e.g., U.S. Patent No. 5,034,506). Some internucleotide linkage analogs include morpholinoates, acetals, and polyamide-linked heterocycles. Other backbone and linkage modifications include, but are not limited to, phosphorothioates, peptide nucleic acids, tricyclic-DNA, oligonucleotide decoys, ribozymes, Spiegelmers (L-containing nucleic acids, aptamers with high binding affinity), or CpG oligomers.

Phosphorothioates (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced with sulfur. Sulfurization of the internucleotide bond significantly reduces the effects of endonucleases and exonucleases (including 5' to 3' and 3' to 5' DNA POL1 exonucleases, nucleases S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterases). In addition, the potential to cross the lipid bilayer is increased. Due to these important improvements, phosphorothioates have found enhanced applications in cell regulation. Phosphorothioates are produced by two main routes: the action of solutions of elemental sulfur in carbon disulfide on hydrogen phosphonates, or the more recent method of sulfurizing phosphite triesters with tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD). The latter method avoids the problems of elemental sulfur being insoluble in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also produce phosphorothioates of higher purity.

Peptide nucleic acid (PNA) is a molecule in which the phosphate backbone of an oligonucleotide is completely replaced by repeated N-(2-aminoethyl)-glycine units and the phosphodiester bond is replaced by a peptide bond. Various heterocyclic bases are connected to the backbone using a methylene carbonyl bond. PNA maintains gaps between heterocyclic bases similar to oligonucleotides, but is an achiral and charge-neutral molecule. PNA typically comprises peptide nucleic acid monomers. The heterocyclic base can be any one of the standard bases (uracil, thymine, cytosine, adenine, and guanine) or any one of the modified heterocyclic bases described below. PNA may also have one or more peptides or amino acid variants and modifications. Therefore, the backbone component of PNA may be a peptide bond, or alternatively, it may be a non-peptide bond. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), etc. Methods for chemical assembly of PNAs are well known.

In some embodiments, nucleic acids include one or more chemically modified heterocyclic bases including, but not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5- and 2-amino-5-(2'-deoxy-β-D-ribofuranosyl)pyridine (2-aminopyridine) and various pyrrolo- and pyrazolo-pyrimidine derivatives, 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, N6-isopentenyl adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, β-D-mannose Q nucleoside, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, hydroxybutoxy nucleoside, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxyacetic acid methyl ester, 2,6-diaminopurine and 2'-modified analogs such as, but not limited to, O-methyl, amino-, and fluorescently modified analogs. Inhibitory RNA modified with 2'-fluoro (2'-F) pyrimidines appears to possess favorable properties in vitro. Furthermore, a recent report demonstrated that 2'-F-modified siRNAs have enhanced activity in cell culture compared to 2'-OH-containing siRNAs. While 2'-F-modified siRNAs are functional in mice, they do not necessarily possess enhanced intracellular activity compared to 2'-OH siRNAs.

In some embodiments, the nucleic acid comprises one or more sugar moiety modifications including, but not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidinoethyl (2'-OGE), 2'-O,4-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME), and 2'-O-(N-(methyl)acetamido) (2'-OMA).

The method of gene therapy generally relies on the introduction of nucleic acid molecules that will change the genotype of the cell into the cell. The introduction of nucleic acid molecules can correct, replace or otherwise change endogenous genes via genetic recombination. The method can include introducing a whole replacement copy of a defective gene, a heterologous gene or a small nucleic acid molecule (such as an oligonucleotide). For example, a corrective gene can be introduced into a non-specific position in the host genome. This method generally requires a delivery system for introducing the replacement gene into the cell, such as a genetically engineered viral vector.

The method for constructing the expression vector containing gene sequence and suitable transcription and translation control element is well known in the art. These methods include in vitro recombinant DNA technology, synthetic technology and in vivo gene recombination. Expression vector usually contains the regulatory sequence essential elements for translating and/or transcribing the inserted coding sequence. For example, the coding sequence is preferably operably connected to a promoter and/or enhancer to help control the expression of the desired gene product. According to the predetermined gene expression control type, the promoter used in the biotechnology belongs to different types. It can be divided into constitutive promoter, tissue-specific or developmental stage-specific promoter, inducible promoter and synthetic promoter usually.

Viral vectors include adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, AIDS virus, neurotrophic virus, Sindbis virus (Sindbis) and other RNA viruses, including these viruses with HIV backbone. Also suitable for use are any virus family with a common property that makes it suitable for use as a vector with these viruses. Usually, viral vectors contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, the necessary reverse terminal repeats for replication and encapsidation, and the promoter for transcribing and replicating the viral genome. When engineered as a vector, one or more viral early genes are usually removed, and gene or gene/promoter cassette is inserted into the viral genome to replace the removed viral DNA.

Gene targeting via target recombination (such as homologous recombination (HR)) is another strategy for gene correction. Gene correction at the target locus can be mediated by a donor DNA fragment homologous to the target gene (Hu et al., Mol. Biotech., 29: 197-210 (2005); Olsen et al., J. Gene Med., 7: 1534-1544 (2005)). One method of targeted recombination includes the use of triplex-forming oligonucleotides (TFOs), which bind to homopurine/homopyrimidine sites in double-stranded DNA as the third chain in a sequence-specific manner. Triplex-forming oligonucleotides can interact with double-stranded or single-stranded nucleic acids. When the triplex molecule interacts with the target region, a structure called a triplex is formed, in which three DNA chains forming a complex are present depending on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind to the target region with high affinity and specificity. Preferably, the triplex-forming molecules bind to the target molecule with a Kd of less than 10 ⁻⁶ , 10 ⁻⁸ , 10 ⁻¹⁰ or 10 ⁻¹² .

Methods for using triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) for targeted gene therapy are described in U.S. Published Application No. 20070219122, and their use for treating infectious diseases such as HIV is described in U.S. Published Application No. 2008050920. The triplex-forming molecule can also be a tail-clip peptide nucleic acid (tcPNA), such as those described in U.S. Published Application No. 2011/0262406. A highly stable PNA:DNA:PNA triplex structure can be formed by strand invasion of double-stranded DNA with two PNA chains. In this complex, both the PNA/DNA/PNA triplex portion and the PNA/DNA double-stranded portion produce a shift in the pyrimidine-rich triplex, resulting in an altered structure that has been shown to strongly stimulate the nucleotide excision repair pathway and activate sites for recombination with donor oligonucleotides. The two PNA chains can also be linked together to form a bi-PNA molecule. The triplex-forming molecules are suitable for inducing site-specific homologous recombination in mammalian cells when used in combination with one or more donor oligonucleotides that provide a corrected sequence. The donor oligonucleotides can be tethered to the triplex-forming molecules or can be separated from the triplex-forming molecules. The donor oligonucleotides can contain at least one nucleotide mutation, insertion, or deletion relative to the target double-stranded DNA.

Double double-stranded forming molecules (such as a pair of pseudo complementary oligonucleotides) can also be induced to reorganize with donor oligonucleotides at chromosome sites. The use of pseudo complementary oligonucleotides in targeted gene therapy is described in U.S. published application No. 2011/0262406. Pseudo complementary oligonucleotides are complementary oligonucleotides that contain one or more modifications such that they do not recognize or hybridize with each other, for example, due to steric hindrance, but each can recognize a complementary nucleic acid chain at the target site and hybridize to a complementary nucleic acid chain. In certain embodiments, pseudo complementary oligonucleotides are pseudo complementary peptide nucleic acids (pcPNA). Compared to the method of requiring the induction recombination of polypurine sequences such as triple helix oligonucleotides and dipeptide nucleic acids in target double-stranded DNA, pseudo complementary oligonucleotides can be more effective and provide increased flexibility.

2. Diagnostic agents

Exemplary diagnostic substances include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Suitable diagnostic agents include, but are not limited to, x-ray imaging agents and contrast agents. Radionuclides can also be used as imaging agents. Examples of other suitable contrast agents include gases or gas-emitting compounds (which are radiopaque). The nanoparticles may further include reagents suitable for determining the location of the administered particles. Reagents suitable for this purpose include fluorescent labels, radionuclides, and contrast agents.

For those embodiments in which one or more therapeutic agents, prophylactic agents, and/or diagnostic agents are encapsulated within polymeric nanoparticles and/or associated with the surface of the nanoparticles, the drug loading percentage is from about 1% to about 80% by weight, from about 1% to about 50% by weight, preferably from about 1% to about 40% by weight, more preferably from about 1% to about 20% by weight, and most preferably from about 1% to about 10% by weight. The above ranges include all values from 1% to 80%. For those embodiments in which the agent is associated with the particle surface, the loading percentage can be higher because the amount of the drug is not limited by the encapsulation method. In some embodiments, the agent to be delivered can be encapsulated within the nanoparticles and associated with the particle surface. Nutritional agents can also be incorporated. These nutritional agents can be vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts or plant hormones.

E. Particle Properties

1. Surface charge and particle size

To aid their diffusion through mucus, the nanoparticles described herein typically have a surface charge that is close to neutral. In certain embodiments, the zeta potential of the nanoparticles is between about 10 mV and about -10 mV, preferably between about 5 mV and about -5 mV, preferably between about 3 mV and about -3 mV, and more preferably between about 2 mV and about -2 mV. As discussed above, the particles described herein contain one or more low molecular weight emulsifiers. The emulsifier can be uncharged, in which case the emulsifier has little or no effect on the particle surface charge. However, in some cases, the emulsifier is positively or negatively charged. In these embodiments, the surface-altering material (e.g., PEG) must be present at a sufficient density to form a corona that shields the positively or negatively charged emulsifier, resulting in an effectively uncharged surface.

Although the particles described herein are referred to as nanoparticles, and therefore have an average diameter generally in the range of 1 nm to (but not including) about 1 micron, more preferably about 5 nm to about 500 nm, and most preferably about 5 nm to about 100 nm. In certain embodiments, the average diameter of the particles ranges from about 100 nm to about 150 nm. However, particles having a size in the micron range can be prepared. The conditions and/or materials used to prepare the particles can be varied to change the size of the particles.

In certain embodiments, the nanoparticles retain their particle size and zeta potential after nebulization or storage at 4°C for at least 1 month, more preferably at least 2 months, and most preferably at least 3 months.

2. Effect of emulsifiers on transport capacity

In some embodiments, the particles are administered to penetrate mucus for drug delivery to mucous membranes. The particles described herein contain surface-altering materials that can enhance transport through mucus. For example, PEG-containing block copolymers can self-assemble to form a dense, sticky, inert PEG coating on the surface of emulsion droplets formed using emulsion methods, but only when low molecular weight (MW) emulsifiers are used instead of conventional higher weight or high weight emulsifiers (such as PVA). Relative to nanoparticles prepared with high MW emulsifiers, low MW emulsifiers produce an average of thousands of times the increase in mean square displacement (<MSD>) of nanoparticles in CVM at a time scale of 1 s.

In addition, the particles described here penetrated CVM at an effective rate less than 10 times slower than the same particles in water. For example, PEG-containing diblock copolymers, poly (lactic acid) -b-PEG5k (PLA-PEG5k, Mn -95 kDa) and poly (ε-caprolactone) -b-PEG5k (PCL-PEG5k, Mn -78 kDa) were also evaluated. Nanoparticles prepared from these two polymers and PVA were immobilized in CVM, while rapid mucus penetration was observed for nanoparticles prepared using the low MW emulsifier CHA, with effective diffusion rates similar to those measured for PLGA-PEG5k nanoparticles.

In some embodiments, the particles described herein (made with a low molecular weight emulsifier) exhibit a transport rate that is at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, or 10000 times greater than particles made with PVA and/or exhibit an effective velocity that is less than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 times slower than the same particles in water.

In the absence of PEG, PLGA/CHA nanoparticles have a highly anionic surface charge and are mucoadhesive. In contrast, the surface charge of mucus-permeable PLGA-PEG5k/CHA nanoparticles is nearly neutral, suggesting the formation of a dense PEG coating that masks the negative charges of PLGA and CHA. The concentration of low-MW emulsifiers has no significant effect on the nanoparticle surface charge and mucus-penetrating properties, likely because the PEG corona completely shields these molecules from the particle surface.

Additionally, the intrinsic charge associated with the emulsifiers (DSS and CHA are negatively charged, CTAB is positively charged, and saponin, vitamin E TPGS, TWEEN20, TWEEN80, and SE are neutrally charged) had little or no effect on the surface charge and mucus penetration properties, further supporting a role for the PEG corona in shielding these low MW emulsifier molecules on the particle surface.

The choice of emulsifier also affects the extent of PEG brush formation during the emulsification process. For example, although both PLGA-PEG5k/PVA nanoparticles and PLGA-PEG5k/CHA nanoparticles have a near-neutral surface charge, only the CHA formulation is mucus permeable. PVA and PEMA differ from other emulsifiers in that they contain linear hydrophobic backbones decorated with hydrophilic side groups. It is possible that when PVA or PEMA stabilizes the oil/water interface, the hydrophobic polymer backbone can polyvalently attach to the oil/water interface, where it comes into close contact with the protruding PEG brush. Therefore, PVA and PEMA can disrupt the organization of PEG molecules on the particle surface, thereby causing the particles to become mucoadhesive. In the case of PLGA-PEG5k/PEMA nanoparticles, a negative surface charge (-42 mV) indicates disruption of the PEG coating, as the charge likely originates from exposed PEMA molecules on the surface.

However, not all low MW emulsifiers are suitable for preparing MPPs. For example, Cremophore EL, TWEEN 80, Vitamin E TPGS, PLURONIC F127, and F68 were unable to fully stabilize the emulsion droplets during particle preparation, resulting in varying degrees of larger aggregate formation. Although the non-aggregated nanoparticles thus prepared were partially mucus permeable, the nanoparticle yield was as low as 30%. Therefore, the ability to produce MPPs from PEG-containing block copolymers by emulsification methods depends crucially on both the MW and emulsifying capacity of the emulsifier. The emulsifying capacity of the emulsifier was estimated by the percentage of non-aggregated PLGA-PEG nanoparticles prepared in an aqueous phase containing 1% emulsifier. The emulsifier must be strong enough to stabilize the emulsion droplets, but small enough to be completely obscured by the PEG corona at the particle surface.

PLGA-PEG/CHA nanoparticles prepared by the emulsification method with a wide range of PEG MW (1, 2, 5, and 10 kDa) all rapidly penetrated mucus. The nanoparticle surface charge was inversely proportional to the PEG MW and varied between -18 mV (1 kDa) and -2.3 mV (10 kDa). The surface PEG density [Γ] (number of PEG per 100 square nanometers), measured by ^1H NMR, decreased with increasing PEG MW. However, the ratio [Γ/Γ*] of the surface PEG density to the theoretical PEG density [Γ*] required to form a brush-like PEG coating was greater than 2, regardless of PEG MW, indicating the presence of a dense, brush-like PEG coating on the surface of the PLGA-PEG (1-10 kDa)/CHA nanoparticles. The formation of a dense PEG brush on the particle surface appears to be required for mucus penetration.

It was confirmed that both hydrophobic and hydrophilic drugs can be effectively encapsulated into PLGA-PEG MPPs. Two model compounds, curcumin (a hydrophobic drug (MW = 368 Da)) and BSA (a hydrophilic protein (MW = 66 kDa)), were encapsulated into PLGA-PEG5k/CHA nanoparticles using an o/w single emulsion and into PLGA-PEG5k/saponin nanoparticles using a w/o/w double emulsion, respectively. The efficiency of encapsulating both hydrophobic curcumin and hydrophilic BSA into MPPs using a low-MW emulsifier was similar to that achieved with conventional particles (CPs) using PVA via an emulsification method.

Nanoparticles loaded with curcumin and BSA diffused rapidly in mucus at τ = 1s, at rates only 6 and 36 times slower than in water. Nanoparticles prepared with PVA, on the other hand, were immobilized in CVM, where transport rates were over 2,000 times slower than in water. A substantial fraction (up to 40% and 30% of curcumin-MPP and BSA-MPP, respectively) was expected to physiologically penetrate the thick mucus layer within 60 minutes, while <1% of the nanoparticles coated with PVA were expected to do so.

III. Pharmaceutical Compositions

The formulations described herein contain an effective amount of nanoparticles in a pharmaceutical carrier suitable for administration to a mucosal surface. The formulations can be administered parenterally (e.g., by injection or infusion), topically (e.g., to the eye), or by pulmonary administration.

A. Pulmonary preparations

Pharmaceutical formulations and methods for pulmonary administration of active agents to patients are known in the art.

The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the bloodstream. The respiratory tract encompasses the upper airway, including the oropharynx and larynx; followed by the lower airway, which includes the trachea, which then branches into bronchi and bronchioles. The upper and lower airways are called conducting airways. The terminal bronchioles then divide into respiratory bronchioles, which then lead to the final respiratory zone, the alveoli, or deep lungs, where gas exchange occurs.

Formulations can be divided into dry powder formulations and liquid formulations. Both dry powder and liquid formulations can be used to form aerosol formulations. As used herein, the term aerosol refers to any formulation of a fine mist of particles, which can be in solution or suspension, regardless of whether it is produced using a propellant.

1. Dry powder formulation

A dry powder formulation is a finely powdered solid formulation containing nanoparticle carriers suitable for pulmonary administration. A dry powder formulation includes at least one or more nanoparticle carriers suitable for pulmonary administration. The dry powder formulation can be administered to a patient via pulmonary inhalation without the use of any carrier other than air or a suitable propellant.

In other embodiments, the dry powder formulation contains one or more nanoparticle gene vectors and a pharmaceutically acceptable carrier. In these embodiments, the nanoparticle gene vectors and pharmaceutical carriers can be formed into nano- or micron-particles for delivery to the lungs.

Pharmaceutical carriers may include fillers or lipids or surfactants. Natural surfactants such as dipalmitoylphosphatidylcholine (DPPC) are most preferred. Synthetic and animal-derived lung surfactants include:

Synthetic lung surfactant

Exosurf - a mixture of DPPC with hexadecanol and tyloxapol added as a spreading agent

Pumactant (artificial lung expander compound or ALEC) - a mixture of DPPC and PG

KL-4 is composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21-amino acid synthetic peptide that mimics the structural features of SP-B.

Venticute-DPPC, PG, palmitic acid and recombinant SP-C

Animal-derived surfactants

Alveofact - extracted from cow lung lavage fluid

Curosurf - extracted from material obtained from minced pig lungs

Infasurf - extracted from calf lung lavage fluid

Survanta - extracted from minced cow lungs, with additional DPPC, palmitic acid, and palmitin

Exosurf, Curosurf, Infasurf, and Survanta are surfactants currently approved by the FDA for use in the United States.

The pharmaceutical carrier may also include one or more stabilizers or dispersants. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride.

Dry powder formulations are typically prepared by blending one or more nanoparticle vectors with one or more pharmaceutically acceptable carriers. Optionally, additional active agents may be incorporated into the mixture as discussed below. The mixture is then formed into particles suitable for pulmonary administration using techniques known in the art, such as lyophilization, spray drying, agglomeration, spray coating, coacervation, cryogenic casting, milling (e.g., air attritor (jet milling), ball milling), high pressure homogenization, and/or supercritical fluid crystallization.

An appropriate particle formation method can be selected based on the desired particle size, particle size distribution, and particle morphology desired for the formulation. In some cases, a particle formation method is selected to produce a population of particles having a desired particle size and particle size distribution for transpulmonary administration. Alternatively, a particle formation method can produce a population of particles having a desired particle size and particle size distribution for transpulmonary administration, and a population of particles having a desired particle size and particle size distribution for transpulmonary administration can be separated from the particle population, for example, by screening.

It is known in the art that particle morphology affects the depth of particle penetration into the lung. Therefore, dry powder formulations are processed into particles with appropriate mass median aerodynamic diameter (MMAD), tap density, and surface roughness to achieve delivery of one or more active agents to the desired lung area. For example, preferred particle morphologies for delivery to the deep lung are known in the art and are described, for example, in U.S. Patent No. 7,052,678 to Vanbever et al.

Particles with a mass median aerodynamic diameter (MMAD) greater than about 5 microns generally do not reach the lungs; instead, they tend to hit the back of the throat and be swallowed. Particles with a diameter of about 3 to about 5 microns are small enough to reach the upper to middle lung regions (conducting airways), but may be too large to reach the alveoli. Smaller particles (i.e., about 0.5 to about 3 microns) are able to effectively reach the alveolar region. Particles with a diameter of less than about 0.5 microns can also be deposited in the alveolar region by sedimentation.

The exact particle size range for effectively achieving delivery to the alveolar region will depend on several factors, including the tap density of the delivered particles. Generally speaking, as the tap density decreases, the MMAD of the particles that can effectively reach the alveolar region of the lung increases. Therefore, in the case of particles with low tap density, particles with a diameter of about 3 to about 5 microns, about 5 to about 7 microns, or about 7 to about 9.5 microns can be effectively delivered to the lungs. The preferred aerodynamic diameter for maximum deposition in the lungs can be calculated. See, for example, U.S. Patent No. 7,052,678 to Van Bevere et al.

Microparticles cannot diffuse through mucus, even if their surface is mucoresistant. However, mucus-penetrating particles can be encapsulated in microparticles to impact the upper lung and subsequently release nanoparticles. In some embodiments, the dry powder formulation consists of a plurality of particles having a median mass aerodynamic diameter of between about 0.05 and about 10 microns, more preferably between about 0.05 and about 7 microns, and most preferably between about 0.05 and about 5 microns. In some embodiments, the dry powder formulation consists of a plurality of particles having a median mass aerodynamic diameter of between about 0.05 and about 3 microns, more preferably between about 0.05 and about 1 micron, and more preferably between about 0.05 and about 0.7 microns. In some embodiments, the dry powder formulation consists of a plurality of particles having a median mass aerodynamic diameter of between about 3 and about 5 microns. In some embodiments, the dry powder formulation consists of a plurality of particles having a median mass aerodynamic diameter of between about 5 and about 7 microns. In some embodiments, the dry powder formulation is comprised of a plurality of particles having a median mass aerodynamic diameter of between about 7 and about 9.5 microns.

In some cases, there may be advantages to delivering particles with a diameter greater than about 3 microns. Phagocytosis of particles by alveolar macrophages decreases dramatically as particle size increases beyond about 3 microns. Kawaguchi, H. et al., Biomaterials, 7:61-66 (1986); Krenis, L.J. and Strauss, B., Proc. Soc. Exp. Med., 107:748-750 (1961); and Rudt, S. and Muller, R.H., J. Contr. Rel, 22:263-272 (1992). By administering particles with an aerodynamic volume greater than 3 microns, phagocytosis and engulfment by alveolar macrophages and clearance from the lungs can be minimized.

In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter of less than 10, 9, 8, 7, 6, or 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns.

In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns and less than about 10 microns, more preferably greater than about 0.03 microns and less than about 7 microns, and most preferably greater than about 0.03 microns and less than about 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns and less than about 3 microns. In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns and less than about 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns and less than about 7 microns. In some embodiments, at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the particles in the dry powder formulation have an aerodynamic diameter greater than about 0.03 microns and less than about 9.5 microns.

In some embodiments, the particles have a tap density of less than about 0.4 g/cm ³ , more preferably less than about 0.25 g/cm ³ , and most preferably less than about 0.1 g/cm ³ . Features that may contribute to low tap density include irregular surface texture and porous structure.

In some cases, the shape of the particles is spherical or ovoid. The particles may have a smooth or rough surface texture. The particles may also be coated with a polymer or other suitable material to control the release of one or more active agents in the lung.

Dry powder formulations can be administered as dry powders using suitable methods known in the art. Alternatively, dry powder formulations can be suspended in a liquid formulation as described below and administered to the lungs using methods known in the art for delivering liquid formulations.

2. Liquid preparations

Liquid formulations contain one or more nanoparticle vectors suspended in a liquid pharmaceutical carrier.

Suitable liquid carriers include, but are not limited to, distilled water, deionized water, purified or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate-buffered saline (PBS), Ringer's solution, and isotonic sodium chloride, or any other aqueous solution suitable for administration to animals or humans.

Preferably, the liquid formulation is isotonic with respect to physiological body fluids and has approximately the same pH, for example, in the range of about pH 4.0 to about pH 7.4, more preferably in the range of about pH 6.0 to pH 7.0. The liquid pharmaceutical carrier may include one or more physiologically compatible buffers, such as phosphate buffer. One of ordinary skill in the art can readily determine the appropriate saline content and pH of an aqueous solution for pulmonary administration.

Liquid formulations may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinyl pyrrolidone, gum tragacanth or lecithin. Liquid formulations may also include one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate.

In some cases, the liquid formulation may contain one or more solvents that are low toxicity organic (i.e., non-aqueous) Class III residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to easily aerosolize the formulation. Any such solvent included in the liquid formulation should not react adversely with one or more active agents present in the liquid formulation. The solvent should be sufficiently volatile to be able to form an aerosol of the solution or suspension. Other solvents or aerosolizing agents (such as freon, ethanol, ethylene glycol, polyethylene glycol, or fatty acids) may also be included in the liquid formulation as needed to increase volatility and/or change the aerosolization behavior of the solution or suspension.

Liquid formulations may also contain small amounts of polymers, surfactants, or other excipients well known to those skilled in the art. In this context, "small amounts" means that there are no excipients that may adversely affect the absorption of one or more active agents in the lungs.

3. Aerosol formulations

The above-described dry powder and liquid formulations can be used to form aerosol formulations for transpulmonary administration. Aerosols for delivering therapeutic agents to the respiratory tract are known in the art. As used herein, the term aerosol refers to any formulation of a fine mist of solid or liquid particles suspended in a gas. In some cases, the gas may be the propellant; however, this is not required. Aerosols can be produced using a number of standard techniques, including, for example, ultrasonication or autoclaving.

Preferably, the dry powder or liquid formulation described above is formulated into an aerosol formulation using one or more propellants. Suitable propellants include air, hydrocarbons (such as pentane, isopentane, butane, isobutane, propane, and ethane), _carbon dioxide, _{chlorofluorocarbons} , fluorocarbons, and combinations thereof. Suitable fluorocarbons include fluorocarbons containing 1-6 hydrogen atoms (such as _CHF2CHF2 , _CF3CH2F , _{CH2F2CH3 ,} and _CF3CHFCF3 ) and fluorinated ethers (such as _CF3 - _{O - CF3} , _CF2HO - _CHF2 , _{and CF3} - _CF2 -O _{- CF2 - CH3 )} . Suitable fluorocarbons also include perfluorocarbons, such _as 1-4 carbon perfluorocarbons, including _CF3CF3 , _CF3CF2CF3 , and _{CF3CF2CF2CF3 .}

Preferably, the propellant includes, but is not limited to, one or more hydrofluoroalkanes (HFAs). Suitable HFA propellants include, but are not limited to, 1,1,1,2,3,3,-heptafluoro-n-propane (HFA227), 1,1,1,2-tetrafluoroethane (HFA134), 1,1,1,2,25,3,3,3-heptafluoropropane (propellant 227), or any mixture of these propellants.

Preferably, the one or more propellants have sufficient vapor pressure to be effective as a propellant. Preferably, the one or more propellants are selected so that the density of the mixture matches the density of the particles in the aerosol formulation to minimize settling or creaming of the particles in the aerosol formulation. The propellant is preferably present in an amount sufficient to propel multiple selected doses of the aerosol formulation from the aerosol can.

4. Devices for pulmonary administration

In some cases, a device is used to administer the formulation to the lungs. Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

Inhalation may be performed through the patient's nose and/or mouth.Administration may be performed by self-administration of the formulation while inhaling or by administering the formulation to a ventilator-dependent patient via a ventilator.

dry powder inhaler

The above-described dry powder formulations can be administered to a patient's lungs using a dry powder inhaler (DPI). DPI devices typically use a mechanism such as gas puffing to form a bulk of dry powder inside a container, which can then be inhaled by the patient.

In a dry powder inhaler, the dose to be administered is stored in a non-pressurized dry powder form, and when the inhaler is actuated, the powder particles are inhaled by the individual. In some cases, a compressed gas (i.e., a propellant) can be used to dispense the powder, similar to a pressurized metered dose inhaler (pMDI). In some cases, a DPI can be breath-activated, meaning that the aerosol is formed in response to inspiration. Typically, a dose of less than tens of milligrams is administered per inhalation of a dry powder inhaler to avoid stimulating a cough.

DPIs function by various mechanical means to administer the formulation to the lungs. In some DPIs, a scraper blade or gate slide on the dry powder formulation is contained in the reservoir, sorting the formulation into a flow path so that the patient can inhale the powder in a single breath. In other DPIs, the dry powder formulation is packaged in a preformed dosage form, such as a blister, tablet dosage form, tablet, or caplet, which is pierced, crushed, or otherwise unpacked to release the dry powder formulation into the flow path for subsequent inhalation. Other DPIs release the dry powder formulation into a chamber or capsule and use a mechanical or electrical stirrer to keep the dry powder formulation suspended in the air until the patient inhales.

Dry powder formulations may be packaged in various forms, such as loose powder, cakes, or in reservoirs for insertion into DPI reservoirs.

Examples of suitable DPIs for administering the above-described formulations include the Inhaler (Astrazeneca, Wilmington, Del.), the Inhaler (Innovata, Ruddington, Nottingham, UK), the Inhaler (Glaxo, Greenford, Middlesex, UK), the Inhaler (Orion, Expoo, FI), the Inhaler (Pfizer, New York, N.Y.), the Inhaler (Microdose, Monmouth Junction, N.J.), and the Inhaler (Dura, San Diego, Calif.).

pressurized metered-dose inhaler

Such liquid formulations can be administered to the patient's lungs using a pressurized metered dose inhaler (pMDI).

A pressurized metered dose inhaler (pMDI) typically comprises at least two components: a canister in which a liquid formulation is maintained under pressure in combination with one or more propellants, and a container for holding and actuating the canister. The canister may contain a single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, from which the contents of the canister can be discharged. Aerosolized medication is dispensed from the pMDI by applying force to the canister to push it into the container, thereby opening the valve and allowing drug particles to be transported from the valve via the container outlet. After discharge from the canister, the liquid formulation is atomized to form an aerosol.

pMDIs typically use one or more propellants to pressurize the contents of the canister and propel the liquid formulation out of the container outlet, forming an aerosol. Any suitable propellant can be used, including those mentioned above. The propellant can take a variety of forms. For example, the propellant can be a compressed gas or a liquefied gas. Chlorofluorocarbons (CFCs) were once commonly used as liquid propellants, but are now banned. They have been replaced by the now widely accepted hydrofluoroalkane (HFA) propellants.

pMDIs are available from many suppliers, including 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough, and Vectura. In some cases, patients administer aerosolized formulations by manually expelling the aerosolized formulation from the pMDI in conjunction with inhalation. In this manner, the aerosolized formulation is entrained in the inhaled air stream and delivered to the lungs.

In other cases, a breath-actuated trigger that simultaneously discharges a dose of the formulation upon sensing inhalation may be used, such as the trigger included in the inhaler (MAP Pharmaceuticals, Mountain View, Calif.). These devices that discharge the aerosol formulation when the user begins to inhale are called breath-actuated pressurized metered-dose inhalers (baMDIs).

sprayer

The above-described liquid formulations can also be administered using a nebulizer. A nebulizer is a liquid aerosol generator that converts the above-described liquid formulations (typically aqueous-based compositions) into a mist or cloud of small droplets, preferably having a mass median aerodynamic diameter of less than 5 microns, which can be inhaled into the lower respiratory tract. This process is called atomization. When the aerosol cloud is inhaled, the droplets carry one or more active agents to the nose, upper airways, or deep lungs. Any type of nebulizer can be used to administer the formulation to the patient, including but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.

Pneumatic (jet) nebulizers use a pressurized gas supply as the driving force for atomizing liquid formulations. Compressed gas is delivered through a nozzle or ejector to create a low-pressure region that entrains the liquid formulation around it and shears it into a thin film or fibril. The film or fibril is unstable and breaks down into small droplets that are carried by the compressed gas flowing into the inhaled breath. A baffle inserted into the droplet plume screens out large droplets and returns them to the main liquid reservoir. Examples of pneumatic nebulizers include, but are not limited to, PARI LC, PARI LCD, Devilbiss, and Boehringer Ingelheim.

Electromechanical nebulizers use electrically generated mechanical force to atomize liquid formulations. The electromechanical driving force can be applied, for example, by oscillating the liquid formulation at ultrasonic frequencies or by forcing the bulk liquid through small holes in a thin film. This force creates a thin liquid film or fibrillar stream that breaks up into small droplets to form a low-speed aerosol stream that can be entrained in the inhaled airstream.

In some cases, the electromechanical nebulizer is an ultrasonic nebulizer in which the liquid formulation is coupled to an oscillator that oscillates at a frequency in the ultrasonic range. The coupling is achieved by direct contact of the liquid with the oscillator (such as a plate or ring in a holding cup) or by placing large droplets on a solid oscillating projector (horn). The oscillation creates a circular support film that breaks up into droplets at its edges to atomize the liquid formulation. Examples of ultrasonic nebulizers include DriveMedical, BeetleOctive Tech, and John Bunn

In some cases, the electromechanical sprayer is a screen sprayer in which the liquid formulation is driven through a screen or membrane with small holes in the range of 2 to 8 microns in diameter to produce thin fibrils that break up into small droplets. In some designs, the liquid formulation is forced through the screen by applying pressure with a solenoid piston rod (such as a sprayer); or by clamping the liquid between a piezoelectric oscillating plate and the screen, which produces an oscillating pump action (such as a sprayer or a sprayer). In other cases, the screen is oscillated back and forth by an upright column of liquid to pump it through the holes. Examples of such sprayers include AeroNeb, AeroNeb, Paritron, and Aradigm.

Electrohydrodynamic aerosol device

The above-described liquid formulations can also be administered using an electrohydrodynamic (EHD) aerosol device. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions. Examples of EHD aerosol devices are known in the art. See, for example, U.S. Patent No. 4,765,539 to Noakes et al. and U.S. Patent No. 4,962,885 to Coffee, R.A.

The electrochemical properties of the formulation can be an important parameter to optimize when delivering a liquid formulation to the lung using an EHD aerosol device, and such optimization is generally performed by one of ordinary skill in the art.

C. Parenteral Formulations

In some embodiments, the nanoparticles are formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation can be administered via any route (e.g., the bloodstream or directly to the organ or tissue to be treated). In some embodiments, the nanoparticles are formulated for parenteral administration to the eye.

As used herein, "parenteral administration" means administration by any method other than through the digestive tract or non-invasive local or regional routes. For example, parenteral administration may include intravenous, intradermal, intraperitoneal, intrapleural, intratracheal, intramuscular, subcutaneous, subconjunctival, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, the compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for preparing solutions or suspensions upon addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions and microemulsions thereof, liposomes, or creams.

The carrier may be a solvent or dispersion medium containing, for example, water; ethanol; one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol); oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by maintaining the desired particle size (in the case of a dispersion), and/or by the use of a surfactant. In many cases, it will be preferred to include an isotonic agent, such as a sugar or sodium chloride.

Solutions and dispersions of the active compound in the form of a free acid or base, or a pharmacologically acceptable salt thereof, can be prepared in water or another solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH adjusters, and combinations thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium and ammonium salts of long-chain alkyl sulfonates and alkylaryl sulfonates, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates, such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates, such as sodium bis-(2-ethylthio)-sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyethylene oxide and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-lauryl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoyl amphoacetate, lauryl betaine, and lauryl sulfobetaine.

The formulation may contain a preservative to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent.

The formulation is typically buffered to pH 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer.

Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinyl pyrrolidone, dextran, carboxymethyl cellulose, and polyethylene glycol.

Sterile injectable solutions can be prepared in the following manner: the active compound in the desired amount is optionally incorporated into an appropriate solvent or dispersion medium together with one or more of the above-listed excipients, followed by filtration sterilization. Typically, dispersions are prepared in the following manner: various sterilized active ingredients are incorporated into a sterile vehicle containing an alkaline dispersion medium and the desired other ingredients from those listed above. In the case of using sterile powders to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which obtain a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution. Powders can be prepared in this way so that the properties of the particles are porous, which can increase the dissolution of the particles. Methods for making porous particles are well known in the art.

Pharmaceutical formulations for ophthalmic administration are preferably in the form of sterile aqueous solutions or suspensions of particles formed from one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate-buffered saline (PBS), and isotonic sodium chloride solution. The formulations may also be sterile solutions, suspensions, or emulsions in non-toxic, parenterally acceptable diluents or solvents such as 1,3-butanediol.

In some cases, the formulation is distributed or packaged in liquid form. Alternatively, the formulation for ocular administration can be packaged as a solid, for example, obtained by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.

Solutions, suspensions or emulsions for ophthalmic administration can be buffered with an effective amount of a buffer necessary to maintain a pH suitable for ophthalmic administration. Suitable buffers are well known to those of ordinary skill in the art, and some examples of suitable buffers are acetate, borate, carbonate, citrate and phosphate buffers.

Solutions, suspensions or emulsions for ophthalmic administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art, and some examples include glycerol, mannitol, sorbitol, sodium chloride, and other electrolytes.

Solutions, suspensions, or emulsions for ophthalmic administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparation. Suitable preservatives are known in the art and include polyhexamethylene biguanide (PHMB), benzalkonium chloride (BAK), stabilized oxychloride complexes (or as), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parahydroxybenzoates, thimerosal, and mixtures thereof.

Solutions, suspensions, or emulsions for ophthalmic administration may also contain one or more excipients known in the art, such as dispersing agents, wetting agents, and suspending agents.

D. Topical Preparations

In other embodiments, the nanoparticles are formulated for topical administration to mucous membranes. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulations can be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The compositions contain one or more chemical permeation enhancers, membrane permeants, membrane transport agents, lubricants, surfactants, stabilizers, and combinations thereof.

In some embodiments, the nanoparticles can be administered as liquid formulations (e.g., solutions or suspensions), semi-solid formulations (e.g., lotions or ointments), or solid formulations. In some embodiments, the nanoparticles are formulated as liquids, including solutions and suspensions, such as eye drops, or as semi-solid formulations, such as ointments or lotions for topical administration to mucous membranes (e.g., eyes), vaginally, or rectally.

The formulation may contain one or more excipients, such as lubricants, surfactants, emulsifiers, penetration enhancers, and the like.

"Emollients" are externally applied agents that soften or soothe the skin and are generally known in the art and listed in compendiums such as "Handbook of Pharmaceutical Excipients", 4th edition, Pharmaceutical Press, 2003. These include, but are not limited to, almond oil, castor oil, carob extract, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, glycol palmitostearate, glycerin, glyceryl monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium chain triglycerides, mineral oil and lanolin alcohols, paraffin oil, paraffin oil and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol, and combinations thereof. In one embodiment, the lubricant is ethylhexyl stearate and ethylhexyl palmitate.

"Surfactants" are surfactants that reduce surface tension and thereby enhance the emulsification, foaming, dispersion, spreading, and wetting properties of a product. Suitable nonionic surfactants include emulsifying waxes, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbates, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glyceryl monostearate, poloxamers, povidone, and combinations thereof. In one embodiment, the nonionic surfactant is stearyl alcohol.

An "emulsifier" is a surface-active substance that promotes the suspension of one liquid in another and promotes the formation of a stable mixture or emulsion of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include gum arabic, anionic emulsifying wax, calcium stearate, carbomer, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glyceryl monostearate, glyceryl monooleate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lanolin, hydrate, lanolin alcohol, lecithin, medium chain triglycerides, methylcellulose, mineral oil and lanolin alcohol, sodium dihydrogen phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamer, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth gum, triethanolamine, triglyceride, and combinations thereof. In one embodiment, the emulsifier is glyceryl stearate.

Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithin, bile salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and imides), macrocycles (such as macrolides), ketones, and anhydrides and cyclic ureas, surfactants, N-methylpyrrolidone and its derivatives, DMSO and related compounds, ionic compounds, azones and related compounds, and solvents (such as ethanol, ketones, amides, polyols (e.g., ethylene glycol). Examples of these classes are known in the art.

i. Lotions, creams, gels, ointments, lotions and foams

As used herein, "hydrophilic" refers to a substance having strongly polar groups that readily interact with water.

"Lipophilic" refers to a compound that has an affinity for lipids.

"Amphiphilic" refers to molecules that have a combination of hydrophilic and lipophilic (hydrophobic) properties.

As used herein, "hydrophobic" refers to a substance that lacks affinity for water; tends to repel water and does not absorb water as well as dissolve in water or mix with water.

A "gel" is a colloid in which a dispersed phase has combined with a continuous phase to produce a semisolid material (eg, a jelly).

An "oil" is a composition containing at least 95% by weight of a lipophilic substance. Examples of lipophilic substances include, but are not limited to, naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides, and combinations thereof.

The "continuous phase" refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes referred to as the external phase. This also refers to the fluid phase of a colloid, within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), then a water-soluble or hydrophilic drug will be dissolved in the continuous phase (as opposed to dispersed). In multiphase formulations (e.g., emulsions), the discontinuous phase is suspended or dispersed in the continuous phase.

An "emulsion" is a composition containing a mixture of immiscible components uniformly blended together. In certain embodiments, the immiscible components include a lipophilic component and an aqueous component. An emulsion is a formulation in which a liquid is distributed in the form of small globules throughout a bulk of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When an oil is the dispersed liquid and an aqueous solution is the continuous phase, it is referred to as an oil-in-water emulsion, while when a water or aqueous solution is the dispersed phase and an oil or oil-containing substance is the continuous phase, it is referred to as a water-in-oil emulsion. Either or both the oil and aqueous phases may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, particularly nonionic surfactants; emulsifiers, particularly emulsifying waxes; and liquid non-volatile, non-aqueous substances, particularly glycols such as propylene glycol. The oil phase may contain other oily, pharmaceutically approved excipients. For example, substances such as hydroxylated castor oil or sesame oil may be used as surfactants or emulsifiers in the oil phase.

An emulsion is a formulation in which a liquid is distributed in the form of small globules throughout a bulk of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When an oil is the dispersed liquid and an aqueous solution is the continuous phase, it is called an oil-in-water emulsion, while when a water or aqueous solution is the dispersed phase and an oil or oil-containing substance is the continuous phase, it is called a water-in-oil emulsion. The oil phase may be composed at least in part of a propellant (such as an HFA propellant). Either or both the oil and aqueous phases may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, particularly nonionic surfactants; emulsifiers, particularly emulsifying waxes; and liquid non-volatile, non-aqueous substances, particularly glycols such as propylene glycol. The oil phase may contain other oily, pharmaceutically approved excipients. For example, substances such as hydroxylated castor oil or sesame oil can be used as surfactants or emulsifiers in the oil phase.

A subset of emulsions is self-emulsifying systems. These drug delivery systems are typically capsules (hard or soft shells) that contain a drug dispersed or dissolved in a mixture of a surfactant and a lipophilic liquid (such as an oil or other water-immiscible liquid). When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents immediately produces minimal emulsion droplets. These are typically within the size range of micelles or nanoparticles. No mixing forces are required to produce the emulsion, as this is typically the case with emulsion formulation processes.

"Lotion" is a liquid formulation of low to medium viscosity. Lotions can contain a finely powdered substance that is soluble in a dispersion medium using a suspending agent and a dispersant. Alternatively, a lotion can have a dispersed phase liquid substance that is immiscible with the vehicle and is typically dispersed with the aid of an emulsifier or other suitable stabilizer. In one embodiment, the lotion is in the form of an emulsion having a viscosity between 100 and 1000 centistokes. The fluidity of the lotion allows for rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin, leaving a thin coating of their medicinal components on the skin surface.

A "cream" is a viscous liquid or semisolid emulsion of the "oil-in-water" or "water-in-oil" type. Creams may contain emulsifiers and/or other stabilizers. In one embodiment, the formulation is in the form of a cream having a viscosity greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are generally preferred over ointments because they are generally easier to spread and easier to remove.

The difference between creams and lotions is the viscosity, which depends on the amount/use of various oils and the percentage of water used to prepare the formulation. Creams are generally thicker than lotions, can have a variety of uses, and generally use a wider variety of oils/butter, depending on the desired effect on the skin. In cream formulations, the water base percentage is about 60%-75% of the total 100%, and the oil base is about 20%-30% of the total, with the remaining percentage being emulsifiers, preservatives, and additives.

An "ointment" is a semisolid formulation containing an ointment base and, optionally, one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., paraffin oil, white petrolatum, yellow ointment, and mineral oil); absorbent bases (hydrophilic paraffin oil, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointments) and water-soluble bases (e.g., polyethylene glycol ointments). Pastes are generally different from ointments in that they contain a greater percentage of solids. Pastes are generally more absorbent and less greasy than ointments made with the same ingredients.

A "gel" is a semisolid system containing a dispersion of small or large molecules in a liquid vehicle that has been rendered semisolid by the action of a thickener or polymeric substance dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component, or both. Some emulsions may be gels or additionally include a gel component. However, some gels are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses such as hydroxypropyl cellulose and hydroxyethyl cellulose; carbomer homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diethylene glycol monoethyl ether, alkylene glycols such as propylene glycol; dimethyl isosorbide; and alcohols such as isopropyl alcohol and ethanol. Solvents are generally selected for their ability to dissolve the drug. Other additives may also be incorporated to improve skin feel and/or soften the formulation. Examples of such additives include, but are not limited to, isopropyl myristate, ethyl acetate, C ₁₂ -C ₁₅ alkyl benzoate, mineral oil, squalane, cyclomethicone, capric/caprylic triglyceride, and combinations thereof.

The foam is composed of an emulsion and a propellant gas. The propellant gas is mainly composed of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA227), but mixtures and blends of these and other HFAs currently approved or approved for medical use are suitable. The propellant is preferably not a hydrocarbon propellant gas that can generate flammable or explosive vapors during the spraying process. In addition, the composition preferably contains a non-volatile alcohol that can generate flammable or explosive vapors during use.

The buffer is used to control the pH of the composition. Preferably, the buffer buffers the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.

Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

In certain embodiments, it may be desirable to provide continuous delivery of one or more noscapine analogs to a patient in need thereof. For topical administration, repeated administrations may be performed, or patches may be used to provide continuous administration of noscapine analogs over an extended period of time.

E. Enteral formulations

Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be manufactured using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells that can encapsulate liquid, solid, and semisolid fill materials using techniques well known in the art.

The formulations may be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

Excipients (including plasticizers, pigments, colorants, stabilizers and glidants) can also be used to form coated compositions for enteral administration. Delayed release dosage formulations can be prepared as described in standard references, such as "Pharmaceutical dosage form tablets", Liberman et al. (New York, Marcel Decker, 1989), "Remington-The science and practice of pharmacy", 20th edition, Lippincott Williams & Wilkins, Baltimore, MD, 2000 and "Pharmaceutical dosage forms and drug delivery systems", 6th edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules, as well as delayed-release dosage forms of tablets, capsules, and granules.

Nanoparticles can be coated, for example, to delay release once the particles have passed the acidic environment of the stomach. Examples of suitable coating materials include, but are not limited to, cellulosic polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic acid resins available under the trade name CERAMIDE (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Diluents (also called "fillers") are often necessary to increase the bulk of a solid dosage form to provide a practical size for tablet compression or bead and granule formation. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, anhydrous starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, and powdered sugar.

Binders are used to impart cohesive qualities to solid dosage formulations and thereby ensure that the tablets or beads or granules remain intact after the dosage form is formed. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums (such as acacia, tragacanth), sodium alginate, celluloses (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, and veegum), and synthetic polymers (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid, and polyvinyl pyrrolidone).

Lubricants are used to facilitate tablet manufacturing. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

Disintegrants are used to promote the disintegration or "breakdown" of the dosage form after administration and typically include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums, or cross-linked polymers such as cross-linked PVP (Vitamin(R) XL from GAF Chemical Corp).

Stabilizers are used to inhibit or retard drug decomposition reactions (including, for example, oxidation reactions). Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; vitamin E, tocopherol and its salts; sulfites such as sodium metabisulfite; cysteine and its derivatives; citric acid; propyl gallate and butylated hydroxyanisole (BHA).

IV. Methods for Making MPP

Techniques for making nanoparticles are known in the art and include, but are not limited to, solvent evaporation, solvent removal, spray drying, phase inversion, cryogenic casting, and nanoprecipitation. Suitable methods for particle formulation are briefly described below. Pharmaceutically acceptable excipients (including pH adjusters, disintegrants, preservatives, and antioxidants) can optionally be incorporated into the particles during the particle formation process. As described above, one or more other active agents can also be incorporated into the nanoparticles during the particle formation process.

1. Solvent Evaporation

In this method, the polymeric components of the nanoparticle gene carrier are dissolved in a volatile organic solvent (such as dichloromethane). The organic solution containing the polymer-drug conjugate is then suspended in an aqueous solution containing a surfactant (such as poly(vinyl alcohol)). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid nanoparticles. The resulting nanoparticles are washed with water and dried overnight in a freeze dryer. Nanoparticles of varying sizes and morphologies can be obtained using this method.

2. Solvent Removal

In this method, the components of the nanoparticle gene carrier are dispersed or dissolved in a suitable solvent. This mixture is then suspended by stirring in an organic oil (such as silicone oil) to form an emulsion. Solid particles are formed from the emulsion, which can then be separated from the supernatant.

3. Spray drying

In this method, the components of the nanoparticle gene carrier are dispersed or dissolved in a suitable solvent. The solution is pumped through a micronizing nozzle driven by a stream of compressed gas, and the resulting aerosol is suspended in a heated cyclone of air, allowing the solvent to evaporate from the droplets, forming particles.

4. Phase conversion

In this method, the components of the nanoparticle gene carrier are dispersed or dissolved in a "good" solvent, and the solution is poured into a strong non-solvent for the polymeric components of the nanoparticle gene carrier, thereby spontaneously generating nanoparticles under appropriate conditions.

5. Low temperature casting

A method for casting nanoparticles at very low temperatures is described in U.S. Patent No. 5,019,400 to Gombotz et al. In this method, the components of the nanoparticle gene delivery vehicle are dispersed or dissolved in a solvent. The mixture is then atomized into a container containing a liquid nonsolvent at a temperature below the freezing point of the solution, which freezes the components of the nanoparticle gene delivery vehicle into very small droplets. As the droplets of components and the nonsolvent are warmed, the solvent in the droplets thaws and is extracted into the nonsolvent, causing the nanoparticles to harden.

6. Nanoprecipitation

In this method, a solution containing one or more nucleic acids is added dropwise to a solution containing the polymeric components of the nanoparticle gene delivery system. As the nucleic acids are complexed by the cationic polymer, nanoparticles precipitate from the solution. The resulting nanoparticles are separated from the solution, for example, by filtration or centrifugation, washed, and dried using a freeze dryer.

In a specific embodiment, the nanoparticles are prepared using emulsification in the method. Generally, the particles are prepared by an o/w single emulsion or w/o/w double emulsion method as described in R.C. Mundargi et al., J. Control Release 125, 193 (2008), M. Li et al., Int. J. Pharm. 363, 26 (2008), C.E. Astete and C.M. Sabliov, J. Biomater. Sci. Polymer ed., 17, 247 (2006), and R.A. Jain, Biomaterials, 21, 2475 (2000). In this procedure, the polymer is dissolved in an organic solvent (e.g., dichloromethane) to form an oil phase. This oil phase is added to an aqueous solution of an emulsifier, typically with probe sonication for a period of time (e.g., 2 minutes) to form an emulsion. This emulsion is then added to a larger volume of the emulsifier under magnetic stirring to evaporate the organic solvent.

The nanoparticles were collected by centrifugation (e.g., 20,000 g for 25 minutes) after filtering through a 1 μm membrane filter and washing thoroughly with water. To prepare the nanoparticles for fluorescence microscopy, a certain amount of AF555-labeled polymer was blended and then subjected to an emulsification process. In a control experiment for the nanoprecipitation method, a PLGA45k-PEG5k acetonitrile solution at a concentration of 25 mg/ml was slowly injected into DI water under magnetic stirring (700 rpm). After complete removal of the organic solvent, the nanoparticles were collected by the same procedure as described above.

The diameter (nm), polydispersity index (PDI), and surface charge (zeta potential, mV) of the nanoparticles were obtained from three replicate measurements by dynamic light scattering on a Zetasizer Nano ZS90 (Malvern Instruments, Southborough, MA). The nanoparticles were dispersed in a 10 mM NaCl solution (pH 7). The morphology of the nanoparticles was characterized by transmission electron microscopy (TEM) on an H7600TEM (Hitachi, Japan).

V. Methods of using MPP

The data described herein highlight a number of potential advantages of mucus-permeable nanoparticles prepared by emulsion methods for mucosal drug delivery applications. First, the predominantly used emulsifier, PVA, can be replaced with a low-MW emulsifier to prepare biodegradable nanoparticles with similar drug encapsulation by emulsion methods. The nanoparticles exhibit higher drug loadings, such as greater than 5% for hydrophobic drugs (e.g., curcumin) and greater than 10% for biomolecules. Surface-altering materials (e.g., PEG) can enhance the delivery of nanoparticles to sites of interest due to their formation of neutral or near-neutral surface charges that enhance in vivo transport through fluids and materials.

For example, the nanoparticles described herein rapidly penetrate the human mucus barrier, while PVA-coated nanoparticles are immobilized. Thus, the most widely used industrial production methods for controlled-release particles can be applied to manufacture MPPs for drug delivery applications.

Second, MPPs prepared by the emulsification method are expected to rapidly penetrate other mucosal surfaces, such as the eyes, nose, lungs, gastrointestinal tract, and more. CVM has similarities to other mucus fluids in chemical content and rheological properties. In fact, MPPs prepared by the emulsification method have been observed to rapidly penetrate normal airway mucus collected during surgery and sputum expectorated by cystic fibrosis (CF) patients.

Third, challenging hydrophilic drugs (including proteins, peptides, and nucleic acids) can be encapsulated into nanoparticles by emulsification. For example, vaccine antigens (such as ovalbumin and tetanus toxoid) can be formulated into biodegradable nanoparticles for vaccination, such as MPP for mucosal vaccination.

Fourth, hydrophobic drugs that are difficult to dissolve in water-miscible organic solvents can be successfully formulated into biodegradable nanoparticles (such as MPPs) through emulsification. Improved pharmacokinetics and therapeutic efficacy of hydrophobic drugs can be expected to be achieved through the delivery of nanoparticles, such as mucosal drug delivery of MPPs.

The present invention will be further understood by reference to the following non-limiting examples.

Examples

Materials and methods

Cholic acid sodium salt, 20, 80, cetyltrimethylammonium bromide (CTAB), dioctyl sodium sulfosuccinate (DSS), polyethylene glycol 35 hydrogenated castor oil (Cremophor EL), and D-α-tocopheryl polyethylene glycol 1000 (vitamin E-TPGS) were purchased from Sigma (St. Louis, MO).

Poly(vinyl alcohol) (Mw = 25 kDa, 88% hydrolyzed, and 6 kDa, 80% hydrolyzed) and poly(ethylene-maleic anhydride, 1:1 molar ratio) with a Mw of approximately 400 kDa were purchased from PolySciences (Warrington, PA).

Sugar ester D1216 (SE) was a gift from Mitsubishi-Kagaku Foods Co. (Tokyo, Japan).

Alexa Fluor 555 cadaverine was purchased from Invitrogen (Grand Island, NY).

Poly(lactic-co-glycolic acid) (PLGA; LA:GA50:50) (MW approximately 15 kDa) with an intrinsic viscosity of 0.15-0.25 dL/g was purchased from Lakeshore Biomaterials (Birmingham, AL). PLGA(LA:GA50:50)-PEG copolymers, PLA-PEG5k, and PCL-PEG5k with PEG MWs of 10, 5, 2, and 1 kDa were custom synthesized by Jinan Daigang Biomaterial Co., Ltd. (Jinan, China) and characterized by ¹ H NMR and gel permeation chromatography (GPC). A Shimadzu instrument equipped with a refractive index detector and two Waters HR4 and HR5 columns was used. Analyses were performed at 35° C. using tetrahydrofuran (THF) as the eluent at a flow rate of 0.5 mL/min. The GPC was calibrated with polystyrene standards (Sigma, St. Louis, MO).

The chemical composition and molecular weight (MW) of the PLGA-PEG block copolymer were characterized by ¹ H NMR. The polymer was dissolved in CDCl ₃ and recorded at 400 MHz using a Bruker 400 REM instrument. ^{The 1 H} NMR spectrum of the copolymer in CDCl ₃ is shown in FIG3 . The peaks of CH (5.22 ppm) from the LA unit, CH ₂ (4.83 ppm) from the GA unit, and CH ₂ CH ₂ (3.65 ppm) from the ethylene oxide unit were integrated, with 15.22, 14.83, and 13.65 being the integrated intensities of the peaks at 5.22, 4.83, and 3.65 ppm, respectively. The ratio of LA:GA was estimated to be 1: _5.22 :(1: _4.83 /2).

The MW of PLGA-PEG was estimated as follows:

(I _3.65 /4)/(I _4.83 /2)-(MW _PEG /44)/(MW _GA /58)

(I _3.65 /4)/(I _5.22 /1)-(MW _PEG /44)/(MW _LA /72)

MW _PLGA-PEG = MW _PEG + (MW _GA + MW _LA ), where MW _PEG is 1, 2, 5, and 10 kDa.

Similarly, the molecular weights of PLA-PEG and PCL-PEG were estimated as follows:

(I _3.65 /4)/(I _5.22 /l)＝(MW _PEG /44)/(MW _LA /72)

MW _PLA-PEG =MW _PEG +MW _LA ;

(I _3.65 /4)/((I _4.06 +I _2.31 )/4)＝(MW _PEG /44)/(MW _CL /114)

MW _PCL-PEG =MW _PEG +MW _CL ),

where the MW _PEG is 5 kDa, I _4.06 and I _2.31 are the integrated intensities of the peaks from PCL at 4.06 and 2.31 ppm, respectively.

The characteristics of various PEG-containing block copolymers are shown in Table 1.

Table 1: Characteristics of PEG-containing block copolymers

^[a] The molar ratio of LA:GA was measured by comparing ^{the 1} H NMR integrated intensities at 5.22 ppm (-CH- on lactide), 1.59 ppm (-CH ₃ on lactide), and 4.83 ppm (-CH ₂ - on glycolide).

^[b] The PEG content in the block copolymer was determined by ¹ H NMR.

^[c] PLGA-PEG molecular weight (Mn) was determined using ¹ H NMR by comparing the integrals at 5.22 ppm (-CH- in lactide), 1.59 ppm (-CH ₃ on lactide), 4.83 ppm (-CH ₂ - in glycolide), and 3.65 ppm (-CH ₂ CH ₂ - in PEG) and taking into account the known Mn of PEG. For PCL-PEG, the integrals at 4.06 ppm (-O-CH ₂ -) and 2.31 ppm (-CH ₂ -CO-) were analyzed.

^[d] Mn, Mw and polydispersity (PDI) were measured using GPC.

^[e] PLGA-PEG10 kDa nanoparticles were prepared by blending PLGA15 kDa with PLGA-PEG10 kDa (21.6% PEG content), where the total PEG content in the nanoparticles was 6 wt%.

The total PEG content within the nanoparticles was determined using a Bruker 400 REM instrument at 400 mHz using 1H NMR. Lyophilized nanoparticles were accurately weighed and dissolved in _CDCl containing 1 wt% hexadeuterated dimethyl sulfoxide (TMS) as an internal standard. The PEG content was determined by comparison with a PEG 5 kDa calibration curve obtained from 1H NMR spectroscopy using TMS as an internal standard.

Tracking of fluorescently labeled nanoparticles in fresh human cervicovaginal mucus (CVM) was performed as previously described. 39-40 Briefly, 0.6 μl of nanoparticles at the appropriate dilution were mixed into 20 μl of mucus and incubated for 1 hour before microscopic examination. Movies were captured with a silicon-intensified objective camera (VE-1000, Dage-MTI) at a temporal resolution of 66.7 ms, mounted on an inverted epifluorescence microscope equipped with a 100× oil-immersion objective. Trajectories of n>150 particles per experiment were extracted using MetaMorph software (Universal Imaging). Tracking movies (20 s) were analyzed using metamorph software (Universal Imaging, Glendale, WI). The time-averaged mean square displacement (MSD) and effective diffusion rate of each particle were calculated as a function of the time scale. Three experiments were performed for each condition. One-sided, unequal variance Student's t-test was used to assess significance (P < 0.05).

ITC experiments were performed at 25°C using a VP-ITC microcalorimeter (MicroCal Inc., USA). The experiments were performed as follows: a 2 mg/ml solution of mucin in DI water was injected at a concentration of 1 mg/ml in water at a stirring speed of 481 rpm into a 2 mL sample cell containing nanoparticles with varying PEG surface densities. A total of 28 injections were performed at an interval of s and a reference power of μcal/s. A 2 μl injection of mucin solution was performed first, followed by 27 injections of 10 μl of mucin solution. Binding isotherms were plotted and analyzed using Origin software, where the ITC measurements were fitted to a single-site binding model. The stoichiometry was applied to calculate the bound content of mucin on the nanoparticle surface, expressed as mg mucin/m².

Example 1. Preparation of nanoparticles

Materials and methods

Biodegradable nanoparticles were prepared by an o/w single emulsion or w/o/w double emulsion method as described in R.C. Mundargi et al., J. Control Release 125, 193 (2008), M. Li et al., Int. J. Pharm. 363, 26 (2008), C.E. Astete and C.M. Sabliov, J. Biomater. Sci. Polymer ed., 17, 247 (2006), and R.A. Jain, Biomaterials, 21, 2475 (2000).

The nanoparticles were characterized for size, surface properties, and drug loading (for drug-encapsulated nanoparticles).The displacement of the nanoparticles was tracked in fresh, undiluted human CVM using multiple particle tracking.

Nanoparticles prepared with different amounts of PEG

PLGA-PEG nanoparticles were prepared using emulsification with different target PEG contents (0 wt%, 2 wt%, 3 wt%, 5 wt%, 8 wt%, 10 wt%, and 25 wt%, referred to as PLGA, PLGA-PEG2%, PLGA-PEG3%, PLGA-PEG5%, PLGA-PEG8%, PLGA-PEG10%, and PLGA-PEG25%). A PEG molecular weight of 5 kDa was chosen because, at the same PEG content, 6 wt% PLGA-PEG nanoparticles with PEG ranging from 1 kDa to 10 kDa rapidly penetrated mucus. The target PEG content was controlled by varying the ratio of PLGA to PLGA-PEG during nanoparticle preparation. The nanoparticle size was controlled to approximately 100 nm by adjusting the polymer concentration and emulsification procedure, and all nanoparticles exhibited monodisperse diameters with a low polydispersity index (less than 0.1) under dynamic light scattering. Based on TEM studies, the nanoparticles were spherical, and the PLGA-PEG25% nanoparticles with the highest target PEG content exhibited less contrast at the particle boundaries, which may be caused by the high content of lower electron density PEG located on the surface.

result

Table 2 shows the characteristics of the particles prepared as described above.

Table 2: Nanoparticle characteristics

^[a] The diameter and polydispersity index (PDI) of the nanoparticles were measured by dynamic laser light scattering.

^[b] The transport rate can also be reflected by the slope of the double logarithmic MSD versus time scale plot (α = 1 indicates unhindered Brownian transport, while smaller α reflects increased resistance to particle movement).

^[c] The ratio of the bulk mean diffusion coefficient of nanoparticles in mucus (D _m ) compared to that in water (D _w ) and the effective diffusivity values were calculated on a time scale of 1 s.

Data are mean ± SD.

Increasing target PEG content causes nanoparticle surface charge to substantially reduce (Table 2), and when PEG content reaches 8 wt % and 8 wt % or more, obtains almost neutral surface charge (about 4mV).The surface charge that reduces reflects that surface PEG coverage increases, because dense PEG coating can effectively shield the surface charge of nanoparticles.However, surface charge (ζ-potential) measurement value can not provide the quantitative information for assessing PEG surface density aspect PEG chain number on particle surface.In addition, surface charge measurement value can be affected by core material and measurement medium.

^1H NMR was used to directly quantify the PEG surface density on the nanoparticles. As shown in Table 3, the surface PEG content on the nanoparticles increased with increasing target PEG content. Table 3 shows the PEG surface density of PLGA-PEG nanoparticles with varying PEG contents. Surface PEG levels were measured by ^1H NMR in D ₂ O compared to standard DSS (1 wt %). The total PEG content in the nanoparticles was measured by ^1H NMR in CDCl ₃ compared to standard TMS (1 wt %). N/A, not applicable.

Table 3: Surface PEG content on nanoparticles

^[a] PEG density [Γ] means the number of PEG molecules per 100 square nanometers calculated by assuming that all PEG chains on the surface are full-length PEG 5 kDa.

^[b] PEG density/full surface coverage [Γ/Γ*]. Full mushroom coverage [Γ*] refers to the number of unconstrained PEG molecules per 100 square nanometers. (Values <1 indicate mushroom coverage with low PEG density, while >1 indicates a brush state; values >>1 indicate a dense brush state with very high PEG density).

The data (mean ± SD) are the average of at least three different batches of samples.

Example 2: Nanoparticles prepared with different emulsifiers

Materials and methods

Alexa Fluor 555 cadaverine (AF555) was chemically bound to the polymer. Nanoparticles were prepared using emulsification. Typically, a mixture of PLGA-PEG5k and AF555-labeled PLGA-PEG5k (50 mg total) was dissolved in 1 mL of dichloromethane (DCM). The oil phase was poured into 5 mL of an aqueous solution containing 1% emulsifier under sonication (VibraCell, Sonics & Materials Inc., Newtown, CT) in an ice-water bath at 30% amplitude for 2 minutes to form an oil-in-water emulsion.

The emulsion was poured into another 40 mL aqueous phase of the emulsifier solution under magnetic stirring at 700 rpm for at least 3 hours to allow the solvent to evaporate. The solvent was further evaporated by placing the solution in a vacuum chamber for 30 minutes. The final nanoparticle suspension was filtered through a 1 μm syringe filter, centrifuged at 20,000 g for 25 minutes, and washed thoroughly with water.

Emulsifiers tested at a concentration of 1% w/v included cholic acid sodium salt (CHA), dioctyl sodium sulfosuccinate (DSS), cetyltrimethylammonium bromide (CTAB), polyvinyl alcohol (PVA), poly(ethylene-maleic anhydride) (PEMA), saponin, TWEEN 20, TWEEN 80, and the sugar ester D1216 (SE). Solutions of CHA at 0.01% to 0.5% w/v also successfully produced nanoparticles. Solutions of F127 and F68, as well as other low MW emulsifiers (such as Cremophor EL and vitamin E TPGS), were also tested, but unstable emulsions produced large, aggregated particles.

Table 4 shows the characteristics of nanoparticles prepared using PLGA-PEG (Mn approximately 83 kDa) and PLGA (Mn approximately 15 kDa) and various emulsifiers (1% w/v).

Table 4. Characteristics of biodegradable nanoparticles prepared by emulsification using PLGA-PEG5k (Mn approximately 83 kDa) and PLGA (Mn approximately 15 kDa) and representative emulsifiers (1% w/v).

^[a] Ratio of bulk mean diffusion coefficients in water ( _Dw ) compared to mucus ( _Dm ) at a time scale of 1 s.

To evaluate the effect of polyethylene glycol molecular weight (PEG MW) on the mucus penetration properties of nanoparticles prepared by emulsification, CHA was selected as a representative low MW strong emulsifier. PLGA-PEG nanoparticles with different PEG MW at approximately 6 wt% PEG content were prepared in 0.5% CHA solution. To achieve an overall 6 wt% PEG content for PLGA-PEG10k nanoparticles, a blend of PLGA-PEG10k (21.6 wt%) and PLGA15k was used.

result

The properties of nanoparticles prepared from various molecular weights of PEG (approximately 6 wt% PEG content) are shown in Table 5.

Table 5. Characteristics of biodegradable nanoparticles (approximately 6 wt% PEG content) prepared by the emulsification method using PEG of various MW.

^[a] PEG density [Γ] indicates the number of PEG molecules per 100 nm2. The surface PEG content was quantified by ^1H NMR of the nanoparticles in _D2O .

^[b] Ratio of PEG density to complete surface coverage [Γ/Γ*]. Complete surface coverage [Γ*] indicates the theoretical number of untethered PEG molecules required to completely coat a 100 nm surface. ([Γ/Γ*] < 1 indicates a mushroom state with low surface PEG density, while > 1 indicates a brush state with high surface PEG density)

Table 6 shows the properties of nanoparticles prepared from various concentrations of CHA and PLGA-PEG5k containing 6 wt% PEG.

Table 6: Characterization of biodegradable nanoparticles prepared by emulsification method using different concentrations of emulsifier (CHA) PLGA-PEG5k containing 6 wt% PEG was used.

Example 3: Preparation of drug-encapsulated nanoparticles

Materials and methods

Curcumin was chosen as a model hydrophobic drug and dissolved in DCM along with the polymer. The procedure was similar to that used to prepare unloaded nanoparticles. The prepared curcumin-nanoparticles were visible in mucus due to the intrinsic fluorescence of curcumin.

BSA is used as a model hydrophilic drug because it represents macromolecular biology. BSA-FITC and BSA (10% ratio of BSA-FITC) are dissolved in 0.2mL 16% w/v aqueous solution at 37°C. This solution is added to 1mL of a DCM solution containing 100mg/ml PLGA-PEG5k in an ice-water bath during probe sonication (30% amplitude, 1min, wherein the pulse is 1s). The resulting W/O main emulsion is immediately added to a second aqueous phase (5mL 1% saponin solution) under sonication (20% amplitude, continuing 2min). The double emulsion is transferred to another 40mL 1% saponin solution under magnetic stirring for 3 hours. The nanoparticles are filtered through a 1μm syringe filter, washed and collected by centrifugation. BSA-FITC allows the possibility of tracking nanoparticles loaded with BSA in mucus.

result

The target drug loadings of curcumin nanoparticles and BSA nanoparticles were 9.1% and 16.7%, respectively.

Example 4. Estimation of emulsification capacity

Materials and methods

PLGA-PEG5k (MW approximately 83kDa) was used as a model polymer and dissolved in DCM at 50mg/ml. 0.5ml of a solution of PLGA-PEG5k in DCM was added to 5ml of an aqueous phase containing 1% (w/v) emulsifier under sonication at 30% amplitude to prepare an emulsion using the same method described above. The formed emulsion was added to another 20ml 1% emulsifier solution under magnetic stirring at 700rpm for 3 hours. The emulsifying ability of each emulsifier was estimated by its ability to prevent the formation of aggregated particles. Aggregated particles were collected by centrifugation at 500g for 20min, and the remaining nanoparticles in the supernatant were collected by centrifugation at 30,000g for 25min. The weight ratio of nanoparticles to aggregated particles was calculated and used as an index to estimate the emulsifying ability of the emulsifier.

Diameter and surface charge

The diameter and ζ-potential (surface charge) of the nanoparticles were measured using a Zetasizer Nano ZS90. The nanoparticles were resuspended in a 10 mM NaCl solution. TEM samples were prepared by dropping a dilute suspension of the nanoparticles onto a TEM grid and allowing it to air dry. Particle morphology was characterized using an H7600 transmission electron microscope (Hitachi, Japan).

Encapsulation efficiency

The encapsulation efficiency of curcumin in nanoparticles was measured by dissolving the lyophilized nanoparticles in DMSO and measuring the absorbance at 430 nm using a Biotek Synergy MX plate reader. The drug content was determined by comparing with a curcumin calibration curve (concentration range 0-50 μg/ml). The absorbance of blank nanoparticles in DMSO at the same polymer concentration was subtracted. The encapsulation efficiency of BSA-FITC was analyzed after alkaline digestion. A known amount of lyophilized nanoparticles was completely hydrolyzed in 1 M sodium hydroxide. The resulting solution was analyzed using a Biotek Synergy MX plate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. Standard solutions containing the same amount of polymer and increasing amounts of BSA-FITC were prepared under the same processing conditions. The amount of BSA in the nanoparticles was determined by comparing with the BSA-FITC calibration curve.

The drug loading (DL) and encapsulation efficiency (EE) were calculated as follows:

result

The results for various emulsifiers are shown in Table 7.

Table 7: Encapsulation of a model hydrophobic drug (curcumin) and a model hydrophilic drug (BSA) using PLGA-PEG5k (6 wt% PEG) in both MPP (CHA and saponin as emulsifiers) and CP (PVA as emulsifier)

^[a] Drug loading (DL%) represents the weight content of the drug in the nanoparticles.

^[b] Drug encapsulation efficiency (EE%) represents the ratio of the final drug loading compared to the theoretical drug loading.

Quantification of surface polyethylene glycol (PEG) density

The surface PEG density on the nanoparticles was determined by 1H NMR using a Bruker 400 REM instrument at 400 MHz. The relaxation time was set at 10 s and the ZG was at 90°. Nanoparticles with different PEG contents were directly prepared in 0.5% CHA D ₂ O solution and suspended in D ₂ O with 1 wt % 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt as an internal standard for ^1H NMR analysis.

A calibration curve for the PEG signal in ¹ H NMR was established by serially diluting known weights of PEG 5 kDa (Sigma, St. Louis, MO) homopolymer into different concentrations in D ₂ O with 1% 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt, and this calibration curve was used to calculate the surface PEG content on the nanoparticles.

0.2 ml of a solution of nanoparticles in D ₂ O was lyophilized and weighed. The surface PEG density was calculated as the number of PEG molecules per 100 square nanometers of surface area on the nanoparticles by assuming that all surface PEG chains were full-length PEG 5kDa. A control ¹ H NMR experiment was also performed with PLGA nanoparticles prepared by the same method, and there was no detectable CHA peak for the PLGA nanoparticles. The PEG density [Γ] is the number of PEG molecules per 100 square nanometers on the nanoparticle surface. It can be calculated by dividing the total PEG content (MPEG, in moles) detected by ¹ H NMR by the total surface area of all nanoparticles as follows:

Where W _NP is the total mass of the nanoparticles, d _NP is the density of the nanoparticles (the density of the nanoparticles was assumed to be equal to that of the polymer, for PLGA 1.21 g/ml), and D is the particle size as measured using dynamic light scattering.

The total surface mushroom coverage [Γ*] is the number of unconstrained PEG molecules occupying 100 ^nm2 of the particle surface area. To determine [Γ*], the surface area occupied by a single PEG chain was estimated. Using random walk statistics, the area occupied by a single PEG chain at the interface given by a sphere of diameter ζ is:

Where m is the molecular weight of the PEG chain. The surface area occupied by one PEG molecule can be determined by (ζ/ ² ). PEG5kDa has an unconstrained molecular sphere with a diameter of 5.4nm and occupies a surface area of ^22.7nm . Therefore, the number of PEG molecules [Γ*] that completely covers a surface area of ^100nm is 4.4.

[Γ/Γ*] can be used as an index to measure the PEG density on the nanoparticle surface, where a value <1 indicates a low PEG density, where the PEG molecules are in a mushroom conformation; and a value >1 indicates a high PEG density, where the PEG molecules are in a brush-like conformation. Similarly, [Γ*] for PEG 10 kDa, 2 kDa, and 1 kDa were 2.2, 11, and 22, respectively. The results are shown in Table 2 above.

Table 8 shows the PEG surface density of PLGA-PEG nanoparticles with different PEG contents. Surface PEG levels were determined by 1H NMR in D2O compared to standard DSS (1 wt%). Total PEG content in the nanoparticles was measured by 1H NMR in CDCl3 compared to standard TMS (1 wt%). N/A, not applicable.

Table 8. PEG surface density of PLGA-PEG nanoparticles with different PEG contents

^[a] PEG density [Γ] means the number of PEG molecules per 100 square nanometers calculated by assuming that all PEG chains on the surface are full-length PEG 5 kDa.

^[b] PEG density/full surface coverage [Γ/Γ*]. Full mushroom coverage [Γ*] refers to the number of unconstrained PEG molecules per 100 square nanometers. (<1 indicates mushroom coverage with low PEG density, while >1 indicates a brush state; values >>1 indicate a dense brush state with very high PEG density).

The data (mean ± SD) are the average of at least three different batches of samples.

The surface PEG density ([Γ, the number of PEG chains per 100 square nanometers) was calculated and compared to the complete surface mushroom coverage ([Γ*], the number of untethered PEG molecules per 100 square nanometers). PLGA-PEG3% nanoparticles exhibited a surface PEG content of 2.6 wt%, with a density of 6.5 PEGs/100 square nanometers, equivalent to [Γ]/[Γ*]=1.5, which gives PLGA-PEG3% a surface PEG coating with a brush conformation. Highly dense PEG coatings with a brush conformation ([Γ]/[Γ*]>3) were obtained at PEG surface densities above 10 PEGs/100 square nanometers (PLGA-PEG5%).

By dissolving lyophilized PLGA-PEG nanoparticles in the NMR solvent CDCl ₃ , the total PEG content within the nanoparticles was measured using ¹ H NMR, and it was found that the total PEG content in the nanoparticles (both surface PEG and PEG embedded in the nanoparticle core) was extremely close to the surface PEG content, as shown in Table 5. Almost all of the PEG chains in the PLGA-PEG nanoparticles prepared by the emulsion method were detected on the particle surface. The emulsion method involves evaporating the organic solvent (dichloromethane) from the emulsion droplets and then solidifying the polymer core. The slow evaporation of the organic solvent provides sufficient time for the hydrophilic PEG chains to diffuse and assemble on the nanoparticle surface, which results in a high distribution ratio of PEG to the surface. However, there is a significant loss of PEG in the process of preparing nanoparticles using the emulsion method, and for PLGA-PEG25% nanoparticles, the PEG loss ratio can be as high as 50%.

Similar to the previous reports, the loss of PEG can be attributed to the formation of micelles by the low molecular weight portion of PLGA-PEG in the copolymer, which has a higher PEG content and higher hydrophilicity. This fraction of very small particles containing polymers with a higher PEG content could not be collected after the centrifugation and washing steps, as confirmed by the increased average molecular weight of the polymer measured by gel permeation chromatography after nanoparticle formation compared to the base polymer. In a control experiment, PLGA-PEG10% nanoparticles (117 nm) prepared by the nanoprecipitation method (solvent diffusion method) showed a total PEG content of 6.5% by weight in the nanoparticles, and only 89% of PEG chains were detected on the surface (equivalent to a surface PEG content of 5.8% by weight).

Example 5. Mucus penetration tracking of nanoparticles

Materials and methods

Human cervicovaginal mucus (CVM) was collected. Briefly, undiluted cervicovaginal secretions with normal vaginal flora were obtained from women using a self-sampling menstrual collection device according to a protocol approved by the Institutional Review Board of the Johns Hopkins University. The device was inserted into the vagina for 60 seconds, removed and placed in a 50 ml centrifuge tube, and centrifuged at 1000 rpm for 2 minutes to collect the secretions.

Tracking of fluorescently labeled nanoparticles in fresh human cervicovaginal mucus (CVM) was performed. Briefly, 0.6 μl of nanoparticles at the appropriate dilution were added to 20 μl of mucus in a custom chamber slide and incubated for 1 hour at room temperature before microscopy. The trajectories of the nanoparticles in CVM were recorded using multiple particle tracking (MPT). 20 s movies were captured with a time resolution of 66.7 ms using a silicon-enhanced objective camera (VE-1000, Dage-MTI) mounted on an inverted epifluorescence microscope equipped with a 100× oil immersion objective (N.A., 1.3). Tracking movies (20 s) were analyzed using metamorph software (Universal Imaging, Glendale, Wisconsin).

The time-averaged mean square displacement (MSD) and effective diffusivity of each particle are calculated as a function of the time scale:

<Δr2(τ)>=[x(t+τ)-x(t)] ² +[y(t+τ)-y(t)] ²

where x and y represent the coordinates of the nanoparticle as a function of time, and τ is the time lag.

Curcumin-loaded nanoparticles and FITC-BSA-loaded nanoparticles were tracked in human CVM in the same manner using fluorescence from encapsulated curcumin or BSA-FITC. Particle penetration into the mucus layer was modeled using Fick's second law and diffusion coefficients obtained from tracking experiments.

result

A comparison of human CVM transport of PLA-PEG and PCL-PEG nanoparticles containing CHA and PVA prepared by emulsification is shown in Figures 1a-h. Figures 1a and 1b show representative trajectories of PLA-PEG and PCL-PEG nanoparticles containing CHA and PVA. Figures 1e and 1d are graphs showing the overall average geometric mean square displacement (MSD) over time. Figures 1e and 1f are graphs showing the logarithmic distribution of the effective diffusivity (Deff) of individual particles on a time scale of 1 s. Figures 1g and 1h are graphs showing the estimated fraction of particles capable of penetrating a physiological 30 μm thick mucus layer over time. Data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as s.e.m. This data demonstrates the immobilization of nanoparticles prepared using PVA and the rapid mucus penetration of nanoparticles prepared using the low-MW emulsifier CHA, with effective diffusivities similar to those measured for PLGA-PEG5k nanoparticles.

The effect of PEG molecular weight on the transport rate of MPP in CVM is shown in Figures 2a and 2b. Figures 2a and 2b show the effect of PEG MW on the transport rate of MPP in human cervicovaginal mucus. Figure 2a is a graph showing the overall mean geometric mean square displacement <MSD> as a function of time scale. Figure 2b is a graph showing the logarithmic distribution of the effective diffusivity (Deff) of individual particles at a time scale of 1 s. The particles were prepared using an emulsion method using PLGA-PEG (6 wt% PEG). The data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as s.e.m. The particles all rapidly penetrated the mucus (see also Table 5).

The surface charge of the nanoparticles was inversely proportional to the PEG MW and varied between -18 mV (1 kDa) and -2.3 mV (10 kDa). The surface PEG density [Γ] (number of PEG per 100 square nanometers) measured by 1H NMR decreased with increasing PEG MW. However, the ratio of the surface PEG density to the theoretical PEG density [Γ*] required to form a brush-like PEG coating [Γ/Γ*] [11] was greater than 2 (Table 5), regardless of PEG MW, indicating the presence of a dense, brush-like PEG coating on the surface of the PLGA-PEG (1-10 kDa)/CHA nanoparticles.

Figures 3a-3c show the transport rates of MPPs and conventional particles (CPs) loaded with curcumin and BSA. Figure 3a is a graph showing the overall mean geometric mean square displacement (MSD) as a function of time. Figure 3b is a graph showing the logarithmic distribution of the effective diffusivity (Deff) of individual particles at a time scale of 1 s. Figure 3c is a graph showing the estimated fraction of particles predicted to be able to penetrate a 30 μm thick mucus layer over time. The data are representative of three independent experiments, with ≥120 nanoparticles tracked for each experiment. Error bars are presented as s.e.m. Nanoparticles loaded with curcumin and BSA diffused rapidly in mucus at rates only 6 and 36 times slower than in water at τ = 1 s (Figure 3a). In contrast, nanoparticles prepared with PVA were immobilized in CVM (Figure 3b), where the transport rate was over 2,000 times slower than in water.

PLGA nanoparticles without a PEG coating were completely immobilized within mucus, where their diffusion rate was 38,000 times slower than that of nanoparticles of the same size in water. The presence of a PEG surface coating on the nanoparticles significantly improved their diffusion through the highly viscoelastic mucus, with PLGA-PEG 3% at a surface PEG density of 6.5 PEG/100 nm² exhibiting increased Dw/Dm values of up to 142. Further increasing the surface PEG density to 10.4 PEG/100 nm², PLGA-PEG 5% nanoparticles diffused only 17 times slower than in water. When the surface PEG density was above 16.4 PEG/100 nm² (PLGA-PEG 8%), over 90% of the nanoparticles diffused. Further increases in the surface PEG density likely would not significantly improve particle diffusion within mucus, as a surface density of 16.4 PEG/100 nm² effectively shields the binding of mucus components. About 50%-70% of the nanoparticles of PLGA-PEG 8%, 10% and 25% were able to penetrate the physiological 30 μm thick mucus layer within 60 minutes, which was much higher than PLGA-PEG 5%, PLGA-PEG 3% (dense coating), PLGA-PEG 2% (low coating) and PLGA (no coating).

Example 6: Stability of nanoparticles in mucus.

Materials and methods

Stabilizing nanoparticles in mucus by minimizing adhesive interactions between the particles and mucus components is a key criterion for their in vivo application as mucus-penetrating drug carriers. The stability of nanoparticles with varying PEG surface densities in the presence of mucin was determined by studying changes in nanoparticle size, an indicator of mucin binding. Mucin extracted from the bovine submandibular gland was chosen as a model mucin because it is a major component of mucus and shares structural and physiological properties with human CVM.

Nanoparticles were incubated with mucin solution (10 mg/ml), and the changes in particle size over time were monitored.

result

PLGA-PEG nanoparticles with a PEG surface density of ≥16.4 PEG/100 nm² were stable in mucin solution, maintaining their hydrodynamic diameter throughout the 3-hour incubation, and at these PEG surface densities, the PEG coating exhibited a highly dense brush conformation ([Γ]/[Γ*]>3). In contrast, PLGA-PEG5% nanoparticles with a surface density of 6.5 PEG/100 nm² exhibited an approximately 5% increase in particle size even after just 5 minutes of incubation with mucin solution, and the PEG surface density on these PLGA-PEG5% nanoparticles already produced a brush PEG coating ([Γ]/[Γ*]>1). Thus, a brush PEG coating alone is insufficient to completely shield mucin binding. There was a progressive increase in particle size at decreasing PEG surface density from a brush conformation to a mushroom conformation. Without a PEG coating, PLGA nanoparticles exhibited a significant size increase from 109±2 nm to 207±9 nm within 5 minutes of incubation in mucin.

Figure 4a is a schematic diagram illustrating the effect of surface PEG coverage ([Γ/Γ*]) on mucus penetration of nanoparticles. The top panel shows PLGA-PEG nanoparticles prepared with surface PEG coatings at increasing coverage. As surface PEG coverage increases, the PEG state changes from a mushroom (adjacent PEG chains do not overlap, [Γ/Γ*] < 1, Figure 4a) to a brush (adjacent PEG chains overlap, 1 < [Γ/Γ*] < 3, Figure 4b) to a dense brush ([Γ/Γ*] > 3, Figure 4c). The middle panel illustrates how PEG coverage determines the adhesive-adhesive interactions after mucus exposure. At low PEG coverage ([Γ/Γ*] < 1), mucin fibers strongly adhere to the nanoparticle core. At intermediate PEG coverage (1 < [Γ/Γ*] < 3), mucin fibers can still be partially absorbed into the nanoparticle core. At high PEG coverage ([Γ/Γ*] > 3), the nanoparticle core is completely shielded by the bioinert PEG corona, resulting in no mucin absorption into the nanoparticle. The lower panel shows that nanoparticles with low PEG coverage are immobilized in mucus, nanoparticles with medium PEG coverage are hampered or even immobilized in mucus, and nanoparticles with high and very high PEG coverage are able to rapidly penetrate mucus.

Claims (32)

1. A nanoparticle formed from an emulsion of one or more core polymers, one or more therapeutic agents, preventive agents, or diagnostic agents, one or more surface-modifying materials comprising polyethylene glycol, and one or more low-molecular-weight emulsifiers under stirring for a sufficient time, wherein the stirring step allows solvent evaporation and allows the surface-modifying material to diffuse and assemble on the surface of the nanoparticle. The molecular weight of the one or more low molecular weight emulsifiers is less than 1500 amu; When the nanoparticles are dispersed in a 10mM sodium chloride solution with a pH of 7, the ξ-potential of the nanoparticles is between 10mV and -10mV. The surface-modifying material containing polyethylene glycol has a surface density/total surface coverage [Γ/Γ*] greater than 3 and is configured with an extended brush; and The nanoparticles penetrate the cervical and vaginal mucus at an effective rate less than 10 times slower than the same nanoparticles in water. 2. The nanoparticles of claim 1, wherein the surface-modifying material comprising polyethylene glycol is a polyethylene oxide block copolymer. 3. The nanoparticles of claim 1, wherein the core polymer and the surface-modifying material are different components. 4. The nanoparticles of claim 1, wherein the surface-modifying material is covalently bonded to the core polymer. 5. The nanoparticles of claim 3, wherein the core polymer is a block copolymer containing one or more blocks of the surface-modifying material. 6. The nanoparticles of claim 4, wherein the core polymer comprises a single block of the surface-modifying material covalently bonded to one end of the core polymer. 7. The nanoparticles of claim 1, wherein the core polymer further comprises one or more polymers not covalently bonded to the surface-modifying material. 8. The nanoparticles of claim 7, wherein the one or more polymers not covalently bonded to the surface-modifying material have the same chemical composition as the one or more core polymers. 9. The nanoparticles of claim 1, wherein the surface-modifying material is polyethylene glycol. 10. The nanoparticles of claim 9, wherein the molecular weight of the polyethylene glycol is from 1 kD to 10 kD. 11. The nanoparticles of claim 9, wherein the molecular weight of the polyethylene glycol is from 1 kD to 5 kD. 12. The nanoparticles of claim 9, wherein the molecular weight of the polyethylene glycol is 5 kDa. 13. The nanoparticles of claim 9 or 10, wherein the density of polyethylene glycol is 0.1 to 100 chains/100 square nanometers when measured by ^1H NMR. 14. The nanoparticles of claim 9 or 10, wherein the density of polyethylene glycol is 1 to 50 chains/100 square nanometers when measured using ^1H NMR. 15. The nanoparticles of claim 9, wherein the density of polyethylene glycol is 5 to 50 chains/100 square nanometers when measured by ^1H NMR. 16. The nanoparticles of claim 9, wherein the density of polyethylene glycol is 5 to 25 chains/100 square nanometers when measured by ^1H NMR. 17. The nanoparticles of claim 1, wherein the molecular weight of the one or more emulsifiers is less than 1300 amu. 18. The nanoparticles of claim 1, wherein the molecular weight of the one or more emulsifiers is less than 500 amu. 19. The nanoparticles of claim 17, wherein the one or more emulsifiers are uncharged, positively charged, negatively charged, or a combination thereof. 20. The nanoparticles of claim 19, wherein the one or more emulsifiers are selected from the group consisting of: sodium cholate, sodium dioctyl sulfosuccinate, hexadecyltrimethylammonium bromide, saponins, TWEEN 20, TWEEN 80, and glycolipids. 21. The nanoparticles of claim 1, wherein the one or more emulsifiers have an emulsifying capacity of at least 50%. 22. A pharmaceutical composition comprising the nanoparticles as described in claim 1 and one or more pharmaceutically acceptable carriers. 23. The use of an effective amount of the nanoparticles as described in claim 1 in the preparation of a medicament for administering one or more therapeutic agents, preventive agents and/or diagnostic agents to patients in need. 24. The application of claim 23, wherein the particles are delivered intraintestinally. 25. The application of claim 23, wherein the nanoparticles are administered extracorporeally via the gastrointestinal tract. 26. The application of claim 23, wherein the nanoparticles are administered via intravenous, subcutaneous, intramuscular, or intraperitoneal injection. 27. The application of claim 25, wherein the nanoparticles are delivered via subconjunctival delivery. 28. The application of claim 23, wherein the nanoparticles are locally delivered. 29. The application of claim 28, wherein the nanoparticles are locally applied to the eye or a compartment thereof. 30. The application of claim 28, wherein the nanoparticles are delivered into the lung airway via intranasal, intravaginal, rectal, or buccal delivery. 31. A method for manufacturing nanoparticles as claimed in claim 1, the method comprising dissolving one or more core polymers in an organic solvent, adding the solution of the one or more core polymers to an aqueous solution or suspension of an emulsifier to form an emulsion, wherein the one or more surface-modifying materials comprising polyethylene glycol and one or more therapeutic agents, preventive agents, or diagnostic agents are dissolved in the organic solvent or the aqueous solution; and The emulsion is added to a second solution or suspension of the emulsifier while being stirred for a sufficient time to achieve the formation of the nanoparticles, wherein the stirring step allows the organic solvent to evaporate and allows the surface-modifying material to diffuse and assemble on the surface of the nanoparticles. 32. A nanoparticle prepared by the method of claim 31.