HK1215380B - Nanoparticle formulations with enhanced mucosal penetration - Google Patents

Description

Nanoparticle formulations for enhanced mucosal penetration

Technical Field

The present invention is in the field of nanoparticle formulations, particularly hypotonic nanoparticle formulations that rapidly deliver mucus-penetrating nanoparticles to mucosal-lined epithelial surfaces and methods of making and using the same.

Government rights

The U.S. Government has certain rights in this invention. This work was supported by National Institutes of Health Grants R01HD062844, R33AI079740, R01CA140746 (J.H. and R.C.), the National Science Foundation (L.M.E.), and NIH/Microbicide Innovation Program 5R21AI079740.

Priority claim

U.S.S.N. 61/588,350, filed January 19, 2012

PCT/US2012/024344, filed February 8, 2012

PCT/US2012/069882, filed December 14, 2012

Background Art

Local delivery of therapeutic agents via biodegradable nanoparticles generally 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 has been limited by the presence of a protective mucus layer.

Mucus is a viscoelastic gel that coats all exposed epithelial surfaces not covered by skin, such as the respiratory, gastrointestinal, nasopharyngeal, and female reproductive tracts and the surface of the eye. Mucus effectively entraps conventional particulate drug delivery systems via steric and/or adhesive interactions. Due to mucus turnover, most therapeutic agents delivered topically to mucosal surfaces suffer from poor retention and distribution, which limits their efficacy.

The nanoparticles that carry drugs and genes that are delivered to the cells covered by the mucus in the eyes, nose, lungs, gastrointestinal tract and female reproductive tract must be evenly distributed to treat or protect these surfaces to greatest extent. However, high viscoelasticity (that is, viscous and solid-like in nature) and adhesive mucus layer can slow down particles or fix particles completely, and thus prevent them from spreading on the mucosal surface. In addition, some mucosal surfaces (such as the mucosal surfaces of the oral cavity, stomach, intestines, colon and vagina) show highly folded epithelial surfaces, and conventional sticky-adhesive particles and many small molecule drugs and therapeutic agents can not reach the epithelial surface. In the absence of maximum distribution and penetration into these deep recesses, many of the epithelia are susceptible and/or untreated. In addition, penetration into the folds that may contain the mucus layer that is much slower to remove allows the residence time at the epithelial surface to be extended.

For drug or gene delivery applications, therapeutic particles must be able to 1) achieve uniform distribution on the mucosal surface of interest and 2) effectively cross the mucus barrier to avoid rapid mucus clearance and ensure effective delivery of their therapeutic payload to the underlying cells (Neves J and Bahia MF Int J Pharm 318, 1-14 (2006); Lai et al. Adv Drug Deliver Rev 61, 158-171 (2009); Ensign et al. Sci Transl Med 4, 138ral79 (2012); Eyles et al. J Pharm Pharmacol 47, 561-565 (1995)).

Biodegradable nanoparticles that penetrate deeply into the mucus barrier can provide improved drug distribution, retention, and efficacy at the mucosal surface. A dense surface coating of low molecular weight polyethylene glycol (PEG) allows the nanoparticles to rapidly penetrate the highly viscoelastic human and animal mucus secretions. The hydrophilic and biologically inert 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.

The vaginal surface is highly folded to accommodate expansion during sexual intercourse and childbirth; these folds or "plicas" are often collapsed by intra-abdominal pressure, hindering drug delivery to the folded surface. For truly effective prevention and treatment, sustained drug concentrations must be delivered to and maintained across the entire susceptible surface. Failure to achieve adequate distribution across the entire vaginal epithelium is a documented failure mode of vaginal microbicides.

Another significant barrier to effective drug delivery into the vagina is the viscoelastic mucus layer secreted by the endocervical lining, which lines the vaginal epithelium. Mucus effectively traps foreign particles and microparticles through both steric and adhesive mechanisms, contributing to their rapid clearance. Although mucoadhesive dosage forms have been proposed to increase vaginal retention time, mucus clearance is rapid (approximately minutes to hours), limiting the retention time of mucoadhesive systems.

Mucosal epithelia use osmotic gradients to induce fluid absorption and secretion. Vaginal products have traditionally been formulated with hypertonic formulations, including yeast infection treatments, most sexual lubricants such as warming gels, and gels designed to prevent sexually transmitted infections (such as HIV). Hypertonic formulations induce rapid, osmotic secretion of fluid into the vagina, and this causes an immediate increase in fluid leakage from the vagina at a rate proportional to the hypertonicity of the formulation. In addition, recent studies of candidate vaginal and rectal microbicides in both animal models and humans have revealed that hypertonic formulations can cause toxic effects that can increase susceptibility to infection. The first successful microbicide trial for HIV prevention found that the antiretroviral drug tenofovir, delivered in the form of a vaginal gel, provided partial protection. Unfortunately, the gel formulation is highly hypertonic, leading researchers to reduce the glycerol concentration in most recent tenofovir clinical trials to reduce toxicity. However, the concentration was not reduced, and the formulation remained significantly hypertonic. There seems to be no evidence to support the use of hypertonic formulations for vaginal drug delivery because, in addition to documented toxic effects, hypertonic formulations also cause rapid osmotic-driven secretion of vaginal fluid, antagonizing fluid flow for drug delivery to the epithelium. This lack of rationale has been ignored by both researchers and manufacturers of vaginal products, with the only notable exception being lubricants intended to support fertility. These products are formulated to be isotonic (osmolarity equivalent to that of plasma) to help maintain sperm motility.

It is therefore an object of the present invention to provide formulations for the rapid and uniform microparticle delivery of various drugs to mucosal-lined epithelial surfaces with minimal toxicity to the epithelium.

Summary of the Invention

Penetration can be used to cause mucus-permeable particles to quickly penetrate deep recesses in highly folded mucosal tissue. Absorption and penetration into deep recesses in mucosal tissue improves the distribution of otherwise poorly distributed entities on the mucosal surface. Rapid absorption and penetration into deep recesses in mucosal tissue results in prolonged retention time of the mucus-permeable particles. Rapid absorption not only increases therapeutic effectiveness and minimizes the time between administration and mucosal protection, but also contributes to user acceptability.

Hypotonic formulations are evaluated for delivery of water-soluble drugs and for drug delivery with viscous (i.e., non-adhesive) mucus-permeable nanoparticles (MPPs). Hypotonic formulations significantly increase the rate at which drugs and MPPs reach the epithelial surface. In addition, hypotonic formulations greatly enhance the delivery of drugs and MPPs to the entire epithelial surface, including deep into the vaginal folds (rugs) that isotonic formulations cannot reach. Hypotonic formulations can cause free drugs to be attracted not only to the epithelium but also to be attracted via the epithelium, reducing vaginal retention. In contrast, hypotonic formulations cause MPPs to accumulate quickly and evenly on the vaginal surface, but they do not pass through the epithelium and therefore remain ideally positioned to achieve sustained mucosal drug delivery. Minimum hypotonic formulations preferably within the range of 20-220 mOsm/kg enable MPPs to be delivered quickly and evenly to the entire vaginal surface with minimal risk of epithelial toxicity. Hypotonic formulations for vaginal drug delivery via MPPs should significantly improve the prevention and treatment of reproductive tract diseases and disorders.

The data also demonstrate that osmolality is higher in the colon, such that vehicles with osmolality higher than plasma osmolality (generally considered isotonic at approximately 300 mOsm/kg) still result in improved distribution in the colon due to rapid, osmotic fluid absorption. The range for improved colonic distribution with low-osmotic vehicles in the colon is approximately 20 mOsm/kg-450 mOsm/kg. In preferred embodiments, the osmolality of formulations for administration to the colon or rectum is between approximately 20 mOsm/kg and 450 mOsm/kg, with sodium ions (Na ⁺ ) contributing at least 30% to an osmolality exceeding 220 mOsm/kg. (i.e., if the osmolality of the formulation is 450 mOsm/kg, then Na ⁺ ions must constitute at least 30% of 450-220=230, or 69 mOsm/kg). Improved distribution of hypotonic-administered MPP (compared to CP) on rectal tissue in patients with induced ulcerative colitis was also demonstrated, including absorption of MPP into ulcerated tissue. Hypotonic administration also resulted in improved distribution of free drug (FITC-labeled tenofovir) in the colon.

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 graphical representations showing the overall mean geometric mean square displacement (<MSD>/μm ² ) as a function of time (time scale/s). Figures 1e and 1f are graphical representations showing the permeation fraction as a function of the logarithmic distribution of the effective diffusivity (D _eff ) of individual particles on a time scale of 1 s. Figures 1g and 1h are graphical representations showing the estimated fraction of particles able to permeate 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 adsorption 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.

Figures 5A and 5B show vaginal coverage of doxorubicin (Dox) administered as a hypotonic (hypo) or isotonic (iso) solution. Mice (A) remained supine for 1 hour followed by tissue collection (non-ambulatory) or (B) were ambulatory for 10 minutes followed by tissue collection (ambulatory). Images represent the average vaginal surface coverage of ambulatory mice dosed with Dox as (C) isotonic (iso) and (D) hypotonic (hypo) solutions. Data are mean ± SEM (n = 5). *P < 0.05 compared to isotonic, Wilcoxon rank sum test.

Figure 6 shows the vaginal retention of doxorubicin administered in isotonic (iso) or hypotonic (hypo) solutions. Mice were kept in a supine position for 10 minutes, followed by tissue collection. Overlay of doxorubicin fluorescence intensity and bright field images of isotonic and hypotonic solutions in whole cervicovaginal tissue. The relative doxorubicin signal based on fluorescence signal quantification adjusted for solution fluorescence represents the mean value calculated for n = 4 mice and is quantified as relative signal ± SEM. *P < 0.05 compared to isotonic, Wilcoxon rank sum test.

Figure 7 shows vaginal HSV-2 infection after treatment with acyclovir monophosphate (ACVp) in a hypotonic (hypotonic) or isotonic (isotonic) solution. ACVp (10 mg/ml) was administered 1 minute or 60 minutes before viral inoculum, with n≥45 mice tested per group, and the infection rate in the control group was approximately 90%. *P<0.05 compared to isotonic, Fisher's exact test.

Figures 8A and 8B demonstrate vaginal retention of MPPs administered as hypotonic or isotonic solutions. Figure 8A , mice remained supine for 1 hour, followed by tissue collection (non-ambulatory). Figure 8B , mice were ambulatory for 10 minutes, followed by tissue collection (ambulatory). Particle retention is calculated as mean ± SEM (n ≥ 5). *P < 0.05 compared to isotonic, Wilcoxon rank sum test.

FIG9 shows the vaginal distribution of fluorescent 100 nm MPP administered as solutions of varying osmolarity in transverse vaginal cryosections and across flattened vaginal tissue. All tissues were collected within 10 minutes of particle administration. All values are in mOsm/kg. The osmolarity of the mucoadhesive CP particles was 20 mOsm/kg. Images are representative of n ≥ 5 mice. Data are calculated as mean ± SEM (n ≥ 3). ^# Reprinted from (4). *P < 0.05, Wilcoxon rank sum test, as compared to hypotonic solution (20-220).

DETAILED DESCRIPTION

Many mucosal surfaces (such as those of the oral cavity, stomach, intestines, colon, and vagina) contain a large number of deeper epithelial folds to accommodate epithelial expansion and the absorption of fluids and nutrients. For these reasons, a significant portion of the epithelial surface is contained in these difficult-to-reach folds. The ability to design particles that are evenly distributed on the mucosal tissue surface holds many important implications for therapeutic agent delivery, imaging, and diagnostic applications. For example, particles that do not achieve even distribution and penetration into deep recesses cannot fully treat or protect mucosal surfaces (Rajapaksa et al., J Biol Chem 285, 23739-23746 (2010).

In the field of vaginal drug delivery, achieving adequate distribution to all target surfaces is a frequently cited problem. The vaginal surface is highly folded to accommodate expansion during sexual intercourse and childbirth, creating folded wrinkles or "rugs." Poor distribution in the folds (even after simulated sexual intercourse) has been proposed as a key factor in the inability of microbicide products to protect against vaginal infections. Other microbicide studies in mice have used larger volumes of test products (up to 40 μl) to promote more complete vaginal distribution. The mouse vagina can accommodate a volume of approximately 50 μl; this relatively large volume of test agent expands and spreads the vaginal epithelium. In contrast, the human vagina can be maintained in the 50 ml range, while typical vaginal products only deliver 2-5 ml. To study vaginal distribution in mice, a smaller volume (5 μl) was used that would more accurately simulate the volume used in humans. Methods that deliver drugs into deeply folded surfaces without expanding the vagina may result in more effective vaginal drug delivery.

The vaginal epithelium is permeable to small molecules and can absorb a variety of drugs. The superficial layer of the vaginal epithelium contains densely packed dead and dying cells (stratum corneum), which protect the deeper living cell layer while allowing fluid to be secreted and absorbed through the epithelium. The vagina has a natural ability to absorb fluid induced by osmotic induction that can be used for drug delivery. Doxorubicin administered as a hypotonic solution coated more than 85% of the vaginal surface of ambulatory mice, while only 25% of the vaginal tissue surface was coated with doxorubicin administered as an isotonic solution. Isotonic fluids do not penetrate into the folds, leaving a striped pattern when the vaginal tissue is flattened. It has been proposed that hypotonic delivery can increase the contraceptive efficacy of the detergent nonoxynol-9 (N9) by improving its mobility through mucus (Dunmire EN and Katz DF. Contraception 55, 209-217 (1997); Owen et al. J Control Release 60, 23-34 (1999). The enhanced penetration of hypotonic N9 solution through mucus was demonstrated in vitro in isolated mucus, with the emphasis on achieving more rapid contact between the detergent and sperm in the mucus.

Delivery to the cervix, vagina, and colon can be particularly challenging, not only because of distribution issues, but also because of "leakage." Most vaginal and rectal formulations are highly osmolar, which causes osmotic secretion of fluid from the mucosal epithelium. This fluid secretion causes dilution and leakage of the formulation and toxicity associated with high osmolarity (Rudolph et al. Mol Ther 12, 493-501 (2005); Bertschinger et al. Journal of Controlled Release 116, 96-104 (2006); Pihl et al. Acta Physiol 193, 67-78 (2008); Noach J Pharmacol Exp Ther 270, 1373-1380 (1994)). Drug absorption using hypotonic drug solutions is known, but the effect of absorption on distribution and retention is not. See, for example, Ayers et al., Journal of Pharmacology and Pharmacotherapy 47, 561-565 (1995); Rajapaksa et al., Journal of Biochemistry 285, 23739-23746 (2010); Rudolph et al., Mol. Ther. 12, 493-501 (2005); Botzinger et al., J. Controlled Release 116, 96-104 (2006); Pihl et al., Acta Physiologica Sinica 193, 67-78 (2008); Noah's J. Pharmacol. Exp. Therapeutics 270, 1373-1380 (1994); Lennemas H. Pharmaceut Res 12, 1573-1582 (1995).

The tonicity of the formulation depends on the osmotic properties of the tissue (e.g., colon vs. vagina), and there is a critical range of moderate hypotonicity for increased absorption and uniform distribution without toxicity. Moderately hypotonic formulations should induce fluid absorption that will reduce "leakage," which is often reported by patients in clinical trials as an adverse side effect. This product leakage results in both reduced user acceptability and rapid removal of the therapeutic agent. For example, a decrease in osmolality from 294 mOsm/kg to 220 mOsm/kg has the following effects: moderately hypotonic fluid increases vaginal surface coverage from 60% to 76%, and substantially all of the hypotonically delivered MPPs are drawn out of the lumen to reach the epithelial surface deep within the folded surface within 10 minutes of administration.

Microparticle formulations that rapidly achieve uniform distribution and transport through mucus can be used to effectively target mucus-lined epithelia in vivo for a wide range of applications, including drug therapy (ranging from small molecule therapeutics such as chemotherapeutics to peptides, proteins, oligonucleotides, DNA, etc.), imaging, and diagnostics. For therapeutic purposes, the molecules encapsulated in the particles can then be released at a predetermined rate over an extended period of time. In general, therapeutic applications of the technology include delivering any drug when standard formulations are not feasible due to inefficient distribution, toxicity, or "leakage," are not 100% effective, or cause undesirable side effects. The method should also improve the penetration and uniform distribution of standard drug formulations (i.e., without drug delivery particles), especially for gene/oligonucleotide delivery; targeted and highly localized chemotherapy delivery for cancer treatment; targeted delivery of anti-inflammatory drugs; treatment or prevention of STDs; penetration in biofilms and other biological coatings/barriers; and targeted delivery of antibiotics for the treatment of bacterial infections.

Most vaginal gels have been formulated with excipients that render the gel hypertonic, such as glycerol or propylene glycol. Unfortunately, recent studies have shown that these hypertonic formulations cause toxicity in the vagina of mice that increases susceptibility to HSV-2 infection (Moench et al. BMC Infect Dis 10, 331 (2010)), likely by becoming hypertonic (Fuchs et al. J Infect Dis 195, 703-710 (2007); Clark MR and Friend DR (2012) Pharmacokinetics and Topical Vaginal Effects of Two Tenofovir Gels in Rabbits. AIDS Res Hum Retroviruses). Furthermore, hypertonic gel formulations have been found to disrupt epithelial integrity in the human colon, and hypertonic tenofovir gel formulations have been found to induce epithelial disruption in exocervical and colorectal explants compared to tissues exposed to vehicle alone (Rohan, PLoS One 5, e9310 (2010)). It has been hypothesized that the primary contributing factor to the dextran sodium sulfate (DSS)-induced experimental irritable bowel disease mouse model is the hypertonicity of the DSS solution. Inflammatory cytokine release in mouse vaginal lavage fluid increased after 7 daily doses of the hypertonic gel vehicle, but not after 7 daily doses of the hypotonic formulation.

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).

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

"Lipophilic" refers to a compound's affinity for lipids.

"Amphoteric" refers to a molecule that combines hydrophilic and lipophilic (hydrophobic) properties.

As used herein, "hydrophobic" refers to a substance's lack of affinity for water; a tendency to repel and not absorb water as well as not dissolve in or mix with water.

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, 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.

The conventional use of the term "isotonic" refers to a fluid that does not cause cell swelling or shrinkage, which typically occurs when the total solute concentration (osmolality) is equal to that of blood (approximately 300 mOsm/kg). Isotonic is defined herein as a formulation that does not cause water to enter or leave the lumen or to be driven osmotically through the epithelium. Hypotonic is defined herein as a formulation that causes water to flow inward from the mucosal surface toward the epithelium, and hypertonic formulations are defined as hypertonic formulations that cause water to flow outward toward mucus-coated surfaces.

As used herein, "mucus-permeable particles" or "MPPs" generally refer to particles that have been coated with a mucosal permeation-enhancing coating. In some embodiments, the particles are particles of an active agent (e.g., a therapeutic agent, a diagnostic agent, a prophylactic agent, and/or a nutraceutical agent) coated with a mucosal permeation-enhancing coating as described below (i.e., a drug particle). In other embodiments, the particles are formed from a matrix material (e.g., a polymeric material) in which the therapeutic agent, diagnostic agent, prophylactic agent, and/or nutraceutical agent is encapsulated, dispersed, and/or associated. The coating material can be covalently or non-covalently associated with the drug particle or polymeric particle.

II. Mucus-penetrating nanoparticles (MPPs)

A. Aggregate particles

1. 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-co-glycolic acid) (PLGA), poly(L-lactic-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- and blends thereof; polyalkylene glycols such as poly(ethylene glycol) (PEG) and polyalkylene oxides (PEO) and block copolymers thereof, such as polyoxyethylene oxide; and 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 halide, such as poly(vinyl chloride) (PVC); polyvinyl pyrrolidone; polysiloxane; polystyrene (PS; cellulose, including derivatized cellulose, such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose and carboxymethyl cellulose; polymers of acrylic acid, 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 copolymers thereof; polyhydroxyalkanoates; polypropylene fumarate; polyoxymethylene; poloxamer; poly(butyric acid); trimethylene carbonate; and polyphosphazene. Examples of preferred natural polymers include proteins (such as albumin), collagen, gelatin, and prolamins (such as zein), as well as polysaccharides (such as alginates). 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.

In some embodiments, the particles can be used as nanoparticle gene carriers. In these embodiments, the particles can be formed by one or more polycationic polymers complexed with one or more negatively charged nucleic acids. The cationic polymer can be any synthetic or natural polymer with at least two positive charges per molecule, and it 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.

B. Coated Drug Particles

In some embodiments, particles of therapeutic agents, diagnostic agents, prophylactic agents, and/or nutritional agents are coated with a mucosal permeation enhancing coating. The particles may be microparticles or nanoparticles. Exemplary therapeutic agents, diagnostic agents, prophylactic agents, and/or nutritional agents are described in more detail below. Drug particles can be coated with a mucosal permeation enhancing coating material using techniques known in the art. The density and morphology of the coating can be evaluated as described below. The mucosal permeation enhancing coating can be covalently or non-covalently associated with the agent. In some embodiments, it is non-covalently associated. In other embodiments, the active agent contains reactive functional groups or is incorporated into a mucosal permeation enhancing coating that can be covalently bound.

C. Materials that promote diffusion through mucus

The microparticles and/or 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 a particle to its 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 or PLURONIC (a polyethylene oxide block copolymer available from BASF).

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 has or contains polyethylene glycol (PEG) or F127. Alternatively, PEG or F127 can be in the form of a block covalently bound (e.g., inside or at one or both ends) to the core polymer for forming the particle. In a particular embodiment, the particle is formed by a block copolymer containing PEG. In a more particular embodiment, the particle is prepared by a block copolymer containing PEG, wherein PEG is covalently bound to the end of the base polymer. Representative PEG molecular weights include 300Da, 600Da, 1kDa, 2kDa, 3kDa, 4kDa, 6kDa, 8kDa, 10kDa, 15kDa, 20kDa, 30kDa, 50kDa, 100kDa, 200kDa, 500kDa and 1MDa and all values within the range of 300 dalton to 1MDa. In a preferred embodiment, the molecular weight of PEG is about 5kD. The PEG of any given molecular weight can be different in other features (such as length, density and branching).

1. Evaluate surface density

The surface density of poly(ethylene glycol) (PEG) on microparticles and/or nanoparticles is a key parameter in determining their successful in vivo administration. (As used herein, reference to PEG on the particle surface is generally presumably to F127. 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. A dense coating of PEG on biodegradable nanoparticles can allow for rapid penetration through mucus due to greatly reduced adhesive interactions between mucus components and the nanoparticles.

In a preferred embodiment, nuclear magnetic resonance (NMR) is 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. Thus, 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 composition. 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 chains/nm². In specific 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. In other specific embodiments, the density is about 0.05 to about 0.5 PEG chains/nm².

The concentration of the surface-altering material (e.g., PEG) can also be varied. In certain embodiments, the density of the surface-altering material (e.g., PEG) is such that the surface-altering material (e.g., PEG) adopts an extended brush configuration. In other embodiments, the mass of the surface-altering moiety 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.

D. Emulsifier

The particles described herein may contain an emulsifier, particularly a low molecular weight emulsifier. The emulsifier is incorporated into the particle during particle formation and thus becomes a component of the finished particle. The emulsifier may be encapsulated within the particle, 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. 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. To aid in their diffusion through mucus, the nanoparticles described herein typically have a near-neutral surface charge. In certain embodiments, the nanoparticles have a zeta potential 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.

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 emulsification methods.

E. 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 therapeutic agents include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antipsychotics, neuroprotectants, antiproliferatives (such as anticancer agents), anti-infectives (such as 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, nutritional agents, vitamins, parasympathomimetics, stimulants, anorexias, and anti-somnia agents. Nutritional agents may also be incorporated. These nutritional agents may be vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts or plant hormones.

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 defective genes that contribute to the development of a disease. Researchers can use one of several methods to correct defective genes: A normal gene can be inserted into a non-specific location within the genome to replace a non-functional gene. An abnormal gene can be exchanged for a normal gene through homologous recombination. An abnormal gene can be repaired through selective reversion mutations, which restore the gene to its normal function. The regulation of a specific gene (the degree to which the gene is turned on or off) can be altered.

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 problem 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 RNAs modified with 2'-fluoro (2'-F) pyrimidine appear to possess advantageous properties in vitro.

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 usually 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 may include introducing a whole replacement copy of a defective gene, a heterologous gene or a small nucleic acid molecule (such as an oligonucleotide). This method usually 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. Three-stranded molecules are preferred because they can bind to the target region with high affinity and specificity. It is preferred that the three-stranded molecules bind to the target molecule with a Kd lower 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 three-stranded molecules can also be tail-clip peptide nucleic acids (tcPNAs), such as those described in U.S. Published Application No. 2011/0262406.

Double duplex-forming molecules (such as a pair of pseudocomplementary oligonucleotides) can also induce recombination with a donor oligonucleotide at a chromosomal site. The use of pseudocomplementary oligonucleotides in targeted gene therapy is described in US Published Application No. 2011/0262406.

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.

III. Pharmaceutical Compositions

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 wt % to about 80 wt %, from about 1 wt % to about 50 wt %, preferably from about 1 wt % to about 40 wt %, more preferably from about 1 wt % to about 20 wt %, and most preferably from about 1 wt % to about 10 wt %. 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.

The formulations described herein contain an effective amount of nanoparticles ("MPPs") in a pharmaceutical carrier suitable for administration to a mucosal surface, wherein the pharmaceutical carrier is adjusted to be hypotonic. Once the desired tissue to be treated is identified, one of ordinary skill in the art can routinely adjust the tonicity of the pharmaceutical carrier based on the preferred tonicity ranges described herein.

Tonicity is the 'effective osmolality' and is equal to the sum of the concentrations of solutes capable of exerting an osmotic force across a membrane. Many different substances can be used to adjust tonicity. For example, USP 29-NF 24 lists five excipients classified as "tonicity" agents, including dextrose, glycerol; potassium chloride; mannitol; and sodium chloride. See, e.g., United States Pharmacopeial Convention. Pharmacopeia 29-National Formulary 24. Rockville MD: United States Pharmacopeial Convention; 2005: 3261; Day, A. Dextrose. In: Rowe RC, Sheskey PJ and Owen SC. Handbook of Pharmaceutical Excipients. 5th ed. Washington, DC: American Pharmaceutical Association; 2005: 231-233; Price JC. Glycerol. In: Rowe RC, Sheskey PJ and Owen SC. Handbook of Pharmaceutical Excipients. 5th ed. Washington, DC: American Pharmaceutical Association; 2005: 301-303; Price JC. =JC.) Glycerol. In: Rowe RC, Shesky PJ and Irving SC, eds. Handbook of Pharmaceutical Excipients. 5th ed. Washington, DC: American Pharmaceutical Association; 2005: 301-303; Armstrong NA. Mannitol. In: Rowe RC, Shesky PJ and Irving SC, eds. Handbook of Pharmaceutical Excipients. 5th ed. Washington, DC: American Pharmaceutical Association; 2005: 449-453; Irving SC. Sodium chloride. In: Rowe RC, Shesky PJ and Irving SC, eds. Handbook of Pharmaceutical Excipients. 5th ed. Washington, DC: American Pharmaceutical Association; 2005: 671-674. Mannitol is an example of a GRAS-listed ingredient accepted for use as a food additive in Europe and is included in the FDA Inactive Ingredients Database (FDA Inactive Ingredients Database; IP, IM, IV, and SC injections; infusions; buccal, oral, and sublingual tablets, powders, and capsules; ophthalmic preparations; topical solutions), in the UK licensed non-parenteral and parenteral drug products, and in the Canadian Natural Health Products Ingredients Database. A 5.07% w/v aqueous solution is isotonic with serum.

Minimally hypotonic formulations, preferably in the range of 20-220 mOsm/kg, allow rapid and uniform delivery of the MPP to the entire vaginal surface with minimal risk of epithelial toxicity. The osmolality in the colon is higher, so vehicles with osmolality higher than the plasma osmolality (generally considered isotonic at about 300 mOsm/kg) result in improvements in distribution in the colon. The range for improving colonic distribution with hypotonic vehicles in the colon is about 20 mOsm/kg-450 mOsm/kg, which makes the formulation effectively hypotonic if the major portion of the solute in the formulation consists of Na ⁺ ions, since these Na ⁺ ions will be actively absorbed (adsorbed) by the epithelium, despite being hyperosmolal relative to the blood.

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: 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 - composed of DPPC, palmitoyl-oleoylphosphatidylglycerol, and palmitic acid, combined with a 21-amino acid synthetic peptide that mimics the structural characteristics of SP-B; Venticute - DPPC, PG, palmitic acid, and recombinant SP-C; Alveofact - extracted from cow lung lavage fluid; Curosurf - extracted from material obtained from minced pig lungs; Infasurf - extracted from calf lung lavage fluid; and 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 affect 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 mucus-inert. However, mucus-penetrating particles can be encapsulated in microparticles to impact the upper lung and subsequently release nanoparticles. In some cases, the shape of the particles is spherical or ovoid. The particles can have a smooth or rough surface texture. The particles can also be coated with polymers or other suitable materials 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, water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers (e.g., phosphate-buffered saline (PBS), Ringer's solution, and isotonic sodium chloride), or any other aqueous solution suitable for administration to animals or humans, adjusted to the desired hypotonicity as indicated by osmotic flow of water from the luminal (mucosal) surface to the serosal surface through the epithelium. At certain mucosal surfaces (e.g., the colon), "hyperosmolar" fluid carriers (defined in the conventional sense, relative to blood osmolarity) can actually exploit hypotonicity in the colon and induce fluid absorption by the epithelium. In fact, isotonic formulations have not yet been defined in the lung and in certain disease states (e.g., cystic fibrosis), where the osmolarity of lung fluid is hyperosmolar (higher than blood osmolarity).

Preferably, the liquid formulation is slightly hypotonic relative 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, which are low-toxic organic (i.e., non-aqueous) Class 3 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 have sufficient volatility to be able to form an aerosol of a 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.

In some cases, the 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 can be performed through the patient's nose and/or mouth. Administration can be performed by self-administering the formulation while inhaling or by administering the formulation to a patient who is on a ventilator via a respirator.

B. Topical and Ophthalmic Formulations

Topical or enteral 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, emulsions, sprays, gels, creams, or ointments.

The carrier can 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 preferable to include an isotonic agent, such as a sugar or sodium chloride, to adjust the tonicity.

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, 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 administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer.

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

Sterile 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 sterilization by filtration. 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 ocular 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, osmolarity (PBS), and isotonicity sodium chloride solution, which are then adjusted to the desired hypotonicity of the eye as determined using MPP to observe osmotic induced flow of water (tears). The formulation can also be a sterile solution, suspension, or emulsion in a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

In some cases, the formulation is distributed or packaged in liquid or semisolid form, such as a solution (eye drops), suspension, gel, cream, or ointment. Alternatively, the formulation for ophthalmic administration may be packaged as a solid, for example, obtained by lyophilization of a suitable liquid formulation. The solid may be reconstituted with an appropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for ocular administration may be buffered with an effective amount of a buffer necessary to maintain a pH suitable for ocular 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 ocular administration may also contain one or more tonicity agents to adjust the tension of the formulation to an appropriate hypotonic range. 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.

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, and emulsions. The compositions may contain one or more chemical permeation enhancers, membrane permeants, membrane transport agents, lubricants, surfactants, stabilizers, and combinations thereof. In some embodiments, the nanoparticles may be administered as liquid formulations (e.g., solutions or suspensions), semisolid formulations (e.g., gels, lotions, or ointments), or solid formulations. A "gel" is a colloid in which a dispersed phase has merged with a continuous phase to produce a semisolid substance (e.g., a jelly).

In some embodiments, the nanoparticles are formulated as liquids, including solutions and suspensions, such as eye drops, or as semisolid formulations, such as ointments or lotions for topical administration to mucous membranes (such as the eye) or vaginally or rectally.

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

"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.

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 consists of an emulsion and either a propellant gas or a gas exhaust component.

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.

D. 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. Formulations can 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," ed. Liberman et al. (New York, Marcel Decker, 1989), "Remington - The science and practice of pharmacy," 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems," 6th ed., 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.

V. Methods of Using MPP-Hypotonic Formulations

The formulation containing the particles is administered to a mucosal surface in a therapeutically effective amount to alleviate one or more symptoms, wherein the formulation is hypotonic to enhance absorption of the particles through the mucosa without causing toxicity. This can be done using single-dose sterile packaging containing a solution or suspension (such as eye drops) or a dry powder, gel, ointment, cream or lotion (for topical administration to the eye area, oral area (buccal, sublingual), vaginal, rectal) or spray applicator, or it can be formulated for oral administration.

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

Examples

Examples 1-6 demonstrate the preparation and characterization of mucus permeable particles ("MPPs"). Examples 7-9 demonstrate the effects of hypotonic formulations on the absorption and toxicity of MPPs administered to mucosal tissues.

Materials and methods

Cholic acid sodium salt, 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:GA 50:50) (MW approximately 15 kDa) with an intrinsic viscosity of 0.15-0.25 dL/g was purchased from Lakeshore Biomaterials (Birmingham, AL). PLGA (LA:GA 50:50)-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 400REM 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 400REM 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 was performed in fresh human cervicovaginal mucus (CVM). 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 ℃. This solution is added to 1mL containing 100mg/ml PLGA-PEG5k in DCM solution in an ice-water bath during probe sonication (30% amplitude, 1min, wherein the pulse is 1s). The gained W/O main emulsion is immediately added to the second aqueous phase (5mL1% 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. Nanoparticles are filtered through a 1 μm syringe filter, washed and collected by centrifugation. BSA-FITC allows tracking the possibility of 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 400REM 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% CHAD 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 the density of the polymer, 1.21 g/ml for PLGA), 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.

Example 7: Effect of osmolarity of the particle solution on the distribution and retention of nanoparticles in vaginal and colonic mucosal tissues.

Mucus-permeable nanoparticles (MPPs), which avoid being adhered to the mucus layer present on the mucosal surface, were used to study this effect at the mucosal surface. A very dense coating of low molecular weight (5 kDa) polyethylene glycol (PEG) was covalently attached to the surface of fluorescent 100 nm carboxyl-modified polystyrene (PS) particles to produce mucus-permeable PS-PEG nanoparticles. Uncoated nanoparticles ("conventional particles" or "CPs") adhered to mucin. CPs did not fully penetrate the mucus layer and instead accumulated in the lumen of rapidly frozen whole mouse colon and vaginal tissue. However, MPPs penetrated the mucus barrier all the way to the underlying epithelium, creating a continuous "layer" of particles in both mouse rectal and vaginal tissue. If the particles are administered in a low osmolarity solvent (such as pure water), the extremely high absorptive capacity of the mucosal surface allows the MPPs, rather than the CPs, to be rapidly "absorbed" through the mucus layer. Administration of the particles in a low osmolarity solution, such as ultrapure (UP) water, allows the fluid to be absorbed by the underlying tissue of the mucus layer in order to reach osmotic equilibrium, thereby being drawn into the MPP by convection.

Recent reports have detailed the negative side effects of administering highly hyperosmolar formulations for vaginal and rectal delivery, which can cause significant toxicity and epithelial erosion (Fuchs et al. J Infect Dis 195, 703-710 (2007); Lacey et al. Int J STD AIDS 21, 714-717 (2007). However, moderately hypoosmolar fluids have not been shown to have the same toxicity profile.

Materials and methods

The distribution of fluorescent 100 nm mucus-permeable PEGylated polystyrene particles administered in (A) 1×PBS or (B) ultrapure water was examined on cross-sections of snap-frozen whole colon tissue. Tissue was excised immediately after administration and stained with DAPI to reveal cell nuclei.

The distribution of fluorescent 100 nm mucus-permeable PEGylated polystyrene particles administered in (A) 1× PBS or (B) ultrapure water was also examined on cross-sections of snap-frozen whole vaginal tissue. Tissue was excised immediately after administration and stained with DAPI to reveal cell nuclei.

Fluorescence of whole mouse vaginal and colorectal tissues was then assessed following initial administration of fluorescent nanoparticles in UP water (low osmolarity) or PBS (isoosmolarity).

To assess nanoparticle retention, 5 μL of red-fluorescent CP or MPP was administered intravaginally. Whole cervicovaginal tissue was obtained at 0, 2, 4, and 6 hours and placed in standard tissue culture dishes. For each condition and time point, >7 mice were used. Fluorescent images of tissue were acquired using a Xenogen IVIS Spectrum imaging device (Caliper Life Sciences). Fluorescent counts per unit area were quantified using Xenogen Living Image 2.5 software.

result

MPPs rapidly begin to line the vaginal and colorectal epithelium when administered as a low osmolarity solution.

The percentage of MPP (PSPEG) and CP (PS) retained in the mouse vagina over time after administration in UP water was measured. After 6 hours, 57% of MPP and 7% of CP remained in the CV tract.

MPP was retained in the mouse vagina at much higher amounts for at least 6 hours.

Biodegradable MPP containing FITC was administered intravaginally in UP water. For comparison, FITC was administered in a standard isosmolarity placebo gel, hydroxyethylcellulose (HEC). After 24 hours, vaginal tissue was excised and flattened between two glass slides. Hypoosmotically delivered MPP appeared to completely coat the epithelium, while FITC was sparsely distributed. MPP significantly improved the epithelial distribution of an otherwise poorly distributed entity.

Example 8: Distribution of MPP in vaginal tissue related to osmolality

The study in Example 7 demonstrated that MPPs, which do not adhere to mucus, were able to diffuse rapidly through human and mouse cervicovaginal mucus (CVM), causing deep penetration into the slower-clearing mucus layer in the folds, coating the entire vaginal surface and remaining in the vagina longer than conventional mucoadhesive nanoparticles (CPs), as published by Enser et al., Sci Transl Med 4, 138ral79 (2012).

A key to improving vaginal distribution and retention using MPPs is to administer nanoparticles in the form of a hypotonic solution. When delivered as a hypotonic solution, MPPs rapidly aggregate across the vaginal surface, arriving much faster than expected based solely on diffusion. When the fluid is pressure-induced to flow through the mucus gel, the MPPs flow through the mucus along with the flowing fluid (i.e., dragged by the solvent). Studies were conducted to determine whether similar effects would occur when the drug and MPPs were delivered to the vagina in a hypotonic formulation. This study investigated whether hypotonic delivery of free drug and hypotonic vaginal delivery by MPPs could provide improved distribution, retention, and protection.

Materials and methods

Animal models

Female 6-8 week old CF-1 mice were purchased from Harlan (Indianapolis, IN). Mice were housed in a reverse light cycle facility (12 hours light/12 hours dark) to select mice in a naturally cycling estrus state. The mouse vagina during the estrus phase of the estrous cycle is most similar to the human vagina. The barrier properties of nanoparticles in the mucus of estrous mice closely mimic the barrier properties of nanoparticles in human CVM. Therefore, mice in estrus, as determined by visual appearance of the vaginal opening, were used for all distribution and retention studies. Mice used for vaginal HSV-2 protection and susceptibility studies were given a subcutaneous flank injection of 2.5 mg of Depo-Provera (Pharmacia & Upjohn Company, New York, NY) in 100 μL of phosphate-buffered saline (PBS) 7 days before the experiment. This treatment is commonly used to increase susceptibility to vaginal HSV-2 infection. All experimental protocols were approved by the Johns Hopkins Animal Care and Use Committee.

Nanoparticle preparation and characterization

Fluorescent carboxyl (COOH)-modified polystyrene (PS) nanoparticles (100 nm in diameter) were purchased from Molecular Probes (Eugene, OR). To make MPPs, PS particles were covalently modified with 5 kDa amine-modified PEG (Creative PEGworks, Winston Salem, NC) as previously described by Nance et al., Science Translational Medicine 4, 149ral19 (2012). Particle size and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, respectively, using a Zetasizer Nano ZS90 (Malvern Instruments, Southborough, MA). Size measurements were performed at 25°C with a scattering angle of 90°. Samples were diluted in 10 mM NaCl solution (pH 7) and measured according to the instrument instructions. Near-neutral zeta potential was used to confirm PEG conjugation, and the particles were tested for mucus permeability in human CVM as previously described in Lai et al., Proc. Natl. Acad. Sci. USA 104, 1482-1487 (2007) and Wang et al., Angew Chem Int Ed Engl 47, 9726-9729 (2008). These particles were previously shown to rapidly penetrate the vaginal mucus of estrous mice. The osmolarity of the solutions was measured using a Wescor Vapro vapor pressure osmometer.

Drug and nanoparticle distribution in the vagina

Doxorubicin (NetQem, Durham, NC) was dissolved at a concentration of 1 mg/ml in PBS (iso-osmolarity relative to blood) or ultrapure water (low osmolarity). For 1 liter of 1×PBS, 800 ml of distilled water, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na ₂ HPO ₄ , and 0.24 g of KH ₂ PO ₄ were added. The pH was adjusted to 7.4 with HCl, and distilled water was added to a total volume of 1 liter.

Doxorubicin was administered vaginally to mice under two different conditions, both in isosmolar and hypoosmolar concentrations. The "non-ambulatory" group was anesthetized with an intraperitoneal injection of Avertin working solution (prepared according to Johns Hopkins ACUC guidelines) and kept in a supine position for 1 hour, followed by tissue collection. The "ambulatory" group was anesthetized with fast-acting inhaled isoflurane, which allowed the mice to wake up immediately and ambulate for 10 minutes, followed by tissue collection. Vaginal tissue was then collected, cut longitudinally, flattened, and clamped between two glass slides sealed with super glue. This procedure completely flattens the tissue, exposing the surface enclosed by the lumen. Tissues were imaged on an epifluorescence microscope (Nikon E6100) at 2× magnification. Doxorubicin is fluorescent (excitation/emission 470/590). Untreated control tissue was imaged to ensure that the fluorescence signal from doxorubicin was sufficiently higher than the tissue's autofluorescence. Low magnification captures a larger portion of the tissue, so only 2-3 images are needed to observe the entire tissue surface. Images were 'thresholded' to draw a region of interest boundary around the fluorescent signal, and the area covered was then quantified using ImageJ software. The average percent coverage was determined for each mouse, and these values were averaged across groups of n=5 mice.

To capture the distribution of MPPs due to immediate fluid absorption dynamics, 20 μl of isotonic (PBS) or hypotonic (ultrapure water) MPP solutions were administered vaginally. The higher solution volume helps ensure that the lumen will be filled with fluid and the particles are diluted (0.01% w/v) so that the concentration gradient will be visually apparent. Mice were anesthetized with isoflurane and sacrificed immediately after particle administration. Vaginal tissue was rapidly excised and quickly frozen in Tissue-Tek O.C.T compound. Cross-sectional slices were obtained at various points along the length of the tissue using a Leica CM-3050-S cryostat. The slice thickness was set to 6 μm to achieve a single cell layer thickness. The slices were then stained with ProLong Gold anti-fluorescence decay reagent and DAPI to observe cell nuclei and preserve particle fluorescence. Fluorescence images of the slices were obtained using an inverted epifluorescence microscope (Zeiss AxioObserver). For the distribution of MPPs at different solution osmolarities, particle solutions (0.08% w/v) were prepared from different ratios of PBS and ultrapure water. Mice were anesthetized with isoflurane and 5 μl of particle solution was administered vaginally. After 10 minutes, the tissues were collected, quickly frozen, sectioned, and stained according to the procedures outlined for observing fluid absorption dynamics. To quantify MPP vaginal tissue coverage, mice were anesthetized with isoflurane and 5 μl of nanoparticle solution was administered vaginally. Within 10 minutes, the tissues were excised, cut longitudinally, and then flattened as described for drug distribution experiments. Control tissues were imaged to ensure that the fluorescence signal was sufficiently higher than the tissue autofluorescence. Tissues were imaged using an inverted epifluorescence microscope (Zeiss Axio Observer) at 10× magnification, and 8 images were obtained for each tissue. Coverage was quantified as previously outlined for drug distribution experiments.

Drug and nanoparticle retention in the vagina

Doxorubicin was dissolved in phosphate-buffered saline (PBS) (iso-osmolarity relative to blood) or ultrapure water (hypo-osmolarity) at a concentration of 1 mg/ml. Mice were anesthetized with an intraperitoneal injection of afetin, followed by intravaginal administration of 5 μl of doxorubicin solution. The mice were kept in a supine position for 10 minutes to ensure that "dripout" of the solution would not affect retention measurements. The entire cervix was then excised and placed in a standard tissue culture dish. Fluorescence images of the tissue were acquired using a Xenogen IVIS Spectrum imaging device (Caripor Life Sciences). To account for potential differences in the fluorescence of the doxorubicin solutions, vials of both isosmolar and hypo-osmolar doxorubicin solutions were included in the images. The intensity ratio of the two solutions was used to normalize tissue fluorescence for each group. Quantification of fluorescence counts per unit area was calculated using Xenogen Living Image 2.5 software. The average values of the isosmolar and hypo-osmolar groups were normalized to the isosmolar group.

Red fluorescent MPPs were suspended at 0.2% (w/v) in PBS (iso-osmolarity) or ultrapure water (hypo-osmolarity). Mice were anesthetized with an intraperitoneal injection of afetin ("non-ambulatory") or by inhaled isoflurane ("ambulatory"), as previously described for drug distribution experiments. 5 μl of iso-osmolar or hypo-osmolar MPP solution was administered intravaginally. After 1 hour for non-ambulatory mice or 10 minutes for ambulatory mice, the entire cervix was excised and placed in a standard tissue culture dish. Fluorescence images were acquired and quantified as described for drug retention. As a reference point for MPP retention, 5 μl of MPP solution was carefully pipetted into the vagina after the entire cervix had been removed and placed in a tissue culture dish. This method was used for MPP (but not doxorubicin) because the nanoparticles do not penetrate tissue; therefore, differential tissue penetration has no potential effect on the fluorescence signal. Retention was then calculated as a percentage of the average reference tissue signal.

HSV-2 infection mouse model

Immediately before use, mice received 20 μl of 10 mg/ml acyclovir monophosphate dissolved in PBS (iso-osmolarity) or ultrapure water (hypo-osmolarity). This drug has been shown to provide partial protection in a mouse model of vaginal HSV-2 infection. Mice were dosed 1 minute or 60 minutes before the viral inoculum. Mice were then challenged with 10 μl of an inoculum containing HSV-2 strain G (ATCC #VR-724, 2.8×10 ⁷ TCID ₅₀ /ml). For protection studies, HSV-2 was diluted 10-fold in Bartel's medium to deliver 10 ID ₅₀ , a dose that typically infects 85%-90% of control mice. For osmotic-induced susceptibility testing, the virus was diluted 10 times with Bartell's medium and further diluted 10 times in Bartell's medium (isosmolarity) or deionized water (low osmolarity) to ID ₅₀ , a dose that infects half of the mice. Mice were assessed after three days of infection after inoculation by culturing PBS vaginal lavage on human foreskin fibroblasts (Diagnostic Hybrids, MRHF batch #440318W). In this model, if the input (challenge) virus is collected for more than 12 hours after the attack, it is no longer detectable in the lavage fluid.

statistics

The Wilcoxon rank-sum test was used to compare data sets. This test is nonparametric and more suitable for situations where a Gaussian distribution is not possible. For studies of HSV-2 infection, statistical significance was determined using Fisher's exact test (two-tailed distribution).

result

Effect of tension on vaginal drug distribution

When mice were non-ambulatory (supine for 1 hour), only 15% of the vaginal tissue area was covered by doxorubicin administered as an isotonic solution, while 88% was covered when administered as a hypoosmolar solution ( FIG. 5A ). When mice were ambulatory for 10 minutes, followed by tissue collection, the isotonic solution delivered doxorubicin to 25% of the vaginal surface area, while the hypoosmolar solution delivered doxorubicin to 86% of the area ( FIG. 5B ). The isotonic solution delivered the drug only to the vaginal surface facing the lumen, rather than to the surface contained within the folds of the vaginal folds, resulting in a "striped" pattern in which unexposed tissue appeared black. In contrast, the hypoosmolar solution distributed doxorubicin to the entire vaginal surface.

Effect of tension on vaginal drug retention

Isotonic and hypoosmolar doxorubicin solutions were administered intravaginally to anesthetized mice to avoid any solution leakage. After 10 minutes in the supine position, the entire reproductive tract was excised and quantitatively analyzed using fluorescence imaging. After only 10 minutes, the relative fluorescence signal of doxorubicin administered as a hypoosmolar solution was half that of the relative fluorescence signal of doxorubicin administered as an isotonic solution ( FIG. 6 ).

Effect of Tension on Preventing Vaginal HSV-2 Infection

The enhanced distribution of drugs administered hypotonically over a shorter period of time and the reduced retention over a longer period of time were tested to determine how they would affect the efficacy of drugs administered vaginally. Acyclovir monophosphate (ACVp, a suitable protective drug that provides partial protection when administered 30 minutes before the viral inoculum in a mouse vaginal HSV-2 infection model) was used because it would likely reveal the benefits of any improved distribution. Similar to doxorubicin, ACVp is water-soluble and acts intracellularly. First, it ensures that hypotonic fluids do not increase susceptibility to infection by administering an ID ₅₀ dose of virus suspended in an isotonic or hypotonic solution (usually infecting approximately 50% of mice). When the virus was administered in an isotonic or hypotonic solution, 60% (9/15) of the mice were infected. This control experiment indicates that the change in infection rate is due to the presence of the drug rather than relying on the hypotonic effect on virus or tissue susceptibility. For the protection study 10 ID ₅₀ , a dose that typically infects approximately 90% of the mice was used.

When 1% ACVp was administered 1 minute before the virus, 49% (22/45) of the mice were infected when ACVp was administered isotonically, and 31% (14/45) of the mice were infected when ACVp was administered hypotonically ( FIG. 7 ). Although this result suggests that hypotonic solutions may have increased vaginal protection, this difference was not statistically significant ( p = 0.1 ). When 1% ACVp was administered 1 hour before the virus, 45% (27/60) of the mice were infected when ACVp was administered isotonically, and 73% (33/45) of the mice were infected when ACVp was administered hypotonically ( FIG. 7 ). This difference was statistically significant, indicating that although hypotonic delivery may have improved immediate protection, osmotic flow through the epithelium appears to remove ACVp from the vagina, resulting in reduced protection.

Role of osmotic-driven convection in vaginal nanoparticle distribution

Hypotonic delivery of free drug results in improved distribution in the vagina, but osmotic absorption of the fluid can cause drugs that can permeate the vaginal epithelium to be removed by solvent drag. In contrast, the vaginal epithelium is essentially impermeable to nanoparticles, and if it is mucus permeable, osmotic flow delivers them to the epithelial surface. MPPs are able to permeate vaginal mucus, coating the entire vaginal surface within 10 minutes after delivery as a hypotonic solution. Even when delivered as a hypotonic fluid, mucoadhesive nanoparticles (CPs) still accumulate in the luminal mucus layer and are not transported to the vaginal epithelium via vaginal mucus by osmotic convection. The dependence of osmotic-driven MPP distribution on tonicity was determined immediately after administration. By freezing the vaginal tissue immediately after MPP administration, a "snapshot" of the initial particle distribution dynamics can be taken.

When MPPs were administered in an isotonic solution, the nanoparticles were found to be distributed throughout the lumen, but when delivered in a hypotonic solution, a gradient of particle concentration was evident, and the MPPs were concentrated at the surface of the vaginal epithelium. From the surface distribution of the MPPs, it was apparent that hypotonicity-induced fluid flow caused the MPPs to be rapidly attracted to the vaginal surface, with no transepithelial absorption.

Effect of tension on vaginal nanoparticle retention

It was hypothesized that vaginal retention of MPP would be improved under hypotonic delivery conditions compared to free drug administered in a hypotonic solution. Since fluid leakage was expected to play a role in nanoparticle retention, both non-ambulatory and ambulatory conditions were compared. In non-ambulatory mice (supine for 1 hour), 69% of MPP administered in an isotonic solution and 83% of MPP administered in a hypotonic solution were retained ( FIG8A ). It is likely that the 1-hour period allowed for fluid absorption and the removal of gravity, reducing leakage. Although a higher percentage of MPP was retained when administered in a hypotonic solution, the difference was not statistically significant.

However, when the mice were ambulatory, the retention of MPP administered in isotonic solution was significantly reduced. After 10 minutes of ambulation, only 22% of MPP administered in isotonic solution was retained, compared to 75% of MPP administered in hypotonic solution (Figure 8B). It seems that the rapid delivery of MPP to the vaginal surface causes increased retention because much of the fluid in the vagina is quickly discharged during ambulation.

Effect of osmolarity on vaginal nanoparticle distribution

It is well known that tension can strongly affect cells, and recent evidence has highlighted the toxicity of strong hypertonic gels to both vaginal and rectal epithelia, especially under repeated exposure. Hypotonic solutions can also cause toxicity. To avoid potential toxic effects, it was investigated whether moderate levels of hypotonicity could still improve particle distribution in the vagina. Within 10 minutes of administration, MPP in the form of hypotonic solutions covering a 10-fold range (20-220 mOsm/kg) delivered MPP to the vaginal surface. Compared to isotonic solutions (294 mOsm/kg) that distributed MPP particles throughout the vaginal lumen and were rarely attracted to the epithelial surface, moderately hypotonic solutions (220 mOsm/kg) caused rapid transport to the epithelial surface. Furthermore, there was a trend toward increased vaginal coverage with increasing hypotonicity: in the most minimally osmotic solution (20 mOsm/kg), MPP coated 88% of the vaginal epithelium after 10 minutes, whereas only 60% of the vaginal surface was coated with MPP administered as a 294 mOsm/kg solution ( FIG9 ). At all tested tensions, MPP reached a greater portion of the epithelial surface than sticky-adhesive (CP) particles, with the least hypotonic solution (220 mOsm/kg) delivering particles to 76% of the vaginal surface, a significant increase compared to the 60% coverage of the isotonic solution (294 mOsm/kg).

In summary, studies investigating the use of hypotonic solutions for convection-enhanced drug delivery to the vagina have shown that while hypotonic delivery of doxorubicin improves vaginal distribution, the drug is absorbed through the epithelium, reducing vaginal retention. In contrast, hypotonic delivery of inert mucus-permeable nanoparticles (MPPs) has been found to improve both distribution and retention. In addition, even minimal hypotonic delivery has been found to significantly improve vaginal distribution of MPPs. The results indicate that hypotonic formulations are more effective for drug delivery to the vagina than traditional hypertonic formulations, and that hypotonic-delivered MPPs offer important prospects for non-toxic, sustained drug delivery to the entire vaginal surface.

Although the distribution of drugs in the form of hypotonic solutions is improved, fluid absorption can potentially cause the drug to be quickly removed by fluid transport via the vaginal epithelium. Using acyclovir monophosphate (ACVp), the trend of improved protection provided by ACVp in the form of hypotonic solutions administered vaginally immediately before the HSV-2 virus was confirmed. The improved protection is likely due to the increase in vaginal coverage of drugs administered in the form of hypotonic solutions. In contrast, when HSV-2 was administered 1 hour after the drug, the protection provided by ACVp administered in the form of hypotonic solutions was significantly reduced compared to ACVp in the form of isotonic solutions. Drug absorption across the epithelium under osmotic-induced fluid flow causes drug clearance and protection reduction after 1 hour. MPP provides a way to achieve improved drag distribution by hypotonic delivery because nanoparticles accumulate on the surface of the vaginal epithelium, and MPP containing ACVp shows better protection when administered vaginally 30 minutes before the HSV-2 virus compared to 10 times higher concentrations of free drugs. MPPs are immediately attracted to the vaginal epithelium when administered as a minimally hypotonic solution.

Fluid absorption and secretion can also affect vaginal retention. It has been previously shown that the leakage rate of vaginal gels in humans increases linearly with increasing hyperosmolarity (Zeitlin et al. Contraception 68, 139-155 (2003)). In this case, retention is reduced by osmotic-induced fluid secretion, causing product leakage. This indicates that hypotonic products will cause reduced fluid absorption and leakage, and may thereby improve vaginal retention. It has been found that this is not necessarily true for drugs that can be absorbed through the vaginal epithelium (unlike MPPs). MPPs are retained longer than mucoadhesive CPs by penetrating into the more slowly cleared mucus layer deep in the vaginal folds. As shown by this study, MPPs are better retained in the vagina of ambulatory mice when administered as a hypotonic solution, as compared to when administered as an isotonic solution, by reducing vaginal leakage. The results indicate that hypotonic gel formulations containing MPPs will likely promote both drug distribution and retention in the human vagina.

Example 9: Comparison of the Effect of Osmolality on Particle Absorption in the Colon

Materials and methods

Studies were conducted as described in Examples 7 and 9 to compare the distribution of MPPs in solutions with varying tonicities in the mouse colon. The studies compared particle absorption in the colon at 20 mOsm, 260 mOsm, 350 mOsm, 450 mOsm, 860 mOsm, and 2200 mOsm solutions. The only reason epithelial convective transport was achieved with a high osmolarity solution was if it was high osmolarity in the presence of Na.

The distribution of CP and MPP of various sizes was also determined after rectal co-administration to mice with TNBS-induced colitis. Fluorescent images of flattened colonic tissue were analyzed after hypotonic rectal administration of a solution containing a mixture of CP (red) and MPP (green) of various sizes (100 nm, 200 nm, 500 nm). Cross-sectional colon cryosections of 200 nm CP and MPP were also prepared (nuclei were stained blue with DAPI).

result

The effective osmolality of the colon appears to be between 400 and 530 mOsm/kg, which is higher than the plasma osmolality (approximately 300 mOsm/kg). This higher range is supported by Billich and Levitan, J Clinical Invest (1969). Vehicles with osmolality of 400 mOsm/kg and below provide improved distribution of MPPs on the surface of colonic tissue.

Example 10: Determination of Toxicity of Hypotonic MPP Formulations

Recent studies indicate that in response to certain vaginal products, the vaginal epithelium can secrete immune mediators that can enhance susceptibility to sexually transmitted infections. Other conditions, such as premature birth, have also been associated with genital tract inflammation. Therefore, it is important that vaginal products do not induce such an immune response, especially after repeated administration. High osmolarity formulations have previously been shown to be toxic to the vaginal and rectal epithelium, which can negate the protective or therapeutic benefits of the administered drug. Therefore, this study was conducted to ensure that low osmolarity formulations did not cause toxicity when administered to the mucosal epithelium.

Materials and methods

Seven daily treatments with pure hypotonic (approximately 20 mOsm/kg) fluid (with Pluronic F127 or MPP) and standard isosmolar concentration of hydroxyethylcellulose were compared to no treatment. A hypertonic gel (vehicle containing 20% glycerol, used in clinical trials) was compared to a conventional hypertonic gel formulation (HEC gel with a 20% increase in water content to offset water absorption) containing MPP based on vaginal cross-sectional images.

DP mouse models were administered 20 μL of each test agent intravaginally once daily for seven days. HEC gel and N9 were provided by T. Moench (Reprotect), and TFV vehicle gel was kindly provided by C. Dezzutti (University of Pittsburgh). On day eight, each mouse was lavaged twice with 50 μL of PBS. Each lavage sample was further diluted with 200 μL of PBS and centrifuged to remove the mucus plug. The supernatant (200 μL) was removed and aliquoted into 50 μL for each of four Quantikine ELISA kits (R&D Systems) (IL-1β, IL-1α, TNF-α, and IL-6). ELISA was performed according to the manufacturer's instructions.

result

Seven daily treatments with pure hypotonic (approximately 20 mOsm/kg) fluid (carrying Pluronic F127 or MPP) and standard isosmolar concentrations of hydroxyethylcellulose did not result in increases in vaginal cytokines (IL-1α/β) compared to no treatment. A hypertonic gel (a vehicle containing 20% glycerol, used in clinical trials) resulted in significant increases in these vaginal cytokines. Based on vaginal cross-sectional images, a hypotonic gel formulation containing MPP was compared to a conventional hypertonic gel formulation. It appeared that after 6 hours, vaginal distribution and retention of MPP were improved with a hypotonic gel (81% retention after 6 hours) (HEC gel in which the water content was increased by 20% to offset water absorption) compared to a hypertonic gel formulation (approximately 20% retention after 6 hours) (HEC gel).

Claims (20)

1. A hypotonic formulation comprising mucus-permeable nanoparticles, said nanoparticles comprising: Enhanced nanoparticle diffusion via a mucus-permeable enhanced coating, the coating comprising polyethylene glycol or a block copolymer containing polyethylene glycol; and Therapeutic agents, preventative agents, or diagnostic agents; The surface density of the mucus-penetrating enhanced coating on the nanoparticles is 0.05 to 100 chains per square nanometer when measured by ^1H NMR. The zeta potential of the nanoparticles suspended in a 10 mM NaCl solution is between 10 mV and -10 mV. The nanoparticles are used to deliver the excipients to mucosal tissue, wherein the excipients are hypotonic to the mucosal epithelium to which they are delivered, resulting in the rapid delivery of the mucosal nanoparticles to the epithelial surface. 2. The formulation of claim 1, wherein the nanoparticles provide sustained mucosal delivery of the agent. 3. The formulation according to claim 1, wherein the mucus-permeable nanoparticles comprise polymeric nanoparticles, the polymeric nanoparticles comprising a core polymer and a mucus-permeability-enhancing coating on their surface, and the nanoparticles containing the therapeutic, preventive, or diagnostic agent for administration to mucosal tissues. 4. The formulation of claim 3, wherein the slime penetration enhancement coating is covalently bonded to the core polymer, wherein the core polymer is a block copolymer containing one or more blocks of the slime penetration enhancement coating, or wherein the core polymer comprises a single block of slime penetration enhancement coating material covalently bonded to one end of the core polymer. 5. The formulation according to claim 1, wherein the nanoparticles comprise the nanoparticles of the therapeutic, preventive, or diagnostic agent for administration to mucosal tissues coated with the mucus penetration-enhancing coating. 6. The formulation according to claim 1, wherein the polyethylene glycol has a molecular weight of 1 kD to 100 kD and the density of the polyethylene glycol is 0.05 to 0.5 chains per square nanometer when measured by ^1H NMR. 7. The formulation according to any one of claims 1 to 6, wherein the mucus penetration-enhancing coating is present in an amount that effectively neutralizes or substantially neutralizes the surface charge of the nanoparticles. 8. The formulation according to any one of claims 1 to 6, for application to the vagina, having an osmolar concentration between 20 and 220 mOsm/kg. 9. The formulation of claim 8, wherein the mucus-permeable nanoparticles comprise an effective amount of a therapeutic agent for vaginal delivery. 10. The formulation according to any one of claims 1 to 6, for application to the colon or rectum, wherein the osmolality is between 20 mOsm/kg and 450 mOsm/kg, and wherein sodium ions constitute at least 30% of the osmolality exceeding 220 mOsm/kg. 11. The formulation of claim 10, wherein the mucus-permeable nanoparticles comprise an effective amount of a therapeutic agent for administration into the rectum and/or colon. 12. The formulation according to any one of claims 1 to 6, wherein the formulation is selected from the group consisting of: solutions, suspensions, gels, ointments, creams, lotions, tablets or capsules, and powders. 13. The use of the formulation according to any one of claims 1 to 6 in the preparation of a medicament for administration to humans or animals in need of one or more therapeutic agents, preventive agents and/or diagnostic agents. 14. The application according to claim 13, wherein the preparation is administered orally. 15. The application according to claim 13, wherein the preparation is administered to the eye or adjacent tissue or compartment thereof. 16. The application according to claim 13, wherein the formulation is administered locally. 17. The application according to claim 13, wherein the formulation is administered into the lung tract. 18. The application according to claim 13, wherein the preparation is administered intranasally. 19. The application according to claim 13, wherein the preparation is administered to the rectum or colon. 20. The application according to claim 13, wherein the formulation is administered buccally, sublingually, or orally.