Mucosal preparations and uses thereof

Abstract

The present invention features a mucosal coating composition comprising (i) water, (ii) at least one mucoadhesive polymer / polysaccharide (e.g., at a concentration of 0.1-20% w / v), and (iii) at least one surfactant (e.g., at a concentration of 0.005-5% w / v), which exhibits prolonged residence time in mucosal tissues and prophylactic protection against transmucosal pathogens. TIFF2025527267000002.tif80156

Description

[Background technology]

[0001] Background of the Invention Over the past few decades, numerous cases of mucosal disease outbreaks, particularly those caused by respiratory pathogens, have occurred, often leading to epidemics or unexpected pandemics, such as COVID-19. These pathogens are common causes of upper and lower respiratory tract infections, for example. One of the main modes of pathogen transmission is through the inhalation of contaminated respiratory droplets and their subsequent deposition in the nasal cavity. Most respiratory viruses have an entry checkpoint in the nasal cavity due to their tissue tropism and receptors. For example, the SARS-CoV-2 virus binds to the angiotensin-converting enzyme receptor located on goblet cells via its receptor-binding domain (RBD), continuing its replication cycle and spreading infection in the respiratory tract. Therefore, there is a need in the art for nasal formulations to combat respiratory pathogens. Summary of the Invention

[0002] The present invention features a mucosal coating composition that includes (i) water, (ii) at least one mucoadhesive polymer / polysaccharide, and (iii) at least one surfactant.

[0003] The present invention further features a mucosal coating composition comprising: (i) water; (ii) at least one mucoadhesive polymer / polysaccharide at a concentration of 0.1-20% w / v (e.g., at a concentration of 1±0.5, 3±2, 7±2.5, 10±2.5, 15±2.5, or 17.5±2.5% w / v); and (iii) at least one surfactant at a concentration of 0.005-5% w / v (e.g., at a concentration of 0.1±0.05, 0.25±0.15, 0.5±0.25, 1±0.25, 1.5±0.5, 2±1, or 3±2% w / v).

[0004] In certain embodiments of the above compositions, after application to a surface, the coating composition forms a barrier that reduces small molecule transport by at least 90% (e.g., at least 95%, 99%, or 99.9%) over a period of 4 hours.

[0005] In specific embodiments of the above compositions, after application to a surface, the coating composition forms a barrier that reduces viral transport by at least 90% (e.g., at least 95%, 99%, or 99.9%) for a period of 4 hours.

[0006] In certain embodiments of the above compositions, after application to a surface, the coating composition increases the capture of atomized particles by at least 1.5-fold (eg, at least 2-fold, 3-fold, or 5-fold).

[0007] In specific embodiments of the foregoing compositions, after application to a surface, the coating composition increases the residence time of the coating, resulting in at least 2-fold (e.g., at least 5-fold, 10-fold, 15-fold, or 30-fold) more coating remaining 8 hours after application compared to a control coating identical in composition except that it lacks surfactant.

[0008] In some embodiments of the above compositions, the mucoadhesive polymer / polysaccharide is present in a concentration of 0.25-20% w / v, hi other embodiments of the above compositions, the mucoadhesive polymer / polysaccharide is present in a concentration of 0.75-20% w / v.

[0009] In some embodiments of the above compositions, the surfactant is present at a concentration of 0.01-5% w / v. In other embodiments of the above compositions, the surfactant is present at a concentration of 0.05-5% w / v. In certain embodiments of the above compositions, the surfactant is present at a concentration of 0.5-5% w / v.

[0010] The present invention features a mucosal coating composition comprising (i) water, (ii) at least one mucoadhesive polymer / polysaccharide at a concentration of 0.1-10% w / v (e.g., at a concentration of 0.5±0.25, 3±2, 5±2.5, or 7.5±2.5% w / v), and (iii) at least one surfactant at a concentration of 0.005-5% w / v (e.g., at a concentration of 0.1±0.05, 0.25±0.15, 0.5±0.25, 1±0.25, 1.5±0.5, 2±1, or 3±2% w / v), wherein the composition is a sprayable solution.

[0011] In any of the above compositions, at least one mucoadhesive polymer / polysaccharide may contain a carboxyl group, a hydroxyl group, a sulfate group, or an acetamide group. For example, at least one mucoadhesive polymer / polysaccharide may be selected from gellan, pectin, HPMC, CMC, xanthan, chondroitin sulfate, alginic acid, hyaluronic acid, and salts or derivatives thereof. In one specific embodiment, at least one mucoadhesive polymer / polysaccharide comprises a polymer of glucuronic acid or galacturonic acid (e.g., pectin, gellan, HPMC, CMC, xanthan, and alginic acid, and salts or derivatives thereof).

[0012] In any of the above compositions, the at least one surfactant may be (a) a hydrophilic surfactant, and (b) a nonionic surfactant or a cationic surfactant. For example, the at least one surfactant may be selected from polysorbate surfactants, sorbitan fatty acid ester surfactants, and benzalkonium chloride. The at least one surfactant may be selected from polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene 20 sorbitan monopalmitate, polyoxyethylene 20 sorbitan monostearate, and polyoxyethylene 20 sorbitan monooleate.

[0013] In any of the foregoing compositions, the composition may further comprise a pathogen-neutralizing agent (e.g., a surfactant, alcohol, an antibacterial agent, and / or an antiviral agent).

[0014] In any of the foregoing compositions, the composition may further comprise a mucoadhesive polysaccharide / polymer possessing antiviral activity. In one specific embodiment, the mucoadhesive polysaccharide / polymer possessing antiviral activity is pectin.

[0015] In any of the foregoing compositions, the composition may further comprise an alcohol containing a tertiary or aromatic hydroxyl group (eg, phenethyl alcohol, benzyl alcohol, or chlorobutanol).

[0016] The present invention features a composition including: (i) water; (ii) at least one mucoadhesive polymer / polysaccharide selected from gellan, pectin, and combinations thereof; (iii) at least one surfactant selected from benzalkonium chloride, polyoxyethylene 20 sorbitan monooleate, and combinations thereof; and (iv) phenethyl alcohol.

[0017] In specific embodiments of the above compositions, the at least one mucoadhesive polymer / polysaccharide comprises gellan at a concentration of 0.1-10% w / v (e.g., at a concentration of 0.5±0.25, 3±2, 5±2.5, or 7.5±2.5% w / v).

[0018] In certain embodiments of the above compositions, the at least one mucoadhesive polymer / polysaccharide comprises pectin at a concentration of 0.5-10% w / v (e.g., at a concentration of 0.75±0.25, 3±2, 5±2.5, or 7.5±2.5% w / v).

[0019] In some embodiments of the above compositions, the at least one surfactant comprises polyoxyethylene sorbitan monooleate at a concentration of 0.01 to 0.5% w / v (e.g., a concentration of 0.025±0.015, 0.075±0.025, 0.1±0.025, 0.25±0.1, 0.35±0.1, or 0.4±0.1% w / v).

[0020] In certain embodiments of the above compositions, the at least one surfactant comprises benzalkonium chloride at a concentration of 0.01 to 1% w / v (e.g., a concentration of 0.025±0.015, 0.075±0.025, 0.1±0.025, 0.25±0.1, 0.35±0.1, 0.5±0.25, or 0.75±0.25% w / v).

[0021] In some embodiments of the above compositions, the composition comprises 0.25 to 1% w / v (e.g., 0.35±0.1, 0.45±0.1, 0.5±0.25, or 0.75±0.25% w / v) phenethyl alcohol.

[0022] In a specific embodiment of the above composition, the composition comprises 0.2% w / v gellan, 0.75% w / v pectin, 0.05% w / v polyoxyethylene 20 sorbitan monooleate (Tween® 80), 0.01% w / v benzalkonium chloride, and 0.25% w / v phenethyl alcohol.

[0023] The compositions of the present invention may exhibit a residence time of 4 to 8 hours when applied to the mucous membranes of the nasal cavity.

[0024] In certain embodiments of the above compositions, the composition has a viscosity of 0.01 to 1 Pa·s. In other embodiments of the above compositions, the composition has a viscosity of 0.01 to 0.1 Pa·s.

[0025] In some embodiments of the above compositions, the at least one mucoadhesive polymer / polysaccharide comprises a polysaccharide having an average MW in the range of 10,000 to 2,000,000 Da. In other embodiments of the above compositions, the at least one mucoadhesive polymer / polysaccharide comprises a polysaccharide having an average MW in the range of 50,000 to 500,000 Da.

[0026] In certain embodiments of the above compositions, the formulation contains less than 0.1% (w / w) solid particles.

[0027] In some embodiments of the above compositions, the formulation further comprises a therapeutic or diagnostic agent (eg, an analgesic, an anti-inflammatory, an antihistamine, naltrexone, or melatonin).

[0028] In certain embodiments of the above compositions, the at least one mucoadhesive polymer / polysaccharide comprises gellan at a concentration of 0.1-0.4% w / v to achieve a sprayable formulation that forms a mucosal coating that reduces viral transport by at least 90% over a 4 hour period.

[0029] In some embodiments of the above compositions, to achieve a sprayable formulation with greater than 90% pathogen neutralization, the at least one mucoadhesive polymer / polysaccharide comprises pectin at a concentration of 0.25% to 2% w / v. For example, to achieve greater than 99% pathogen neutralization, the composition may comprise 0.75±0.05% w / v pectin and benzalkonium chloride at a concentration of 0.1% or less.

[0030] In certain embodiments of the compositions, the at least one surfactant comprises polyoxyethylene sorbitan monooleate at a concentration of 0.01-0.05% w / v to achieve a formulation that forms a mucosal coating that enhances respiratory droplet capture by more than three-fold and increases nasal residence time compared to uncoated surfaces. For example, the mucosal coating enhances respiratory droplet capture by more than 15-fold 8 hours after application compared to an identical coating without the surfactant. The mucosal coating can be formulated to be non-toxic to either mucosal membranes or epithelia.

[0031] In some embodiments of the compositions, at least one surfactant comprises benzalkonium chloride at a concentration of 0.005-0.02% w / v to achieve a formulation that achieves greater than 90% pathogen neutralization and forms a mucosal coating that is non-toxic to mucosa and epithelia. For example, to achieve greater than 99% pathogen neutralization, the composition may comprise 0.010±0.005% w / v benzalkonium chloride and a gellan concentration of 0.2% w / v or less. In another embodiment, to achieve greater than 99% pathogen neutralization, the composition comprises 0.010±0.005% w / v benzalkonium chloride and a pectin concentration of 1% w / v or less.

[0032] The invention features a method for reducing the risk of exposure to an infectious pathogen at a mucosal membrane of a subject, comprising topically applying a composition of the invention to the mucosal membrane of the subject. The method can include applying the composition to the oral cavity, throat, vagina, nasal cavity, anus, or a wound of the subject.

[0033] The invention further features a method of reducing the risk of exposure to an infectious pathogen in a subject, comprising topically applying to the skin of the subject a composition of the invention.

[0034] In specific embodiments of the above methods, the subject is a mammal (e.g., a human, a dog, a cat, a horse, or a livestock animal). In some embodiments of the above methods, the formulation further comprises a therapeutic or diagnostic agent (e.g., an analgesic, an anti-inflammatory, an antihistamine, naltrexone, or melatonin).

[0035] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. [Brief explanation of the drawings]

[0036] [Figure 1]Figure 1 shows a schematic diagram of the functional features of the Pathogen Capture and Neutralizing Spray (PCANS). PCANS is a "drug-free" formulation containing diverse classes of compounds, including biopolymers, surfactants, and alcohols, that are listed in the U.S. Food and Drug Administration's (FDA) inactive ingredient database (IID) or generally recognized as safe (GRAS) list and are present as excipients in commercially available nasal / topical formulations. Aqueous solutions of PCANS administered using a pocket-sized nasal device undergo a phase transition to form a hydrogel layer on the nasal epithelium. PCANS, composed of mucopolysaccharides, surfactants, and alcohols, interacts with divalent ions in nasal fluid and entangles with mucin chains, forming a cross-linked layer. PCANS enhances the capture of pathogen-laden respiratory droplets from inhaled air by preventing rebound. PCANS achieves this by reducing the interfacial tension of the nasal lining, similar to that of pulmonary surfactant in the alveoli. Second, PCANS forms a physical barrier on the nasal mucosa to prevent pathogen invasion / colonization. Finally, PCANS consists of a "non-drug" agent that rapidly neutralizes trapped pathogens. [Figure 2]Figures 2a-2k are a set of graphs illustrating the results from a biopolymer screen to identify sprayable concentrations and also determine which biopolymers form an efficient physical barrier against pathogen penetration. Viscosity as a function of shear rate up to 40 s-1 at 25 °C for various concentrations of (a) gellan, (b) pectin, (c) hydroxypropyl methylcellulose (HPMC), (d) carboxymethylcellulose (CMC), (e) Carbopol, and (f) xanthan gum in water. The sprayable viscosity window is shown below the dashed line. (g) Storage modulus (G') of 0.4% (w / v) gellan, 2% (w / v) pectin, 0.5% (w / v) HPMC, 0.5% (w / v) CMC, 0.2% (w / v) Carbopol, and 0.2% (w / v) xanthan gum with and without simulated nasal fluid (SNF). Amplitude sweep measurements were performed at 37°C by varying the oscillatory strain from 0.005% to 10% at a frequency of 1 Hz. ****P<0.0001, *P<0.05. ns, non-significant. (h) The amount of influenza A virus (IAV) passing through cell strainers (approximately 70 μm pore size) coated with simulated nasal fluid (SNF) or a simulated mucus / SNF or biopolymer / SNF mixture within 4 h. Viral particle passage was quantified by assessing the viral titer in the chamber below the strainer using a plaque assay performed in MDCK host cells. Results are expressed as plaque-forming units (PFU / mL). **P<0.01, *P<0.05 (compared to mucus / SNF). ns, non-significant. Passage rate of the fluorescent dye rhodamine B isothiocyanate through (i) cell strainers coated with SNF or cell strainers coated with simulated mucus / SNF or biopolymer / SNF mixtures (****P<0.0001 compared to mucus / SNF), and (j) strainers coated with SNF or gellan / SNF at various gellan concentrations (****P<0.0001 compared to 0.05% w / v gellan / SNF).(k) Drip length ratio of free brilliant green dye or mucoadhesive polymer mixed with brilliant green dye in porcine mucosal tissue. Drip length from the sprayed area was measured as the distance traveled by the biopolymer or free dye from the point of deposition over 4 hours. The drip length ratio for each biopolymer was calculated relative to the drip length of the free dye. ****P<0.0001 (compared to free dye). For g and h, P values were determined using two-way ANOVA with Tukey's multiple comparison test. For i–k, P values were determined using one-way ANOVA with Tukey's post hoc analysis. Data for a–f are from a single experiment (triplicates). Data for g–k are mean ± SD of technical replicates (n=3, each experiment performed at least twice). [Figure 3] Figures 3a-3c show that the in situ gelation and mucoadhesive characteristics of gellan combined with pectin are not altered. (A) Storage modulus of 0.2% w / v gellan, 0.75% w / v pectin, and their combinations, without and with simulated nasal fluid (SNF). (B) Drip length modulus of mucoadhesive polymers in porcine mucosal tissue. (C) Photographic images illustrating the drip length of various mucoadhesive polymers or their combinations mixed with dyes sprayed onto the mucosa. Drip length was measured from the distance traveled by the dyed formulation from the target area indicated by the red circle. [Figure 4] Figure 4 shows that the physical barrier properties against the passage of influenza A virus or free dye were evaluated by coating a mucoadhesive polymer onto a cell strainer. After 4 hours, the amount of virus that passed or the diffused dye (fluorescence intensity) was quantified from the acceptor compartment. [Figure 5] Figures 5a-5d show the concentration-dependent effect of gellan on viscosity and spray characteristics. (a) Viscosity measured as a function of shear rate up to 40 s at 25 °C for various concentrations of gellan. Quantitative measurements of (b) plume angle and (c) spray coverage were performed to identify the gellan concentration with the greatest coverage. (d) Representative images of plume angle and spray pattern. [Figure 6]Figures 6a–6u are a set of graphs and schematic diagrams showing results from screening mucopolysaccharides, surfactants, and alcohols from the IID and GRAS inventories to identify effective neutralizing agents against respiratory pathogens. (a) The table summarizes the various components and their concentrations used to determine their neutralizing potency against respiratory pathogens. Each component was individually evaluated for virus neutralization potency. (b, g) Viral loads of IAV and SARS-CoV-2 in host cells after 10 or 60 minutes of incubation with various biopolymers, (c, h) various surfactants, and (d, i) various alcohols. Viable virus titers were quantified using a plaque assay in MDCK host cells for IAV and a focus-forming assay in Vero E6 cells for SARS-CoV-2 virus. Results are expressed in plaque-forming units (PFU / mL) or focus-forming units (FFU / mL). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 (compared to 10 min incubation with PBS). ns, non-significant. (e) Viral load in host cells after 10 min incubation of IAV and (j) SARS-CoV-2 with various concentrations of pectin and BKC, respectively. **P<0.01, *P<0.05 (compared to PBS). (f) Viral load in host cells after 10 min incubation of IAV and (k) SARS-CoV-2 with pectin (0.75% w / v) + polyethyleneimine and BKC (0.01% w / v) + bovine serum albumin, respectively. **P<0.01 (compared to PBS). ns, non-significant. (l) Pectin (yellow) binds to the receptor-binding site of IAV (purple) in the distal part of the hemagglutinin monomer (purple) through hydrophobic interactions with Ser227 and Glu190, and hydrogen bonds with Ser228, Ser186, and Thr187. Blue and red dots in the hydrogen bond map represent carbon and oxygen atoms, respectively. (m) Chemical interactions of BKC (green) with the ACE2-binding motif (red) in the spike protein of SARS-CoV-2.The interaction map reveals hydrogen bonds between BKC and Tyr505 and Gly496. (n) Aromatic π-π interactions between BKC (green) and the Phe23 (purple) and Thr11 (brown) membrane helices within the transmembrane domain. Interaction analysis shows 10 hydrophobic bonds with Phe23 and 8 hydrophobic bonds with Phe26. (o) Viral load in host cells after incubating IAV with pectin (0.75% w / v) for 10 minutes in the presence of various concentrations of BKC. ****P<0.0001 (compared to PBS). Viral load in host cells after incubating SARS-CoV-2 with BKC (0.01% w / v) for 10 minutes in the presence of various concentrations of (p) gellan and (q) pectin. ****P<0.0001 (compared to PBS). Effect of detergents (r,t) and alcohols (s,u) on the Gram-negative bacteria Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) using the colony-forming unit (CFU) plate count method after 30 and 60 minutes of exposure. Viable bacterial colonies are expressed as CFU / mL. *P<0.05 (compared to PBS). For b–d, P values were determined using two-way ANOVA with Tukey's post-hoc analysis. For e, f, j, k, o–q, P values were determined using one-way ANOVA. Data for b–k and o–u are presented as mean ± SD of technical replicates (n=3, each experiment performed at least twice). Ser, serine; Thr, threonine; Glu, glutamic acid; Phe, phenylalanine; Tyr, tyrosine; Gly, glycine. [Figure 7] Figure 7 shows the interaction analysis of sialic acid with the receptor-binding domain of influenza virus A. Sialic acid (yellow) binds to the receptor-binding site of the hemagglutinin monomer (purple) through hydrophobic interactions with Ser227 and Glu190 and hydrogen bonds with Ser288, Ser186, and Thr187. [Figure 8]Figures 8a–8k are a set of schematics, graphs, and micrographs demonstrating the enhanced capture of respiratory droplet-mimicking aerosols and the prolonged residence time of PCANS in the nasal cavity. (a) Experimental design for measuring the capture of respiratory droplet-mimicking aerosols. A twin impinger was used to simulate the aerodynamics of the human airway. Mucus or a solution of gellan (0.02% w / v) and pectin (0.75% w / v) (G+P) without or with various concentrations of Tween-80, Tween-20, or BKC was coated onto the inner surface of the throat region of the impinger using a nasal drop device. Droplets with an aerodynamic mass median diameter greater than 5 μm and loaded with rhodamine B-loaded liposomes (approximately 400 nm in size) were generated using a jet nebulizer and administered to the impinger under vacuum (15 L / min). Droplet capture was determined by quantifying the fluorescence intensity of rhodamine B within the biopolymer / surfactant mixture or mucus layer. (b) Fold increase in fluorescence intensity compared to mucus. ****P<0.0001, *P<0.05 (compared to mucus). (c) Transepithelial electrical resistance (TEER) across human nasal epithelial cell (RPMI-2650)-based monolayers at various time points after treatment with medium alone or medium containing Triton-X (0.1% w / v) or various concentrations of Tween-80. After 4 hours, the surfactant-containing medium was replaced with fresh medium, and impedance recovery was examined. ****P<0.0001, ***P<0.001 (compared to untreated control). ns, non-significant. (d) Experimental design for measuring respiratory droplet-mimicking aerosol capture using a 3D-printed human nasal cavity model (Koken cast). The inner surface of the nasal cavity was coated with mucus, G+P solution, or PCANS (final formulation) using a nasal drop device. The model throat was connected to a vacuum pump to simulate respiratory airflow (15 L / min). The nostrils were then exposed to atomized rhodamine B-loaded liposomes for 1 minute. Droplet capture was determined by quantifying the fluorescence intensity of rhodamine B in the nasal cavity. (e) Fold increase in fluorescence intensity compared to mucus. **P<0.01 (compared to mucus). ns, non-significant.(f) Experimental outline for evaluating the nasal residence time of PCANS in mice. 10 μL of free DiR or DiR-loaded PCANS (PCANS / DiR) was administered intranasally to each nostril of C57Bl / 6 mice. Mice were euthanized at different time points over 24 h, and nasal cavities were collected and imaged using an in vivo imaging system (IVIS). (g) Representative images of excised nasal cavities at different time points. (h) Quantification of intranasal fluorescence intensity at different time points. (i) Fold change in total flux after 8 h in the nasal cavity compared to G+P. *P<0.05 (compared to G+P). ns, non-significant. (i) Experimental design for determining the biocompatibility of PCANS in the mouse nasal cavity. 10 μL of PCANS or PBS was administered to each nostril of C57Bl / 6 mice once daily for 14 consecutive days. Animals were euthanized on day 15, and nasal cavities were analyzed histologically. (j) Representative images of H&E-stained sections of nasal turbinates from mice taken with a 4x objective. The insets show healthy olfactory epithelium (i) and (iii) and lamina propria (ii) and (iv) taken with a 20x objective. For b, e, and i, P values were determined by one-way ANOVA with Tukey's post-hoc analysis. For b, each surfactant concentration was compared individually. For c, P values were determined by two-way ANOVA with Tukey's multiple comparison test. Data for b, c, and e are presented as mean ± SD of biological replicates (n = 3, each experiment performed at least twice). Data for h and i are presented as mean ± SEM (n = 5 mice / group). [Figure 9] 9a-9b show the effect of tween-80 concentration on cell viability after 24 and 48 hours in human nasal epithelium (RPMI-2650). [Figure 10] 10a-10b show the in vitro release kinetics of (A) tween-80 and (B) IgG and lysophosphatidylcholine (LPC) from a nasal formulation composed of 0.2% w / v gellan and 0.75% w / v pectin. [Figure 11]Figures 11a-11b show nasal retention of NIR dye-loaded formulations. (A) Concentration-dependent effect of Tween-80 on the retention time of nasal formulations composed of gellan and pectin 8 hours after administration. (B) Screening of nonionic surfactant (Span-20) and cationic surfactant (BKC) on formulation retention after 8 hours. Total flux measured from the nasal cavity. [Figure 12] Figure 12 shows in vivo imaging demonstrating the biodistribution of DiR-loaded PCANS. The left panel shows the localization of the formulation in the nasal cavity after 2 hours, and the right panel shows the systemic clearance of PCANS after 24 hours. Lu - lung, H - heart, St - stomach, Li - liver, Sp - spleen, and K - kidney. [Figure 13]Figures 13a-13s are a set of graphs and schematic diagrams showing that multifunctional PCANS exhibit barrier properties, broad-spectrum pathogen neutralization, and long-term storage stability. (a-b) The amount of various viruses that passed through cell strainers coated with simulated nasal fluid (SNF) or with a mixture of simulated mucus / SNF or PCANS / SNF within 4 hours. Virus passage was quantified by plaque assay in MDCK cells (IAV), Vero E6 cells (SARS-CoV-2), and Hep-2 cells (RSV and adenovirus). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. ns, non-significant. (e) The amount of E. coli and (f) K. pneumoniae that passed through cell strainers coated with SNF or with a mixture of simulated mucus / SNF or PCANS / SNF within 4 hours. Bacterial passage was quantified using the CFU plate count method. ****P<0.0001, ***P<0.001, **P<0.01. ns, non-significant. (g-k) Viral titers for IAV, SARS-CoV-2, adenovirus, and RSV after treatment with PBS or PCANS. IAV and SARS-CoV-2 were incubated with PCANS for 10 minutes, while adenovirus and RSV were treated for 30 minutes. Antibacterial activity of PCANS against (k) Escherichia coli and (l) Klebsiella pneumoniae using the CFU plate count method after 30 and 10 minutes of incubation, respectively. ***P<0.001, **P<0.01, *P<0.05. (m) Aerosol characteristics of PCANS. Droplet size distribution of PCANS was analyzed using a laser diffraction system. Representative images of single time delay plume angle and ellipticity taken using a high-speed digital camera and laser light sheet. (n) Experimental design to determine the stability of PCANS under accelerated temperature conditions (40°C). PCANS was stored in amber glass bottles. Aliquots were taken at different time points to investigate spray characteristics and pathogen neutralization efficacy.(o) Plume angle, (p) ellipticity, (q) mean droplet diameter, and (r) spray deposition area over 60 days. ***P<0.001, **P<0.01 (compared to day 0). ns, non-significant. (s) Percent reduction in IAV and SARS-CoV-2 viral load in each host cell after 10 minutes of incubation with aliquots of PCANS at different time points during the stability study. *P<0.05 (compared to day 0). ns, non-significant. For a-f and o-s, p-values were determined using one-way ANOVA with Tukey's post-hoc analysis. For g-l, p-values were determined using a two-tailed t-test. Data are presented as the mean ± SD of biological replicates (n=3, each experiment performed at least twice). [Figure 14]Figures 14a-14p show that prophylactic intranasal administration of PCANS prevents viral infection in a mouse model. (a) Experimental outline of the prophylactic efficacy study. C57Bl / 6 mice received a single dose (10 μl) of PCANS or PBS 15 minutes before intranasal inoculation with 250 PFU influenza A / PR / 8 / 34. One cohort of animals was followed for weight change and survival over 10 days. Animals from a second cohort were euthanized on days 2 or 4 post-infection to measure lung virus titers, inflammatory cell counts in bronchoalveolar lavage (BAL) fluid, and inflammatory cytokine levels in lung homogenates. Hematoxylin and eosin (H&E)-stained lung tissue sections from euthanized animals were assessed for inflammation. (b) Survival and (c) weight change of mice over 10 days post-infection. P=0.0007 (Kaplan-Meier survival curve compared to the PBS-treated group). *P<0.01 (body weight change curve compared to the PBS-treated group). (d) Viral titers from lung homogenates of mice on days 2 and 4 post-infection, and (e) the rate of reduction in intrapulmonary viral load, as quantified by plaque assay performed in MDCK cells. **P=0.001. (f-i) Number of inflammatory cells in BAL on days 2 and 4 post-infection. ****P<0.0001, **P<0.01. (j) IL-6, (k) TNF-α, and (l) IL-1β levels in lung tissue. ****P<0.0001, **P=0.005. ns, non-significant; nd, not detected. (m) Representative images of H&E-stained lung tissue sections from virus-challenged mice prophylactically treated with PBS or PCANS. Histological images were taken using 10x and 40x objectives. Scale bar: 100 μm. Higher magnification insets illustrate the varying degrees of inflammatory infiltrate. Scale bar: 20 μm. (n) Experimental outline for assessing time-dependent nasal protection by PCANS. Mice received a single dose of PCANS 2 or 4 hours before intranasal inoculation with 100 PFU influenza A / PR / 8 / 34. Animals were euthanized 2 days post-infection, and lung virus titers were measured.(o) Virus titers quantified from lung homogenates and (p) percent reduction in pulmonary viral load on day 2 postinfection for animals challenged 2 or 4 hours after prophylactic treatment. **P<0.01, *P<0.05, ns not significant. For b, P values were determined using the Gehan-Breslow-Wilcoxon test. For c, P values were determined using one-way analysis of variance with Brown-Forsythe. For d and f-l, P values were determined using two-way analysis of variance with Tukey's post-hoc analysis. For o, P values were determined using one-way analysis of variance with Tukey's post-hoc analysis. Data for c are presented as mean ± SEM (n = 6 mice / group). Data for d-l are presented as mean ± SEM (n = 4 mice / group). Data for o–p are presented as mean ± SEM (n = 6 mice / group). [Figure 15] FIG. 15 shows the bacteriostatic activity of a formulation composed of 0.2% w / v gellan and 0.75% w / v pectin, containing the preservatives phenylethyl alcohol and benzalkonium chloride. DETAILED DESCRIPTION OF THE INVENTION

[0037] definition As used herein, "increasing the capture of nebulized particles" or "increasing the capture of respiratory droplets" refers to the ability of the coating composition of the present invention to capture nebulized particles in an ex vivo (e.g., in vitro) model using a twin impinger chamber to simulate the aerodynamics of the human airway. The coating composition of the present invention (e.g., after coating with SNF, 100 μL of the coating composition is deposited) or a control coating of identical composition except that it does not contain surfactant is deposited on the inner surface of the chamber. Fluorescent dye-loaded particles are nebulized (e.g., resulting in droplets with a mass median aerodynamic diameter of greater than 5 μm). Nebulization is performed at a rate of 30 L / min for 30 seconds. The capture efficiency is determined by quantifying the fluorescence intensity compared to that observed for the control. The coating of the present invention can increase the capture of nebulized particles by at least 1.5-fold, 2-fold, 3-fold, or 5-fold compared to the control.

[0038] As used herein, the term "residence time" refers to the length of time that a mucous membrane coated according to the method of the present invention provides a physical barrier that traps particles and droplets. The residence time can be measured using the method described in Example 4.

[0039] As used herein, the term "increasing residence time" refers to an increase in the length of time that the mucosa coated according to the method of the present invention provides a physical barrier that traps particles and droplets. The increase in residence time is measured using the in vivo method described in Example 4 (e.g., by quantifying the amount of coating material remaining in the nasal cavity, e.g., 8 hours after administration). Mice are treated (e.g., intranasally) with a coating composition of the present invention (e.g., loaded with a fluorescent dye) compared to a control coating of identical composition except that it does not contain surfactant (e.g., 10 μL / nostril). The coating of the present invention can increase residence time by at least 2-fold, 5-fold, 10-fold, 15-fold, or 30-fold compared to the control. For example, the fluorescent signal in the nasal cavity can be quantified after 8 hours.

[0040] As used herein, the term "pathogen neutralization" refers to the ability of the compositions and methods of the present invention to inhibit pathogen entry into host cells or inhibit pathogen growth, either by destabilizing the pathogen cell membrane through chemical interactions or by preventing receptor-mediated pathogen binding / fusion. Pathogen neutralization for viruses was measured by adding 50 μL of a specific agent or coating composition of the present invention to 50 μL of a virus suspension (10 μL), as described in Examples 2 and 5. 4 ~10 6 PFU / mL) for 10, 30, or 60 minutes, followed by centrifugation for 1 minute, followed by infection of target cells and evaluation of the supernatant using a plaque-forming or focus-forming assay. Pathogen neutralization for bacteria is assessed by measuring bactericidal activity as described in Examples 2 and 5. 50 μL of each agent or coating composition of the present invention is applied to bacteria (10 8 CFU / mL) for 30 or 60 min, followed by centrifugation for 1 min, and then assessing the bacterial load in the supernatant using a colony formation assay.

[0041] As used herein, the term "reducing small molecule transport" refers to the ability of a coating composition of the present invention to reduce the transport of rhodamine B isothiocyanate dye (e.g., 0.1 mL) at a concentration of 0.1 mM through a strainer (70 micron pore size) coated with SNF (e.g., 15 μL) plus a coating composition of the present invention (e.g., 15 μL) compared to a strainer (70 micron pore size) coated with SNF alone (e.g., 15 μL). The dye that passes through the cell strainer is quantified by measuring fluorescence intensity, for example, after 4 hours. The reduction in small molecule transport is based on the relative performance of tests coated with the composition / SNF and tests coated with SNF alone. The coating of the present invention can reduce small molecule transport by at least 90%, 95%, or 99.9% over a 4-hour period.

[0042] As used herein, the term "reducing viral transport" refers to (i) reducing the viral transport of influenza A virus (e.g., 0.1 mL; 1 x 10) in a strainer (70 micron pore size) coated with SNF (e.g., 15 μL) plus a coating composition of the present invention (e.g., 15 μL). 5 This refers to the ability of the coating composition of the present invention to reduce the transport of virus (PFU / mL) compared to (ii) a strainer (70 micron pore size) coated with SNF alone (e.g., 15 μL). The amount of virus that passes through the cell strainer is quantified, for example, after 4 hours, using a plaque assay performed on MDCK host cells. The percentage reduction in virus transport is based on the relative performance of coated and uncoated tests. The coating of the present invention can reduce virus transport by at least 90%, 95%, or 99.9% over a 4-hour period.

[0043] As used herein, the term "reduced risk of exposure to infectious pathogens" refers to the trapping and / or neutralization of infectious microorganisms (e.g., bacteria, viruses, and fungi) in mucosa coated with a composition of the present invention compared to an uncoated mucosa exposed to the same infectious pathogen under the same conditions. The methods of the present invention may be used to reduce the number of infectious pathogens that reach the mucosa of a subject, thereby reducing the risk of exposure to infectious pathogens.

[0044] As used herein, the term "polysorbate surfactant" refers to a class of nonionic surfactants derived from PEGylated sorbitan esterified with fatty acids. Common trade names for polysorbates include Alkest, Canarcel, and Tween. Polysorbate surfactants include, but are not limited to, polyoxyethylene 20 sorbitan monolaurate (Tween 20), polyoxyethylene (4) sorbitan monolaurate (Tween 21), polyoxyethylene 20 sorbitan monopalmitate (Tween 40), polyoxyethylene 20 sorbitan monostearate (Tween 60), and polyoxyethylene 20 sorbitan monooleate (Tween 80).

[0045] As used herein, the term "sorbitan fatty acid ester surfactants" refers to a class of nonionic surfactants derived from sorbitan esterified with fatty acids. Sorbitan fatty acid ester surfactants include, but are not limited to, sorbitan monolaurate (Span-20 (Atlas / ICI), Cril 1 (Croda), Arlancel 20 (ICI)); sorbitan monopalmitate (Span-40 (Atlas / ICI), Cril 2 (Croda), Nikkol SP-10 (Nikko)); sorbitan monooleate (Span-80 (Atlas / ICI), Cril 4 (Croda), Cril 50 (Croda)); sorbitan monostearate (Span-60 (Atlas / ICI), Cril 3 (Croda), Nikkol SS-10 (Nikko)); sorbitan trioleate (Span-85 (Atlas / ICI), Cril 45 (Croda), Nikkol Examples of sorbitan isostearate include sorbitan monoisostearate (Crill 6 (Croda), Nikkol SI-10 (Nikko)), sorbitan sesquistearate (Span-65 (Atlas / ICI), Cril 35 (Croda), Nikkol SS-30 (Nikko)), sorbitan monoisostearate (Crill 6 (Croda), Nikkol SI-10 (Nikko)), and sorbitan sesquistearate (Nikkol SS-15 (Nikko)).

[0046] Detailed Description In this study, we describe a nasal spray that integrates the unique properties of different classes of compounds, including polysaccharides, surfactants, and alcohols, from the U.S. Food and Drug Administration's (FDA) Inactive Ingredients Guide (IIG) inventory for nasal, ocular, and topical routes. We created a stable nasal spray formulation, called PCANS, for the prophylactic treatment of upper respiratory tract infections, surpassing the benchmark of conventional nasal sprays against a single airborne pathogen. Furthermore, we established the minimum gel strength of the nasal spray to provide a protective physical barrier that prevents pathogen penetration into the underlying nasal epithelium (Figure 1). We employed a biomimetic approach to capture foreign particles by reducing the interfacial tension of PCANS, as observed by pulmonary surfactant in the alveoli. As illustrated in Figure 1, we enhanced the wettability of PCANS to promote the interaction of differently charged viral particles and allow them to be naturally washed away by mucociliary clearance. As described below, we have demonstrated long-term nasal retention of PCANS and prophylactic protection in animal models. Furthermore, PCANS provides a platform with excellent shelf life and the ability to target relevant airborne pathogens.

[0047] The methods and compositions of the present invention include polymers and polysaccharides that can act as a physical barrier and / or kill / neutralize pathogens. The following is a non-exhaustive list of polymers and polysaccharides that can act as a physical barrier and / or kill / neutralize pathogens: cellulose, crystalline / carboxymethylcellulose sodium / potassium / calcium, hydroxyethylcellulose, hypromellose, methylcellulose, ethylmethylcellulose, pectin, amidated pectin, xanthan, guar gum, karaya gum, hyaluronic acid, collagen, gelatin, sodium alginate, gellan, kappa carrageenan, lambda carrageenan, iota carrageenan, starch, glucomannan, chitin, chitosan, carboxymethylchitosan, glycosaminoglycans, dextran, levan, polygalactosamine, amylose, amylopectin, propane-1,2 alginate. -diol, alginic acid; sodium alginate; potassium alginate; ammonium alginate; calcium alginate; locust bean gum, tragacanth, acacia, ammonium phosphatide, linear polymer of 1-vinyl-2-pyrrolidone and polyethylene glycol, thiolated poly(acrylic acid), poloxamer, cellulose acetophthalate, methylcellulose, hydroxyethylcellulose, poly(amidoamine) dendrimers, poly(dimethylsiloxane) and poly(vinylpyrrolidone), chitosan-iminothiolane, poly(acrylic acid)-homocysteine, chitosan-thioethylamidine, alginate-cysteine, poly(methacrylic acid)-cysteine, sodium carboxymethylcellulose-cysteine, polyoxWSR, carbophil, carbomer, poly(dimethylaminoalkyl methacrylate), polyethylene glycol, poly(dimethylaminoalkyl acrylate), and copolymers poly(dimethylaminoalkyl methacrylate-co-trimethylaminoalkyl methacrylate) and poly(dimethylaminoalkyl acrylate-co-trimethylaminoalkyl acrylate), cationic oligomers or polymers including cationic polysaccharides, cationic copolymers of saccharides and synthetic cationic monomers, cationic polyalkyleneimines, cationic ethoxypolyalkyleneimines, cationic poly[N-[3-(dialkylammonio)alkyl]N'[3-(alkyleneoxyalkylenedialkylammonio)alkyl]urea dichloride], vinylcaprolactam / VP / dialkylaminoalkyl alkylate copolymers, polyquaternium-10 Polyquaternium-2, Polyquaternium-4, Polyquaternium-5, Polyquaternium-6, Polyquaternium-7, Polyquaternium-10, Polyquaternium-11, Polyquaternium-16, Polyquaternium-22, Polyquaternium-24, Polyquaternium-28, Polyquaternium-32, Polyquaternium-37, Polyquaternium-39, Polyquaternium-40, Polyquaternium-41, Polyquaternium-42, Polyquaternium-43, Polyquaternium-44, Polyquaternium-45, Polyquaternium-46, Polyquaternium-47, Polyquaternium-48, Polyquaternium-49, Polyquaternium-50, Polyquaternium-51, Polyquaternium-52, Polyquaternium-53, Polyquaternium-54, Polyquaternium-55, Polyquaternium-56, Polyquaternium-57, Polyquaternium-58, Polyquaternium-59, Polyquaternium-60, Polyquaternium-61, Polyquaternium-62, Polyquaternium-63, Polyquaternium-64, Polyquaternium-65, Polyquaternium-66, Polyquaternium-67, Polyquaternium-68, Polyquaternium-69, Polyquaternium-70, Polyquaternium-71, Polyquaternium-72, Polyquaternium-73, Polyquaternium-74, Polyquaternium-75, Polyquaternium-76, Polyquaternium-77, Polyquaternium-78, Polyquaternium-79, Polyquaternium-80, Polyquaternium-81, Polyquaternium-82, Polyquaternium-83, Polyquaternium-84, Polyquaternium-85, Polyquaternium-86, Polyquaternium-87, Polyquaternium-88, Polyquaternium-89, Polyquaternium Polyquaternium-42, Polyquaternium-43, Polyquaternium-44, Polyquaternium-46, Polyquaternium-47, Polyquaternium-51, Polyquaternium-53, Polyquaternium-55, Polyquaternium-57, Polyquaternium-58, Polyquaternium-59, Polyquaternium-60, Polyquaternium-63, Polyquaternium-64, Polyquaternium-65, Polyquaternium-68, and mixtures thereof).

[0048] The methods and compositions of the present invention include surfactants. The following is a non-exhaustive list of surfactants that can maximize respiratory droplet capture, extend the residence time of the formulation in the nasal cavity, and / or kill / neutralize pathogens: polyoxyethylene esters of fatty acids, polyoxyethylene mercaptans and polyoxyethylene alkylamines, polyethoxyethanol, polyoxyethylene oleyl ether, sorbitan monolaurate, sorbitan monooleate, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80, and onyxo. l, nonoxynol-9, laureth-9, poloxamer-124, octoxynol-9, octyl glucoside, and lauramide DEA, sodium stearoyl-2-lactylate; calcium stearoyl-2-lactylate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monopalmitate; Quillaja extract; polyglycerol esters of dimerized fatty acids from soybean oil; oxidatively polymerized soybean oil, saponin, 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol, sodium salt , dipalmitoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, palmitic acid, sodium, potassium, and calcium salts of fatty acids; mono- and diglycerides of fatty acids; acetate esters of mono- and diglycerides of fatty acids; lactate esters of mono- and diglycerides of fatty acids; citrate esters of mono- and diglycerides of fatty acids; monoglycerides of fatty acids and tartrate esters of diglycerides; monoacetyltartaric and diacetyltartaric esters of mono- and diglycerides of fatty acids; mixtures of acetate and tartrate esters of mono- and diglycerides of fatty acids; sucrose esters of fatty acids; sucroglycerides; polyglycerol esters of fatty acids; polyglycerol esters of polycondensed castor oil fatty acids; propane-1,2-diol esters of fatty acids, sorbitan monolaurate, benzalkonium chloride, and other cationic surfactants.

[0049] The methods and compositions of the present invention may include agents that can kill / neutralize pathogens.The following is a non-exhaustive list of other agents that can kill / neutralize pathogens: phenethyl alcohol, benzyl alcohol, citric acid monohydrate, eucalyptol, menthol, methylparaben, phenylmercuric acetate, polyethylene glycol 3350, polyethylene glycol 300, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 4000, phenol, propylparaben, sodium hydroxide, sorbitol, trisodium citrate dihydrate, propylene glycol, ascorbic acid, aloe, povidone, povidone k12, Povidone k15, povidone k30, benzoic acid, crospovidone, polyvinyl alcohol, sodium iodide, lysine acetate, tromethamine, sodium carbonate monohydrate, peppermint oil, sodium saccharin, cyclodextrin, amylmetacresol, chlorohexagluconic acid, polyhexamethylene biguanide, 2,4-dichlorobenzyl alcohol, hexylresorcinol; and metals, including but not limited to manganese (Mn), mercury (Hg), silver (Ag), zinc (Zn), tin (Sn), iron (Fe), copper (Cu), aluminum (Al), nickel (Ni), and cobalt (Co). Non-limiting examples of metal compounds suitable for use herein include salicylate, fumarate, benzoate, glutarate, lactate, citrate, malonate, acetylate, glycolate, thiosalicylate, adipate, succinate, gluconate, aspartate, glycinate, tartrate, malate, maleate, ascorbate, chloride, sulfate, nitrate, phosphate, fluoride, iodide, metal compounds called pidolate, and mixtures thereof. Acetate, ascorbate, chloride, benzoate, citrate, gluconate, glutarate, lactate, malate, malonate, salicylate, succinate, sulfate, and mixtures thereof are preferred metal compounds.

[0050] The methods and compositions of the present invention may include a therapeutic agent incorporated into the drug delivery formulation. The following is a non-exhaustive list of therapeutic agents that may be incorporated into the drug delivery formulation: antibiotics, such as streptomycin and vanamycin; antiviral and antiretroviral agents, such as tenofovir, emtricitabine, and ribaribine; antibacterial agents, such as tetracycline, quinoline, and kanamycin; antiparasitic agents, such as quinacrine and chloroquine; antihistamines, such as cetirizine, chlorpheniramine, and diphenhydramine; hormones, such as insulin, progesterone, steroids, such as corticosteroids and estrogens; nonsteroidal anti-inflammatory analgesics, such as salicylic acid, ibuprofen, and naproxen; opioid analgesics, such as morphine, oxycodone, fentanyl, codeine, and hydromorphone; narcotic antagonists, such as naltrexone, naloxone, and bupivacaine. anticholinergics, such as atropine, tiotropium, and ipratropium; beta-adrenergic agonists, such as salbutamol, salmeterol, and formoterol; cardiac agents, such as nitroglycerin, papaverine, and digitalis; vaccines against diseases such as hepatitis, mumps-measles-rubella, influenza, COVID-19, MERS, and polio; cell growth agents, such as fibronectin, epidermal growth factor, interleukin-1, and human growth hormone; anti-cell growth agents and chemotherapeutic agents, such as cisplatin, 5-fluorouracil, paclitaxel, doxorubicin, docetaxel, tumor necrosis factor, fibroblast growth factor antagonists, and various monoclonal antibodies;and hypnotics, sedatives, immunosuppressants, mucolytics, decongestants, such as phenylephrine, oxymetazoline, naphazoline, ephedrine, pseudoephedrine, propylhexedrine, and phenylpropanolamine, anticonvulsants, antidepressants, antispasmodics, antipruritics, vasodilators, moisturizers, herbal medicines, aloe, essential oils, antidiuretics, antihypertensives, sleep promoters, melatonin, benzodiazapines, midazolam, diazepam, and diamorphine, dietary supplements, naphazoline hydrochloride, menthol, cannabinoids, vitamins, substance use disorders drugs used in neuroleptic malignant disorders, benzodiaepines, barbiturates, disulfiram, naltrexone, acamprosate, clonidine, nicotine, bupropion, buprenorphine, contraceptives, cholesterol-lowering drugs, nasal hydration promoting agents, nutritional supplements, xylometazoline, fluticasone, and other biological compounds, such as DNA and RNA nucleic acid sequences and oligonucleotides, peptides, lipids, polysaccharides, proteins, enzymes, antibodies, or ribozymes, interferons, toll-like receptor agonists, such as nucleoside analogs, imidazoquinolines and their derivatives, and retinoic acid and its derivatives, and combinations thereof; [Example]

[0051] result Example 1: Utilizing biopolymers to limit pathogen entry through a physical barrier We selected mucoadhesive biopolymers that are listed on the FDA IID or GRAS list and present as excipients in commercially available nasal / topical formulations. Specifically, we selected gellan, pectin, hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose sodium salt (CMC), carbopol, and xanthan gum. These biopolymers were screened for their ability to impart physical barrier properties to PCANS. Because a metered-dose spray device is used to administer PCANS, we first identified the nebulizable concentration of each biopolymer by performing rheological measurements (Figure 2a-f). Dynamic viscosity curves were generated using a rotational rheometer by varying the shear rate up to 40 s-1, which is within the lower limit of the shear rate encountered when dispensing formulations through a nasal spray device. Concentrations that exhibited a viscosity of less than 0.1 Pa.s were considered "nebulizable." Next, we determined the mechanical strength of each biopolymer at its highest nebulizable concentration before and after the addition of simulated nasal fluid (SNF). SNF was added to mimic the physiological environment within the nasal cavity. Mechanical strength was measured using a rotational rheometer and quantified as storage modulus (G'), which represents the amount of structure present in the material. In the presence of SNF, gellan exhibited the highest G' compared to other biopolymers, indicating its superior mechanical strength (Figure 2g). Gellan showed a 100-fold increase in G' in the presence of SNF (Figure 2g), consistent with its ability to undergo in situ gelation under physiological conditions. Monovalent and divalent cations present in SNF complex with the glucuronic monomer units of gellan to form crosslinked hydrogels. Compared to gellan, the other biopolymers showed minimal or no increase in storage modulus, suggesting insufficient in situ gelation. We also observed that the addition of pectin to gellan did not impair either its in situ gelation ability or its storage modulus in the presence of SNF (Figure 3a).To investigate the physical barrier properties of biopolymers, we devised a membrane penetration assay that assessed IAV transport through cell strainers coated with SNF (approximately 70 μm pore size) or with simulated mucus / SNF or biopolymer / SNF mixtures (Figure 4). After 4 h, the virus titer in the chamber below the strainer was quantified by performing a plaque assay in Madin-Darby canine kidney (MDCK) host cells. Consistent with its superior mechanical strength, gellan / SNF reduced IAV particle transport by more than 4-log-fold (99.99%) compared with strainers coated with SNF alone or mucus / SNF (Figure 2h). Xanthan / SNF, CMC / SNF, and HPMC / SNF also significantly reduced IAV transport, although not as efficiently as gellan / SNF. Interestingly, pectin / SNF interfered with IAV transport with similar efficiency to gellan / SNF, despite having significantly lower mechanical strength compared to gellan / SNF. Carrageenan, a biopolymer previously reported and used in commercially available chemopreventive nasal drops, was used as a control; it did not reduce IAV transport in the presence of SNF.

[0052] The reduction in IAV particle transport by anionic biopolymers may be the result of physical barrier properties and / or electrostatic interactions between the negatively charged polymer chains and the positively charged IAV capsid. To separate the effects of physical barrier properties from electrostatic interactions, we studied the transport of rhodamine B isothiocyanate, a low-molecular-weight anionic dye that reduces electrostatic interactions with anionic biopolymers. Gellan / SNF resulted in a 100% reduction in dye transport, confirming its excellent physical barrier properties (Figure 2i). Other biopolymers did not reduce dye transport, indicating poor physical barrier properties. This also indicates that the reduction in IAV transport by pectin was primarily mediated through electrostatic interactions between pectin chains and the viral capsid. Interestingly, gellan / SNF reduced rhodamine B dye transport in a concentration-dependent manner (Figure 2j). Gellan at a concentration of 0.2% w / v also reduced the transport of E. coli by more than 8-log fold (100%), suggesting a broad physical barrier property that limits both viral and bacterial transport. In conclusion, gellan inhibited the transport of Rhodamine B dye, E. coli, and IAV by 100% at concentrations of 0.2% w / v and above.

[0053] To ensure maximum coverage of the nasal cavity, we evaluated the spray characteristics of gellan at concentrations of 0.2% w / v and above using a hydraulic spray nozzle. Plume shape and spray coverage were measured using a high-speed image acquisition system. Increasing gellan concentration resulted in a significant decrease in the angle of the spray's ejected plume (defined as the "plume angle") and coverage area (Figure 5). Henceforth, we used 0.2% w / v gellan due to its superior physical barrier properties, plume angle, and coverage area compared to other concentrations.

[0054] Next, we evaluated the retention ability of gellan and other biopolymers in mucosal tissues upon spraying. Mucosal retention was measured as the drip length, defined as the distance traveled by the biopolymer from the point of deposition in the intestinal mucosa of a vertically placed sheep over 4 hours. To visualize dripping, the biopolymers were mixed with brilliant green dye. The drip length ratio of each biopolymer compared to the drip length of the free dye was calculated. Gellan (0.2% w / v) showed excellent mucosal retention with a drip length of zero (Figure 2k and Figures 3b and c). Other biopolymers, including carrageenan used as a control, showed drip lengths of more than 95%, indicating poor mucosal retention. The excellent mucosal retention of gellan is attributed to its ability to tightly entangle with mucin glycoproteins in mucosal tissues during the sol-gel transition.

[0055] Example 2: Identification of pathogen-specific neutralizing agents To confer broad-spectrum pathogen-neutralizing capacity to PCANS, we screened agents from three different compound classes, including biopolymers, surfactants, and alcohols. These compounds were selected based on their previously reported ability to neutralize different types of pathogens. To maximize the safety and clinical translatability of PCANS, we selected only agents that are on the FDA IID or GRAS list and present as excipients in commercially available nasal / topical formulations (Figure 6a). We first evaluated the neutralizing ability of these agents against viruses. Neutralization was studied in vitro by incubating each agent individually with either IAV or SARS-CoV-2 for 10 or 60 minutes, followed by centrifugation for 1 minute, infecting target cells, and evaluating the supernatants using plaque- or focus-forming assays. We selected IAV and SARS-CoV-2 because of their high global prevalence as respiratory viruses and because they have different capsid proteins and electrical charges. Biopolymers other than gellan and carrageenan were evaluated at their highest sprayable concentrations. Gellan was evaluated at 0.2% w / v due to its superior physical barrier properties compared to 0.1% w / v and superior spray pattern compared to 0.4% w / v. Carrageenan, used as a control, was evaluated at 0.16% w / v because this concentration is present in commercially available chemoprophylactic nasal sprays. Surfactants and alcohols were evaluated at the highest concentrations previously used in humans via the nasal route. Compared to carrageenan, pectin demonstrated superior neutralization of IAV, regardless of incubation time, demonstrating a 4-log (99.99%) reduction in intracellular viral titers compared to PBS (Figure 6b). Incubation with Carbopol for 10 min did not reduce IAV titers, but a 4-log (99.99%) reduction was observed after 60 min of incubation. Gellan showed similar neutralization of IAV as carrageenan, resulting in only a 1-log (90%) reduction in viral load within host cells.For SARS-CoV-2, both pectin and carrageenan showed a less than 1-log reduction in the viral load within host cells (Figure 6g). Gellan showed a 4-log (99.99%) reduction in viral titer, but only at a 1-hour incubation time. Among surfactants, Tween 80 and benzalkonium chloride (BKC) showed a 1-log reduction in IAV titer within host cells, regardless of incubation time (Figure 6c). Rapid neutralization of SARS-CoV-2 was observed with BKC, resulting in a 5-log (>99.99%) reduction in the viral load within the host (Figure 6h). Alcohols did not neutralize SARS-CoV-2, and minimal neutralization was observed against IAV, resulting in a less than 1-log (90%) reduction in viral load for chlorobutanol and phenethyl alcohol (PEA) (Figures 6d and 6i). Overall, this extensive screening identified pectin and BKC as the most effective agents for rapid neutralization of IAV and SARS-CoV-2, respectively. The neutralizing potency of pectin and BKC was found to be dose-dependent (Figures 6e and 6j). Minimum concentrations of 0.75% w / v and 0.01% w / v for pectin and BKC, respectively, were required to achieve a >4-log (>99.99%) reduction in viral load over a 10-minute incubation period.

[0056] To elucidate the virus-neutralizing mechanisms of pectin and BKC, we performed in silico modeling to determine their binding affinity to the receptor-binding domains (RBDs) of IAV and SARS-CoV-2, respectively. For IAV, anionic pectin targets the RBD located in the distal portion of the positively charged hemagglutinin, thereby avoiding viral entry into host cells (Figure 6l). Pectin exhibited stronger RBD binding compared to the host ligand sialic acid present in mucins, through distant hydrogen bonds with Se228, Ser186, and Thr187, and hydrophobic bonds with Ser227 and Glu190 (Figure 7). BKC was found to exhibit hydrophobic interactions with the ACE2-binding motif of the SARS-CoV-2 spike protein (Figure 6m). BKC also exhibits hydrophobic interactions with Phe23 and Phe26 within membrane helices via π-π stacking (Figure 3m). Because aromatic stacking of Phe23 and Phe26 is essential for stabilizing the helix-helix interface of envelope transmembrane proteins, this interaction may distort the helical structure of adjacent helices. BKC may fit into the pentameric ion channel at the N-terminus of the transmembrane domain through its interaction with Thr11, blocking ion influx / efflux (Figure 6n). To determine the role of electrostatic interactions in the neutralization of IAV and SARS-CoV-2 mediated by pectin and BKC, we performed neutralization assays by pretreating pectin and BKC with charge-cancelling counterions. As expected, anionic pectin lost its neutralizing activity in the presence of positively charged polyethyleneimine and failed to significantly reduce viral load compared to PBS (Figure 6f). Similarly, pretreatment of BKC with negatively charged bovine serum albumin attenuated its ability to reduce SARS-CoV-2 titers within host cells (Figure 6k).

[0057] Next, we investigated whether ionic interactions between anionic gellan or pectin and cationic BKC affect the neutralization potency of pectin or BKC when coexisting in a formulation. Notably, the neutralization efficacy of pectin (0.75% w / v) against IAV remained preserved with dose-dependent increases in BKC up to 0.1% w / v (Figure 6o). The neutralization efficacy of BKC (0.01% w / v) against SARS-CoV-2 was unaffected by gellan or pectin at concentrations of 0.2% w / v or 0.75% w / v, respectively, but decreased at higher concentrations (Figures 6p, q). These results further emphasize that the concentration of each agent is important for efficient neutralization.

[0058] Finally, we screened surfactants and alcohols to determine their neutralizing ability against bacteria, including E. coli and K. pneumoniae. Neutralization was determined by measuring bactericidal activity. Each agent was individually incubated with either E. coli or K. pneumoniae for 30 or 60 minutes, followed by 1 minute of centrifugation. The bacterial load in the supernatant was then assessed using a colony formation assay. BKC was more effective than nonionic surfactants, resulting in a 4-log (99.99%) and 7-log (99.99%) reduction in colony-forming units (CFUs) of E. coli and K. pneumoniae, respectively, after a 30-minute incubation period (Figure 6r, t). Alcohol had negligible bactericidal effect after a 30-minute or 60-minute exposure period (Figure 6s, u). Taken together, our data on physical barrier properties, spray pattern, mucosal retention, and neutralization indicate that gellan, pectin, and BKC are the three key ingredients for formulating PCANS. However, we also incorporated phenethyl alcohol (PEA) because it is commonly added to nasal formulations as a stabilizer to prevent the growth of gram-negative bacteria and fungi and ensure a long shelf life.

[0059] Example 3: Surfactants enhance respiratory droplet capture We identified surfactants that reduce the interfacial tension of PCANS and reduce the rebound / leakage of respiratory droplets. We evaluated surfactants listed on the IID list, including Tween-20, Tween-80, and BKC. Screening was performed using a twin impinger, a glass instrument that can be used to determine the deposition of aerosolized particles in different regions of the respiratory tract (Figure 8a). Simulated mucus or a biopolymer mixture of gellan (0.2% w / v) and pectin (0.75% w / v), without or with various concentrations of surfactant, was sprayed onto the oropharyngeal region of an SNF-coated impinger (Figure 8a). To mimic large pathogen-laden respiratory droplets, a jet nebulizer was used to generate droplets with an aerodynamic mass median diameter greater than 5 μm and loaded with rhodamine B-loaded liposomes (approximately 400 nm in size). Droplet capture was determined by quantifying the fluorescence intensity of rhodamine B within the biopolymer / surfactant mixture or mucus layer. Surfactant-free biopolymer mixtures exhibited fluorescence intensity similar to that of mucus (Figure 8b). Combining the biopolymer mixture with Tween-80 or Tween-20 at concentrations greater than 0.005% w / v or BKC at concentrations greater than 0.01% w / v resulted in a significant increase in fluorescence intensity compared to mucus or the biopolymer mixture alone, suggesting increased droplet capture by the surfactant. Compared to Tween-20, BKC and Tween-80 resulted in a significantly higher fold increase in fluorescence intensity when added to the biopolymer mixture at concentrations greater than 0.05% w / v (Figure 8b). At a concentration of 0.05% w / v, both the BKC-containing biopolymer mixture and the Tween-80-containing biopolymer mixture exhibited similar fluorescence intensities, fourfold higher than those of mucus or the surfactant-free biopolymer mixture. Because 0.01% w / v is the most commonly used concentration of BKC in commercially available nasal formulations and also exhibited excellent neutralizing activity against SARS-CoV-2, we decided to use this concentration in PCANS, even though BKC did not increase respiratory droplet capture at this concentration.To confer respiratory droplet capture capacity, we decided to proceed with Tween-80 and determined a safe concentration that would not impair either the permeability or metabolic activity of the nasal epithelium. To that end, we performed an in vitro assay to evaluate the transepithelial electrical resistance (TEER) across human nasal epithelial cell (RPMI-2650) monolayers upon treatment with various concentrations of Tween-80. Regardless of the concentration evaluated in this study, a transient decrease of less than 15% in TEER was observed in the monolayers immediately after the addition of Tween-80. However, TEER rapidly returned to its original value within less than 1 hour after replacing the Tween-80-containing medium with fresh medium (Figure 8c). The decrease in TEER with Tween-80 was significantly less than that with Triton-X (negative control), which resulted in a permanent change in TEER. Second, we evaluated the effect of various concentrations of Tween-80 on the metabolic activity of RPMI-2650 cells after 24 or 48 hours of incubation. Cells incubated with 0.01% w / v or 0.05% w / v Tween-80 exhibited metabolic activity similar to that of cells incubated in medium. However, Tween-80 (0.5% w / v) significantly reduced the metabolic activity of RPMI cells (Figure 9). Therefore, we decided to use 0.05% w / v as the final concentration of Tween-80 in PCANS. We also evaluated the release kinetics of Tween-80 from a composite mucoadhesive polymer consisting of 0.2% w / v gellan and 0.75% w / v pectin in the presence of SNF. Sustained release of Tween-80 from the gel was observed over a 4-hour period (Figure 10A). We demonstrated the feasibility of the nasal platform to deliver biologics such as immunoglobulin G (IgG) and the lipid biomolecule lysophosphatidylcholine (LPC) over a 4-hour period (Figure 10B).Overall, based on data on physical barrier properties, spray pattern, mucosal retention, neutralization, droplet capture, and nasal epithelial cytotoxicity, we selected gellan, pectin, BKC, PEA, and Tween-80 as the final components of PCANS. We then tested the respiratory droplet capture capacity of the final formulation using a 3D-printed model (Koken cast) of an anatomically complex human nasal cavity (Figure 8d). Consistent with the twin impinger results, there was no significant difference in fluorescence intensity between the gellan and pectin mixture and mucus (Figure 8e). Meanwhile, PCANS exhibited twofold higher fluorescence than mucus, suggesting its ability to increase capture of pathogen-laden respiratory droplets from inhaled air.

[0060] Example 4: Enhanced nasal residence time of PCANS We evaluated the residence time of PCANS in the nasal cavity of mice (Figure 4f). PCANS (10 μL) mixed with a fluorescent dye (DiIC18(7) (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) (DiR) was administered to both nostrils of C57 / BL6 mice. Free DiR was used as a control. Mice were euthanized at different time points over 24 h, and the nasal cavities were collected and imaged using an in vivo imaging system (IVIS) to quantify the fluorescent signal from DiR. Free DiR produced negligible fluorescent signals even 15 min after administration. This suggested its rapid clearance (Fig. 8g, h). Interestingly, mice administered DiR-loaded PCANS showed significant fluorescence for up to 8 hours, suggesting prolonged nasal retention of PCANS (Fig. 8g, h). We hypothesized that the prolonged retention of PCANS was due to the presence of surfactants, including Tween-80, BKC, and Span-20, which have previously been shown to reduce ciliary beat frequency in the nasal cavity. To test this hypothesis, we performed nasal administration of DiR-loaded PCANS without or with surfactants. The nasal retention times of DiR-loaded mixtures of gellan and pectin were compared. The addition of BKC, Span-20, or Tween-80 significantly enhanced the nasal retention of the gellan and pectin mixture 8 hours after nasal administration (Figures 8i and 11A). However, Tween-80 resulted in significantly higher nasal retention than BKC. Span-20 exhibited higher nasal retention than Tween-80, but also required significantly higher concentrations (Figure 11A). The ability of Tween-80 to enhance the nasal retention of the gellan and pectin mixture was found to be concentration-dependent. (Fig. 11B). However, considering the irreversible nasal epithelial permeability and cytotoxicity of Tween-80 at concentrations above 0.5% w / v, we maintained the concentration of 0.05% w / v in PCANS for subsequent experiments. Notably, nasal administration of DiR-loaded PCANS showed fluorescent signals only in the nasal cavity and stomach, suggesting no systemic absorption (Fig. 12). PCANS was completely eliminated after 24 h (Figs. 8g, h, and Fig. 12), resulting in negligible fluorescent signals in both the nasal cavity and stomach.To confirm the safety of PCANS, a repeat-dose toxicity study was conducted in healthy mice that received either PCANS or PBS intranasally once daily for 14 consecutive days (Figure 8j). Hematoxylin and eosin (H&E)-stained sections of the nasal cavity from both PBS- and PCANS-treated mice showed no evidence of inflammation or other overt toxicity, as evidenced by a clear lamina propria (Figure 8k). This indicates the safety of PCANS for daily administration.

[0061] Example 5: Broad-spectrum pathogen neutralization, barrier properties, and storage stability of PCANS After identifying the final components of PCANS along with their optimal concentrations, we sought to demonstrate its physical barrier properties and neutralization capacity against a broad spectrum of respiratory pathogens, including enveloped viruses (IAV, SARS-CoV-2, RSV), non-enveloped viruses (adenovirus), and bacteria (E. coli and Klebsiella pneumoniae). Physical barrier properties were evaluated by determining pathogen transport on cell strainers coated with SNF or on cell strainers coated with simulated mucus / SNF or PCANS / SNF mixtures. PCANS / SNF prevented transport of all pathogens by more than 4-log fold (>99.99%) (Figures 13a-f), suggesting its broad-spectrum physical barrier properties. For all pathogens except RSV, the mucus / SNF mixture prevented significantly less pathogen transport compared to PCANS / SNF. PCANS efficiently neutralized all tested pathogens within a 10-minute incubation period, resulting in a greater than 3-log (>99.9%) reduction in intracellular pathogen burden (Figure 13g-l). We also evaluated the spray characteristics of PCANS sprayed by a standard, commercially available VP3 multi-dose nasal pump (Aptar, USA). Droplet distribution data showed that 10% of PCANS droplets had a size greater than 10 μm and 90% had a size less than 200 μm, which is desirable for maximizing nasal deposition while minimizing deep lung deposition (Figure 13m). PCANS produced a wide plume angle within the ideal range of 35–55°, an ellipticity close to 1, and a circular area coverage of up to 8%, comparable to commercially available nasal sprays (Figure 13m).

[0062] Storage stability is a critical attribute governing the clinical applicability of a formulation. We tested the storage stability of PCANS for 60 days at 40°C, following the International Conference on Harmonization (ICH) guidelines for stability testing under accelerated storage conditions (Figure 13n). No substantial variation was observed in spray characteristics, including plume angle, ellipticity, coverage area, and droplet size distribution, over the 60-day period (Figures 13o-r). PCANS also showed no change in neutralizing activity over the 60-day period, resulting in a greater than 99.99% reduction in intracellular influenza A and SARS-CoV-2 viral loads after 10 minutes of incubation (Figure 13s). Collectively, these data confirm the storage stability of PCANS. The bacteriostatic activity of the formulation was assessed by a microplate assay, which utilizes optical density to quantify bacterial growth. Results showed nearly 100% bacteriostatic activity with the addition of phenylethyl alcohol to the BKC-containing formulation (Figure 15). This data highlights the significance of phenylethyl alcohol for the long-term stability of the formulation, preventing unwanted bacterial growth promoted by air and moisture exposure due to the intermittent opening of the spray bottle.

[0063] Example 6: In vivo prophylactic protection by PCANS against influenza Next, in a proof-of-concept study, we investigated the preventive efficacy of PCANS against respiratory infection in vivo. PR8, a mouse-adapted strain of H1N1 influenza virus, was used to induce infection. PR8 is a highly pathogenic strain that induces severe respiratory infections in mice and can be lethal at a dose of 10 PFU. To demonstrate in vivo efficacy, healthy mice were prophylactically administered PCANS or PBS (10 μl) into both nostrils on day 0 (FIG. 14a). Fifteen minutes later, the animals were challenged intranasally with PR8 (250 PFU), a dose previously used by other groups. Remarkably, all mice in the PCANS-treated group survived for at least 10 days after infection, whereas the PBS-treated group showed 100% mortality by day 8 (FIG. 14b). Over a 10-day period, no discernible change in body weight was observed in PCANS-treated animals, whereas significant weight loss was observed for PBS-treated animals 3 days after infection (Fig. 14c). PCANS also suppressed lung virus titers to undetectable levels on days 2 and 4 postinfection, resulting in a greater than 5-log-fold (>99.99%) reduction compared to PBS-treated mice (Fig. 14d-e). Compared to healthy mice, PR8-infected PBS-treated mice showed significant differences in the levels of inflammatory cells, including leukocytes, neutrophils, lymphocytes, and macrophages, in bronchoalveolar lavage (BAL) fluid (Fig. 14f-i). Prophylactic treatment of mice with PCANS restored the levels of inflammatory cells in BALF to normal. Furthermore, cytokine profiles from lung homogenates showed significant reductions in IL-6 and TNFα levels in PCANS-treated mice compared to the PBS-treated group (Fig. 6j-l). However, no reduction in IL-1β levels was observed. Histological examination of lung sections revealed a substantial reduction in leukocyte infiltrates in PCANS-treated mice compared with the PBS-treated group, which showed the presence of abundant infiltrates in the bronchi and alveoli (Fig. 14m). Overall, a significant reduction in pulmonary inflammation scores was observed for the PCANS-treated group compared with the PBS-treated mice.PCANS also protected mice against PR8 challenge administered 2 and 4 hours after prophylactic treatment, as evidenced by a significant reduction in lung viral titers observed on day 2 compared to the PBS-treated group (Figures 14n-p). Specifically, an average 60% and 78% reduction in lung viral titers was observed for the 2- and 4-hour challenge groups, respectively. However, no statistically significant difference was observed in the PCANS-mediated reduction between animals challenged 2 and 4 hours later. Overall, our data clearly suggest the ability of nasally administered PCANS to prevent respiratory infections. In conclusion, PCANS represents a promising chemoprevention approach against respiratory infections. In addition to its ability to serve as a first line of defense against respiratory pathogens for which no vaccine is available and emerging variants, our approach could also be used as an additional layer of protection provided by existing vaccines.

[0064] The above results were obtained using the following method.

[0065] method Preparation of biopolymer solutions and PCANS. Biopolymer solutions were prepared by adding biopolymers (0.2–2% w / v) to ultrapure deionized sterile water (Invitrogen). The solution was then mixed with slight heating at 60°C to obtain a homogeneous mixture. Biopolymers, including gellan (Gelzan), pectin, carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), carrageenan, xanthan gum, and carbopol, were purchased from Sigma-Aldrich. To prepare PCANS, a 0.4% w / v gellan solution and a 1.5% w / v pectin solution were mixed in a 1:1 ratio, followed by the addition of Tween-80 (Sigma-Aldrich) to obtain a final concentration of 0.05% w / v. The solution was then supplemented with benzalkonium chloride (BKC) (Sigma-Aldrich) and immediately mixed by pipetting several times to obtain a 0.01% w / v solution. Finally, 0.25% w / v phenethyl alcohol (Sigma Aldrich) was added and the pH of the solution was adjusted to 5.5. For cell culture experiments and in vivo efficacy studies, the individual components of PCANS were sterile filtered using 0.2 μm PVDF syringe filters (EMD Millipore) and combined as described above.

[0066] Preparation of simulated nasal fluid (SNF) and simulated mucus: SNF was prepared by dissolving 1.32 g of sodium chloride (150 mM), 447 mg of potassium chloride (39.9 mM), and 88.5 mg of calcium chloride (5.3 mM) in 150 mL of ultrapure deionized sterile water and filtering through a 0.2 μm filter. Healthy simulated mucus was prepared by dissolving 0.6 mg of porcine gastric mucin type II (Sigma-Aldrich), 0.8 mg of porcine gastric mucin type III (Sigma-Aldrich), and 0.32 mg of bovine serum albumin (Sigma-Aldrich) in 10 mL of ultrapure deionized water containing 20 mM HEPES buffer and 38 mM sodium chloride solution. The mixture was vigorously stirred with slight heating to obtain a homogeneous solution.

[0067] Rheological measurements. The dynamic viscosity behavior of the biopolymer solutions was evaluated using a rotational rheometer (Discovery HR-2, TA Instruments) using a 40 mm diameter cone with a 1° geometry angle. To mimic the strains encountered by the formulation passing through the nozzle of a spray device during actuation, the samples were spun at 25 °C for 40 s. -1 The biopolymer solutions were subjected to a linear shear rate increase up to 1000 kJ / min. The viscosity of the biopolymer solutions was measured in triplicate during the shear rate increase. The sol-gel transition of the biopolymer solutions with and without SNF was evaluated by rotational rheology. The mechanical strength in terms of storage modulus was determined by applying an amplitude sweep with an oscillatory strain varying at 1 Hz at 37°C.

[0068] Ex vivo mucosal retention study. Tissues harvested from sheep were dissected to expose the mucosal surface and cut into 75 × 26 mm sections. The mucosal tissues were then placed face up on glass slides and positioned at a 45° angle to match the spray operating angle. First, the tissues were moistened with SNF using a standard nasal spray device, and excess liquid was removed with a sterile wipe. Brilliant Green dye (Sigma-Aldrich)-loaded polymer solution was sprayed while the spray nozzle tip was held 5 cm from the slide surface. Four hours after spraying, the slides were examined for slippage / drip. The distance traveled by the polymer solution down the slide from the bottom edge of the formulation deposited on the mucosal tissue was measured as the drip length. The drip length of the free dye was considered to be 100%.

[0069] Cell culture. Madin-Darby canine kidney cells (ATCC®) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Invitrogen) in T-175 flasks (CELLTREAT) at 37°C and 5% CO2. Hep2 and Vero E6 cells (ATCC®) were cultured in EMEM supplemented with 10% FBS and 1% penicillin-streptomycin in T-75 flasks at Integrated Biotherapeutics (IBT) Bioservices at 37°C and 5% CO2. Human nasal epithelial cells (ATCC®) were cultured in EMEM supplemented with 10% FBS and 1% penicillin-streptomycin in T-175 flasks at 37°C and 5% CO2.

[0070] Generation of recombinant SARS-CoV-2 expressing NanoLuc luciferase. All replication-competent SARS-CoV-2 experiments were performed in a BSL-3 facility at the Boston University National Emerging Infectious Diseases Laboratories. Recombinant SARS-CoV-2 virus expressing Neon Green fluorescent protein (rSARS-CoV-2 mNG) 58 was kindly provided by Pei-Yong Shei's laboratory. To propagate the virus, 1 × 10 7Vero E6 cells were seeded into a T-175 flask. The next day, 10 μL of rSARS-CoV-2 mNG virus stock was diluted with 10 mL of OptiMEM and added to the cells, which were then incubated at 37°C for 1 hour. After incubation, 15 mL of DMEM containing 10% FBS and 1% penicillin / streptomycin was added to the cells. The following morning, the medium was removed, the cells were washed with 1x PBS, and 25 mL of fresh DMEM containing 2% FBS was added. The virus was incubated for an additional 48 hours. The supernatant was collected after 72 hours, filtered through a 0.22 μm filter, and stored at -80°C. The virus stock was thawed and concentrated by ultracentrifugation (Beckman Coulter Optima L-100k; SW32 Ti rotor) on a 20% sucrose cushion (Sigma-Aldrich, St. Louis, MO) at 25,000 × g for 2 hours at 4 ° C. The medium and sucrose were then discarded, the pellet was dried at room temperature for 5 minutes, and the virus pellet was resuspended in 100 μL of cold 1 × PBS overnight at 4 ° C. The next day, the concentrated virus was combined, aliquoted, and stored at -80 ° C.

[0071] In vitro physical barrier assay. A 70 μm pore-size mesh cell strainer was coated with 15 μL of mucus or biopolymer solution or PCANS. The formulation was evenly spread using a sterile stainless steel spatula with a tapered end. To facilitate in situ gelation, 15 μL of SNF was added to cover the entire surface of the strainer. The strainer was placed in a 6-well plate containing 0.9 mL of serum-free DMEM (for virus / bacteria passage) or ultrapure deionized water (for rhodamine B isothiocyanate passage) per well, and 0.1 mL of diluted virus (approximately 1 × 105 PFU / mL) / bacteria (1 × 107 CFU / mL) stock or rhodamine B isothiocyanate (1 mg / mL) was added to the upper compartment of the strainer. After 4 hours of incubation at 37°C, the medium or deionized water was collected from the lower reservoir, and the viral titers that passed through the hydrogel layer were quantified using the following methods: plaque assay performed on MDCK cells for IAV, crystal violet staining performed on Hep-2 cells for RSV, immunostaining performed on Vero E6 cells for adenovirus, focus-forming assay in Vero E6 cells for SARS-CoV-2, and colony-forming unit (CFU) plate counting for bacteria. The passage of the dye through the biopolymer solution / mucus was quantified by measuring fluorescence intensity using a microplate reader.

[0072] In vitro neutralization assay with influenza A. The neutralizing activity of various excipients and PCANS was evaluated by plaque assay. MDCK cells were seeded into 6-well plates at a density of 2-3 million cells per well and then incubated at 37°C to achieve approximately 80-90% confluency one day before infection. On the day of infection, HKx31 influenza A virus (H3N2, 5 x 10) was added to the infection medium (serum-free DMEM containing 3 mg / mL TPCK-trypsin). 4 ~1×10 550 μL of virus (PFU / mL) (BEI Resources) was pretreated with 50 μL of PCANS, biopolymer solution, surfactant solution, alcohol solution, or PBS. The sample was vortexed for 10 seconds and incubated at 37°C for 10 or 60 minutes. After incubation, the sample was centrifuged at 1000 RPM for 1 minute, and the supernatant was subjected to 10-fold serial dilutions up to the 8th dilution using infection medium. MDCK cells were then exposed to the pretreated virus dilutions for 1 hour. After infection, an overlay growth medium (50:50) containing 2x DMEM with 2% agarose was poured onto the cell monolayer and incubated for 72 hours. The overlay was removed, and the cells were then fixed using 1 mL of 10% formalin and left at room temperature for 1 hour. Subsequently, 1% crystal violet was added for 5–15 minutes. The wells were washed with water, dried, and PFU were counted to determine the virus titer.

[0073] In vitro neutralization assay with SARS-CoV-2. On the day before the infection experiment, 8 × 10 4 Vero E6 cells / well were seeded in 24-well plates. To perform the neutralization assay, 50 μL of PCANS, biopolymer solution, surfactant solution, alcohol solution, or PBS was added to 8 × 10 cells / well in 50 μL of infection medium (OptiMEM (Gibco) containing 3 mg / mL TPCK-trypsin). 4The cells were mixed with 1000 μL of SARS-CoV-2 mNG, vortexed, briefly centrifuged, and then incubated at 37°C for 10 or 60 minutes. After incubation, the samples were centrifuged at 1000 RPM for 1 minute and serially diluted 10-fold in infection medium up to the eighth dilution. 200 μL of each dilution was then plated into 24-well plates and incubated at 37°C for 1 hour, after which 800 μL of 1.2% Avicel (Dupont) was added. After a 24-hour incubation period at 37°C, Avicel was removed, and the cells were washed with 1x PBS and fixed in 10% neutral buffered formalin for 3 hours. Focus-forming units (FFU) per mL were determined by counting NeonGreen-expressing foci using an Evos M5000 fluorescence microscope (Thermo Scientific).

[0074] In vitro neutralization assay with adenovirus and respiratory syncytial virus. The broad-spectrum neutralization potency of PCANS was evaluated against adenovirus type 5 (ADV-5, ATCC, VR-2554™) and respiratory syncytial virus strain A2 (RSV-A2, ATCC, VR-1540™) using a plaque assay. Briefly, on the day before infection, 1 x 10 vaccinia ... 5 Vero E6 cells / well or 1.5 × 10 5 Hep-2 cells / well were seeded in a 24-well plate. On the day of infection, 50 mL of PCANS was added to 50 mL of infection medium containing 1 × 10 6 PFU / mL of ADV-5 and 2 × 10 6The cells were mixed with 100 PFU / mL of RSV-A2 and incubated at 37°C for 30 minutes. The pretreated mixture was serially diluted 10-fold in infection medium after incubation. Cells were washed with serum-free medium before infection, and 200 mL of each dilution was transferred to the cells for 1 hour of incubation, followed by the addition of 1 mL of overlay medium containing methylcellulose. After 72 hours of incubation, the overlay layer was removed, and the cells were fixed using 10% formalin. Immunostaining for Vero E6 cells and crystal violet staining for Hep-2 cells were then performed. Plaques were counted using a plaque reader (Bioreader-600-Va).

[0075] In vitro neutralization assay with bacteria. The neutralization potency of the components and PCANS was studied against Gram-negative bacteria, including Escherichia coli (E. coli) and Klebsiella pneumoniae. Overnight bacterial cultures were prepared in 5 mL of tryptic soy broth (TSB, Sigma-Aldrich) medium. On the day of the experiment, 10 8 OD equivalent to CFU / mL 600nm Bacterial suspensions were prepared to achieve a RI = 0.2. 50 μL of bacterial suspension in TSB medium was incubated with 50 μL of PCANS, biopolymer solution, surfactant solution, or alcohol solution at 37°C for 10 or 60 minutes. After incubation, the sample / bacterial mixture was serially diluted 10-fold with 1x PBS, and 10 mL of each dilution was plated onto pre-poured LB (Luria Broth) agar plates (HiMedia Laboratories Pvt Ltd) and subsequently incubated at 37°C and 5% CO2 for 16–18 hours. The plates were then counted for CFU.

[0076] Bacteriostatic assessment. An overnight culture of E. coli was prepared by inoculating a colony grown on agar into tryptic soy broth at 37°C, and the OD 600nm =0.2~10 7Appropriate dilutions were made to achieve CFU / mL. Equal volumes of the formulation, consisting of 0.2% w / v gellan, 0.75% w / v pectin, 0.01% w / v BKC, and 0.25% w / v phenylethyl alcohol, were incubated with the E. coli suspension for 24 hours. The absorbance was measured, and the percent reduction in bacterial growth was expressed relative to the untreated control.

[0077] TEER assay and in vitro cytotoxicity of Tween-80. RPMI 2650 cells were seeded into the apical compartment of a Transwell insert (6.5 mm polyester membrane, approximately 0.4 μm pore size, Corning) at a density of 1.5 × 10 cells / cm in 0.1 mL EMEM. The basolateral compartment of the insert was filled with 0.6 mL EMEM medium supplemented with 10% FBS and then incubated at 37°C. On day 4, the medium was removed from the top of the insert, and the medium volume in the bottom well was reduced to 200 μL. The medium was replaced every two days, and TEER was measured. Impedance was measured using an epithelial volt-ohm meter (World Precision Instrument). Cells were grown at the air-liquid interface until a monolayer with consistent impedance was formed around day 12. On day 12, TEER was measured, and then the cells were treated with surfactant. 200 μL of medium containing Triton X-100 (0.1% w / v) or various concentrations of Tween-80 was added to the inserts. The plates were incubated at 37°C for 4 hours. After incubation, the wells were replenished with fresh medium, and TEER was measured after 4, 5, 12, and 24 hours. The cytotoxic effects of various concentrations of Tween-80 were also studied in RPMI 2650 cells. Briefly, 20,000 cells / well were seeded into a 96-well plate and incubated overnight at 37°C to achieve 70–80% confluency. 0.2 mL of Tween-80 (0.01, 0.05, and 0.5% w / v) solution in EMEM medium was added to the wells, followed by incubation for 24 and 48 hours. The metabolic activity of RPMI 2650 cells was measured using an XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide) assay kit (ATCC) according to the manufacturer's protocol.

[0078] Respiratory droplet capture. The inner surface of the oropharyngeal region of a glass twin impinger (Copley Scientific) (indicated by the red arrow in Figure 4a) was coated with SNF using a VP3 nasal pump (Aptar) and then nebulized with a mixture of gellan (0.2% w / v) and pectin (0.75% w / v) without or with various concentrations of Tween-20, Tween-80, or BKC. Droplets with a mass median aerodynamic diameter of greater than 5 μm and loaded with rhodamine B-loaded liposomes (approximately 400 nm in size) were generated using a jet atomizer. The atomized droplets were administered to the impinger under vacuum at a flow rate of 15 L / min for 3 minutes. The gel was collected, and fluorescence intensity was quantified at excitation and emission wavelengths of 543 nm and 580 nm. Rhodamine B-loaded liposomes were prepared using the thin-film hydration method. 59 The lipids were synthesized using a 10 mg / mL lipid stock solution. Briefly, lipid DSPE-PEG(2000) amine (Avanti Polar lipids), cholesterol (Sigma), and hydrogenated L-α-phosphatidylcholine (soybean) (HPC, Avanti Polar lipids) were dissolved in chloroform in a 1:1:3 molar ratio to prepare a 10 mg / mL lipid stock solution. 2 mL of the lipid stock solution was added to a round-bottom flask containing 0.8 mL of rhodamine B isothiocyanate from a 1 mg / mL stock. The organic solvent was then evaporated using a rotary evaporator for 5 minutes to form a thin lipid layer. The lipid film was then hydrated using 10 mL of ultrapure water (Invitrogen), silica glass beads were added to the flask, and the lipid was suspended in the solution with vigorous shaking using a rotary evaporator for 45 minutes at 40 °C. The hydrated lipid suspension was sonicated (Probe sonicator) for 1 minute at 30% amplitude with 2-second pulses on and off, and liposome size was then analyzed using a Zeta analyzer (Malvern).

[0079] To emulate the capture of pathogen-laden droplets in the human nasal cavity, a 3D-printed transparent silicone human nose model (Koken Co., Ltd.) was used. SNF was deposited into the anterior region of the Koken model using a nasal pump (Aptar), followed by a mixture of gellan (0.2% w / v) and pectin (0.75% w / v) or PCANS in a single actuation. The Koken model was connected to a vacuum pump with an air flow rate of 15 L / min, and rhodamine B-loaded liposomes were then nebulized for 1 minute. After nebulization, the model was disassembled to collect the formulation and the captured dye-loaded droplets. Droplet capture was measured by quantifying fluorescence intensity at excitation and emission wavelengths of 543 nm and 580 nm.

[0080] Spray Characterization. Multidose nasal vials were filled with water, gellan solution, or PCANS. A pump (140 μL) with a 1.8 cm insertion depth (Aptar) was used to study spray characteristics, including plume shape, spray plume, and droplet size distribution. Three replicate measurements were performed for each sample. Plume shape and spray pattern were measured using a Spray-View® measurement system (Proveris Scientific, Hudson, MA) at a distance of 30 mm from the actuator nozzle orifice. This acquisition system utilizes a high-speed digital camera and laser light sheet to capture images. Data were analyzed using Viota® image processing software. Actuation parameters, including velocity, acceleration, and hold time, as well as camera and laser settings, were kept the same for all samples. Plume shape measures the angle of the plume as it exits the nozzle orifice. To quantify the sample spray pattern, ellipticity and plume area were evaluated. Ellipticity is defined as the ratio of the maximum to minimum cross-sectional diameter of the spray plume. A uniform, circular plume with an ellipticity close to 1 can be considered the optimal condition for nasal sprays. Droplet size analysis of the samples was performed using a Malvern Spraytec® laser diffraction system. The FDA recommends reporting size distribution data measurements at thresholds of D(v, 0.1), D(v, 0.5), and D(v, 0.9), which correspond to the 10%, 50%, and 90% droplet sizes by volume distribution, respectively. It is suggested to have droplet populations with D(v, 0.1) greater than 10 mm, D(v, 0.5) between 30 and 70 mm, and D(v, 0.9) less than 200 mm. Droplet populations smaller than 10 mm tend to induce non-target lung deposition, while droplets larger than 200 mm tend to drip / slide from the nasal cavity.

[0081] Storage Stability Study. PCANS (15 mL) was filled into actuator-capped sterile multi-dose nasal sprays (Aptar). Nasal vials were stored at accelerated temperature conditions (40°C). Aliquots were withdrawn at different time points and evaluated for neutralizing activity against IAV and SARS-CoV-2 using the plaque-forming and focus-forming assays described above, respectively. Aliquots were collected from three different vials. Similarly, 5 mL aliquots were used to evaluate spray characteristics, including spray pattern, plume shape, and droplet size distribution.

[0082] Mice. Animal experiments were performed in accordance with ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital. Experiments were performed on 6- to 8-week-old C57BL / 6 mice (Jackson Laboratories, USA). Mice were maintained under pathogen-free conditions and randomly assigned to various experimental groups regardless of gender. Animal group size in experiments was determined based on the minimum number of animals required to achieve statistical significance of P < 0.05 between different test groups. Experiments on the mouse model of influenza infection were performed at biosafety level 2 in accordance with ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital.

[0083] In vivo biodistribution and nasal retention. The nasal retention time of the formulations was determined in mice. Briefly, C57BL / 6 mice were administered 10 μL of free DiR (Thermofisher) or PCANS mixed with DiR at a final concentration of 10 μg / ml into each nostril. Mice were euthanized at different time points, and nasal cavities were collected and imaged using IVIS (Bruker's In-Vivo Xtreme optical and x-ray in vivo imaging system) at excitation and emission wavelengths of 680 / 700 nm. Vital organs, including the lungs, liver, spleen, kidneys, and heart, were also imaged at 2 and 24 h. To determine the mechanism behind the long retention time, animals were intranasally instilled with a mixture of gellan (0.02% w / v) and pectin (0.75% w / v) mixed with DiR, with or without BKC and Tween-80. After 8 hours, animals were euthanized and nasal cavities were collected and imaged using a Perkin Elmer IVIS Lumina II, and total flux was calculated as (p / sec / m 2 / sr).

[0084] In vivo prophylactic activity of PCANS. Under brief anesthesia using isoflurane, mice were intranasally instilled with 10 mL of PCANS or PBS into each nostril. 15 minutes later, animals were challenged intranasally with 250 PFU of PR8. One cohort of animals was followed for 10 days for weight change and survival. Animals were euthanized when their body weight dropped to 20% of the normal range. Animals from a second cohort were euthanized on either day 2 or day 4 postinfection, and lung virus titers, inflammatory cell counts in bronchoalveolar lavage (BAL) fluid, and inflammatory cytokine levels in lung homogenates were measured. BAL fluid was isolated by gently injecting saline solution into the bronchioles via a catheter inserted through the trachea. Total cells and immune cell types from the collected BAL fluid were quantified using a Diff-quik kit according to the manufacturer's protocol. For lung virus titer and cytokine profiling, the left lung was homogenized and centrifuged at 2000 g for 10 minutes at 4°C to collect the supernatant. The resulting supernatant was used for further downstream assays. Virus titers were enumerated using a plaque assay with MDCK cells, as previously described. Cytokine profiling was performed using IL-6, TNF-α, and IL-1β ELISA kits (BioLegend) according to the manufacturer's protocol. Histopathology of the right lung was determined using hematoxylin-eosin staining. To evaluate the time-dependent protection of PCANS, animals were challenged with 100 PFU of PR-8 via the intranasal route 2 or 4 hours after treatment with PCANS or PBS. Animals were euthanized on day 2 postinfection, and lung virus titers were quantified using a plaque assay.

[0085] statistics Statistical analysis and graphing were performed using Graphpad Prism. For comparisons of multiple groups, one-way analysis of variance with Tukey's post-hoc analysis was used. For analysis of data involving two variables, two-way analysis of variance with Tukey's multiple comparison test was used. To evaluate the efficacy of PCANS, Kaplan-Meier survival curves were used to generate survival plots, and the Gehan-Breslow-Wilcoxon test was used to analyze the statistical significance of the results. P values for weight change were determined using one-way analysis of variance with Brown-Forsythe post-hoc analysis. A P value of less than 0.05 was considered statistically significant.

[0086] Other Aspects All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0087] While the invention has been described with respect to specific embodiments thereof, it will be understood that it is capable of further modifications, and that any variation, use, or adaptation described herein, including departures from the invention as may be applied to the essential features described above, which generally follows from the principles described herein and is within the scope of the claims, and which are within the scope of known or customary practice in the art to which the invention pertains, is encompassed by this application and falls within the scope of the claims.

[0088] Other embodiments are within the scope of the claims.

Claims

1. (i) water; (ii) at least one mucoadhesive polymer / polysaccharide; (iii) at least one surfactant; A mucosal coating composition comprising:

2. (i) water; (ii) at least one mucoadhesive polymer / polysaccharide at a concentration of 0.1-20% w / v; (iii) at least one surfactant at a concentration of 0.005 to 5% w / v; A mucosal coating composition comprising:

3. 3. The mucosal coating of claim 1 or 2, wherein after application to a surface, the coating composition forms a barrier that reduces small molecule transport by at least 90% for 4 hours.

4. 3. The mucosal coating of claim 1 or 2, wherein after application to a surface, the coating composition forms a barrier that reduces viral transport by at least 90% for 4 hours.

5. 5. The mucosal coating of claim 1, wherein after application to a surface, the coating composition increases the capture of atomized particles by at least 1.5 times.

6. 6. The mucosal coating of any one of claims 1 to 5, wherein the coating composition increases the residence time of the coating after application to a surface, thereby resulting in at least twice as much coating remaining 8 hours after application as compared to a coating of identical composition but lacking surfactant.

7. The mucosal coating of any one of claims 1 to 6, wherein the mucoadhesive polymer / polysaccharide is present in a concentration of 0.25 to 20% w / v.

8. 8. The mucosal coating of claim 7, wherein the mucoadhesive polymer / polysaccharide is present in a concentration of 0.75 to 20% w / v.

9. 9. The mucosal coating of any one of claims 1 to 8, wherein the surfactant is present at a concentration of 0.01 to 5% w / v.

10. 10. The mucosal coating of claim 9, wherein the surfactant is present at a concentration of 0.05 to 5% w / v.

11. 11. The mucosal coating of claim 10, wherein the surfactant is present at a concentration of 0.5 to 5% w / v.

12. (i) water; (ii) at least one mucoadhesive polymer / polysaccharide at a concentration of 0.1-10% w / v; (iii) at least one surfactant at a concentration of 0.005 to 5% w / v; 1. A mucosal coating composition comprising:

13. The mucosal coating of any one of claims 1 to 12, wherein at least one mucoadhesive polymer / polysaccharide comprises a carboxyl group, a hydroxyl group, a sulfate group, or an acetamide group.

14. 14. The mucosal coating of claim 13, wherein the at least one mucoadhesive polymer / polysaccharide is selected from gellan, pectin, HPMC, CMC, xanthan, chondroitin sulfate, alginic acid, hyaluronic acid, and salts or derivatives thereof.

15. 15. The mucosal coating of claim 14, wherein the at least one mucoadhesive polymer / polysaccharide comprises a polymer of glucuronic acid or galacturonic acid.

16. 16. The mucosal coating of claim 15, wherein the at least one mucoadhesive polymer / polysaccharide is selected from pectin, gellan, HPMC, CMC, xanthan, and alginic acid, and salts or derivatives thereof.

17. At least one surfactant is (a) a hydrophilic surfactant, and (b) Nonionic surfactants or cationic surfactants The mucosal coating of any one of claims 1 to 16, wherein

18. 18. The mucosal coating of claim 17, wherein the at least one surfactant is selected from polysorbate surfactants, sorbitan fatty acid ester surfactants, and benzalkonium chloride.

19. 19. The mucosal coating of claim 18, wherein the at least one surfactant is selected from polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene 20 sorbitan monopalmitate, polyoxyethylene 20 sorbitan monostearate, and polyoxyethylene 20 sorbitan monooleate.

20. 20. The mucosal coating of any one of claims 1 to 19, further comprising an agent that neutralizes a pathogen.

21. 21. The mucosal coating of claim 20, wherein the agent is selected from a surfactant, an alcohol, an antibacterial agent, and an antiviral agent.

22. 22. The mucosal coating of any one of claims 1 to 21, comprising a mucoadhesive polysaccharide / polymer that possesses antiviral activity.

23. 23. The mucosal coating of claim 22, wherein the mucoadhesive polysaccharide / polymer possessing antiviral activity is pectin.

24. 22. The mucosal coating of claim 21, comprising an alcohol containing a tertiary or aromatic hydroxyl group.

25. 25. The mucosal coating of claim 24, wherein the alcohol is selected from phenethyl alcohol, benzyl alcohol, and chlorobutanol.

26. (i) water; (ii) at least one mucoadhesive polymer / polysaccharide selected from gellan, pectin, and combinations thereof; (iii) at least one surfactant selected from benzalkonium chloride, polyoxyethylene 20 sorbitan monooleate, and combinations thereof; (iv) phenethyl alcohol and A composition comprising:

27. 27. The composition of any one of claims 1 to 26, wherein the at least one mucoadhesive polymer / polysaccharide comprises gellan at a concentration of 0.1 to 10% w / v.

28. 28. The composition of any one of claims 1 to 27, wherein the at least one mucoadhesive polymer / polysaccharide comprises pectin at a concentration of 0.5 to 10% w / v.

29. 29. The composition of any one of claims 1 to 28, wherein the at least one surfactant comprises polyoxyethylene sorbitan monooleate at a concentration of 0.01 to 0.5% w / v.

30. 30. The composition of any one of claims 1 to 29, wherein the at least one surfactant comprises benzalkonium chloride at a concentration of 0.01 to 1% w / v.

31. 31. The composition of any one of claims 1 to 30, comprising 0.25 to 1% w / v phenethyl alcohol.

32. 32. The composition of any one of claims 1 to 31, comprising 0.2% w / v gellan, 0.75% w / v pectin, 0.05% w / v polyoxyethylene 20 sorbitan monooleate (Tween® 80), 0.01% w / v benzalkonium chloride, and 0.25% w / v phenethyl alcohol.

33. 33. The composition of any one of claims 1 to 32, which exhibits a residence time of 4 to 8 hours when applied to the mucous membrane of the nasal cavity.

34. 34. The composition of any one of claims 1 to 33, having a viscosity of 0.01 to 1 Pa s.

35. 35. The composition of claim 34, having a viscosity of 0.01 to 0.1 Pa s.

36. 36. The composition of any one of claims 1 to 35, wherein the at least one mucoadhesive polymer / polysaccharide comprises a polysaccharide having an average MW in the range of 10,000 to 2,000,000 Da.

37. 37. The composition of claim 36, wherein the at least one mucoadhesive polymer / polysaccharide comprises a polysaccharide having an average MW in the range of 50,000 to 500,000 Da.

38. 38. The composition of any one of claims 1 to 37, wherein the formulation contains less than 0.1% (w / w) solid particles.

39. 33. The composition of any one of claims 1 to 32, wherein the formulation further comprises a therapeutic or diagnostic agent.

40. 40. The composition of claim 39, wherein the formulation comprises an analgesic, an anti-inflammatory, an antihistamine, naltrexone, or melatonin.

41. 41. The composition of any one of claims 1 to 40, wherein the at least one mucoadhesive polymer / polysaccharide comprises gellan at a concentration of 0.1 to 0.4% w / v to achieve a sprayable formulation that forms a mucosal coating that reduces viral transport by at least 90% over a period of 4 hours.

42. 41. The composition of any one of claims 1 to 40, wherein the at least one mucoadhesive polymer / polysaccharide comprises pectin at a concentration of 0.25% to 2% w / v to achieve a nebulizable formulation with greater than 90% pathogen neutralization.

43. 43. The composition of claim 42, comprising 0.75±0.05% w / v pectin and benzalkonium chloride at a concentration of 0.1% or less to achieve greater than 99% pathogen neutralization.

44. A mucosal coating that enhances respiratory droplet capture by more than three times compared to uncoated surfaces and increases nasal residence time 44. The composition of any one of claims 1 to 43, wherein the at least one surfactant comprises polyoxyethylene sorbitan monooleate at a concentration of 0.01 to 0.05% w / v to achieve a formulation that forms

45. Mucosal coating that achieves over 90% pathogen neutralization and is non-toxic to mucosa and epithelium 42. The composition of any one of claims 1 to 41, wherein the at least one surfactant comprises benzalkonium chloride at a concentration of 0.005 to 0.02% w / v to achieve a formulation that forms:

46. 46. The composition of claim 45, comprising 0.010±0.005% w / v benzalkonium chloride and a gellan concentration of 0.2% w / v or less to achieve greater than 99% pathogen neutralization.

47. 46. The composition of claim 45, comprising 0.010±0.005% w / v benzalkonium chloride and pectin at a concentration of 1% w / v or less to achieve greater than 99% pathogen neutralization.

48. 48. A method of reducing the risk of exposure to an infectious pathogen in a mucosa of a subject, comprising topically applying to the mucosa of the subject the composition of any one of claims 1-47.

49. 49. The method of claim 48, wherein the composition is applied to the oral cavity, throat, vagina, nasal cavity, anus, or a wound.

50. 48. A method of reducing the risk of exposure to an infectious pathogen in a subject, comprising topically applying to the skin of the subject the composition of any one of claims 1-47.

51. The method of any one of claims 48 to 50, wherein the subject is a mammal.

52. The method of any one of claims 48 to 50, wherein the subject is a human, dog, cat, or livestock.

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