Native neutrophil-mediated transtympanic drug delivery to minimize antibiotic exposure in otitis media treatment background of the invention

Abstract

A composition includes a therapeutic compound, a liposome shell, and a pharmaceutically acceptable carrier comprising a stimuli-responsive hydrogel encapsulating the therapeutic compound. The liposome shell includes a plurality of hydroxyl-containing lipids.

Description

NATIVE NEUTROPHIL-MEDIATED TRANSTYMPANIC DRUG DELIVERY TO MINIMIZE ANTIBIOTIC EXPOSURE IN OTITIS MEDIA TREATMENT BACKGROUND OF THE INVENTION

[0001] Embodiments described herein relate generally to drug delivery, and more particularly, to native neutrophil-mediated transtympanic drug delivery to minimize antibiotic exposure in otitis media treatment.SUMMARY OF THE INVENTION

[0002] According to one or more embodiments, a composition includes a therapeutic compound, a liposome shell, and a pharmaceutically acceptable carrier comprising a stimuli- responsive hydrogel encapsulating the therapeutic compound. The liposome shell includes a plurality of hydroxyl-containing lipids.

[0003] According to other embodiments, a method of treating a patient with a disease includes administering to the patient an effective amount of the composition, and treating the disease in the patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1A illustrates a nature neutrophils-mediated non-invasive delivery strategy to infected middle ear according to embodiments, and specifically a schematic illustration of an artificial non-invasive drug delivery process, of which recruited neutrophils recognize and opsonize DSPG / sLip / Cip-iC3b complex, cany' them across the ty mpanic membrane and expel the cargo by the formation of neutrophils extracellular traps according to some embodiments;

[0005] FIG. IB illustrates DOTAP and DSPG dlipid structures, and the reaction between hydroxy group and C3b protein under bio-circumstance according to some embodiments;

[0006] FIG. 1C illustrates protein coronas (incubating liposomes with middle ear fluids sampled from infected chinchillas) characterized by western blot (n = 3) according to some embodiments;

[0007] FIG. ID illustrates the tympanic membrane (excised from NTHi-infected chinchilla) penetrating ability of DSPG / sLip and DOTAP / sLip, showing the cell nucleus 101, elastase 102 on the surface of neutrophils, and Dil labeled liposomes 103, according to some embodiments;

[0008] FIG. IE illustrates co-localization of liposomes with neutrophils in middle ear fluid inwhich the dates are means ± SD, and the scale bar is 20 jam, according to some embodiments;

[0009] FIG. 2 A illustrates characterization of liposomes-P407 hydrogel formulations and release kinetics, and in particular, cryo-EM characterizations of DOTAP / sLip / Cip-P407[15%] and DSPG / sLip / Cip-P407[15%] (49,000 times magnification) (double arrows indicate the bilayer of lipids and single arrows indicate the encapsulated ciprofloxacin crystals in liposomes), according to some embodiments;

[0010] FIG. 2B illustrates theology of P407[15%], DOTAP / sLip-P407[15%] and DSPG / sLip-P407[15%] (n = 3) according to some embodiments;

[0011] FIG. 2C illustrates release kinetics of ciprofloxacin from liposomes-hydrogel formulations under infinite sink conditions, in which the concentrations of ciprofloxacin were detected by HPLC (n = 3), according to some embodiments;

[0012] FIG. 2D illustrates the size distributions of solutions in reservoirs at the time point of 4 and 48 hours according to some embodiments;

[0013] FIG. 2E illustrates ciprofloxacin released from liposomes-hydrogel formulations at the time point of 4 and 48 hours, detected by HPLC (ciprofloxacin) and TECAN (Dil) respectively (n = 3, dates are means ± SD), according to some embodiments;

[0014] FIG. 3A illustrates cumulative ex vivo transfer of ciprofloxacin across the TM into a receiving chamber, and in particular, a schematic illustration of ex vivo experiments, according to embodiments;

[0015] FIG. 3B illustrates permeation percentages of ciprofloxacin across the tympanic membranes of healthy chinchillas at 37°C (n = 3) according to some embodiments;

[0016] FIG. 3C illustrates permeation percentages of ciprofloxacin across the infected ty mpanic membranes excised from NTHi infected chinchillas at 37°C from (n = 3 ~ 4, purple line indicates the formulation prepared with plain DSPG / sLip, free ciprofloxacin and P407, dates are median ± quartiles) according to some embodiments;

[0017] FIG. 4A illustrates in vivo efficacy and pharmacokinetics of DSPG / sLip / Cip- P407[15%], and specifically, schematic illustration of in vivo experiments, according to embodiments;

[0018] FIG. 4B illustrates percentage of infected ears in animals with otitis media from NTHitreated with DOTAP / sLip / Cip-P407[15%], Cip-P407[15%] and DSPG / sLip / Cip-P407[15%] (day 0 indicates status immediately before administration of formulations) according to some embodiments;

[0019] FIG. 4C illustrates a time course of bacterial CFU from middle ear fluid sampled from chinchillas with otitis media from NTHi treated with DOTAP / sLip / Cip-P407[15%], Cip- P407[15%] and DSPG / sLip / Cip-P407[15%] (loglO CFU is set to 0 instead of minus infinity for the purpose of this illustration) according to some embodiments;

[0020] FIG. 4D illustrates concentrations of ciprofloxacin over time in the middle ear fluid of the same chinchillas as in Fig. 4B and Fig. 4C (dates are median ± quantiles (n= 4)) according to some embodiments;

[0021] FIG. 5A illustrates in vivo efficacies and ciprofloxacin pharmacokinetics of DSPG / sLip / Cip-P407[15%] without complement proteins, and in particular, a schematic illustration of in vivo experiment with cobra venom factor (CVF) pre-treated chinchillas according to some embodiments;

[0022] FIG. 5B illustrates the level of C3b over time in middle ear fluid and serum sampled from CVF pre-treated chinchillas follow the scheme of Fig. 1A (day of -1 indicates status immediately before injection of CVF) according to some embodiments;

[0023] FIG. 5C illustrates percentage of infected ears in CVF pre-treated chinchillas with OM from NTHi after treatment with DSPG / sLip / Cip-P407[15%] according to some embodiments;

[0024] FIG. 5D illustrates a time course of bacterial CFU from middle ear fluid of the same animals in Fig. 5C treated with DSPG / sLip / Cip-P407[15%] according to some embodiments;

[0025] FIG. 5E illustrates concentrations of ciprofloxacin over time in the middle ear fluid of the same chinchillas as in Fig. 5C and Fig. 5D according to some embodiments;

[0026] FIG. 5F illustrates percentage of ciprofloxacin in middle ear fluid at day 1 calculated from Fig. 5E (dates are median ± quantiles (n = 4)) according to some embodiments;

[0027] FIG. 6A illustrates in vivo impact on the tympanic membrane, and in particular, representative photomicrographs of hematoxylin and eosin (H&E)-stained sections of tympanic membrane cross-sections in untreated NTHi infected and uninfected tympanic membranes, and treated tympanic membrane cross-sections after 7 days of otitis media treatedwith Cip-P407[15%], DOTAP / sLip / Cip-P407[15%], DSPG / sLip / Cip-P407[15%] and DSPG / sLip / Cip-P407[15%] (CVF) (operated with CVF pre-treated chinchilla following Fig.5 A scheme)) according to some embodiments;

[0028] FIG. 6B illustrates the thickness of the tympanic membrane was calculated with ImageJ as the same tympanic membrane with Fig. 6A (scale bar, 50 pm. data are means ± SD (n = 3)) according to embodiments;

[0029] FIG. 7 A illustrates the existence of neutrophils on the infected tympanic membrane, and in particular, showing tympanic membranes that were excised from NTHi-infected chinchillas (72 hours), fixed with formalin, and stained with hematoxylin and eosin (H&E) stain (white arrows indicate neutrophils close to the stratum comeum layer, scale bar, 10 pm) according to some embodiments;

[0030] FIG. 7B illustrates the tympanic membranes of Fig. 7A, with white arrows that indicate neutrophils close to the stratum comeum layer, according to embodiments;

[0031] FIG. 8 illustrates the existence of neutrophils in middle ear fluid according to embodiments, and in particular, middle ear fluid was sampled from NTHi-infected chinchillas (72 hours) and stained with the Wright-Giemsa method (white arrows indicate neutrophils, scale bar, 10 pm);

[0032] FIG. 9 illustrates the cleavage process of complement 3 protein through alternative activation pathway according to embodiments, in which C3 is composed of two polypeptide chains (a and 0) linked by disulfide bonds, and chain a fragments contain molecular weights of approximately 67, 46, and 40 kDa);

[0033] FIG. 10 illustrates characterization of C3 level in middle ear fluid according to embodiments, in which middle ear fluid was sampled from NTHi-infected chinchilla and characterized by western blot, and iC3b fragments were detected by anti-complement antibody;

[0034] FIG. 11 A illustrates characterization of C3 level in serum and protein corona according to embodiments, in which serums were sampled from NTHi-infected chinchillas, and the level of C3 in serum was characterized by w estern blot (n = 3);

[0035] FIG. 11B illustrates characterization of the level of C3b in protein coronas produced by incubating liposomes with serum (n = 3) according to some embodiments;

[0036] FIG. 12A illustrates size distribution and zeta potential of liposomes according to embodiments, and in particular, size distribution and zeta potential (Q of DOTAP / sLip and DOTAP / sLip / Cip (dates are means ± SD);

[0037] FIG. 12B illustrates size distribution and zeta potential (Q of DSPG / sLip and DSPG / sLip / Cip according to some embodiments;

[0038] FIG. 12C illustrates the statistical results of size (nm) and zeta potential ( , mv) of liposomes (n = 3);

[0039] FIG. 13 A illustrates rheology characterization of formulations according to embodiments, and in particular, rheology of hydrogels with or without liposomes containing 10% P407 (dates are means ± SD (n = 3));

[0040] FIG. 13B illustrates rheology of hydrogels with or without liposomes containing 20% P407 according to some embodiments;

[0041] FIG. 14 illustrates ex vivo cumulative ciprofloxacin in reservoirs after 48 hours according to embodiments, and in particular, comparing DSPG / sLip / Cip with DOTAP / sLip / Cip penetrating capability across uninfected and infected TM (the fold ratio is DSPG / sLip / Cip divided by DOTAP / sLip / Cip. and dates are means ± SD (n = 3 ~ 4));

[0042] FIG. 15 A illustrates in vivo efficacy of ciprofloxacin trans the ty mpanic membrane according to embodiments, and in particular, the percentage of ciprofloxacin trans the tympanic membrane in NTHi-infected chinchillas (dates are median ± interquartile (n = 4));

[0043] FIG. 15B illustrates the percentage of ciprofloxacin trans the tympanic membrane in CVF pre-treated NTHi-infected chinchillas according to some embodiments;

[0044] FIG. 16A illustrates auditory brainstem results of DSPG / sLip / Cip-P407[15%J according to embodiments, and in particular, auditory7brainstem result wave up to 10 ms (black and orange arrows indicate wave II absolute latency and amplitude, respectively, non-treated animals w ere set as control, 0 min indicates status immediately before administration, dates are means ± SDs (n = 3));

[0045] FIG. 16B illustrates wave II latency with specified time points after being administered with DSPG / sLip / Cip-P407[15%] through the external auditory canal according to some embodiments;

[0046] FIG. 16C illustrates amplitude with specified time points after being administered with DSPG / sLip / Cip-P407[15%] through the external auditory canal according to some embodiments;

[0047] FIG. 17A illustrates cytotoxicity of fibroblast cells according to embodiments, and in particular, fibroblast cells viabilities at day 1 and day 3 treated with Cip, P407[15%], Cip- P407[15%], DOTAP / sLip / Cip-P407[15%], and DSPG / sLip / Cip-P407[15%], respectively (total dose of ciprofloxacin was 0.5 mg for all groups, data are means ± SD (n = 3), GFP (green fluorescent protein), live cells (green), TRITC (tetramethyl rhodamine isothiocyanate), dead cells (red), scale bar, 50 pm);

[0048] FIG. 17B illustrates LIVE / DEAD assay of fibroblasts, which was done to confirm the data in FIG. 17A, according to some embodiments;

[0049] FIG. 18A illustrates cytotoxicity of PC 12 cells. (A) PC 12 cells viabilities at day 1 and day 3 treated with Cip, P407[15%], Cip-P407[l 5%], DOTAP / sLip / Cip-P407[15%], and DSPG / sLip / Cip-P407[15%]. respectively according to embodiments (total dose of ciprofloxacin was 0.5 mg for all groups, data are means ± SD (n = 3), GFP (green fluorescent protein), live cells (green), TRITC (tetramethyl rhodamine isothiocyanate), dead cells (red), scale bar, 50 pm);

[0050] FIG. 18B illustrates LIVE / DEAD assay of PC 12 cells, which was done to confirm the data in FIG. 18A, according to some embodiments;

[0051] FIG. 19 illustrates the ex vivo neutrophils uptake of the liposomes according to embodiments, and in particular, neutrophils originated from BALB / c mice bone marrow7;

[0052] FIG. 20 illustrates in vivo efficacy of PG / sLip / Cip according to embodiments.

[0053] FIG. 21 A illustrates western blot of the liposomes’ protein corona after incubating with BALB / c mice serum (i-ii) (****P < 0.0001) and the quantify of iC3b fragments in the protein corona of DOTAP / sLip and DSPG / sLip, respectively, according to some embodiments;

[0054] FIG. 21B illustrates ex vivo uptake of the opsonized liposomes by the innate neutrophils harvested from the bone marrow7of BALB / c mice (identify of the neutrophils was confirmed using flow cytometry, where neutrophils were gated at CDllb+ / LY6G+ (i); the liposomes were fluorescently labeled using 3,3'-Dioctadecyloxacarbocyanine (DiO), andincubated with the neutrophils ex vivo for 1 h (ii) and 4 h (iii), respectively, with the fraction of liposome-containing neutrophils quantified (iv); ***P = 0.0003 and ***P = 0.0002) according to some embodiments;

[0055] FIG. 21 C illustrates in vivo uptake after intravenous injection (50 mg lipid per kg body weight) of the two types of liposomes by BALB / c mice peripheral neutrophils (i) at Ih (ii) and 4 h (iii), respectively, with the fraction of liposome-containing neutrophils quantified (iv); ***P = 0.0008 and **P = 0.0058, according to some embodiments;

[0056] FIG. 22A illustrates cumulative ex vivo transfer of ciprofloxacin across the TM into a receiving chamber according to embodiments, and in particular, a diagram of the ex vivo protocol for assessing ciprofloxacin (Cip) permeation across the TM (a colony -forming unit (CFU) value of 0 indicates healthy animals);

[0057] FIG. 22B illustrates permeation (%) of Cip across the TMs of healthy chinchillas over 48 hours (n = 3) according to some embodiments; and

[0058] FIG. 22C illustrates permeation (%) of Cip across the TMs of chinchillas with AOM over 48 hours (n = 4) (DSPG / sLip-Cip-P407 (purple line) indicates a mixture of DSPG / sLip, free Cip, and P407; *P = 0.0146, *P = 0.0177, *P = 0.0170 and **P = 0.0097 (from left to right), calculated using one-way analysis of variance (ANOVA) with Turkey’s test; *P < 0.05; **P < 0.01, all formulations contained 0.5 mg Cip; in all formulations containing P407, its concentration is 15% (w / v), data are presented as mean ± s.d.) according to some embodiments.DETAILED DESCRIPTION OF THE INVENTION

[0059] Certain exemplary embodiments will now' be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the systems and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the systems and methods specifically described herein and illustrated in the accompanying drawings are nonlimiting exemplary embodiments and that the scope of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure.Accordingly, aspects and features of every embodiment may not be described with respect to each embodiment, but those aspects and features are applicable to the various embodiments unless statements or understandings are to the contrary.

[0060] As used herein, the term “patient” or “user” refers to any subject, including but not limited to, plants, pathogens, animals (e.g., pets, farm animals, etc.), and humans. The patient may have a condition and / or disease or suspected of having a condition and / or disease and as such is being treated with a drug. In some instances, the patient is a mammal, such as a human, a premature neonate, neonate, infant, juvenile, adolescent, or adult thereof. In some instances, the term “patient,” as used herein, refers to a human (e.g., a man, a woman, or a child). In some instances, the term “patient,” as used herein, refers to plants, pathogen, or laboratory animal of an animal model study. The patient or subject may be of any age, sex, or combination thereof.

[0061] The term “treating” refers to administering a therapy in an amount, manner, or mode effective (e.g., a therapeutic effect) to improve a condition, symptom, disorder, or parameter associated with a disorder, or a likelihood thereof.

[0062] The terms “essentially” or “substantially” as used herein mean to a great or significant extent, but not completely.

[0063] The term “about” as used herein refers to any values, including both integers and fractional components that are within a variation of up to plus or minus 10% of the value modified by the term “about.” In some embodiments, “about” means plus or minus 1%, 2%. 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the stated value.

[0064] Acute otitis media (AOM) is the most common reason U.S. children take oral antibiotics, causing severe systemic side effects. The local delivery7of antibiotics to the middle ear requires crossing the tympanic membrane (TM), which is minimally permeable to nearly all molecules.

[0065] Described herein are compositions and methods that leverage an innate immune response to achieve an unprecedented transtympanic delivery efficiency and complete eradication of AOM in vivo. The compositions and methods harness neutrophils as endogenous micromotors for the targeted delivery7of the antimicrobial cargo to the infected middle ear. Theneutrophil hitchhiking is enabled by a simple liposome, which is designed to trigger enhanced opsonization by a complementary protein and subsequently phagocytosis by neutrophils. This delivery strategies achieve an unprecedented transtympanic delivery ratio that is, in some embodiments, two-orders-of-magnitude greater than existing liposome-free systems. In one or more embodiments, compositions and methods eradicate AOM in 100% of the chinchillas infected by nontypable Haemophilus influenzae (NTHi). The strategy of leveraging the complement system to harness neutrophils is unprecedented, which bypasses the need for costly targeting biomolecules for the treatment of this prevalent disease, thus accelerating the progression along the discovery to deployment pathway to reduce pediatric antibiotic usage globally.

[0066] AOM is the primary reason for pediatric antibiotic usage, accounting for 24% of the antibiotic prescriptions written to U.S. children. Approximately 80% of all children experience at least one episode of AOM by school age, making AOM the most common reasons for pediatrician visits. An estimated 709 million episodes are reported globally every year, leading to an annual cost (hospitalization, emergency department / urgent care visits, and outpatient visits) of $ 4.3 billion in the U.S. alone.

[0067] AOM is predominantly caused by bacterial infections, among which the pathogen non-typeable Haemophilus influenzae (NTHi) accounts for 30-52% of the total AOM episodes globally. The current mainstay treatment for AOM comprises oral antibiotics for 5-10 days.

[0068] However, oral antibiotics cause acute side effects including diarrhea and vomiting in more than 50% of U.S. children and are correlated with long-term health issues, such as asthma and obesity. As a result, approximately 1 / 3 of families in the U.S. discontinue the medication prematurely, particularly if symptoms resolve. The poor patient compliance speeds up the development of antibiotic resistance, which ranks among the biggest threats to global health by the World Health Organization. Therefore, minimizing the antibiotic exposure during AOM treatment is pivotal to reducing the global pediatric antibiotic usage and mitigating the development of drug resistance.

[0069] Accordingly, compositions and methods described herein are fresh concepts to target the delivery of therapeutic compounds such as antibiotics to the infected middle ear byhitchhiking on the patient’s innate neutrophils, overcoming the foregoing challenges. In contrast to existing immune-cell mediated therapies, which rely on circulating immune cells, thus calling for systemic drug administration, we avoid the systemic exposure entirely by employing neutrophils that readily cross the impermeable barrier of the tympanic membrane (TM). The methods thus harness the neutrophils as endogenous micromotors to traffic the antibiotic cargo that is placed in the patient's outer ear canal across the TM and into their infected middle ear (see Fig. 1 A).

[0070] In one or more embodiments, compositions include a therapeutic compound, a liposome shell encapsulating the therapeutic compound, with the liposome shell comprising a plurality of hydroxyl-containing lipids, and a pharmaceutically acceptable carrier comprising a stimuli-responsive hydrogel.

[0071] In other embodiments, methods of making compositions described herein include combining the therapeutic compound with the plurality of hydroxyl-containing lipids to form liposomes comprising the hydroxyl-containing lipids and the therapeutic compound, and combining the liposomes with the pharmaceutically acceptable carrier comprising the stimuli- responsive hydrogel.

[0072] Non-limiting examples of therapeutic compounds include a small molecule or a macromolecule. For example, the therapeutic compound is, but is not limited to, a peptide, a glycopeptide, a protein, a hormone, a synthetic nanoparticle, a nucleic acid, or any combination thereof. In some embodiments, the therapeutic compound is an antibiotic. In one or more embodiments, the therapeutic compound is a penicillin, a cephalosporin, a tetracycline, an aminoglycoside, a macrolide, a sulfonamide, a trimethoprim, a quinolone, a nitrofurantoin, a vancomycin, or any combination thereof. Yet, in some embodiments, the therapeutic compound is ciprofloxacin.

[0073] In one or more embodiments, the therapeutic compound has a molecular weight of less than 4,000 Daltons, or about 100 to about 3,500 Daltons, including any value therewithin or any subranges therebetween. In some embodiments, the therapeutic compound has a molecular weight about or in any range between about 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 Daltons.

[0074] In some embodiments, the therapeutic compound is at least 0.01 wt% of the total composition (e g., 0. 1-70 wt%, including any value therewithin or any subranges therebetween). In one or more embodiments, the therapeutic compound is about or in any range between about 0.01, 5. 10. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70 wt% of the total composition

[0075] The TM, while only ~ 100-pm mean thick, has been a longstanding bottleneck in transtympanic drug delivery, as it is largely impermeable to most molecules, including antibiotics. Existing approaches for the local delivery into the middle ear often rely on painful disruption of the TM (e g., surgically placement of a tympanostomy tube). While chemical permeation enhancers (CPE) have enabled non-invasive transtympanic delivery of antibiotics, the delivery efficiency (i.e., the fraction of antibiotics delivered across an intact TM) of CPEbased systems have been capped at 2% to 3.8%. As a result, antibiotics often need to be formulated at their solubility limit, exacerbating the local exposure to high drug concentrations, yet the eradication of pathogens is incomplete in a chinchilla AOM model. Elamessing the TM- traversing capability of innate neutrophils, methods include employing an antibiotic-loaded liposome that achieves an unprecedented transtympanic drug delivery efficiency of 14.4% or more in some embodiments (a 4-fold of the best-in-class formulation reported to date), and in turn completely eradicates OM in all chinchillas infected by NTHi.

[0076] The liposomes hitchhike on neutrophils by leveraging the complement cascade. During AOM, neutrophils, the most abundant leukocyte in the bloodstream, undergo chemotaxis, migrating from the bloodstream toward the middle ear. Along the way, neutrophils phagocytize foreign substances opsonized by the complement system, most notably fragments of the complement component 3 (C3). i.e., C3b and iC3b 17. whose levels will be high in the middle ear fluid (MEF) of AOM patients. As the exposed thioester groups on C3b are known to react with hydroxyl (OH) groups on foreign substances, some embodiments include making the liposome using a OH-bearing phospholipid, for example, l,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG) (Fig. IB). The C3 fragments covalently linked to the liposome serve as biomarkers for enhanced neutrophil phagocytosis and trafficking to the infected middle ear.

[0077] In one or more embodiments, the liposome shells of the compositions and methods comprise a plurality' of hy droxyl-containing lipids that comprises a cationic lipid, a neutral lipid.a negatively charged lipid, or any combination thereof. In some embodiments, the liposome shell comprising the plurality of hydroxyl-containing lipids comprises a poly(ethylene glycol)- phospholipid, a sterol, or any combination thereof.

[0078] In one or more embodiments, the liposome shell comprising the plurality of hydroxyl- containing lipids are negatively charged. In some embodiments, hydrogel formulations containing positively and negatively charged liposomes outperformed positively charged ones in terms of in vitro release, permeation across intact TM ex vivo, in vivo AOM treatment efficacy, and tissue-level biocompatibility using an established chinchilla model.

[0079] Non-limiting examples of the plurality of hydroxyl-containing lipids include 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l- aminium; N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide;1.2-dioctanoyl-sn-glycerol, l-O-l’-(Z)-octadecenyl-sn-glycerol; phosphatidylserine; 1-olelyl- 2-lysophosphatidate, 1 ,2-Dimyristoyl-sn-glycero-3-phosphoglycerol; 1 ,2-dipalmitoyl-sn- glycero-3-phosphoglycerol; l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol);1.2-dioleoyl-sn-glycero-3-phosphoglycerol, l,2-distearoyl-sn-glycero-3-phosphoglycerol, cholesterol, poly(ethylene glycol)-distearoylphosphatidylethanolamine, or any combination thereof.

[0080] Still in other embodiments, the liposome shell comprising the plurality of hydroxyl- containing lipids comprises a phospholipid - polyethylene glycol conjugate. In some embodiments, the liposome shell compnsing the plurality of hydroxyl-containing lipids comprises a phospholipid conjugate.

[0081] In one or more embodiments, the liposome shell comprising the plurality of hydroxyl- containing lipids comprises about 30% to about 70% molar ratio of a phospholipid, including any value therewithin or any subranges therebetween; about 0% to about 50% molar ratio of a sterol, including any value therewithin or any subranges therebetween, e.g. greater than 0% and less than 50%; and about 0% to about 10% molar ratio of a phospholipid - polyethylene glycol conjugate, including any value therewithin or any subranges therebetween, e.g. greater than 0% and less than 50%.

[0082] In some embodiments, the liposome shell comprising the plurality' of hydroxyl-containing lipids comprises about or any range between about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% molar ratio of a phospholipid.

[0083] In other embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises about or any range between about 0%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and about 50% molar ratio of a sterol.

[0084] Yet in other embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises about or any range between about 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 8%, and 10% molar ratio of a phospholipid - polyethylene glycol conjugate.

[0085] Still yet, in some embodiments, the liposome shell or the hydroxyl-containing lipids is at least 1 wt% of the total composition (e.g. 1-70 wt% including any value therewithin or any subranges therebetween). In other embodiments, the liposome shell is about or any range between about 1, 5, 10, 15, 20, 25. 30. 35, 40, 45, 50, 55, 60, 65, and 70 wt% of the total composition. Yet, in other embodiments, the hydroxyl containing lipids are about or any range between about 1, 5, 10, 15, 20. 25. 30. 35, 40, 45, 50, 55, 60, 65, and 70 wt% of the total composition.

[0086] In some embodiments, the stimuli-responsive hydrogel is a temperature-dependent hydrogel, a shear-thinning hydrogel, a cross-linkable hydrogel, or a combination thereof. In other embodiments, the stimuli-responsive hydrogel comprises an amphiphilic block copolymer. A non-limiting example of the amphiphilic block copolymer comprises a triblock copolymer. In one or more embodiments, the triblock copolymer comprises a poly(ethylene oxide) (PEO) block and a polypropylene oxide) (PPO) block. In other embodiments, the stimuli-responsive hydrogel comprises a poloxamer.

[0087] In some embodiments, the stimuli-responsive hydrogel is an amount of about 0. 1% to about 30% w eigh t / volume (w / v) of the total composition, including any value therewithin or any subranges therebetween. In other embodiments, the stimuli-responsive hydrogel is an amount about or any range between about 0.1, 1, 5, 10, 15, 20, 25, and 30% weight / volume (w / v). In one or more embodiments, compositions include a pharmaceutically acceptable carrier comprising a stimuli-responsive hydrogel, and the pharmaceutically acceptable carrier or the stimuli-responsive hydrogel comprises a polymer and a solvent (e.g. w ater), wherein the carrieror the hydrogel is about 0.1% to about 30% weight / volume (w / v) of the total composition, including any value therewithin or any subranges therebetween. In some embodiments, the pharmaceutically acceptable carrier is about or any range between about 0.1, 1, 5, 10, 15, 20, 25, and 30% weight / volume (w / v).

[0088] In some embodiments, the pharmaceutically acceptable carrier or the stimuli- responsive hydrogel comprises one or more polymers and a solvent (e.g. water) wherein the one or more polymers are at least 5 wt% of the carrier or hydrogel (e.g. 5-40 wt% of the of the carrier or hydrogel, including any value therewithin or any subranges therebetween), and the solvent has at least 50 wt% of water (e.g. 60-95 wt% of the of the carrier or hydrogel, including any value therewithin or any subranges therebetween). In one or more embodiments, the pharmaceutically acceptable carrier comprises one or more polymers and a solvent (e.g. water), wherein the one or more polymers are about or any range between about 5. 10. 15, 20, 25, 30, 35, and 40 wt% of the pharmaceutically acceptable carrier. In other embodiments, the stimuli- responsive hydrogel comprises one or more polymers and a solvent (e.g. water), wherein the one or more polymers are about or any range between about 5, 10, 15, 20, 25, 30, 35, and 40 wt% of the stimuli-responsive hydrogel.

[0089] Methods of making the compositions include combining the therapeutic compound with the plurality of hydroxyl-containing lipids to form liposomes comprising the hydroxylcontaining lipids and the therapeutic compound; and combining the liposomes with the pharmaceutically acceptable carrier comprising the stimuli-responsive hydrogel. Combining the therapeutic compound with the plurality7of hydroxyl-containing lipids to form the liposomes comprises encapsulating the therapeutic compound with the plurality of the hydroxyl-containing lipids. The resulting liposomes comprise the therapeutic compound arranged in, tethered to, or both arranged in and tethered to, lipid bilayers of the liposomes.

[0090] In some embodiments, encapsulating the therapeutic compound comprises adding the plurality of hydroxyl-containing lipids to a solvent; removing the solvent from the plurality of hydroxyl-containing lipids to form a film; hydrating the film to form a lipid suspension; and combining the lipid suspension with the small molecule therapeutic compound. Anon-limiting example of a solvent includes water.

[0091] Methods of treating a patient with a disease include administering to the patient an effective amount of any one of the compositions described herein; and treating the disease in the patient.

[0092] Non-limiting examples of the disease include an ear infection, chronic inflammatory reaction, a chronic infection, an acute inflammatory reaction, and acute infection, or any combination thereof. In some embodiments, the disease is otitis media, a skin infection, a skin bum, or a combination thereof.

[0093] In one or more embodiments, the effective amount of the composition is a single dose.

[0094] In other embodiments, administering to the patient the effective amount of the composition comprises non-invasive administration to an external auditory canal onto the tympanic membrane of the patient.

[0095] In some embodiments, the compositions have a drug delivery efficiency (e.g. a transtympanic drug delivery efficiency) of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%. at least 70%. or at least 80% (e.g. 10%- 30% for a transtympanic drug delivery efficiency or higher for transcutaneous delivery efficiency, including any value therewithin or any subranges therebetween), and wherein a drug delivery efficiency is calculated as a percentage of an amount of drug delivered to a target location in the body of a patient (e.g. middle ear), divided by the total amount of drug administered to the patient.

[0096] In other embodiments, the methods have a drug delivery efficiency (e.g. a transty mpanic drug delivery efficiency) of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%. at least 40%. at least 50%. at least 60%, at least 70%, or at least 80% (e.g. 10%- 30% for a transtympanic drug delivery efficiency or higher for transcutaneous delivery efficiency, including any value therewithin or any subranges therebetween), and wherein a drug delivery efficiency is calculated as a percentage of an amount of drug delivered to a target location in the body of a patient (e.g. middle ear), divided by the total amount of drug administered to the patient.EXAMPLESExample 1

[0097] Non-limiting example described as follows illustrate ciprofloxacin in a liposome shell, as it is commonly used in AOM treatment when the TM is perforated.

[0098] The ciprofloxacin encapsulating DSPG liposomes (DSPG / sLip / Cip) were loaded into a thermal responsive hydrogel made of Poloxamer 407 (P407). Importantly, the formulation was a solution under room temperature for the ease of administration into the outer ear canal, and gelled firmly upon reaching the TM.

[0094] As such, these results demonstrate that a single dose (e.g., applied in the pediatrician’s office) could sustain the release of the antibiotics over the 7-day treatment, avoiding the premature discontinuation of the treatment and development of drug resistance. The neutrophilbased mechanism enables a record-low amount of antibiotics for the entire course, i.e., 0.5 mg ciprofloxacin, which corresponds to 6.25% that in the best-in-class transtympanic formulationl3. The local administration and high transtympanic delivery efficiency combined minimized antibiotic exposure during AOM treatment.

[0095] The strategy of leveraging the complement system to harness the innate neutrophils as drug delivery’ vehicles is unprecedented. To illustrate the critical role played by the complement system, cobra venom factor (CVF) infected chinchillas (which has been used to deplete complement proteins22,23, prior to receiving the liposomes) were treated.

[0096] Only 25% of the animals cleared their AOM, in stark contrast to the 100% cure rate in ones without the CVF treatment. Capitalizing on the innate complement system enables the targeted delivery without the requirement for the targeting biomolecules, such as antibodies, that are prohibitively costly to deploy for such a prevalent disease. The transtympanic delivery’ platform thus holds enormous potential to reduce pediatric antibiotic usage globally, thus mitigating acute and long-term health issues related to systemic antibiotic exposure early in life.

[0097] Poloxamer 407 (P407) hydrogels are suitable for this system because P407 polymers can provide excellent fluidity during application followed by in situ gelation. As a novel reverse thermal gelation polymer, P407 polymer can achieve administration with fluid and transform to non-flow hydrogel to maintain topical sustained drug delivery'. Additionally, hydrogels often have excellent biocompatibility' in humans.

[0098] A hydrogel drug delivery system was designed to contain liposomes (with ciprofloxacin encapsulated in liposomes) and P407. The design of the formulation was that hydrogel containing liposomes (loaded with ciprofloxacin) should flow readily to the tympanic membrane through the external auditory canal, and then promptly become gel to maintain topical sustained release throughout the course of treatment (Fig. 1 A).

[0099] The formulations were named lipid / sLip / DiI / Cip-P407[z%], where z = 10. 15 or 20% (w / v) of P407, lipid denotes DOTAP or DSPG with a concentration of 10 mg / ml, sLip refers to stealth liposomes (here, synthesized w ith 5% molar ratio of polyethylene glycol), Dil refers to Tetramethylindocarbocyanine Perchlorate dye (a hydrophobic compound that can incorporate itself into the lipid bilayers of liposomes) with a concentration of 100 pg / ml, and the concentrations of ciprofloxacin w ere 2.5 mg / ml in all formulations. If a component was absent from a formulation, it is omitted from the above nomenclature.

[0100] Chinchillas provided the animal model for the middle earNTHi infection. The disease could be produced by very small inoculation injected into the middle ear and remained localized to the middle ear in most cases. Neutrophils were found on the tympanic membrane (FIGS. 7A and 7B) and in the middle ear fluid (FIG. 8) when OM occur. The results were consistent with previous studies.

[0101] Because of the lack of protein data resources on Chinchilla Lanigera, the anti-guinea pig complement antibody was used to detect complement protein in chinchilla. According to the cleavage process of C3 protein (FIG. 9). the molecular weight of iC3b fragments were estimated, and the results were consistent with the western blot (FIG. 1C and FIGS. 10, 11 A, and 1 IB). The existence of C3 and its fragments were verified in the middle ear fluid (MEF) sampled from NTHi-infected chinchillas (infected for 72 hours, consistent with in vivo experiment) (FIG. 10). The level of C3 fragments in serum sampled from infected animals was characterized as well (FIGS. 11 A and 1 IB). The results provided the requisite components to elucidate the drug delivery strategy.

[0102] The structure of the tympanic membrane is similar to skin in the sense that the outmost stratum comeum layer is lipid lamellae with high proportion of negatively charged lipids, indicating that the tympanic membrane could act as negatively charged membrane. Therefore,positively charged particles were commonly considered to possess better transmembrane capability.

[0103] Here, novel positively surface-charged DOTAP liposomes were used as a control group to study if the strategy was effective (structure of lipids, see FIG. IB). DSPG lipid has a hydroxyl group at one end as FIG. IB shows. The hydroxy groups stay at the surface of liposomes after automatic re-assembling because of the hydrophobic force. Whether DSPG / sLip could react with C3b fragments compared with DOTAP / sLip were also analyzed.

[0104] DOTAP and DSPG liposomes were well prepared and characterized, exhibiting sizes around 130 nm and surface charges of approximately +20 mv or -20 mv, respectively (FIGS. 12A, 12B, 12C).

[0105] Protein coronas produced by liposomes incubating with MEF were analyzed and results demonstrated that DSPG / sLip could strongly react with C3b fragments in MEF and deposit these complement biomarkers on their surface more effectively than DOTAP / sLip (P < .01, **) (FIG. 1C). Protein coronas of liposomes with serum sampled from infected chinchillas were characterized as well (FIGS. 11 A and 11B).

[0106] Whether DSPG / sLip could transport through the tympanic membrane and arrive at the infected middle era by hijacking neutrophils was studied. DSPG / sLip / DiI-P407[15%] and DOTAP / sLip / DiI-P407[15%] were applied onto the tympanic membrane through the external auditory canal with the chinchillas of OM.

[0107] Animals were euthanized after being treated for 24 hours, and the tympanic membrane and middle ear fluid were collected and stained with Hoechst and elastase on the surface of neutrophils. Immunocytochemistry results showed that DSPG / sLip could diffuse the tympanic membrane deeper than DOTAP / sLip (FIG. ID), and DSPG / sLip were found engulfed by neutrophils and existed in the middle ear fluid (FIG. IE).Example 2

[0108] Whether liposomes (loaded with ciprofloxacin) could maintain an intact structure in 15% P407 with the help of Cryo-EM was studied. High-resolution pictures (49,000 times magnification) showed that both DOTAP and DSPG liposomes structures were intact. The phospholipid bilayers membrane and the encapsulated ciprofloxacin crystals were clearlyvisible (FIG. 2A).

[0109] Next, the impacts of liposomes on the gelation behaviors of P407 was assessed. As for the hydrogel with 20% P407 (FIGS. 13A and 13B), the sol-gel transition temperature was around 24°C, and the storage (G’) and loss (G”) moduli (measured by linear oscillatory shear rheology at 100 rad-1, 1% strain, and 1°C min-1) were around 15 and 1.5 kPa at body temperature, respectively. This means that the formulation with 20% of P407 would be very hard to be extruded through a catheter to mimic application in vivo to the tympanic membrane, presumably because the friction generated during injection was enough to heat the formulations and clog the catheter.

[0110] However, the formulations with 10% P407 were still fluid at body temperature (FIGS. 13A and 13B). The material did not form a gel with 10% of P407.

[0111] Finally, 15% of P407 was analyzed and showed that the G’ and G” moduli were below 100 Pa at room temperature and behaved as a liquid. When the temperature was raised to body temperature, G' was around 3 kPa. Also, the addition of liposomes (both DOTAP and DSPG liposomes) did not significantly alter the rheology performance of the hydrogel (FIG. 2B). Example 3

[0112] The formulations included two components: liposomes, which were expected to transport the drug across the tympanic membrane, and P407 hydrogel, which was expected to slow flux yet prolong treatment for the duration that is needed to clear the infection. The effect of the hydrogel on the transport rate of liposomal ciprofloxacin was studied by in vitro diffusion from the bulk hydrogel matrix.

[0113] In vitro release experiments showed that the addition of 15% P407 slowed the release of the drug. The release kinetics of ciprofloxacin (detected by High-Performance Liquid Chromatography, HPLC) from DOTAP / sLip / DiI / Cip-P407[15%] and DSPG / sLip / Dil / Cip- P407[15%] performed similarity (FIG. 2C), as groups could release over 90% ciprofloxacin over the time course of 48 hours.

[0114] Solutions in reservoirs at 4 and 48 hours were evaluated by the dynamic light scattering method (DLS), and the size distribution results showed that the size of the solutions was the same as the initial liposomes (FIG. 2D). This indicated that ciprofloxacin was releasedfrom P407 with the shield of liposomes.

[0115] The Dil fluorescence of the solutions (at 4 hours and 48 hours) in reservoirs with TEC AN was measured. The released percentage of liposomes was plotted and calculated from Dil (FIG. 2E). By comparing the released percentages computed from HPLC (ciprofloxacin concentration) and TECAN (Dil concentration) results, it was concluded that liposomes could release from hydrogel with intact structure and no drug leakage. The results demonstrated that liposomal ciprofloxacin could release from hydrogel without leakage from liposomes, providing evidence of local, sustained release during treatment.Example 4

[0116] In vitro experiments were performed in infinite sink conditions, which were unlikely to exist on the tympanic membrane surface. The ex vivo permeation across the tympanic membrane was also studied.

[0117] Drug transport across the tympanic membrane was evaluated in auditor}7bullae excised from healthy and NTHi-infected chinchillas following the procedure shown in FIG. 3 A. As for healthy bullae, DOTAP / sLip / Cip performed better permeation ability than DSPG / sLip / Cip (no significant difference), though they were both under 5% accumulative ratio after 48 hours (FIG. 3B).

[0118] The results showed that positively charged liposomes (here, the DOTAP liposomes) could penetrate more deeply through barriers (such as skin or ear drum) than negatively charged liposomes (here, the DSPG liposomes). However, when OM occurs, the tympanic membrane is under edema conditions, and the thicknesses of the tympanic membrane changes, manifesting with an incompact structure that ultimately makes the tympanic membrane easier to be penetrated. The transportation behavior varies depending on whether the infection is present.

[0119] The permeation ability of the system through the infected tympanic membrane was studied, as shown in FIG. 3C, and manifested a reverse trend compared to healthy tympanic membranes (FIG. 3B). DSPG / sLip / Cip showed higher permeating ability than DOTAP / sLip / Cip (P < .05, *) after 48 hours. Formulations with P407 exhibit slower Cip diffusion across the TM for both DSPG / sLip / Cip-P407[15%] and DOTAP / sLip / Cip-P407[15%], DSPG / sLip / Cip-P407[15%] showed faster diffusion than DOTAP / sLip / Cip- P407[15%] (P < .01, **). Notably, plain DSPG / sLip without ciprofloxacin encapsulated combined with an equivalent amount of free ciprofloxacin and P407 (nomenclature as DSPG / sLip-Cip-P407[15%]) demonstrated no enhanced tympanic membrane permeation (below 2% accumulative ratio at 48 hours).

[0120] The results further showed that it was the DSPG liposomal vehicles that effectively performed the enhanced transportation function. The diffusion ability of negatively surface- charged liposomes (DSPG / sLip) and positively surface-charged liposomes (DOTAP / sLip) manifested a reverse trend on the normal and infected tympanic membrane (FIG. 14).

[0121] It is believed that permeation across infected tympanic membranes were not a result of simple molecular force-dominated diffusion (e.g., charge / size-dominated), but is heavily mediated by the immune response. In the case of DSPG / sLip, it leverages neutrophilhitchhiking, which dominated the permeation across infected tympanic membranes. The results provided proof that our the drug delivery system could implement natural immune cells to facilitate active transportation across the infected tympanic membrane. However, the ex vivo model could not be used to demonstrate the presumed principal delivery strategy, because the proteins and immune cells could not be active for a period of long time, and the ty mpanic membranes degraded after 48 hours at 37 °C.Example 5

[0122] OM due to NTHi was established in chinchillas after direct inoculation of the bacteria into the middle ear following the scheme shown in FIG. 4A. The ears were then treated with 200 pL of test formulations (customized with total 0.5 mg ciprofloxacin) deposited through the external canal onto the tympanic membrane. OM was defined as nonzero colony-forming units (CFU) in middle ear fluid aspirated through the dorsal aspect of the auditory bullae, not through the tympanic membrane.

[0123] In chinchillas treated with DOTAP / sLip / Cip-P407[15%] and Cip-P407[l 5%], only 25% of chinchillas had cleared the infection (FIG. 4B). The reason was attributed to the low total administered ciprofloxacin (0.5 mg) and poor permeability.

[0124] In contrast, OM w as 100% cleared from day 1 to day 7 treated with DSPG / sLip / Cip-P407[ 15%] (FIG. 4B). The time course of the average number of colonies (CFU / ml) in middle ear fluid (MEF) is shown in FIG. 4C.

[0125] The time courses of ciprofloxacin concentrations in MEF were detected by HPLC and shown in FIG. 4D. The minimum inhibitory concentration (MIC) of ciprofloxacin for treating NTHi is > 0.12 pg / ml (range, 0.12 to 32 pg / ml).

[0126] The concentration of ciprofloxacin in middle ear fluid peaked at day 1 (72 pg / ml in animals treated with DSPG / sLip / Cip-P407[15%], 3.5 pg / ml with DOTAP / sLip / Cip-P407[15%] treatment, and 1.8 pg / ml with Cip-P407[15%] treatment). To facilitate comparison, it was assumed the total volume of middle ear fluid was 1 ml.

[0127] The percentage of ciprofloxacin delivered into the middle ear on day 1 achieved 14.4% in animals treated with DSPG / sLip / Cip-P407[15%], whereas the percentages of DOTAP / sLip / Cip-P407[15%] and Cip-P407[15%] were 0.7% and 0.4% respectively (FIG. 15A). DSPG / sLip / Cip-P407[15%] demonstrated 20.6-fold higher delivery efficacy than DOTAP / sLip / Cip-P407[15%]. as can be denoted as strong potential cure regimen for acute OM of children. Notably, the artificial drug delivery’ system presented here that could achieve extremely high delivery efficacy (14.4% on day 1) has never been accomplished by other studies.Example 6

[0128] Cobra Venom Factor (CVF) is the complement-activating protein derived from cobra venom. CVF is a three-chain protein that functionally resembles C3b, the activated form of complement component C3. Hence, CVF is often implemented to exhaust C3 in somatic circulation. C3b is also the effector assumed to be the key signal molecule that mediates neutrophils’ carry ing liposomes across the tympanic membrane to the infected middle ear (FIG. IB), due to the inherent chemotaxis effect of neutrophils when infection occurs. Here, a CVF- exhausted NTHi-infected chinchilla model was designed to test the role of C3b in the process of drug delivery.

[0129] Referring to the normal infected chinchilla model, chinchillas were injected with CVF (500 mg / kg) through the thigh vein after 48 hours (day -1) inoculation of the NTHi into the middle ear (FIG. 5 A). The level of C3b was examined in chinchilla serums sampled at day -1to day 7 to testify the complement depleting efficacy of CVF (FIG. 5B).

[0130] The western blot results showed that C3b was successfully depleted from the bloodstream after injection between day 0 and day 3, and with a slight recovery at day 7, but remained well below normal level compared to day -1. Furthermore, the level of C3b in middle ear fluid post-injection of CVF was calculated, and the result demonstrated that CVF could also deplete the C3b in MEF without impacting the virulence of NTHi located in the middle ear.

[0131] In animals pre-treated with CVF, DSPG / sLip / Cip-P407[15%] were deposited through the external canal onto the tympanic membrane following the same procedure mentioned above. Infection was detectable in 75% of CVF pre-injected animals on day 7 (FIG. 5C), and the time course of the average number of colonies (CFU / ml) in middle ear fluid from chinchillas in both with and without CVF pre-injected groups is shown in FIG. 5D.

[0132] To address the reason of decline antimicrobial efficacy of DSPG / sLip / Cip-P407[15%], ciprofloxacin level in MEF of CVF pre-inj ection animals was analyzed (FIG. 5E and FIG. 15B). The ciprofloxacin level at day 1 was far below non-CVF pre-inj ection group, which was 2.4 pg / ml versus 72 pg / ml. With the complement depleted. DSPG / sLip / Cip-P407[15%] could no longer perform enhanced delivery7efficacy, and the percentage of ciprofloxacin in the middle ear fluid on day 1 was only 0.5% (P < .05. *), as shown in FIG. 5F.

[0133] These results demonstrate that the declined curative effect of DSPG / sLip / Cip- P407[ 15%] in CVF depleted animals was attributed to the reduced immune function due to the exhaustion of complement proteins, but also the blockade drug delivery7path mediated by neutrophils. Without C3b. DSPG / sLip / Cip-P407[15%] could no longer be recognized and engulfed by neutrophils, which should be endogenous carriers to facilitate liposomes to be transported to infection sites following the chemotaxis effect. In conclusion, C3b was the molecule that performed a vital role in the process of drug delivery.Example 7

[0134] The effect of DSPG / sLip / Cip-P407[15%] on hearing sensitivity was assessed by auditory brainstem responses (ABRs) on healthy chinchillas. Placement of 200 pl of DSPG / sLip / Cip-P407[15%] on the tympanic membrane did not cause an apparent shift of theABR threshold (worsening of hearing) (FIGS. 16A, 16B, 16C). Nevertheless, the tympanic membrane did return to normal histologically, including the thickness and inflammation condition of the tympanic membrane (FIGS. 6A and 6B).

[0135] Cytotoxicity of the liposomes-hydrogel materials was evaluated in two representative cell lines in the auditory system: human dermal fibroblasts (hFBs) (FIGS. 17A and 17B) and pheochromocytoma cells (PC12, frequently used to test neurotoxicity) (FIGS. 18A and 18B). While the P407 hydrogel showed minimal cytotoxicity on day 1 and day 3, the addition of ciprofloxacin (totaling 0.5 mg to be consistent with the in vivo experiments) increased the cytotoxicity for two cell types on day 3. The liposomal formulation, DSPG / sLip / Cip-P407, showed reduced cytotoxicity compared to Cip-P407[l 5%] .

[0136] Nevertheless, the liposomal drug delivery system demonstrated excellent biocompatibility in the ear. Tympanic membranes excised at day 7 after treatment with DSPG / sLip / Cip-P407[15%] were histologically comparable to healthy untreated tympanic membranes (FIG. 6A), which were around 10 pm thickness and without tissue injuries, necrosis, or inflammatory cells (FIG. 6B). Infected-untreated tympanic membrane sampled at day 7 were 6 times thicker than uninfected-untreated one and exhibited an acute inflammatory response with diffuse edema and dense infiltration by inflammatory cells (FIG. 6A).

[0137] DSPG / sLip / Cip-P407[15%] reversed the prominent inflammatory response caused by bacteria. DOTAP / sLip / Cip-P407[15%] and Cip-P407 [15%] could not eradicate all bacteria and eliminate the edema and inflammation of tympanic membranes, neither could DSPG / sLip / Cip- P407[15%] when the animal was treated by CVF (FIG. 6A) (here, animals were treated with CVF to deplete the complement proteins, thereby blocking the neutrophil hitchhiking process). Histology of tympanic membranes was consistent with in vivo curative efficacy that DSPG / sLip / Cip-P407[15%] eradicated NTHi after 1 week of treatment (FIG. 4C).

[0099] Upon reaching the site of infection, neutrophils release neutrophil extracellular traps (NETs), which provides a potential pathway to expel the cargo into the extracellular space and eradicate the pathogens. There have not been any prior methods or compositions that could enhance the permeability of the ty mpanic membrane to accomplish transmembrane delivery. However, it has been demonstrated as described herein, a new non-invasive drug deliverystrategy that accomplishes the foregoing. Most drug delivery' systems implement active drug delivery' strategies based on the de novo design of systems themself disregarding the changing biological internal environment when the body encounters an inflammatory issue. Yet, the methods described herein propose a new strategy of non-invasive drug delivery' that takes this fact into account.

[0100] Methods and compositions utilize natural immune cells to target lesions. In some embodiments, when OM occurs, neutrophils are recruited from the bloodstream to the middle ear following the chemotaxis effect. As motile cells, neutrophils can migrate following the direction of a gradient of chemoattractants (C5a, chemokines, etc.), also known as the chemotaxis effect. Considering the motile and targeting characteristics of neutrophils, artificial liposomes are designed that specifically adsorb C3b in some embodiments, which is efficiently recognized by neutrophils through the Clr and C3r receptors.

[0101] First, we elucidated the existence of C3b proteins in middle ear fluid sampled from NTHi-infected chinchillas. Next, DSPG liposomes (which are abundant with OH groups on their surfaces) were demonstrated that can react with C3b as reported, while DOTAP liposomes cannot. The deposition of C3b to the surface of DSPG liposomes informed the preliminary foundation for transporting across the ty mpanic membrane.

[0102] P407 thermo-reverse hydrogels were adopted to maintain the sustained release of DSPG / sLip / Cip at the tympanic membrane. The released DSPG liposomes from hydrogels then adsorb the C3b protein, which substantially serves as the bio-identity guide for neutrophils to liposomes. After neutrophils engulfed liposomes, they carried them to the infected middle ear and spitted liposomes by the neutrophils extracellular trap (NET) effect. The total transportation efficacy of ciprofloxacin across the ty mpanic membrane to the middle ear is as high as 14.4% after being treated for 24 hours.

[0103] The high delivery efficacy makes it possible that a 1 / 4 dose of ciprofloxacin (previous report) can completely eradicate all the pathogens in the middle ear, which further decreases the potential risk of systemic antibiotic resistance. Furthermore, studies show that liposomes bind more readily with biofilms40,41. Transportation of liposomal ciprofloxacin to the middle ear possibly has advantages over free ciprofloxacin, taking the stubborn biofilms into account.

[0104] Nevertheless, the delivery system introduced here could have broad applications for treating other diseases other than otitis media. There are many barriers in humans that limit the delivery of drugs to lesion sites. The immune cells-based drug delivery system described here can be a potential solution to conquer bio-barriers. The strategy embraces the intrinsic characteristics of natural immune cells and enhanced therapeutical nanoparticles, which can follow the path of immune systems to hidden sites that traditional delivery systems do not effectively reach.

[0105] Despite the recognized correlation between results obtained with the chinchilla animal model and those from human trials, it is possible that there might be discrepancies in humans. In clinical otopathogens acquired from children with OM, there are many other pathogens aside from NTHi or even frequently with co-infection. Although we detected no hearing deficit in chinchillas on day 7 after administration, the gel deposited on the tympanic membrane still need extra time to be completely degraded.

[0106] A non-invasive. single-dose, local antibiotic treatment regimen has been developed with demonstrated its efficacy in the standard chinchilla animal model. Ciprofloxacin was used as an antimicrobial molecule because of its broad spectrum of activity against relevant bacteria and its effectiveness in OM in children with myringotomy tubes.

[0107] Liposomes were widely investigated as nanocarriers to ameliorate the in vivo behaviors of poor bioavailability and solubility drugs. As the most widely used and investigated nano vehicles, the FDA has approved many liposomes-based nanodrugs. Pol oxamer 407 (P407) is FDA approved polymer to be used as the polymeric carrier. The whole delivery system performs highly in terms of biocompatibility as anticipated. Nevertheless, the system only exerts function under infection, further demonstrating the safety of the system. The delivery system is likely compatible with other drugs due to the versatility encapsulating ability’ of liposomes, including tricky hydrophobic drugs, should a drug other than ciprofloxacin be necessary.Example 8

[0108] To provide direct evidence that the hydroxylated liposomes (DSPG / sLip) were internalized by innate neutrophils, a BALB / c mouse model was adopted to analyze theneutrophil uptake of liposomes. This mouse model was chosen because it is well-established for neutrophil harvesting and characterization. These experiments could not be performed in chinchillas because many key antibodies that are required to label neutrophils are currently missing for chinchillas.

[0109] Opsonization of the hydroxylated liposomes by the complement cascade in BALB / c mice was illustrated by incubating the two types of liposomes with mouse serum for 1 hour at 37°C, followed by western blot to analyze their protein corona (FIG. 21 A). On the hydroxylated liposomes, i.e., DSPG / sLip. several iC3b fragments were identified, i.e., a’-67 and a' -40 (whereas a’ -46 is an intermediate fragment that is not consistently captured). In contrast, the protein corona of DOTAP / sLip did not contain those complement protein fragments. The amount of the C3b fragments displayed on the surface of DSPG / sLip was 40-fold that on DOTAP / sLip (****P < 0.0001).

[0110] To illustrate neutrophils’ internalization of liposomes, fresh neutrophils were isolated from the BALB / c mouse bone marrow and incubated with opsonized liposomes, labeled with 3,3 '-dioctadecyloxacarbocyanine (DiO), for 1 hour and 4 hours, respectively. Using flow cytometry, the harvested neutrophils were gated at CDl lb+ / LY6G+ to confirm the cell type (50) (Fig. 21B, i), and the fraction of neutrophils (CDllb+ / LY6G+) that contain liposomes (DiO) was quantified for each type of liposome (FIG. 21B, ii to iv). Neutrophils incubated with PBS (no liposome) were shown in FIG. 21B, ii to iii as a control group, which showed DiO signals below the threshold (i.e.. 103). At the end of the 1-hour incubation, 86.0 ± 2.0% neutrophils internalized DSPG / sLip (FIG. 21B, iv), as show n by the fraction of the incubated neutrophils that exhibited DiO signal above 103 (FIG. 21B, ii); whereas for DOTAP / sLip, only 25.0 ± 6.3% of the incubated neutrophils internalized the liposomes. Those internalization fractions remained similar after 4 hours of incubation, reaching 88.1 ± 2.6% for DSPG / sLip and 20.5 ± 21.5% for DOTAP / sLip, respectively. This result indicated that DSPG / sLip / DiO led to distinctly high internalization by neutrophils both at 1 hour (P = 0.0003) and 4 hours (P = 0.0002), compared to DOTAP / sLip / DiO.

[0111] To assess the in vivo neutrophil internalization efficiency, mice were injected intravenously with the two types of liposomes (DiO labeled), and flow' cytometry wasperformed on peripheral blood (with erythrocytes removed) at 1 hour and 4 hours post-injection (FIG. 21C). Again, the cell type was confirmed (i.e., CDllb+ / LY6G+) and the fraction of neutrophils (CDllb+ / LY6G+) that contain liposomes (DiO) for each type of liposomes was calculated (FIG. 21C, ii to iv). Neutrophils harvested from the peripheral blood of mice that received saline injection (without liposomes) were shown in as a control group, in which the DiO signal remained below the threshold (i.e.. 103). At 1 hour post-injection, 29.6 ± 8.2% neutrophils internalized DSPG / sLip (FIG. 21 C, ii to iv). Note this fraction was lower than that in the ex vivo experiment (FIG. 21B) because the opsonized liposomes could be eliminated by a variety of immune mechanisms, thus diluting its interaction with neutrophils specifically. For DOTAP / sLip, 7.6 ± 0.9% of the sampled neutrophils internalized the liposomes. These fractions decreased slightly at 4 hours post-injection, due to the further clearance of the injected liposomes by the immune system. As such, DSPG / sLip / DiO enhanced the in vivo neutrophil internalization to 4-fold that of DOTAP / sLip / DiO at 1 h post-injection and 5-fold at 4 h postinjection.Example 9

[0112] The ex vivo uptake of the liposomes was analyzed (FIG. 19). The neutrophils originated from BALB / c mice bone marrow. Neutrophils (106 cells) were incubated with complement protein (iC3b) opsonized liposomes (sLip, 0.5 pM lipids), i.e.. TAP / sLip and PG / sLip, for 1 hour at 37°C. Neutrophil’s nuclei were labeled with 4',6-diamidino-2- phenylindole (DAPI); neutrophil’s surface antigen, i.e.. LY6G. was stained with antibody (APC); liposomes were fluorescently labeled using 5 -Carboxy fluorescein (FAM); PG / sLip readily captured by neutrophils (nuclei. LY6G) by recognizing the complement protein fragments on the liposomes, whereas neutrophils barely captured TAP / sLip.

[0113] In vivo efficacy of PG / sLip / Cip was also studied (FIG. 20). Otitis media (OM) chinchillas (infected with non-typeable Haemophilus influenzae) were treated with PG / sLip / Cip (incorporated in poloxamer 407 (P407)) or without treatment for 7 days. The mucosae (including the middle ear bone) were collected and photographed with the scanning electron microscope (SEM). At day 7 (lower panel), the PG / sLip / Cip treated animals were observed with sparse and deformed pathogens, compared to the untreated group, which hadbiofilm in the middle ear.Example 10

[0114] The effectiveness of the transtympanic drug delivery strategy was demonstrated by quantifying the drug flux across intact TM in health or disease using excised auditory bullae, as shown in FIG. 22A. The intactness of excised TM was confirmed using electrical impedance measurements following a widely adopted procedure (19, 52-54). TMs with microperforations were excluded from the reported results. Each test formulation contains 0.5 mg ciprofloxacin (Cip) (at a concentration of 2.5 mg / ml).

[0115] Among the test formulations, DSPG / sLip / Cip showed the greatest permeability in an infected TM (FIG. 22C). Over 48 hours, 7.6 ± 3.0% of the Cip contained in this formulation permeated across TM cumulatively, representing 2.5-fold that delivered by DOTAP / sLip / Cip (3.1 ± 1.4%; P = 0.0146). This comparison contrasted with their transtympanic delivery efficiency in healthy ears (FIG. 22 2B), where DOTAP / sLip / Cip transported 4.2 ± 1.7% of the Cip contained in the formulation and DSPG / sLip / Cip 3.5 ± 2.2%.Example 11

[0138] Animal maintenance and cells. Healthy adult chinchillas weighing 400 - 600 g were bought from Mutation Chinchilla Breeder Association (NY). All animals were raised and cared along with the protocols approved institutionally and nationally. Experiments were proceeded and permitted by the Institutional Animal Care and Use Committee (IACUC) in conjunction with the Cornell University Center of Animal Resources and Education (CARE) Animal Use Guidelines.

[0139] Materials and reagents. L-a-phosphatidylcholine, hydrogenated (Soy) (HSPC, 840058), l,2-distearoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (sodium salt) (DSPG, 840465), l,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP, 890890), Cholesterol, and 1 ,2-distearoyl-sn-gly cero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (mPEG2000-DSPE, 880120) were bought from Avanti Polar Lipids Co. Ltd (Birmingham, AL); Sephadex® G-50 (9004-54-0), Ciprofloxacin hydrochloride (86393-32-0), l,l'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (41085-99- 8), Cobra Venom Factor (Naja naja kaouthia, 233552), Anti-Neutrophil Elastase Antibody(EM481001) and Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Poloxamer 407. 9003-11-6) were purchased from Sigma Co. Ltd (St. Louis, MO). Complement C3 Polyclonal Antibody (PAI-29715), Rabbit anti-Goat IgG (H+L) Secondary Antibody and HRP (Catalog # 31402) were from ThermoFisher Co. Ltd (US); 4-20% precast polyacrylamide gel (#4561094), 2X Laemmli Sample Buffer (#1610737) and Precision Plus Protein Dual Color Standards (#1610374) were from Bio-Rad Laboratories, Inc. (US).

[0140] Preparation and characterization of liposomes. Liposomes were prepared through thin film hydration and extrusion method. The negatively charged liposomes composed of DSPG (52% molar ratio), Cholesterol (43% molar ratio), and mPEG2000-DSPE (5% molar ratio) were dissolved in a mixed solvent (chloroform / ethanol / deionized water) completely under vacuum distillation to remove solvent until the thin film was formed. The film was dried overnight under vacuum and hydrated with 0.32 M ammonium sulfate at 60°C until the film was fully dropped off. The suspension solution was extruded through polycarbonate membranes with 400 nm, 200 nm, and 100 nm for at least 8 times for each. Ciprofloxacin was encapsulated into liposomes by classic ammonium sulfate gradient method under 60°C water bath. Positively charged liposomes with the formulation of 32% DOTAP, 20% HSPC. 43% Cholesterol and 5% mPEG2000-DSPE were made following the same method above except dissolved only with chloroform when preparing the film. Dil dye (100 pg / ml) was added into the lipids before vacuum distillation.

[0141] Preparation and characterization of proteins. Middle ear fluids (MEF) and serums were sampled from NTHi-infected chinchillas (for 72 hours). Liposomes (100 pl) were incubated with 100 pl MEF or serum at 37°C for 1 hour to form protein corona, then added with 1 ml pre-chilled PBS. The mixtures were centrifuged with 12000 g for 30 min at 4°C, the precipitates were collected and washed with 1 ml PBS for 3 times. The precipitates which were protein corona were added with PBS (contain 5% 2-ercaptoethanol) and equal volume of 2X laemmli sample buffer, then heated at 95°C for 5 min to make sure proteins fully denaturation. 4-20% precast polyacry lamide gel was used to separate proteins, then proteins were transferred from gel to 0.45 pm Nitrocellulose Membrane under 100 mv for 70 min. Complement C3 Polyclonal Antibody and Rabbit anti-Goat IgG Secondary Antibody (HRP) were used to detectcomplement 3 proteins. The level of complement 3 proteins in serum and middle ear fluid were characterized by the same method above.

[0142] Preparation and characterization of P407 hydrogels. P407 hydrogel formulations with different proportions (10, 15, 20%) were prepared by dissolving powdered P407 polymers into aqueous solutions, which were deionized water or liposomes (DSPG or DOTAP liposomes). Gelation temperature was tested by linear oscillatory shear rheology measurements (100 rad- 1, 1% strain, and 1°C min-1). Gelation temperature was the point that the temperature at which storage moduli (G’) became bigger than loss moduli (G”). The changes of G’ and G " over temperatures from 15°C to 45°C were recorded with triplicates. Cryogenic transmission electron microscopy (cryo-EM) was adopted to analyze the stability' of liposomes in hydrogel. DOTAP and DSPG liposomes were 10 times diluted with deionized water and captured by cryo-EM with 49000 times magnification.

[0143] In vitro release studies. The release kinetics of ciprofloxacin from formulations w ere tested by transwell diffusion assay. Transwell membrane inserts (8 pm pore size and E l-cm2; Costar) and 24-well plate (REF353504, notched for use with cell culture inserts, Coming) were used as the donor and acceptor chamber, respectively. Pre-warmed membrane inserts were added with 200 pl each formulation and placed at 37°C for 10 min to form a solid hydrogel. Subsequently, membrane inserts were immersed into wells pre-added with 2 ml warm PBS, then the plates were incubated in an oven at 37°C. PBS in receiving wells was sampled at 0.5, 1, 2, 4. 6, 12, 24 and 48 hours, and mixed with methanol (50%) to ensure completely drug dissolution. HPLC w as used to detect ciprofloxacin concentration in samples (A = 275 nm). Expenments were performed in triplicates.

[0144] Ex vivo TM permeation studies. Chinchilla auditor}' bullae were harvested from healthy and NTHi infected chinchillas (infected for 48 hours). Bullae with external auditory canal facing up were kept in 12-well plate with 3 ml pre-warmed PBS in each well, all formulations (200 pl) were deposited carefully onto the tympanic membranes through auditory canals. At each time point (1, 6, 12, 24 and 48 hours), 200 pl aliquots of the PBS-receiving media were sampled and equal volume fresh pre-warmed PBS were added sequentially. Aliquots were added with methanol (50%) and quantified by HPLC.

[0145] NTHi infected OM chinchilla model. All procedures were implemented in accordance with the Cornell University CARE and approved by the IACUC. Once grown to the mid-log phase, isolates of Non typeable Haemophilus influenzae (NTHi) were diluted in Hanks’ balanced salt solution (HBSS), and then the mixed solution (200 pl, 100-200 CFU) was inoculated directly into each middle ear through the bullae under aseptic conditions. Once pathogens were introduced, assessments of each middle ear were performed by using otoscopy daily, and body weights were recorded as well. Once the signs of middle ear infection occurred, including the presence of fluid in the auditory bullae, and bulging tympanic membrane. Tympanic membranes of the animals to receive the formulation were observed with the speculum of an otoscope, after which the liquid hydrogel (containing 0.5 mg ciprofloxacin) was injected through the speculum using a soft catheter at day 0. Middle ear fluids (MEFs) were sampled at different time points (day 0, day 1, day 3 and day 7), and steaked onto a chocolate agar plate. MEFs were diluted with HBSS and plated on chocolate agar plates (10 pl) with 102, 103, 104 and 105 diluted to count colonies existing in MEFs. The lower limit for detecting organisms by using this dilution method in the MEF was 100 CFU / ml. As for the cobra venom factor blocked (CVF) animal model. CVF was injected with 500 pg per kilogram body weight 48 hours after NTHi inoculation. Other experiment procedures were the same as above, except that serum was sampled at different time points (day -f, day 0. day f , day 3 and day 7).

[0146] To study the immunocytochemistry of the interaction between liposomes and neutrophils, 200 pl DPSG / sLip / DiI-P407[15%] or DOTAP / sLip / DiI-P407[15%] were applied onto the tympanic membrane (NTHi-infected chinchillas) through the external auditory canal. After 24 hours of administration, the tympanic membranes and middle ear fluids w ere sampled and stained with Hoechst for cell nucleus, anti-elastase antibody for neutrophil. Zeiss 710 confocal microscope w as used for imaging.

[0147] Histopathology. Chinchillas of in vivo experiments were euthanized on day 7, tympanic membranes were excised and subsequently fixed with 10% neutral buffered formalin overnight. The obtained fixed tympanic membranes were decalcified as well as in archived paraffin blocks, then sectioned with 5-pm thickness following standard staining technologywith H&E by the Section Anatomic Pathology Histology Laboratory at Cornell University (Ithaca, New York, USA). Specimens were imaged with microscopy (10 times magnification), and the thickness of tympanic membranes was measured by Image J software.

[0148] Statistical analysis. Dates were shown with means and SDs and compared by paired or unpaired Student’s t-test. Otherwise, dates were described with median ± quartiles. Comparisons between groups were conducted by Two-way ANOVA test using GraphPad Prism version 9.4.1 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com.

[0149] Cells and Materials. Fibroblast cells (PCS-201-012) and PC12 cells (CRL-1721) were purchased from the American Type Culture Collection company (ATCC) and cultured under recommended medium under 5% CO2 atmosphere according to ATCC instructions; CCK-8 Cell Counting Kit (DBOC00128) were bought from VitaScientific Co. Ltd (US); LIVE / DEAD™ Viability / Cytotoxicity Kit (L3224) were from ThermoFisher Co. Ltd (US).

[0150] Characterization of liposomes. The size and zeta potential of liposomes prepared were characterized by Zetasizer Nano ZS90 (Malven Instruments, Malvern. UK) with 50 times diluted with deionized water (DW). The loading efficiencies of ciprofloxacin within liposomes were quantified by reversed-phase high performance liquid chromatography (HPLC, Schimadzu).

[0151] Preparation and characterization of protein corona. DOTAP / sLip or DSPG / sLip were incubated with equivalent volume of serum (sampled from NTHi -infected chinchillas) at 37°C for 1 hour. The formed protein coronas were separated by centrifugation at 4°C with 12.000 g for 30 min and washed with chilled PBS for 3 times. The collected protein coronas were diluted with chilled PBS and denaturalized with sampling buffer for subsequent Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under constant 20 mA each page. Western blot was used to study the components of protein coronas, C3b fragments were detected by anti-complement antibody.

[0152] ABR measurements. ABR experiments were proceeded by a custom-designed stimulus generation and measurement system LabView (Laboratory Virtual Instrument Engineering Workbench, National Instruments, Austin, Texas, USA). The results were collected from subdermally placed platinum-iridium needle electrodes (Astro-Med GrassInstruments, West Warwick, Rhode Island, USA). The active, reference and ground electrode were placed at the cranial vertex, ventrolateral side, and dorsum of chinchilla, respectively. ABR signals were passed to an amplifier (AC amplifer model CP511, Astro-Med Grass Instruments), with a gain of 200,000 and filtered with low- and high-pass filters of 3,000 Hz and 300 Hz. Chinchillas were anesthetized and administered 200 pl DSPG / sLip / Cip-P407[15%] onto tympanic membrane (n = 3). The measurements were taken at 10 mins, 1 day, 3 days and 7 days after administration, with a stimulus frequency of 21 Hz and 1000 acquisitions.

[0153] Cytotoxicity evaluation. Human derived dermal fibroblasts (hFB) and pheochromocytoma cell line (PC 12) were cultured according to ATCC recommended medium in 37°C incubator with 5% CO2 condition. Cells were seeded in 24-well carrier cell plate with 50,000 cells per well and cultured overnight for subsequent use. Transwell membrane inserts (8 pm pore size and 1. l-cm2; Costar) were added with 200 pl sterilized formulations and placed at 37°C for 10 min to form hydrogels. Porous membrane inserts were pipetted into each well with 0.5 ml renewal fresh medium separately. After 24 hours and 72 hours of incubating, CCK8 kit and LIVE / DEAD kit were utilized to analyze and detect living cells and dead cells following product protocols.

[0154] Statistical analysis. Dates were shown with means and SDs and compared by paired or unpaired Student’s t-test. Otherwise, dates were described with median ± quartiles. Comparisons between groups w ere conducted by Two-way ANOVAtest using GraphPad Prism version 9.4.1 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com.

[0155] Isolation of Mouse Neutrophils and ex vivo cell uptake evaluation. Obtaining a sufficient quantity of neutrophils from the peripheral blood of mice was challenging; therefore, neutrophils were isolated from bone marrow. Male BALB / c mice were euthanized and immersed in 70% ethanol for 10 min to ensure full sterilization. The femur and tibia, along with attached muscle tissue, were carefully separated, follow ed by sterilization of the collected bones using 70% ethanol and subsequent washing with pre-chilled buffer (0.5% BSA and 2 mM EDTA in PBS, pH 7.2). Bone marrow cells were harvested by thorough flushing of the bone cavity with the aforementioned buffer and subsequent centrifugation at 400 g, 4°C for 5 minutes. The resulting pellet was suspended in 10 ml of 0.2% sterile NaCl solution to lyse thered blood cells, with osmotic pressure restored by mixing with 10 ml of 1.6% sterile NaCl solution after 30 seconds. The cell suspension was filtered through a 70 pm sterile cell strainer and centrifuged at 400 g, 4°C for 5 min. The pellet was suspended with 1 ml PBS for subsequent purification. In a 15 ml sterile tube, 5 ml Histopaque®-1077 (1.077 g / ml), 1 ml bone marrow cell suspension, and 5 ml Histopaque®-ll 19 (1.119 g / ml) were carefully layered from the bottom to the top without agitation. Neutrophils were collected by centrifugation at 1000 g, 4°C for 30 min, and washed twice with RPMI-1640 medium. To assess the purity of the isolated neutrophils, cells were stained with Anti-Mouse CD 11b Antibody (APC (Allophycocyanin)) and Anti-Ly-6G Antibody (BV421 (Brilliant Violet reg 421)) at 0.2 pg of 1 :1 mixed antibodies per 106 cells in 100 pl PBS on ice. The purity of neutrophils was approximately 70% as confirmed by flow cytometry.

[0156] The isolated neutrophils were aliquoted to 106 in 100 pl PBS (supplemented with 10% mouse serum). DiO-labeled liposomes were incubated with B ALB / c mouse serum with 1 : 1 at 37°C for 1 hour to form protein corona. The opsonized liposomes (10 pM lipid) were mixed with 106 neutrophils in 0.1 ml PBS and incubated at 37°C for 1 hour or 4 hours. Cell pellets were collected using a centrifuge (5425R, Eppendorl) and washed with PBS twice to fully remove unreacted liposomes. A total amount of 0.2 pg of antibodies, i.e., Anti-Mouse CDllb Antibody (APC (Allophycocyanin)) and Anti-Ly-6G Antibody (BV421 (Brilliant Violet reg 421)), mixed with a 1 : 1 ratio, w as incubated with 106 neutrophils at 4°C for 1 hour. Cell pellets were washed with PBS and fixed with 4% paraformaldehyde (preserving the samples and avoiding any potential contamination during analysis) for subsequent analyses using Flow7Cytometry (FACSymphony A3, BD Biosciences).

[0157] Mouse in vivo neutrophil uptake studies. Adult healthy BALB / c mice were injected intravenously with DiO-labeled liposomes through the tail vein at a dose of 50 mg lipids per kg bodyweight. Peripheral blood samples were collected through the retro-orbital vein into tubes with pre-chilled PBS supplemented with 10 mM EDTA. Blood cell pellets were collected by centrifugation (5425R, Eppendorf) and subsequently mixed with mouse red blood cell lysis buffer with moderate vortex. Cell pellets were collected by centrifugation and washed twice with PBS. A total amount of 0.2 pg of antibodies, i.e., Anti-Mouse CDllb Antibody (APC(Allophycocyanin)) and Anti-Ly-6G Antibody (BV421 (Brilliant Violet reg 421)), mixed with a 1 : 1 ratio, was incubated with 106 neutrophils at 4°C for 1 hour. Cell pellets were washed with PBS and fixed with 4% PFA (preserving the samples and avoiding any potential contamination during analysis) for subsequent analyses using Flow Cytometry (FACSymphony A3, BD Biosciences). The present disclosure may comprise one or more of the following features and combinations thereof.

[0158] A composition includes a therapeutic compound; a liposome shell encapsulating the therapeutic compound, the liposome shell comprising a plurality of hydroxyl-containing lipids; and a pharmaceutically acceptable carrier comprising a stimuli-responsive hydrogel.

[0159] In some embodiments, the therapeutic compound is small molecule or a macromolecule.

[0160] In some embodiments, the therapeutic compound is a small molecule, peptide, a glycopeptide, a protein, a hormone, a synthetic nanoparticle, a nucleic acid, or any combination thereof.

[0161] In some embodiments, the stimuli-responsive hydrogel is a temperature-dependent hydrogel, a shear-thinning hydrogel, a cross-linkable hydrogel, or a combination thereof.

[0162] In some embodiments, the stimuli-responsive hydrogel comprises a copolymer.

[0163] In some embodiments, the copolymer comprises a block copolymer.

[0164] In some embodiments, the block copolymer comprises a polyethylene oxide) (PEO) block and a polypropylene oxide) (PPO) block.

[0165] In some embodiments, the stimuli-responsive hydrogel comprises a poloxamer segment.

[0166] In some embodiments, the stimuli-responsive hydrogel is an amount of about 0. 1% to about 50% weight / volume (w / v).

[0167] In some embodiments, the therapeutic compound is an antibiotic.

[0168] In some embodiments, the therapeutic compound has a molecular weight of about 50 to about 3,500 Daltons.

[0169] In some embodiments, the therapeutic compound is a penicillin, a cephalosporin, a tetracycline, an aminoglycoside, a macrolide, a sulfonamide, a trimethoprim, a quinolone, anitrofurantoin, a vancomycin, or any combination thereof.

[0170] In some embodiments, the therapeutic compound is ciprofloxacin.

[0171] In some embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises a cationic lipid, a neutral lipid, a negatively charged lipid, or any combination thereof.

[0172] In some embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises a poly(ethylene glycol)-phospholipid, a sterol, or any combination thereof.

[0173] In some embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N- (4-hydroxybutyl)hexan-l-aminium; N-(2-hydroxyethyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminium bromide; 1,2-dioctanoyl-sn-glycerol, l-O-T-(Z)- octadecenyl-sn-gly cerol; phosphatidylserine; 1 -olelyl-2-ly sophosphatidate, 1 ,2-Dimyristoyl- sn-gIycero-3-phosphoglycerol; l,2-dipalmitoyl-sn-glycero-3-phosphogly cerol; l-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(l'-rac-gly cerol); l,2-dioleoyl-sn-glycero-3-phosphogly cerol, l,2-distearoyl-sn-glycero-3-phosphoglycerol, cholesterol, poly(ethylene glycol)- distearoylphosphatidylethanolamine, or any combination thereof.

[0174] In some embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises a phospholipid - polyethylene glycol conjugate.

[0175] In some embodiments, the liposome shell comprising the plurality of hydroxylcontaining lipids comprises about 30% to about 70% molar ratio of a phospholipid; about 0% to about 50% molar ratio of a sterol; and about 0% to about 10% molar ratio of a phospholipid - polyethylene glycol conjugate.

[0176] In some embodiments, a method of making the composition includes combining the therapeutic compound with the plurality of hydroxyl-containing lipids to form liposomes comprising the hydroxyl-containing lipids and the therapeutic compound; and combining the liposomes with the pharmaceutically acceptable carrier comprising the stimuli-responsive hydrogel.

[0177] In some embodiments, combining the therapeutic compound with the plurality ofhydroxyl-containing lipids to form the liposomes comprises encapsulating the therapeutic compound with the plurality of the hydroxyl-containing lipids.

[0178] In some embodiments, the liposomes comprise the therapeutic compound arranged in, tethered to. or both arranged in and tethered to, lipid bilayers of the liposomes.

[0179] In some embodiments, encapsulating the small molecule therapeutic compound comprises adding the plurality of hydroxyl-containing lipids to a solvent; removing the solvent from the plurality of hydroxyl-containing lipids to form a film; hydrating the film to form a lipid suspension; and combining the lipid suspension with the small molecule therapeutic compound.

[0180] A method of treating a patient with a disease includes administering to the patient an effective amount of any one of the compositions; and treating the disease in the patient.

[0181] In some embodiments, the disease is an ear infection, chronic inflammatory reaction, a chronic infection, an acute inflammatory reaction, and acute infection, or any combination thereof.

[0182] In some embodiments, the disease is otitis media, a skin infection, a skin bum, or a combination thereof.

[0183] In some embodiments, the effective amount of the composition is a single dose.

[0184] In some embodiments, administering to the patient the effective amount of the composition comprises non-invasive administration to an external auditory canal onto the tympanic membrane of the patient.

[0185] The figures provided herein are not necessarily to scale, although a person skilled in the art will recognize instances where the figures are to scale and / or what a typical size is when the drawings are not to scale. While in some embodiments movement of one component is described with respect to another, a person skilled in the art will recognize that other movements are possible. Additionally, a number of terms may be used throughout the disclosure interchangeably but will be understood by a person skilled in the art. Further, to the extent features, sides, or steps are described as being “first’' or “second,” such numerical ordering is generally arbitrary, and thus such numbering can be interchangeable. Still further, in the present disclosure, like-numbered components of various embodiments generally havesimilar features when those components are of a similar nature and / or serve a similar purpose. Lastly, the present disclosure includes some illustrations and descriptions that include prototypes, bench models, or experimental design. A person skilled in the art will recognize how to rely upon the present disclosure to integrate the techniques, systems, devices, and methods provided for into a product in view of the present disclosures.

[0186] While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific exemplary embodiments of the disclosure have been shown by way of example. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular disclosed forms; the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. Although this disclosure refers to specific embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the subject matter set forth in the accompanying claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: a therapeutic compound; a liposome shell encapsulating the therapeutic compound, the liposome shell comprising a plurality of hydroxy 1-containing lipids; and a pharmaceutically acceptable carrier comprising a stimuli-responsive hydrogel.

2. The composition of claim 1, wherein the therapeutic compound is small molecule or a macromolecule.

3. The composition of claims 1 or 2, wherein the therapeutic compound is a small molecule, peptide, a glycopeptide, a protein, a hormone, a synthetic nanoparticle, a nucleic acid, or any combination thereof.

4. The composition of any one of claims 1-3, wherein the stimuli-responsive hydrogel is a temperature-dependent hydrogel, a shear-thinning hydrogel, a cross-linkable hydrogel, or a combination thereof.

5. The composition of any one of claims 1-4. wherein the stimuli-responsive hydrogel comprises a copolymer.

6. The composition of claim 5, wherein the copolymer comprises a block copolymer.

7. The composition of any one of claims 1-6, wherein the block copolymer comprises a poly(ethylene oxide) (PEO) block and a polypropylene oxide) (PPO) block.

8. The composition of any one of claims 1-7, wherein the stimuli-responsive hydrogel comprises a poloxamer segment.

9. The composition of any one of claims 1-8, wherein the stimuli-responsive hydrogel is an amount of about 0.1% to about 50% weight / volume (w / v).

10. The composition of any one of claims 1-9, wherein the therapeutic compound is an antibiotic.

11. The composition of any one of claims 1-10, wherein the therapeutic compound has a molecular weight of about 50 to about 3,500 Daltons.

12. The composition of any one of claims 1-11, wherein the therapeutic compound is a penicillin, a cephalosporin, a tetracycline, an aminoglycoside, a macrolide, a sulfonamide, a trimethoprim, a quinolone, a nitrofurantoin, a vancomycin, or any combination thereof.

13. The composition of any one of claims 1-12, wherein the therapeutic compound is ciprofloxacin.

14. The composition of any one of claims 1-13, wherein the liposome shell comprising the plurality of hydroxyl-containing lipids comprises a cationic lipid, a neutral lipid, a negatively charged lipid, or any combination thereof.

15. The composition of any one of claims 1-14, wherein the liposome shell comprising the plurality of hydroxyl-containing lipids comprises a polyethylene glycol)-phospholipid, a sterol, or any combination thereof.

16. The composition of any one of claims 1-15, wherein the liposome shell comprising the plurality of hydroxyl-containing lipids comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium; N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide; 1,2-dioctanoyl-sn-glycerol, 1-0-1’- (Z)-octadecenyl-sn-glycerol; phosphatidylserine; l-olelyl-2-lysophosphatidate, 1,2- Dimyristoyl-sn-glycero-3-phosphoglycerol; l,2-dipalmitoyl-sn-glycero-3-phosphoglycerol; 1- palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol); 1.2-dioleoyl-sn-glycero-3- phosphoglycerol, l,2-distearoyl-sn-glycero-3-phosphoglycerol, cholesterol, poly(ethylene glycol)-distearoylphosphatidylethanolamine, or any combination thereof.

17. The composition of any one of claims 1-16, wherein the liposome shell comprising the plurality of hydroxyl-containing lipids comprises a phospholipid - polyethylene glycol conjugate.

18. The composition of any one of claims 1-17, wherein the liposome shell comprising the plurality7of hydroxyl-containing lipids comprises about 30% to about 70% molar ratio of a phospholipid; about 0% to about 50% molar ratio of a sterol; and about 0% to about 10% molar ratio of a phospholipid - polyethylene glycol conjugate.

19. A method of making the composition of any one of claims 1-18, the method comprising:combining the therapeutic compound with the plurality of hydroxyl-containing lipids to form liposomes comprising the hydroxyl-containing lipids and the therapeutic compound; and combining the liposomes with the pharmaceutically acceptable carrier comprising the stimuli- responsive hydrogel.

20. The method of claim 19, wherein combining the therapeutic compound with the plurality of hydroxyl-containing lipids to form the liposomes comprises encapsulating the therapeutic compound with the plurality of the hydroxyl-containing lipids.

21. The method of claims 19 or 20. wherein the liposomes comprise the therapeutic compound arranged in, tethered to, or both arranged in and tethered to, lipid bilayers of the liposomes.

22. The method of claim 20, wherein encapsulating the small molecule therapeutic compound comprises adding the plurality of hydroxyl-containing lipids to a solvent; removing the solvent from the plurality of hydroxyl-containing lipids to form a film; hydrating the film to form a lipid suspension; and combining the lipid suspension with the small molecule therapeutic compound.

23. A method of treating a patient with a disease, the method comprising: administering to the patient an effective amount of any one of the compositions of claims 1-18; and treating the disease in the patient.

24. The method of claim 23, wherein the disease is an ear infection, chronic inflammatory reaction, a chronic infection, an acute inflammatory reaction, and acute infection, or any combination thereof.

25. The method of claims 23 or 24, wherein disease is otitis media, a skin infection, a skin bum, or a combination thereof.

26. The method of any one of claims 16-18, wherein the effective amount of the composition is a single dose.

27. The method of any of claims 23-26, wherein administering to the patient the effective amount of the composition comprises non-invasive administration to an external auditory canal onto the tympanic membrane of the patient.

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