Targeted delivery of therapeutic cargo to tissues using thermosensitive hydrogels

Abstract

Compositions and methods are provided for transplantation of cells to the body, utilizing a thermosensitive polymer that forms a gel when introduced onto tissue or into a cavity of the tissue, such as the eye.

Description

ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254TARGETED DELIVERY OF THERAPEUTIC CARGO TO TISSUES USING THERMOSENSITIVE HYDROGELSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No 63 / 684,776 filed August 19, 2024, which application is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract EY026877 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] There is a major clinical need for better ways to transplant of cells to diseased or damaged organs of the body. An important example is the cornea, the thin and transparent dome-shaped tissue located at the outermost part of the eye, which not only acts as a barrier to regulate the entry and exit of substances from the outside but also plays a role in securing vision through the refraction of light as the first point of contact for light. Consisting of five layers (three cell layers and two protein layers), the cornea is a tissue without blood vessels, so the monolayered corneal endothelial cells located at the innermost layer regulate the hydration level and preserve the thickness and transparency of the cornea. The sensory function of the eye depends on the transparency of the cornea. Transparency depends on both the outer and inner integrity of the corneal epithelium and corneal endothelium (CE), respectively.

[0004] Corneal endothelial cells, with limited proliferative capacity, have the characteristic of decreasing in number throughout life. When cell loss and death occur due to external impacts or burns, failure to maintain moisture and supply nutrients leads to corneal swelling, ultimately resulting in corneal opacity. Various pathologies cause CEC dysfunction, including viral infection, intraocular surgery and Fuchs' endothelial dystrophy (FED). FED, the most common etiology of corneal endothelial dysfunction, is a primary indication for corneal transplantation. CEC loss can also occur as a result of injury including intraocular surgery and / or the presence of an intraocular implant, such as in the case of pseudophakic bullous keratopathy or CEC loss in the setting of a glaucoma tube shunt in the anterior chamber.

[0005] Corneal endothelial cell transplantation methods have been developed to address the loss of these cells. A desirable method for corneal endothelial cell transplantation is transplanting donated corneas. However, due to the significantly low number of cornealATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 donations and the fact that most donated corneas are aged, they often fail to fulfill their role after transplantation, making them unsuitable for use as grafts.

[0006] To complement this, various methods have been tested, such as directly injecting cultured cells or injecting them in the form of cell sheets. However, direct injection of cultured cells requires patients to maintain a certain posture for an extended period of time after surgery to induce proper attachment of the cells, causing discomfort, and does not guarantee the survival rate, attachment rate, or functional expression and maintenance of the cells.

[0007] Therefore, there is an urgent need to develop biocompatible materials and transplantation methods that can serve as carriers for cultured corneal endothelial cells.SUMMARY

[0008] Compositions, kits and methods are provided for use in delivery of therapeutic cargo to the posterior region of the cornea in a patient. Delivery of the therapeutic cargo aids in wound healing and tissue regeneration, particularly in repair, regeneration, and / or reconstruction of lamellar or partial defects of wounded corneal tissue.

[0009] In some embodiments, the therapeutic cargo comprises cells. In some embodiments, the cells are corneal endothelial cells, corneal stromal cells, or progenitors thereof. In other embodiments, the cells being delivered replace or replenish corneal epithelial cells or corneal stromal cells. An effective dose of cells may be, for example, from about 103to about 109cells.

[0010] In some embodiments the therapeutic cargo comprises a molecular agent. A molecular agent may comprise a ROCK inhibitor. A molecular agent may comprise a growth factor or growth factors. A molecular agent may comprise a biomolecule or biomolecules. A therapeutic cargo may comprise exosomes. The therapeutic cargo may comprise a combination of these agents, which may also be combined with cells.

[0011] The methods of the disclosure provide an efficient and patient-friendly treatment, which can be performed in a clinical setting. A benefit of the method includes administration in the absence of an air bubble. A benefit of the method also includes a short time frame, where the patient maintain a prone position for less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, as little as 1 minute, and may remain prone for a period of time from about 1 to about 30 minutes, from about 10 to about 20 minutes, from about 5 to about 15 minutes. In other embodiments, the patient remains in the supine position, for example in the delivery of epithelial or stromal cells rather than cells that are internal to the eye, like the endothelium. In some embodiments, a contact lens is used to cover, protect, and / or shape the suspension of cells that are applied to the external surface of the eye. In still some other embodiments, an air or gas bubble used to help secure or flatten cells delivered into the eye.

[0012] The methods of the disclosure use a flowable, thermosensitive biomaterial composition comprising a thermosensitive polymer and an effective dose of cargo for delivery to theATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 cornea. The thermosensitive polymer is selected to be flowable at room temperature, but at a concentration that will form a gel at body temperature, e.g. gel forming at a temperature above about 30°C, above about 32°C, above about 35°C, or at around 37°C. In some embodiments, the thermosensitive polymer comprises PF127 hydrogel. In some embodiments the thermosensitive polymer is mixed or conjugated with additional polymers or biopolymers, including without limitation collagen and a collagen derivatives.

[0013] Cells suitable as therapeutic cargo for treating corneal tissue may include, for example, one or more of human corneal stromal stem cells, mesenchymal cells, keratocytes, keratinocytes, corneal endothelial cells, and corneal epithelial cells, and limbal epithelial cells, and transient amplifying cells. In some embodiments the cells are human corneal endothelial cells. In still other embodiments, the cells delivered are retinal cells including but not limited to retinal ganglion cells, retinal pigment epithelial cells, or stem cells being delivered to the inner posterior surface of the eye. In yet other embodiments, the cells being delivered are delivered to the posterior external aspect of the eye such as behind the eye, near and / or around the optic nerve.

[0014] In an embodiment, a method of providing therapeutic cargo to the cornea of an individual is provided. Such methods may comprise introducing a composition comprising a thermosensitive polymer and an effective dose of cells into the limbus of the eye; and maintaining the individual in a prone position for a period of time from about 10 to 30 minutes. In some embodiments the cells are corneal endothelial cells. In some embodiments the cells are provided at a dose of from about 103to 109cells, 104to 108cells, 105to 107cells. In some embodiments the method comprises making an incision in the eye, and introducing the composition with a cannula or a sprayer. In some embodiments the composition is injected.

[0015] In an embodiment, the instant disclosure includes a kit for making a corneal construct for use in treating or reconstructing a surgically incised or wounded corneal area in a mammalian subject. A kit will comprise a flowable, thermosensitive biomaterial composition comprising a thermosensitive polymer and an effective dose of cells for delivery to the cornea.

[0016] These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGS. 1A-1 F. Thermoresponsive behavior of 20% PF127 under various temperature conditions. Photographs show the macroscopic gelation behavior of PF127 solutions in upright, inverted, and horizontal positions: (A) after incubation at 4 °C for 30 minutes (sol state), (B) after incubation at 37 °C for 30 minutes (gel state), (C) after incubation at 37 °C for 30 minutes followed by resting at room temperature (RT) for 5 minutes, and (D) after incubation at 37 °C for 30 minutes, followed by the addition of 37 °C water and observation forATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-2545 minutes. (E) Viscosity profiles of 20% PF127, PEGDA, and cell culture media as a function of temperature. (F) Temperature-dependent changes in storage modulus (G') and loss modulus (G") of 20% PF127.

[0018] FIGS. 2A-2G. Cytocompatibility of 20% Pluronic F127 with human corneal endothelial cells. (A) Live & dead staining of CEndoC after 3 days of suspension culture in either cell culture media or 20% PF127 and (B) F-actin staining after 24 hours cell culture. (C, D) Representative immunofluorescence images of ZO-1 , Na7K+-ATPase, N-cadherin, and Aquaporin-1 on days 3 and 7. (E) Quantitative comparison of cell circularity based on ZO-1 expression in PF127 versus media groups on day 7. (F) Western blot analysis and (G) quantification of functional proteins expression, ZO-1 , Na7K+-ATPase, N-cadherin, and Aquaporin-1 .

[0019] FIGS. 3A-3F. Cell retention capability of PF127 and associated cellular viability in CEndoCs. Schematic illustration of the cell seeding process using 20% PF127, (A) 30-minute incubation in the inverted position followed by (B) two PBS washes, (C) 24 hours, and (D) tile scan imaging after 3 days of culture. (E) Representative confocal images of each condition corresponding to the schematic steps. (F) Quantification of fluorescence intensity from confocal images, showing significantly enhanced cell retention in the PF127 group compared to the media control.

[0020] FIGS. 4A-4D. Optimization of incubation time after PF127+cell mixture injection using endothelium removed rabbit cornea and the artificial anterior chamber. (A) Schematic illustration of the overall process. The PF127 and cell mixture was injected into the anterior chamber, and the cornea was positioned upside down followed by incubation at 37 °C. During this process, the sol-gel transition of PF127 helped facilitate broad spreading and retention of cells, promoting attachment to the stromal surface. Digital images demonstrate effective spreading of trypan blue-stained PF127 and cell mixture on the posterior corneal surface after 30 minutes of incubation. (B) Optimization of the incubation period in the upside-down position. After injecting PF127 and cell mixture, corneas were incubated at 37 °C for 10, 20, 30, or 60 minutes in the inverted position, followed by 7 days of culture. Cell tracker fluorescence imaging at high and low magnifications was used to assess cell localization and attachment. (C) Quantitative comparison of cell attachment and spreading area from the cell tracker images. (D) H & E staining and immunofluorescence analysis (CMFDA, Na7K+-ATPase, and DAPI ) of transplanted corneas.DETAILED DESCRIPTION

[0021] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose ofATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 describing particular embodiments of the invention only, and is not intended to be limiting. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

[0022] In describing embodiments of the present invention, the following terms will be employed, and are intended to be defined as indicated below. As used in this specification and the appended claims, the singular for“s ”a“" ”an" a“d "”he" include plural referents unless the content clearly dictates otherwise.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0024] The practice of the present invention will employ, unless otherwise indicated, conventional methods of medicine, pharmacology, chemistry, biochemistry, molecular biology and recombinant DNA techniques, within the skill of the art. Such techniques are explained fully in the literature. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.

[0025] As used herein, about, or approximately mean within 50 percent, preferably within 20 percent, more preferably within 5 percent, of a given value or range.

[0026] A value which “substantially different” from another value can mean that there is a statistically significant difference between the two values. Any suitable statistical method known in the art can be used to evaluate whether differences are significant or not“

[0027] "Statistically significant" difference means a significance is determined at a confidence interval of at least 90%, more preferably at a 95% confidence interval.

[0028] The terms "treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease.

[0029] The terms "reconstructing" and "reconstruction," and the like are used herein to generally refer to rebuilding, healing and regenerating an injured matter or tissue.

[0030] The term "subject" or "mammalian subject" refers to any mammalian subject for whom treatment or therapy is desired, particularly humans. "Mammal" for purposes of treatmentATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as non-human primates, dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is a human.

[0031] The term "therapeutically effective amount" or "effective amount" means the amount of a compound, agent, composition, construct that when administered to a mammalian subject for treatment is sufficient, in combination with another agent, or alone in one or more doses or administrations, to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, agent, composition, construct, the defect or disease to be treated, and its severity and the age, weight, etc., of the mammalian subject to be treated.

[0032] As used herein, a “biocompatible” substance (for example polymer) is one that does not generally cause significant adverse reactions (e.g. toxic or antigenic responses) to cells, tissues, organs or the organism as a whole, of example, whether it is in contact with the cells, tissues, organs or the organism as a whole, for example, whether it is in contact with the cells, tissues, organs or localized within the organism, whether it degrades within the organism, remains for extended periods of time, or is excreted whole. A biocompatible substance (e.g., a biocompatible polymer) may be selectively compatible in that it exhibits biocompatibility with certain cells, tissues, organs or even certain organisms. For example, the biocompatible substance may be selectively biocompatible with vertebrate cells, tissues and organs but toxic to cells from pathogens or pathogenic organisms. In some circumstances, the biocompatible substance may also be toxic to cells derived from tumors and / or cancers.

[0033] The term "biopolymer" refers to a biocompatible polymers comprising polymers that can be found naturally in organisms, as well as chemical and physical modifications of such polymers, and include, but are not limited to, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, laminin, fibronectin, extracellular matrix constituents, glycosaminoglycans, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin, polysaccharides, synthetic polyamino acids, prolamines, combinations thereof, and other such molecules. In some embodiments, a biopolymer is a conjugated version of the native biopolymer, where the conjugate confers additional functionality.

[0034] As used herein, the term “cell” in the context of the applications of the present disclosure encompasses mammalian cells of any genus or species, particularly human cells. The types of cells that may be incorporated into the polymeric biomaterial include progenitor cells of the same type as those from the tissue site, and progenitor cells that are histologically different from those of the tissue site such as embryogenic or adult stem cells, that can act to accelerate the healing, regenerative or reconstructive process. The compositions comprisingATTORNEY NAME: STAN-2210W0 CLIENT REFERENCE: S24-254 cells can be administered in the form of a solution or a suspension of the cells mixed with the polymeric solution, such that the cells are substantially immobilized within the application site upon gelation. This serves to concentrate the effect of the cells at the site of application; and may provide for release of the cells over a course of time

[0035] Corneal cells may be used, for example limbal epithelial stem cells, corneal stromal stem cells, keratinocytes, keratocytes, corneal endothelial cells, etc., which may be isolated, harvested, and / or propagated from cadaveric donor corneal tissue, from small limbal biopsies from patients (either autologous from a healthy eye, or from another living patient's eye as a donation); generated by in vitro culture, etc. Corneal stem cells also include corneal stromal stem cells, which are quiescent, mesenchymal cells. It has been suggested that corneal stromal stem cells are a subpopulation of stromal cells that can differentiate into keratocytes.

[0036] Corneal endothelial cells (CECs) are of particular interest. These cells play a pivotal role in maintaining the clarity and function of the cornea by regulating fluid balance and preventing edema. The unique nature of human corneal endothelial cells makes them a crucial element in the success of corneal transplantation procedures. Human corneal endothelial cells are a monolayer of hexagonal-shaped cells located at the posterior surface of the cornea. Despite their low proliferative capacity in vivo, these cells exhibit remarkable functional efficiency in maintaining corneal transparency. Morphologically, human corneal endothelial cells form a tightly packed hexagonal mosaic, ensuring optimal coverage of the corneal surface.

[0037] Corneal endothelial cells (CECs) facilitate the function of maintaining the transparency of the cornea. CE develops when neural crest cells migrate and localize between the corneal epithelium and the lens at 6 weeks of human gestation. CE is the innermost layer of the cornea with a thickness of 5 pm. It consists of CECs that form a monolayer of hexagonal cells on the posterior corneal surface. Markers of CECs include:ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254

[0038] Successful culture of human corneal endothelial cells (HCECs) utilizes an environment that promotes cell attachment, survival, and proliferation while maintaining their characteristic properties. HCECs can be isolated from donor corneas, typically from cadaveric eyes unsuitable for transplantation. These cells are located on the posterior surface of the cornea. The corneal endothelium can be peeled or gently scraped from the Descemet's membrane. Enzymatic digestion using collagenase or dispase is commonly used to dissociate the endothelial cells from the membrane. The cells are cultured in a suitable basal medium, which may be supplemented with growth factors, such as bFGF, a Rock inhibitor, e.g. Y-27632, Netarsudul, Riparsudil, etc. HCECs have a limited capacity to be passaged and can undergo senescence. Generally, cells can be passaged 3-5 times before significant changes in phenotype are observed. HCECs can be cryopreserved using standard techniques with dimethyl sulfoxide (DMSO) as a cryoprotectant for future use. Healthy HCECs should exhibit a characteristic hexagonal shape in a monolayer, and express markers including Na+ / K+- ATPase, ZO-1 (Zonula occludens-1 ), and Aquaporin-1 .

[0039] Therapeutically effective amounts of the cells seeded in a hydrogel of the instant disclosure will vary depending e.g., on the condition to be treated, typical survival of the particular cell type within the hydrogel construct (e.g., including the average lifespan of cells of the particular cell type), etc. Usually a dose of at least about 104, at least about 105, at least about 5 x 105cells, at least about 106cells or more, e.g. at least about 107cells, at least about 5 x 107cells are provided per eye. The dose may be from about 105to about 107cells / eye, or around about 106cells / eye.

[0040] The term "gel" or "hydrogel," as used herein, refers to a crosslinked network of hydrophilic biopolymers. Hydrogels of the instant disclosure will generally be made by combining a first flowable composition containing reactive groups of one nature and a second flowable composition containing reactive groups of a different nature, and possibly more flowable compositions with reactive groups of further different nature. The flowable compositions may be combined in situ, particularly where the network is covalently linked.

[0041] The term "biopolymer" refers to a biocompatible polymers comprising polymers that can be found naturally in organisms, as well as chemical and physical modifications of suchATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 polymers, and include, but are not limited to, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, larnnin, fibronectin, extracellular matrix constituents, glycosaminoglycans, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hylauronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin, polysaccharides, synthetic polyamino acids, prolamines, combinations thereof, and other such molecules.

[0042] Thermosensitive gels. Thermosensitive hydrogels are a class of smart materials that undergo physical or chemical changes in response to temperature variations. For the purposes of the present disclosure, thermosensitive gels are flowable, or liquid, at room temperature and below, but form a hydrogel at body temperature, e.g. at around 30-37°C. The changes in the hydrogel are typically reversible, allowing the material to return to its original state once the temperature returns to its initial value.

[0043] Poly(N-isopropylacrylamide) (variously abbreviated PNIPA, PNIPAM, PNIPAAm, NIPA, PNIPAA or PNIPAm) is a temperature-responsive polymer that was first synthesized in the 1950s. It can be synthesized from N-isopropylacrylamide which is commercially available. It is synthesized via free-radical polymerization and is readily functionalized making it useful in a variety of applications. PNIPA dissolves in water, however, when these solutions are heated in above their cloud point temperature, they undergo a reversible lower critical solution temperature (LCST) phase transition from a soluble hydrated state to an insoluble dehydrated state.

[0044] In dilute solution, it undergoes a coil-to-globule transition. PNIPA possesses an inverse solubility upon heating. It changes hydrophilicity and hydrophobicity abruptly at its LCST. At lower temperatures PNIPA orders itself in solution in order to hydrogen bond with the already arranged water molecules. The water molecules must reorient around the nonpolar regions of PNIPA which results in a decreased entropy. At lower temperatures, such as room temperature, the negative enthalpy term from hydrogen bonding effects dominates the Gibbs free energy, causing the PNIPA to absorb water and dissolve in solution. At higher temperatures, the entropy term dominates, causing the PNIPA to release water and phase separate which can be seen in the following demonstration.

[0045] Pluronic F127 is a triblock copolymer consisting of polyethylene oxide (PEG) and polypropylene oxide (PPO) segments. The PEO-based non-ionic surfactants, Pluronics® (known alternatively as Poloxamers®), are composed of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock coploymers. Pluronics’ attractiveness can be attributed to their safety, bioadhesiveness, stability and ability to form gels at low concentrations at body temperature. Gelation properties may be readily tuned using physical blends of Pluronics or other excipients. It forms a gel at body temperature and is liquid at lower temperatures.ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254

[0046] Dilute solutions of block copolymers self-assemble to form micelles in ‘good’ solvents upon reaching a critical micellar temperature (CMT) as a result of PPO-block dehydration. These micelles have a relatively compact core and a heavily solvated corona. At sufficient concentrations, the triblock micelles associate above a critical gelation temperature (CGT) and order onto a lyotropic liquid crystalline (LLC) phase, a complex liquid phase with characteristics of both anisotropic crystalline solids and isotropic liquids. These micellar liquids can be described as colloidal dispersions of hard spheres which undergo a hard sphere crystallisation to form the gel structure above CGT. The variety of mesophases reported for the same triblocks by different groups are largely attributed to polymerisation by-products, which affect the self-assembly leading to formation of different phases before and after polymer purification.

[0047] During drug administration, the polymeric matrices are subjected to shear that alters the spatial arrangement of the particles making up the colloidal crystal, leading to the macroscopic viscoelastic response recorded for this class of materials.

[0048] In one embodiment, the thermosensitive gel is Pluronic F127. The concentration may be, for example, from about 5% to about 40%, from about 10% to about 30%, from about 15% to about 20%. The concentration may be at least about 10%, at least about 15%, at least about 20%. The concentration may be less than about 40%, less than about 30%, less than about 20%.

[0049] In some embodiments, the PF127 hydrogel is mixed or conjugated with another polymer or biopolymer. In an example of a PF127 composite, the 2ndpolymer is collagen or a collagen derivative to provide cell-adhesion properties to the material. A second polymer may be present, for example at a concentration from about 5% to about 40%, from about 10% to about 30%, from about 15% to about 20%. The concentration may be at least about 5%, at least about 10%, at least about 15%. The concentration may be less than about 40%, less than about 30%, less than about 20%.

[0050] The thermosensitive gel may also comprise suitable therapeutic factors. Suitable growth factors and cytokines include, but are not limited to stem cell factor (SCF), granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor- 1 , steel factor, vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFP), platelet derived growth factor (PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), fibroblast growth factor (FGF) hepatocyte growth factor, nerve growth factor, keratinocyte growth factor, insulin-like growth factor (IGF-1 ), interleukin (IL)-3, IL-la, IL-ip, IL-6, IL-7, IL-8, IL-1 1 , and IL-13, colony- stimulating factors, thrombopoietin, erythropoietin, fit3-ligand, and tumor necrosis factor a. Examples of growth factors include EGF, bFGF, HNF, NGF, PDGF, IGF-1 and TGF. These growth factors can be mixed with theATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 membrane materials comprising the compositions. The bioactive agents can also have pro- angiogenic activities, e.g., VEGF, PDGF, prominin-1 polypeptide, and variants thereof that have pro-angiogenic activities, i.e., promote neovascularization and angiogenesis.

[0051] Other thermo-sensitive polymers or biopolymers can be used including single composition or mixtures of polymers, can be utilized for this invention. This includes mixtures of covalently or non-covalently crosslinkable polymers or biopolymers with PF127, for other covalently or non-covalently crosslinkable materials in the absence of PF127. For instance, crosslinkable hyaluronic acid can be used in conjunction with PF127 to achieve a similar result of targeting a suspension of cells to the anterior or posterior surface of the cornea or another location in the eye or the body.

[0052] As used herein, the term “under physiological conditions” encompasses those conditions that are compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, osmolarity, osmolality etc.

[0053] The term "biocompatible" refers to the absence of stimulation of a severe or escalating biological response towards administration of a composition, and is distinguished from a mild, transient inflammation which typically accompanies surgery or implantation of foreign objects into a living organism.

[0054] The term “polymer,” as used herein, refers to a molecule consisting of individual monomers joined together. Polymers that are contemplated herein can be naturally occurring, synthetically produced, or produced using recombinant methodologies.

[0055] The term "transparent," as used herein, refers to at least 70%, 80, or 90% transmission of white light.METHODS

[0056] In an embodiment, a method of providing therapeutic cargo to the cornea of an individual is provided. Such methods comprise introducing a composition comprising a thermosensitive polymer and an effective dose of cargo into the limbus of the eye; and maintaining the individual in a prone position for a period of time from about 10 to 30 minutes, or as little as 1 or 5 minutes. In some embodiments the cargo comprises cells. In some embodiments the cells are corneal endothelial cells. In some embodiments the cells are provided at a dose of from about 105to 107cells. In some embodiments the method comprises making an incision in the eye, and introducing the composition with a cannula. In some embodiments the composition is injected.

[0057] In an embodiment, the thermosensitive hydrogel is injected into other spaces or potential spaces of the eye, such as the suprachoroidal space, the vitreous, the subretinalATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 space, the subconjunctival space, the subtenon’s space, as well as the retro-orbital or periorbital space.

[0058] In an embodiment, the therapeutic cargo comprises a molecular agent. In some embodiments, the therapeutic cargo being delivered is a ROCK inhibitor. A ROCK inhibitor of interest may be selected from fasudil, ripasudil, netarsudil, and belumosudil, Y-27632, GSK 269962, Acalabrutinib, Brigati nib, Capmatinib, Dasatinib, Imatinib, and Thiazovivin.

[0059] In some embodiments, the therapeutic cargo is exosomes or secreted factors (secretome) derived from, for example, mesenchymal stromal cells (MSCs), derived from bone marrow, the cornea, or other sources.

[0060] In some embodiments, the therapeutic cargo being delivered are biomolecules, for example, recombinant proteins, growth factors, mRNA, siRNA, etc. In some embodiments, the therapeutic cargo stimulates the regeneration / proliferation of either the host and / or injected corneal endothelial cells. In some embodiments, the therapeutic cargo includes cells, which are optionally combined with other therapeutic agents. In other embodiments, the therapeutic cargo comprises one or a combination of molecular agents.

[0061] In some embodiments, the release of the cargo or cells proceeds in a sustain or slow- release manner. When therapeutic cargo is injected in the absence of cells, the hydrogel can be formed as a meniscus of material in the inferior angle of the eye. For example, if the solution is injected into the anterior chamber with the head in the upright position, the solution will settle by gravity at the inferior irido-corneal angle of the eye, where the therapeutic cargo is then released slowly over time to modulate the regeneration of the corneal endothelium without being in the visual axis.

[0062] The composition comprising cells and thermosensitive polymer may be formulated with a pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, which are readily available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available.

[0063] The compositions of the disclosure address a need for effective compositions and methodologies to treat corneal transplantation. Ocular and corneal defects may be caused by, e.g., neurotrophic keratopathy, recurrent corneal erosion, corneal ulcer, corneal burns, exposure keratopathy, physical trauma, retinal disease, retinal degeneration, optic nerve damage, optic nerve degeneration, and other disorders. Corneal endothelial cell disorders include Fuchs’ Endothelial Dystrophy and Pseudophakic Bullous Keratopathy.

[0064] Delivering (cultured) cells such as corneal limbal epithelial cells, corneal stromal stem cells (CSSCs) or corneal endothelial cells to the site of corneal injury or limbus may minimize the fibrotic response and enhance the regeneration of the corneal tissue. Delivery of cornealATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 cells, such as keratocytes and keratinocytes as described herein, may be instrumental in repairing and regenerating corneal tissue.

[0065] The cornea is a highly specialized transparent tissue and, as the most anterior ocular tissue, protects the eye by acting as a physical barrier. It is comprised of three cellular layers: the outer layer being the stratified squamous corneal epithelium, the center layer being the corneal stroma, and the inner layer being the corneal endothelium. The corneal stroma makes up the majority of the corneal tissue. The extracellular matrix (ECM) of the corneal stroma has a lamellar, highly organized structure that facilitates the transparency of the cornea, whereby each lamella is composed of tightly organized collagen fibrils. Keratocytes are mesenchymal- derived cells that are quiescent in the mature cornea and that are arranged within the corneal stroma. Upon injury to the cornea, the keratocytes become activated, and several changes in the corneal stroma occur. Upon an initial apoptotic phase, keratocytes lose their quiescence, start to divide and develop either into phenotypes that start to secrete extracellular matrix for corneal regeneration or into phenotypes that induce fibrotic scar formation at the site of injury. Unlike in uninjured stromal tissue, the extracellular matrix in scar tissue is disorganized and opaque, and may seriously impair visual acuity and lead to blindness.

[0066] Delivering cells such as corneal endothelial cells, corneal stromal stem cells (CSSCs), mesenchymal stromal cells, corneal fibroblasts, keratocytes, corneal epithelial cells or limbal corneal epithelial cells to the site of corneal injury may minimize the fibrotic response and enhance the regeneration of the corneal tissue. Delivery of corneal cells, such as keratocytes and keratinocytes, and other cells, within injectable polymeric precursor compositions that gel in-situ upon cross-linking, as described herein, may be instrumental in repairing and regenerating corneal tissue. The cells can then be encapsulated into the corneal construct to provide a scaffold for proliferation and reepithelialization of the corneal defect.KITS

[0067] The present invention also provides kits comprising separate containers holding compositions comprising a suitable thermosensitive polymer, and optionally seeded with living cells to be delivered to the cornea. Compositions can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. The kit can further comprise a container comprising pharmaceutically acceptable excipients or formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The kit can also comprise a package insert containing written instructions describing methods for care of a corneal wound as described herein.ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254

[0068] Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic.

[0069] The kit can further comprise a container comprising pharmaceutically acceptable excipients or formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The kit can also comprise a package insert containing written instructions describing methods for care of a corneal wound as described herein.EXPERIMENTAL PROCEDURES

[0070] The following methods and materials were used in the examples that are described further below. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of what the inventors regard as their invention.

[0071] Reasonable efforts have been made to ensure accuracy with respect to numbers used, e.g. in the context of temperature, amount and such, but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degree Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used throughout the specification, e.g. s or sec for second(s), min for minute(s), h or hr for hour(s), aa for amino acid(s), nt for nucleotide(s), kb for kilobase(s), i.v. for intravenous(ly), and the like.

[0072] Limitations of existing direct injection methods for corneal endothelial cells include discomfort as patients need to maintain a prone position for more than three hours to induce natural cell attachment due to gravity. Continuous hindrance of corneal endothelial cell attachment due to the flow of aqueous humor and delayed recovery time occur. The disclosure herein provides a solution to this problem.Materials and Methods

[0073] Preparation of Thermosensitive hydrogel. Thermosensitive hydrogel, the Pluronic F127 (PF127, P2443, Sigma Aldrich) is dissolved in the distilled water in 20% (w / v) for 3 hours or until fully dissolved at the room temperature and placed at 4°C to keep in fresh. For the cell and tissue culture, it was sterilized under UV light for 30 minutes and stored at 4°C.

[0074] Gelation property of thermosensitive hydrogels. To test the gelation property of the thermosensitive hydrogel, prepared hydrogel solution was transferred into the vial. To compare the behavior of thermosensitive hydrogel according to the different temperatures, the vials were stored at 37°C for 30 minutes, took it out and waited for 5 minutes. The sample withATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254 the water inside was incubated at 37°C for 30 minutes and placed at room temperature for 5 minutes (FIG. 1 ). The images from right, upsided down, laid down position, and with / without the water in each temperature were taken.

[0075] Isolation and culture of primary rabbit corneal endothelial cell. Rabbit eyeballs were purchased from Visiontech Inc. (Sunnyvale, TX, USA). To isolate the corneal endothelial cells (CECs), Descemet’s membrane was separated and placed in a 2 mg / mL collagenase (C0130, Sigma Aldrich) solution to incubate at 37°C for 1 h. It was centrifuged at 1000 rpm for 3 min and resuspended with Dulbecco's Modified Eagle Medium (DMEM, Thermofisher Scientific) supplemented with the 10 nM ROCK inhibitor (Sigma Aldrich), 2 ng / ml of fibroblast growth factor (FGF, Sigma Aldrich), 10% Fetal Bovine Serum, and 1 % Penicillin streptomycin. The collected cells and the descemet’s membranes were seeded on the tissue culture plate (TCP) coated with the FNC coating mix (Athena) and the culture medium was changed every 2-3 days.

[0076] Cell viability test. To test the viability of the CECs, we performed a Live & Dead assay kit following the manufacturer’s protocol. Briefly, the resuspended CECs with the 20% PF127 were cultured on the TCP for 24 h. The culture medium was then changed to a Live & Dead solution mixed with a fresh culture medium. After a 15 min incubation, the live and dead cells were observed via a confocal microscope.

[0077] Preparation of cell / hydrogel mixture and injection in Ex vivo culture system. To prepare the cell / hydrogel mixture, the harvested cells after trypsinization were labeled with the cell tracker (C7025, Thermo Fisher Scientific), resuspended in cold PF127 solution to achieve a concentration of 1 x106cells / 30 pl, and kept on ice to maintain the solution state. The control group was prepared in the same manner, with the cells resuspended in PEGDA (475629, Sigma Aldrich) to achieve the same cell density. To verify the spreading pattern of the injected hydrogel, a 2.5 mm incision was made at the edge of the cornea of a whole rabbit eyeball using a knife (8065921501 , Alcon). Subsequently, 30 pl of trypan blue-stained PF127 solution was injected into the anterior chamber through an anterior chamber Rycroft cannula (Ambler, 23 Gauge) while the eyeball was fixed in an upright position. After incubating in an inverted position at 37°C for 10 minutes, the eyeball was repositioned upright to compare the spreading distribution of the PF127 hydrogel.

[0078] For the cell injection test using the cell / hydrogel mixture, an ex vivo culture system was established by securing the cornea, with the Descemet’s membrane removed, in an artificial anterior chamber (K20-2125, Corza Medical). An incision was made at the edge of the cornea fixed in the artificial anterior chamber, and the cell / hydrogel mixture was injected. The cornea was incubated in an inverted position at 37°C for 10 minutes, then repositioned upside for cultivation at 37°C.ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254

[0079] Immunofluorescence staining. To investigate the protein expressions in the CECs, the samples were rinsed with PBS and fixed using 4% paraformaldehyde (15710, Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min at RT followed by washing 3 times using PBS. The samples were then permeabilized using 0.2% triton X-100 (93443, Sigma Aldrich) for 5 min followed by washing 3 times with PBS. The samples were subsequently incubated with 3% bovine serum albumin (BSA, A2153, Sigma Aldrich) dissolved in PBS for 1 h at RT and then incubated in a primary antibody dissolved in a 1 % BSA solution overnight at 4 °C. The following day, the samples were rinsed 3 times with PBS and incubated with a secondary antibody dissolved in a 1 % BSA solution for 1 h at RT. For DAPI staining, the DAPI solution was diluted in PBS and incubated for 5 min at RT. The images were photographed using a confocal microscope (LSM T-PMT, ZEISS, Jena, Germany). The primary and secondary antibodies used in this study were ZO-1 (339188, Thermo Fisher Scientific), Na+ / K+-ATPase (sc-48345, Santa Cruz Biotechnology, Dallas, Texas, USA), N-cadherin (ab98952, Abeam), aquaporin-1 (ab219055, Abeam, Cambridge, UK), phalloidin-555 (ab176756, Abeam), and DAPI (62248, Thermo Fisher Scientific). For tracking the injected cells, pre-labeled corneal endothelial cells with the cell tracker were visualized using the confocal microscope.

[0080] Attached cell circularity calculation. To calculate cell circularity, images visualized through a confocal microscope were analyzed using Imaged software. A line was drawn representing the longest distance within the cell membrane, and a perpendicular line intersecting this line was also drawn. The ratio of the lengths of these two lines was calculated. A value closer to 1 indicates a more circular cell morphology.Results

[0081] The thermo-responsible property of PF127 at different temperatures was tested to determine its suitability for in vivo use, shown in FIG. 1 . At low temperatures, the PF127 solution behaves like water, flowing downward when inverted. After incubation at 37°C for 30 minutes, it transforms into a hydrogel state, remaining in place even when inverted. The hydrogel property is maintained even after being kept at room temperature for 5 minutes, staying in position when inverted. (D) It was confirmed that even in an environment similar to the anterior chamber filled with warm water, the hydrogel does not mix with the water or flow downward.

[0082] The ability of PF127 for cells was tested, as shown in FIG. 2. After seeding a 20% PF127 solution and cell mixture onto TCP and culturing for 24 hours, the Live / Dead assay results showed that almost all cells were live (green).

[0083] Protein expression in corneal endothelial cells cultured with PF127 and control medium was determined, shown in FIG. 3. Similar levels of cell attachment were observed on day 1 in corneal endothelial cells seeded in either control (media) or 20% PF127 (left column).ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254Immunofluorescence staining of the cytoskeletal protein F-actin shows comparable expression patterns with the control. Comparable expression levels of junctional proteins ZO- 1 , N-cadherin, the ion channel Na+ / K+ATPase, and aquaporin-1 were confirmed, indicating normal hexagonal cell shape cultured. The quantification of cell circularity shows that the cells of control group had more circular shape on day 3, however, both showed comparable circularity on day 7.

[0084] The direct injection method using PF127 for corneal endothelial cells in an air bubble- free environment involves the following steps: maintaining an upright position; direct injection of the PF127 / cell mixture into the anterior chamber using a cannula; holding a prone position for 10 minutes or less; and confirmation of even spreading of the PF127 / cell mixture across the damaged endothelial area.

[0085] The morphological transition of injected PF127 pre- and post-incubation was determined, FIG. 4. Immediate post-injection position of PF127 is settled at the bottom at the bottom, and after a 10-minute incubation at body temperature, is witnessed to distribute evenly around the central region of the eye.

[0086] An ex vivo study utilizing the endothelium-removed rabbit cornea and artificial anterior chamber was performed. A mixture of PF127 and cell-tracker labeled corneal endothelial cells was injected into the chamber. There was a broad distribution of cells centered around the cornea post PF127 injection, demonstrating superior injection efficacy when compared with PEG.

[0087] The present technology can be used to deliver cells an area of geographic atrophy of the macula, for instance, in the case of macular degeneration. In another embodiment, the thermosensitive material can be used to help tamponade (through the formation of a thin, solid gel membrane) over a retinal tear or retinal hole. This can be used in conjunction with gases or liquids used in retina surgery to tamponade and flatten the retina during the repair of a retina detachment or retinal tear, as well as laser treatments to seal off breaks in the retina.

Claims

ATTORNEY NAME: STAN-2210W0CLIENT REFERENCE: S24-254What is claimed is:1 . A composition for use in delivery of therapeutic cargo to the posterior region of the cornea in a mammalian subject in need thereof, comprising: a thermosensitive polymer in a concentration effective to form a hydrogel at a temperature above about 32°C; and an effective dose of therapeutic cargo.

2. The composition of claim 1 , wherein the polymer is a triblock copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO) segments.

3. The composition of claim 1 or claim 2, wherein the polymer is Pluronic-F127.

4. The composition of any of the preceding claims, wherein the polymer is present at a concentration of from about 10% to about 30%.

5. The composition of any of the preceding claims, further comprising a second polymer or biopolymer.

6. The composition of any of the preceding claims, wherein the therapeutic cargo is comprised of cells.

7. The composition of claim 6, wherein the cells are one or more of corneal stromal stem cells, mesenchymal cells, keratocytes, keratinocytes, endothelial cells, epithelial cells, limbal epithelial cells, and transient amplifying cells.

8. The composition of claim 6, wherein the cells are corneal endothelial cells.

9. The composition of any of claims 6-8, wherein the cells are in vitro cultured cells.

10. The method of any of the preceding claims, comprising a dose of from about 103to 109cells.11 . The composition of any of the preceding claims, wherein the therapeutic cargo comprises a ROCK inhibitor.

12. The composition of any of the preceding claims, wherein the therapeutic cargo comprises exosomes.ATTORNEY NAME: STAN-2210W0 CLIENT REFERENCE: S24-25413. The composition of any of the preceding claims, wherein the therapeutic cargo comprises a growth factor or growth factors.

14. The composition of any of the preceding claims, wherein the therapeutic cargo comprises a biomolecule or biomolecules.

15. The composition of any of the preceding claims, wherein the therapeutic cargo comprises a combination af agents and / or cells.

16. A method of providing therapeutic cargo to the eye of an individual, the method comprising: administering a composition of any of the preceding claims onto or into the eye of the individual; and maintaining the individual in a suitable position for a period of time from about 30 seconds to 60 min that allows the cells to settle by gravity to the individual's eye tissue surface.

17. The method of claim 16, wherein the administration is performed in the absence of an air bubble.

18. The method of claim 16 or 17, comprising making an incision in the eye, and administering the composition with a cannula.

19. The method of claim 16 or 17, comprising injection of the composition.

20. The method of any of claims 16-19, wherein the individual is human.

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