Hydrogels for controlled release of therapeutic factors

Abstract

A physiologically acceptable nanocluster, e.g. a protein nanocluster such as albumin, is used as a "hub" to crosslink polymers to form a hydrogel. In addition to the protein, the nanocluster may comprise a drug of interest. The cross-linking utilizes bio-orthogonal chemistry, e.g. click chemistry such as a strain-promoted alkyne azide cycloaddition (SPAAC) to form the hydrogel in situ, in the absence of an external energy source such as light, heat, or a chemical catalyst such as copper or initiator.

Description

ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 HYDROGELS FOR CONTROLLED RELEASE OF THERAPEUTIC FACTORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 656,007, filed June 4, 2024, which application is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under contract EY033363 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] Inadequate or fibrotic healing of corneal defects caused by traumatic injuries can lead to progressive thinning, perforation, and scarring leading to severe vision loss. A promising approach to promote corneal regeneration involves the application of in situ-forming hydrogels to stromal defects. These hydrogels, when applied to defects, can facilitate the regeneration of corneal layers as well as protect the defects from exposure to deteriorating environments. To be effective for corneal repair, the hydrogels must meet certain criteria: they should be transparent, biocompatible, adhesive to native tissues, and structurally stable until the cornea is fully repaired. One effective strategy involves the use of UV light for crosslinking of the hydrogel components, but this approach comes with potential ocular toxicities caused by UV light exposure. Host-guest interactions are a promising option, but these interactions may lack strong mechanical properties as they are reversible. Alternatively, chemical reactions can also be employed to crosslink the components of hydrogels into structurally sound networks. Nonetheless, many crosslinking chemistries also have a limitation of nonspecific side reactions with biological molecules, which have the potential to cause further damage to the cells and tissue in the wound bed.

[0004] Bio-orthogonally crosslinked hydrogels would be a progressive approach for enhancing selectivity and biosafety, primarily due to their lack of cross-reactivity with cells, proteins, and glycosaminoglycans. These hydrogels employ unique chemical reactions for crosslinking, such as copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain- promoted azide-alkyne cycloaddition (SPAAC). Many bio-orthogonal reactions exhibit a time- course for gelation ranging from just a few minutes to several hundred minutes, and those on the shorter end of that spectrum would be well-suited for in situ application.

[0005] Nanoparticle-crosslinked hydrogels have emerged as a promising strategy for drug delivery and tissue engineering. Nanoparticles can serve as crosslinking nodes that improveATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 the mechanical properties of hydrogels. The large surface area and size of nanoparticles might contribute to effective conjugation with branches of hydrogel, conferring potential benefits such as controllable drug release, solubilizing effects, and stimuli-responsiveness. However, this strategy can be applicable to only a limited range of nanomaterials because the crosslinking density depends mainly on characteristics of nanoparticles, the frameworks of hydrogels, and the types of chemical reactions involved. Additionally, nanoparticle-crosslinked hydrogels for ocular applications are more constricted by their color and transparency.

[0006] In conjunction with the use of hydrogels, the delivery growth factors have merged as a valuable strategy to augment the re-epithelialization, transparency, and overall corneal regeneration. Nerve growth factor (Cenegermin, or Oxervate) was recently FDA-approved for the treatment of neurotrophic keratopathy. Meanwhile, other growth factors such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF) have been studied extensively for the purpose of corneal tissue regeneration after injury. Addressing inherent challenges in using growth factors, such as a poor stability, short half-life, and rapid diffusion, hydrogel networks can serve as effective carriers for these molecules, which can improve their therapeutic potential. Two primary fabrication methods, physical mixture and conjugation, are commonly employed. While physically mixed hydrogels offer a straightforward and convenient approach, their drawback lies in a fast release rate, limiting their suitability for long-term applications. On the other hand, growth factor conjugation to hydrogel frameworks is a promising alternative with the challenge of slow release and dependency on matrix degradation. Given that corneal re-epithelialization typically spans a couple of days, an advanced strategy for controlled growth factor release becomes crucial to improve real-time control over dosing and, in turn, the efficiency of corneal regeneration. SUMMARY

[0007] In situ-forming, bio-orthogonally hydrogels cross-linked with a nanocluster are provided. Nanocluster (NC)-crosslinked bio-orthogonal hydrogels (NCColHA hydrogels) have improved mechanical properties; and further can be used to encapsulate forms of therapeutic cargo, including where two or more different cargoes are encapsulated. The NC-crosslinked hydrogels are useful in the repair of corneal defects, for example, and the release of therapeutic agents to a tissue of interest, including to the eye.

[0008] In some embodiments, a physiologically acceptable nanocluster, e.g. a protein nanocluster such as albumin, is used as a “hub” to crosslink polymers to form a hydrogel. In addition to the protein, the nanocluster may comprise a drug of interest. The cross-linking utilizes bio-orthogonal chemistry, e.g. click chemistry such as a strain-promoted alkyne azide cycloaddition (SPAAC) to form the hydrogel in situ, in the absence of an external energy source such as light, heat, or a chemical catalyst such as copper or initiator.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0009] In some embodiments, a nanocluster is decorated with a click chemistry reactant, e.g. a strain-promoted alkyne azide cycloadditions (SPAAC) reactant; and is reacted with a counterpart reactant present on a polymer for hydrogel formulation, for example one or more of a collagen, PEG, hyaluronic acid, etc., or a combination thereof. In some embodiments the nanoclusters are comprised of albumin, e.g. human serum albumin. In some embodiments the nanocluster comprises DBCO as a reactant. The degree of substitution be from about 1 to 100, e.g. from about 10-75, and may be from about 25 to 50, e.g. about 30. The one or more polymers may be conjugated, for example, with azide, with a degree of substitution of 1 to 100, e.g. from about 10-80, and may be from about 25 to 75, e.g. about 50-60. In some embodiments the polymers are collagen and hyaluronic acid.

[0010] In an embodiment a hydrogel is formed by equally mixing albumin nanoclusters, hyaluronic acid and collagen in a 1:1:1 ratio (v / v / v). In some embodiment the nanoclusters are conjugated to DBCO, and the HA and collagen are conjugated to azide. The resulting hydrogel has a G’ value of over 20,000 Pa.

[0011] In some embodiments the biopolymers comprise therapeutic cargo, e.g. drugs, polypeptide factors, etc. conjugated by a photocleavable linker, including without limitation an o-nitrobenzene derivative. In some embodiments two, or more cargo are conjugated. The cargo is released with mild UV light, e.g. around about 365 nm; 2 - 5 mW / cm2. By irradiating UV light multiple time, the concentration of released drugs, growth factors, etc. can be maintained at a therapeutic level. Optionally, a drug of interest is milled into the nanocluster as an additional, or primary therapeutic agent.

[0012] In some embodiments a hydrogel of the disclosure comprises an in situ-forming bio- orthogonally crosslinked hydrogel (PC-HACol hydrogel) containing one or more therapeutic factors tethered to the hydrogel via photocleavable linkages. The gel optionally comprises a nanocluster. In some embodiments a single species of photocleavable linker is used. In some embodiments two or more species of photocleavable linker is used. The linkers may join one, or a plurality of therapeutic factors. In some embodiments, the linker is cleaved when exposed to mild ultraviolet (UV) light, e.g. at about 2 – 5 mW / cm2. Following exposure to light, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more of the therapeutic factor is released within about 24 hours, depending on UV intensity and irradiation time.

[0013] The photo-responsive hydrogel is formed with a click chemistry reactant, e.g. a strain- promoted alkyne azide cycloadditions (SPAAC) reactant; that is reacted with a counterpart reactant present on a polymer for hydrogel formulation, for example one or more of a collagen, PEG, hyaluronic acid, etc., or a combination thereof. The degree of substitution on the reactants be from about 1 to 100, e.g. from about 10-75, and may be from about 25 to 50, e.g. about 30. The one or more polymers may be conjugated, for example, with azide, with aATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 degree of substitution of 1 to 100, e.g. from about 10-80, and may be from about 25 to 75, e.g. about 50-60. In some embodiments the polymers are collagen and hyaluronic acid. The one or more therapeutic factors are grafted to one or both of the reactants via a photocleavable (PC) linker.

[0014] In some embodiments a hydrogel comprises two or more different linkers, where the linkers may cleave at different wavelengths, using, for example, any of the wavelengths disclosed above. In an embodiment, a hydrogel comprises two or more different therapeutic agents. The two or more therapeutic agents may be linked to the hydrogel with linkers cleaved at different wavelengths, where, for example, release may be sequential, staggered, simultaneous as required. In other embodiments, a single therapeutic agent is linked to the hydrogel with linkers cleaved at different wavelengths, where, for example, release may be sequential, staggered, simulataneous as required

[0015] In some embodiments the therapeutic factor is EGF. It is shown that hydrogels comprising photocleavable EGF significantly promote re-epithelialization of a corneal injury, without hyperplasia and stromal regeneration and with return to baseline corneal thickness compared to treatment conditions without UV irradiation and without any gel treatment.

[0016] In some embodiments, the hydrogel is a composite of two or more polymers, such as collagen and PEG or collagen and HA, where the two polymers are covalently linked to each other. Collagen and PEG or collagen and HA can be covalently crosslinked through a nanocluster, where an azide moiety on one of the molecules reacts with an alkyne moiety on the other molecule (e.g. Collagen-alkyne and HA-azide, or Collagen-alkyne and bifunctional or multifunctional PEG-azide).

[0017] In some embodiments the water content of the hydrogel is up to 90% weight / volume, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%. The ratio of first polymer to second polymer, e.g. collagen to HA, weight / weight may be about 1:25; 1:20; 1:15; 1:10: 1:5; 1:1, etc. The amount of time for cross-linking may be up to 30 seconds, up to 1 minute, up to 90 seconds, up to 2 minutes, up to 3 minutes, up to 4 minutes, up to 5 minutes, and usually less than about 60 minutes.

[0018] For corneal implants, optical transparency is desirable. At wavelengths between 300 and 800 nm, transmittance of greater than about 70%, about 80%, about 90%, about 95% is desirable. Cross-linked hydrogels have generally improved transparency relative to non-cross- linked gels.

[0019] In some embodiments the hydrogel composition is applied to a corneal defect in a flowable liquid state and allowed to form a smooth contour on its surface as it gels. The treatment can promote epithelial overgrowth, promote tight junction formation in the epithelium, and decrease myofibroblast activity in the wounded stroma. The lower refractiveATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 index of the gel may lead to under-power of corneal surface if the gel assumes the same contour as the host’s normal cornea. The focal power or focal length can be matched to naïve cornea by changing the radius curvature. In some embodiments the curvature of central cornea is adjusted by changing the volume of applied hydrogel, to compensate the slightly lower refractive index of the hydrogel to get the same focal length. Alternatively the gel can be used in conjunction with a contact lens.

[0020] Compositions, kits and methods are provided for use as in-situ forming tissue constructs, also referred to as a defined hydrogel structure, that can be cellularized to aid in wound healing and tissue regeneration, particularly in repair, regeneration, and / or reconstruction of lamellar or partial defects of wounded corneal tissue. The compositions, kits and methods also find use in the repair, regeneration, and / or reconstruction of skin, subcutaneous tissue, nerve, muscle, bone, cartilage, vitreous, tendon, ligament, fat, retinal, conjunctival, scleral, cardiac, adrenal, and other types of tissue.

[0021] In an embodiment, a flowable biomaterial composition for use as an in-situ-forming corneal construct, i.e. a defined hydrogel structure, is provided, which finds use in treating or reconstructing a surgically incised or wounded corneal area in a mammalian subject in need thereof. The flowable biomaterial may comprise cells or therapeutic agents, or both, that aid in treating or reconstructing a surgically incised or wounded area, where the cells or agent are entrapped or encapsulated in the defined hydrogel structure. Cells of interest include regenerative cells, such as a stem cell, including without limitation corneal stem cells. Cells suitable for treating corneal tissue may include, for example, one or more of corneal stromal stem cells, mesenchymal cells, keratocytes, keratinocytes, endothelial cells, and epithelial cells, and limbal epithelial cells, and transient amplifying cells.

[0022] In another aspect, a method of treating or reconstructing a surgically incised or wounded corneal site in a mammalian subject is provided, by administering a flowable biomaterial that forms a defined hydrogel structure at the site under ambient conditions without the need for an external stimulus such as light. The defined hydrogel structure is effective in treating or reconstructing the wounded corneal area. In some embodiments the flowable biomaterial is applied to an existing cavity, which can be highly irregular in shape, e.g. a pathologic cavity such as an ulcer. In some embodiments a cavity is debrided to eliminate necrotic material and create fresh wound edges. In some embodiments a cavity of specific shape and dimensions created, e.g. with surgical instruments, or a laser, for example to remove tissue that is scarred, fibrotic, opacified, etc.

[0023] In another aspect, the flowable biomaterial, or a defined hydrogel structure derived therefrom as described above, is provided, which optionally comprises cells, therapeutic agents, etc. The cytocompatible hydrogel structure is suitable for use in tissue repair or regeneration.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0024] In yet another aspect, 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 biomaterial that forms a defined hydrogel structure at the site under ambient conditions without the need for an external stimulus such as light. The defined hydrogel is effective in treating or reconstructing a wounded corneal area. The flowable biomaterial may be provided as a single composition, or may be provided as two compositions in separate containers.

[0025] The gels in the present invention can serve as, but not be limited to, tissue scaffolds, tissue substitutes, optical elements (e.g. corneal or lens tissue), scaffold fillers, tissue or scaffold adhesives, nerve guidance conduits, wound dressings, tissue fillers, tissue spacers, or as delivery vehicles for cells, tissues, and / or pharmaceutical agents.

[0026] 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

[0027] FIGS.1A-1F. Characterization of NCColHA hydrogel. a, Schematic illustration for the application of NCColHA hydrogel. i) Administration of NCColHA hydrogel to injured cornea ii) Crosslinking of polymers via NCs. iii) PRD-embedded and HGF-coated NCs b, Rheological properties of NCColHA hydrogel under different fabrication conditions at 37 °C. The DBCO / HSA ratio indicates the molar ratio of DBCO to HSA when the concentrations of NCs, HA-N3, and Col-N3 were 40 mg / mL, 30 mg / mL, and 9 mg / mL, respectively. Hydrogels with varying concentrations of HA-N3 were prepared at a constant concentration of NCs (40 mg / mL), Col-N3(9 mg / mL), and a DBCO / HSA ratio of 30. Hydrogels with different concentrations of Col-N3were prepared under the same conditions with concentrations of NCs (40 mg / mL), HA-N3(30 mg / mL), and a DBCO / HSA ratio of 30. c, Changes in storage modulus and tan δ values over time. d, SEM images of the surface of lyophilized NCColHA hydrogel and mixture of HA-N3 and Col-N3. The length of yellow and orange scale bars is 100 µm and 10 µm, respectively. e, Biodegradability assessment of NCColHA hydrogels with varying DBCO / HSA ratios in a solution containing ≥ 5 CDU / mL of collagenase and 5 units / mL of hyaluronidase. Physically mixed gel consisted of the mixture of the unmodified components. f, Accumulative release patterns of HGF in a 1 % BSA solution and PRD in PBS.

[0028] FIGS. 2A-2E. Transparency and swelling behavior of NCColHA hydrogel. a, Digital images of the hydrogel placed on the background images. b, Transmittance of NCColHA hydrogels under different fabrication conditions. Transmittance was measured in visible light ranging from 400 to 800 nm. c, The swelling ratio of NCColHA hydrogel in PBS at 37 °C. The physically mixed gel consisted of a mixture of the unmodified components. d, Digital images of NCColHA hydrogel on the lettered background pre-incubation and post-incubation for 48 hATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 in PBS at 37 °C. e, TGA analysis of NCColHA hydrogel pre-incubation and post-incubation for 72 h in PBS at 37 °C. The inserted bar chart indicates the water content of hydrogel components (w / w). n.s.: not significant.

[0029] FIGS. 3A-3F. Biocompatibility and in vitro cell proliferation. a, Confocal microscopic images of cells growing on the 6-FAM-labeled NCColHA hydrogel. The NCColHA hydrogel was spread on the culture dishes, and then CECs were seeded on the hydrogel-coated surface. The cells were incubated for two days. The green, blue, and gray colors indicate 6- FAM, DAPI, and bright field, respectively. The scale bar length is 100 µm. Cell proliferation studies b, when the cells were cultured in NCColHA-pre-added condition (n = 5) and c, when the NCColHA hydrogel was added to the cells after one day of incubation (n = 4). Cell viability was measured using CCK-8. d, Live and dead cell analysis when the cells were cultured on the NCColHA hydrogel. The blue and red colors indicate live and dead cells, respectively. The scale bar length is 100 µm. e, The cell counts of live and dead cells observed in d (n = 9). f, Cell migration assay when the cells were treated with NCColHA hydrogel (n = 3). The yellow dotted lines indicate the borders of the cells. The scale bar length is 400 µm. n.s.: not significant.

[0030] FIGS. 4A-4B. Immunohistochemistry of ex vivo rabbit cornea after treatment of NCColHA hydrogel. a, Entire corneal defects after administration of NCColHA hydrogel on day 1, 3, and 5. The control group indicates the corneal defects with PBS treatment on day 5. The orange arrows indicate the re-epithelialized layers on the hydrogel. The scale bar length is 200 µm. The magnified image indicates the edge of corneal defects on day 5. H, E, and S indicate hydrogel, epithelial layer, and stroma, respectively. The scale bar length of the magnified image is 20 µm. b, Epithelial biomarkers on day 5. The cyan, red, green, and white colors indicate the CD44, CK14, phalloidin, and ZO-1, respectively. The scale bar length is 20 µm.

[0031] FIGS. 5A-5E. In vivo rabbit cornea regeneration (n ≥ 4). a, The representative OCT images of the defected cornea after treatment of NCColHA hydrogel. The red stars and orange arrows indicate the NCColHA hydrogel and the regenerated epithelial layers, respectively. The images were taken on day 0, 7, 14, 28, and 56. b, Representative photographs of the defected eyes after fluorescein staining. c, Representative digital images of the defected eyes. d, Thickness of the regenerated epithelium, stroma, and whole cornea on day 56 (n ≥ 4). e, The horizontal slides of the regenerated cornea components (Scale of x-axis: 100%). n.s.: not significant.

[0032] FIGS. 6A-6G. Immunohistochemical analyses of in vivo rabbit cornea on day 56. a, Wounded area of NCColHA hydrogel-treated cornea. The scale bar length is 200 μm. b-d, Epithelial layers of b) NCColHA hydrogel-treated cornea, c) Normal cornea, and d) PBS- treated cornea. e-f, α-SMA staining of e) PBS-treated cornea and f) NCColHA hydrogel-ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 treated cornea. g, CD44 expression in the epithelium of NCColHA hydrogel-treated cornea. The length of scale bar in b-g is 40 μm.

[0033] FIGS.7A-7I. Characterization of PC-HACol hydrogel. a) Schematic illustration of PC- HACol hydrogel treatment. i) The gelation and photo-responsive release of PC-HACol hydrogel. ii) The application of hydrogel pre-solution to corneal defects. iii) Wound healing processes by PC-HACol hydrogel. b,c) Storage (G’) and loss (G’’) modulus of PC-HACol hydrogel with varying (b) HA-PEG-DBCO concentration and (c) Col-N3 concentration. Col-N3 concentration in (b) was fixed at 9 mg / mL1, and HA-PEG-DBCO concentration in (c) was fixed as 70 mg / mL1. Concentrations are expressed is mg / mL1. d) The changes in Tan δ and G’ values of the PC-HACol hydrogel over time at 37 ℃ after mixing. e,f) Transmittance of PC- HACol hydrogel with varying (e) HA-PEG-DBCO concentration and (f) Col-N3 concentration. g) Digital image of PC-HACol hydrogel on the background plastic with letters. h) SEM images of PC-HACol lyophilized before and after gelation for 30 min. Scale bar: 100 µm. i) Enzymatic biodegradation profiles of PC-HACol hydrogel in collagenase solution (≥ 2 CDU / mL) or hyaluronidase solution (5 U / mL) or the mixture of collagenase (≥ 2 CDU / mL) and hyaluronidase (5 U / mL).

[0034] FIGS.8A-8E. In vitro EGF release from HACol hydrogels. a) Structures of PC-HACol and Non-PC-HACol hydrogels. In this structure, ‘HA’ and ‘EGF’ indicates hyaluronic acid and epidermal growth factor, respectively. b) Scheme for photo-cleavable release of EGF under mild UV irradiation. c) In vitro EGF release profiles from hydrogels in 1% BSA solution (n ≥ 3). Physical hydrogel was prepared by physically mixing the native Col, HA, and EGF. PC-HACol and Non-PC-HACol hydrogels were irradiated by UV lights four times with two different conditions. The hydrogels were irradiated at 12 and 60 h with low-strength UV light (2 mW / cm2for 10 min) and at 36 and 84 h with stronger UV light (5 mW / cm2for 10 min). d,e) The released amount of EGF with varying (d) irradiation time at 2 mW / cm2(n ≥ 3) or (e) UV strength for 10 min (n ≥ 3). The amount of EGF was measured at 12 h after irradiation. The released amount of EGF at 0 min in (d) is equal to that at 0 mW / cm2in (e). n.s.: not significant.

[0035] FIGS. 9A-9D. In vitro cell proliferation and biocompatibility using CECs. a) Cell proliferation when co-cultured with PC-HACol hydrogel under or without UV irradiation (2 mW / cm2for 5 min) (n = 4). The cell viability was measured by CCK-8 assay. b) Scratch assay for 48 h (n = 4). CECs were treated with PC-HACol hydrogel, and one group was subjected to UV irradiation (5 mW / cm2for 10 min), while the other group received no irradiation. The bar charts indicate the normalized wounded area in square micrometers relative to the area at 0 h. Scale bar: 400 µm. c) The biocompatibility of PC-HACol hydrogel against CECs. CECs were seeded and cultured on the PC-HACol hydrogel-coated cell culture dishes for 48 h. After incubation, live cells were stained with Calcein-AM (blue color) and dead cells were stainedATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 with ethidium homodimer-1 (red color). Scale bar: 100 µm. d) Bar charts depicting the populations of live and dead cells from (c) (n = 6).

[0036] FIGS. 10A-10B. Ex vivo re-epithelialization in rabbit eyes (n = 3). a) Representative bright field (defected area) and fluorescein-stained images (Day 0 – Day 6) of corneal defects. 2 PC-HACol hydrogel was applied to the wound and irradiated by UV light (2 mW / cm for 5 min). UV light was applied either once on Day 0 or three times on Day 0, 1, and 2, using the same irradiation conditions. PC-HACol hydrogel without UV irradiation group was used as a control group. b) Fluorescein-stained areas were measured for each group.

[0037] FIGS.11A-11F. In vivo corneal wound healing in rats (n = 4). a) OCT images of PC- HACol hydrogel-treated corneas for 7 days. The images were taken on days 0, 1, 3, 5, and 7. The orange arrows indicate the applied PC-HACol hydrogels on the defects. b) Bright field images of defected eyes. c) Fluorescein-stained images of the defects. The green colors in the center of eyes indicate the stained area. d) Fluorescein-stained area calculated from (c). e) Average thickness of the cornea in defects. The thickness was normalized by dividing it by the thickness of the normal cornea. f) Thickness of regenerated epithelium in defects on day 7. Normal group indicates the thickness of the normal cornea. *p < 0.05, compared to the other groups; n.s.: not significant.

[0038] FIGS. 12A-12F. Immunohistochemical analyses of corneas harvested on day 7. α- SMA expression patterns of a) PBS group, b) PC-HACol group, and c) PC-HACol+UV group. d) ALDH3A1 expression of PC-HACol+UV group. e) F-actin expression in stromal and epithelial layers of PC-HACol+UV group. f) CK12 expression of PC-HACol+UV group in epithelial layers. Scale bar: 20 μm.

[0039] FIGS.13A-13E. Characterization of NCs. a) Scheme for the fabrication of HSA NCs via ball-milling technique. b) Concentration-dependent size change profile of NCs in PBS (n = 3). c) The representative size distribution plots at 2, 10, and 20 mg / mL. d) Zeta potential values of NCs and NCs-DBCO (n = 3). The mean value was marked at the bottom of each bar. e) PRD content of NCs (%, w / w; n = 4). The mean value was marked at the top of each bar.

[0040] FIGS.14A-14B. Compressive stress-strain curves of hydrogels. The physical hydrogel was composed of unmodified HA-NH2, collagen, and NCs. The stress was measured using Rheometer-DMA (TA instrument; New Castle, DE, USA).

[0041] FIG.15. Fourier-transform infrared (FT-IR) spectroscopy. All samples were prepared by lyophilization. a) Infrared spectrum ranging from 400 to 4000 cm-1. b) Transmittance ranging from 2000 to 2200 cm-1.

[0042] FIG.16. SEM analysis of NCColHA hydrogel with higher magnification. Scale bar: 500 nm.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0043] FIGS. 17A-17B. Interfaces between the NCColHA hydrogel-coated area and the uncoated area. The cells grew well on the hydrogel. The scale bar length is 100 µm. The cells were incubated for three days.

[0044] FIG.18. Cytotoxicity of Col-N3, HA-N3, and NCs-DBCO (n = 4). CECs (1 × 104cells) were cultured for one day, and each component solution was added into the cells. The treated cells were incubated for one day. The cell viability was determined using CCK-8.

[0045] FIG.19. Structure of PC-HACol hydrogel.

[0046] FIG.20. Mechanism of UV-induced cleavage of linker

[0047] FIG. 21. Normalized amount of released EGF. The amount of released EGF was normalized by the remaining amount of EGF in the hydrogel at the time of irradiation.

[0048] FIGS. 22A-22B. Cytotoxicity of UV irradiation against CECs. a) Optical microscopic images of the cell morphology and population after UV irradiation. Scale bar: 100 µm. b) Cell viability after UV irradiation (n = 5). The cells were incubated for one day after irradiation. The cell viability was measured by CCK-8 assay.

[0049] FIG.23. Bright field images of all tested rats.

[0050] FIG.24. Scar area of eyes on day 7 (n = 4). The scar area was calculated by ImageJ software, based on bright field images. n.s.: not significant.

[0051] FIG.25. ALDH3A1 expression of PBS group on day 7. Scale bar: 20 μm. DETAILED DESCRIPTION

[0052] The invention described below relates to injectable precursor compositions and methods for in-situ forming tissue constructs that find use in partial or total repair, regeneration, and / or reconstruction of wounded tissue in a mammalian subject or host organism. Other purposes of the instant disclosure include, but are not limited to, the use for effective transplantation of cells into the host organism to encourage recellularization of wounded tissue, the delivery of bioactive agents, biomolecules, and / or pharmaceutical agents (either singular or combinations of agents / molecules), and the use as a tissue model for the in-vitro study of cellular responses and interplay.

[0053] 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 of 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.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0054] 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 forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0055] 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.

[0056] 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. See, e.g. S.S. Wong and D.M. Jameson Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation (CRC Press, 2Supnd / Sup edition, 2011); G.T. Hermanson Bioconjugate Techniques (Academic Press, 3Suprd / Sup edition, 2013); B. Bowling Kanski's Clinical Ophthalmology: A Systematic Approach, 8e (Saunders Ltd., 8Supth / Sup edition, 2015); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition). All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.

[0057] 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.

[0058] A value which is "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.

[0059] "Statistically significant" difference means a significance is determined at a confidence interval of at least 90%, more preferably at a 95% confidence interval.

[0060] 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.

[0061] The terms "reconstructing" and "reconstruction," and the like are used herein to generally refer to rebuilding, healing and regenerating an injured matter or tissue.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0062] The term "subject" or "mammalian subject" refers to any mammalian subject for whom treatment or therapy is desired, particularly humans. "Mammal" for purposes of treatment 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.

[0063] 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.

[0064] 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.

[0065] Nanocluster and albumin nanocluster. Protein nanoclusters are aggregates of protein molecules that exhibit unique properties due to their nanoscale size and collective behavior. The parameters of a protein nanocluster can be described in terms of various physical, chemical, and biological characteristics. An albumin nanocluster is a type of nanocluster composed primarily of albumin, a protein commonly found in blood plasma. Albumin nanoclusters leverage the biocompatibility, non-toxicity, and functional versatility of albumin for various biomedical applications. These nanoclusters typically range in size from a few nanometers to a few hundred nanometers and can encapsulate drugs, imaging agents, or other therapeutic molecules.

[0066] Albumin is naturally abundant in the human body, making albumin nanoclusters highly biocompatible and less likely to cause immune reactions or toxicity. Albumin nanoclusters can provide a stable environment for encapsulated drugs or agents, protecting them from degradation and enhancing their shelf life. The surface of albumin nanoclusters can be easily modified with reactants, ligands, antibodies, or other moieties. Examples include Abraxane, a nanocluster of paclitaxel bound to albumin nanoparticles.

[0067] Photocleavable linkers. Physiologically acceptable photocleavable linkers are designed to operate under conditions that are compatible with living systems, minimizing toxicity and avoiding damage to biological tissues. Linkers that release upon irradiation at 365 nm (near-UV light) are particularly useful because 365 nm light is less damaging compared toATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 shorter UV wavelengths. Examples include o-nitrobenzyl derivatives, where the core structure is based on the o-nitrobenzyl group, which can be modified to attach different functional groups. Upon exposure to 365 nm light, the o-nitrobenzyl group undergoes a photochemical reaction, leading to the cleavage of the bond and the release of the attached molecule.

[0068] O-nitrobenzyl derivatives are photoactive compounds that cleave under UV irradiation, typically with a wavelength around 365 nm, but can also be tuned to longer wavelengths. Examples include o-Nitrobenzyl alcohol, cleaved at a wavelength of ~300–365 nm, o- Nitrobenzyl acetate (300–365 nm); o-Nitrobenzyl carbonate (300–365 nm); o-Nitrobenzyl phosphate (300–365 nm); o-Nitrobenzyl carbamate (300–365 nm); o-Nitroveratryloxycarbonyl (NVOC) (350–365 nm); 6-Nitroveratryloxycarbonyl (NVOC) (360 nm); MNVOC (Methylnitroveratryloxycarbonyl) (350–370 nm).

[0069] Alternatives include 4,5-Dimethoxy-2-nitrobenzyl (DMNB), which is a modified version of the o-nitrobenzyl group with methoxy groups that improve solubility and reduce toxicity; 2- nitrobenzyl alcohols and ethers; 4-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc); p- Hydroxyphenacyl (pHP); Nitrodibenzofuran (NDBF); etc. DEACM (7-diethylaminocoumarin) is cleaved at 400–450 nm.

[0070] These linkers allow precise control over the release of the attached molecule using light, providing spatial and temporal resolution. Operating at 365 nm reduces the risk of damaging biological tissues compared to shorter UV wavelengths. In other embodiments, shorter or longer wavelength are used, e.g. from about 300-450 nm, and may be around 300, 325, 350, 375, 400, 425, 450 nm., or ranges within these values.

[0071] In some embodiments a hydrogel comprises two or more different linkers, where the linkers may cleave at different wavelengths, using, for example, any of the wavelengths disclosed above. In an embodiment, a hydrogel comprises two or more different therapeutic agents. The two or more therapeutic agents may be linked to the hydrogel with linkers cleaved at different wavelengths, where, for example, release may be sequential, staggered, simulataneous as required. In other embodiments, a single therapeutic agent is linked to the hydrogel with linkers cleaved at different wavelengths, where, for example, release may be sequential, staggered, simulataneous as required.

[0072] The photocleavable linker may be joined to the hydrogel through a homo- or heterobifunctional linker having a group at one end capable of forming a stable linkage to the hydrophilic head group, and a group at the opposite end capable of forming a stable linkage to the targeting moiety. Illustrative entities include: azidobenzoyl hydrazide, N-[4-(p- azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-γ-maleimidobutyryloxysuccinimide ester, N- hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'- dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 MAL; succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP); N, N'-(1,3-phenylene) bismaleimide; N, N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1- carboxylic acid N-hydroxysuccinimide ester (SMCC); m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), and succinimide 4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS. The succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue.

[0073] Other reagents useful for this purpose include: p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4-disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl- p-diisocyanate (which reacts principally with amino groups); disdiazobenzidine (which reacts primarily with tyrosine and histidine); O-benzotriazolyloxy tetramethuluronium hexafluorophosphate (HATU), dicyclohexyl carbodiimde, bromo-tris (pyrrolidino) phosphonium bromide (PyBroP); N,N-dimethylamino pyridine (DMAP); 4-pyrrolidino pyridine; N-hydroxy benzotriazole; and the like. Homobifunctional cross-linking reagents include bismaleimidohexane ("BMH").

[0074] Exemplary therapeutic factors that can be used include growth factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF); hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), insulin-like growth factors (IGF), nerve growth factor (NGF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), and their derivatives, as well as other biomolecules with pro-regenerative effects such as thymosin beta 4, neuropeptides, such as substance P (SP) and calcitonin gene-related peptide; extracellular matrix proteins, such as fibronectin, collagen, laminin, and fibrin; axon guidance proteins, such as netrins (e.g., netrin-1), ephrins, and cell adhesion molecules; and other biomolecules that play various roles in tissue regeneration, such as beta-thymosins (e.g., thymosin beta-4). Other types of molecules or biomolecules may also be used, such as anti- vascular endothelial growth factor (anti-VEGF) therapeutic agents to prevent vascularization, leakage, or growth. Anti-VEGF therapeutic agents (e.g., bevacizumab, ranibizumab, aflibercept, or similar agents) may be useful, for example, in the treatment of certain cancers or proliferative conditions, including wet macular degeneration or diabetic retinopathy. Additionally, therapeutic factors may include antibiotic agents, for example, anti-viral, anti- fungal, and anti-protozoal agents, antifibrotic agents, anti-inflammatory agents (steroids andATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 non-steroidal agents), chemotherapeutic (anti-oncologic) agents, anti-angiogenic agents, or anti-thrombotic agents, and pro-thrombotic agents.

[0075] In one embodiment, a therapeutic factor such as an anti-VEGF agent is photocleavable incorporated into a material that is injected into the eye such as into the vitreous cavity, suprachoroidal space, subretinal space, subconjunctival or subtenon’s space, anterior chamber, or lens capsule. When a patient is asleep, no therapeutic agent is released, but when they open their eyes and are exposed to light (or certain wavelengths of light), said factor is released. This allows for daily dosing of an agent. The use of sunglasses or goggles that block the activating wavelength(s) of light can further modulate the release pattern as desired by the patient and / or prescribed by their physician. In another embodiment, a contact lens is loaded with the photocleavable agent, which is released upon exposure to the certain wavelength(s) of light either by eye-opening or by removal of a light-blocking element like goggles or sunglasses. In other embodiments, it is injected into the subsurface of the skin and is activated by longer wavelengths able to penetrate deeper into the skin. Finally, in other embodiments, the material with photocleavable therapeutic agent is injected or positioned deep into an anatomic location (such as the orbit, or retro-orbital space, within the skull, abdominal cavity, bone, thorax, etc… away from natural or ambient light, but alongside it is a remotely activatable light source such as a small LED that can be triggered to light up by an external wireless or wired controller. Turning the light source on within the body would lead to on-demand release of the therapeutic agent where light otherwise does not exist.

[0076] The dose of factor released from the hydrogel may be at least about 0.01 µg, at least about 0.05 µg; at least about 0.1 µg, at least about 0.5 µg, at least about 1 µg, at least about 2.5 µg, at least about 5 µg; at least about 10 µg; at least about 25 µg; at least about 50 µg; at least about 100 µg; at least about 250 µg; at least about 500 µg; at least about 750 µg; at least about 1 mg; at least about 10 mg; at least about 100 mg or more. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight, selection, and activity of the active agent. The dosage may also be varied for localized administration.

[0077] 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. The flowable nature of the material allows it to fill defects in tissues such as corneal stroma, and regardless of the irregularity of the underlying stroma, creates a smooth outer contourATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 which depending on the viscosity and volume of the fluid applied, can match the surface contour of the native / normal cornea after crosslinking. This contour can be modulated by the placement of a hard or soft (or hybrid hard / soft) contact lens, where the posterior surface of the contact lens prescribes a curvature to the underlying gel within the corneal defect.

[0078] 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, collagen- like peptide, collagen-mimetic peptides, peptide sequences, gelatins, elastins, elastin-like peptides, 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.

[0079] Naturally occurring polymers include, but are not limited to, proteins and carbohydrates. The term “bio-polymer” also includes derivatised forms of the naturally occurring polymers or peptides that have been modified to facilitate cross-linking to a synthetic polymer of the invention. Additionally, the term “bio-polymer,” as used herein, includes proteins produced using recombinant methodologies, such as, for example, recombinant collagen.

[0080] Combinations of biopolymers can be used. Combinations may be combined in different ratios, e.g. where two biopolymers are used, a ratio may be 1:50; 1:10, 1:5, 1:3, 1:2, 1:1; 2:1; 3:1; 5:1; 10:1; 50:1; etc. For example, collagen can be crosslinked in the presence of uncrosslinked hyaluronic acid and / or chondroitin sulfate to form a polymer network of collagen and hyaluronic acid (and / or chondroitin sulfate). In another example, HA or chondroitin sulfate can be crosslinked in the presence of uncrosslinked collagen to form a polymer network of collagen and hyaluronic acid (and / or chondroitin sulfate). In another embodiment, hyaluronic acid can be crosslinked in the presence of collagen to form a polymer network. In another embodiment, collagen and hyaluronic acid can be crosslinked to each other to form a copolymeric network. In another embodiment, collagen and PEG can be crosslinked to each other to form a copolymeric network. In still another embodiment, collagen can be crosslinked in an independent process from the crosslinking of hyaluronic acid (either simultaneously or in sequence).

[0081] In preparing hydrogels in accordance with the present invention, the ratio of polymers containing reactive groups of one nature and nanoclusters containing reactive groups of a different nature to each other can range from about 0.1 to about 3.0, from about 0.7 to about 3.0, from about 1.0 to about 2.0, 1:1; from about 0.1 to about 10, or from about 0.5 to about 5.0.

[0082] When used as tissue constructs in tissue engineering for replacing or restoring tissue and organ function, as contemplated herein, hydrogels of the present invention may containATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 mammalian cells, such as stem cells such as corneal stromal stem cells, or somatic cells such as keratocytes and keratinocytes, in order to repair tissue or to promote tissue repair, reconstruction and regeneration. The hydrogels of the present invention can be prepared with enhanced mechanical as well as structural properties and resistance to degradation, can be made visually transparent and because of their cytocompatibility support cell overgrowth, in- growth and encapsulation of cells.

[0083] A hydrogel in accordance with the present invention comprises an assembly of polymers and is suitable for use in a variety of applications, including, but not limited to, clinical, therapeutic, prophylactic, or cosmetic applications. The hydrogel material can be used to replace, restore, and / or augment tissue and / or organ function in a mammalian subject in need thereof. Various biomedical, biotechnological, and / or pharmaceutical applications include, for example, corneal substitutes, therapeutic lenses, cell and / or drug delivery carriers, and tissue engineering scaffolds. Besides benefitting therapeutically in the treatment of a disease, disorder or traumatic injury of an eye and, and enhancing corneal regeneration and reconstruction, hydrogels in accordance with the present invention can be used in ophthalmic devices to enhance optical power or comfort.

[0084] Hydrogels that form in situ are adaptable to complicated defect sites when compared to structurally preformed hydrogels. With structures that form in situ and which are contemplated herein, two or more solutions containing the macromeric, precursor compositions of the hydrogel are injected or otherwise delivered to the site where the hydrogel is to be used and crosslinking is initiated. The precursor compositions can be manipulated and formed when the crosslinked solution is over a strain threshold. In most cases, the hydrogel does not require a catalyst to crosslink, thus avoiding biocompatibility problems. The precursor materials are substantially bioorthogonal and will crosslink in the presence of gelatins, collegens, lipids, carbohydrates or polymer nanofibers. Because of their crosslinking reaction kinetics, the hydrogels of the present invention can encapsulate and transport highly sensitive cells and other biological additives. Moreover, many of the hydrogels of the present invention have no known toxic byproducts.

[0085] Polymeric hydrogels can be defined as two- or multicomponent systems consisting of a three-dimensional network of polymer chains, and water that fills the space between macromolecules. A hydrogel is a network of polymer chains that are water-soluble, sometimes found as a colloidal gel where water is the dispersion medium. Hydrogels are superabsorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels possess also a degree of flexibility that is very similar to natural tissue, due to their considerable water content.

[0086] As used herein, the term “reactive group” means a molecule or molecular moiety within one composition that specifically reacts with another reactive moiety in another compositionATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 under physiological conditions and, when brought into sufficient proximity under appropriate conditions, is able to link the two molecules or moieties by a chemical bond, e.g., a covalent bond. Reactant groups of interest are usually inolved in bioorthogonal chemistry, i.e. chemical reactions that can occur inside of living systems without interfering with native biochemical processes. The reaction must be selective between endogenous functional groups to avoid side reactions with biological compounds, and have to be non-toxic and must function in biological conditions taking into account pH, aqueous environments, and temperature.

[0087] In general, the methods of the invention utilize compositions of modified biopolymers that react with a modified nanocluster or a photocleavable linker to form stable hydrogel structures. Biopolymers and nanoclusters for these purposes have been modified by the addition of reactive groups.

[0088] In one embodiment, reactive groups are copper-free click chemistry reactants, which form covalent bonds upon mixing. See Click Chemistry: Diverse Chemical Function from a Few Good Reactions Hartmuth C. Kolb, M. G. Finn, K. Barry Sharpless Angewandte Chemie International Edition Volume 40, 2001, P.2004, herein specifically incorporated by reference). Copper-free click chemistry is an alternative approach to traditional click chemistry that proceeds at a lower activation barrier and is free of cytotoxic transition metal catalysts. The absence of exogenous metal catalysts makes these reactions suitable for the in vivo applications. Strain–promoted alkyne–azide cycloaddition reaction (SPAAC) is a form of copper-free click chemistry that involves the reaction between an strained alkyne and an azide.

[0089] Examples of suitable Click chemistry reactions include, without limitation, Azide– Alkyne Cycloaddition (CuAAC): reaction between Azide and Terminal alkyne; Thiol–Ene / Thiol–Yne Reaction: reaction between thiol and alkene / alkyne; Thiol-Epoxy reaction: reaction between Thiol and epoxide; Amine-Epoxy reaction: reaction between primary or secondary amine and epoxide; Michael Addition: reaction between thiol and maleimide / acrylate; Diels– Alder Reaction: reaction between Diene + Dienophile; Oxime / Hydrazone / Schiff-base formation: reaction between Aldehyde / ketone and Amine / hydrazine; Tetrazine–Trans- cyclooctene (TCO) Ligation: reaction between Tetrazine and TCO; Isonitrile–Tetrazine Ligation: reaction between Isonitrile and Tetrazine; SuFEx (Sulfur Fluoride Exchange): reaction between Sulfonyl fluoride and nucleophile; Oxanorbornadiene (OND) Cycloaddition: reaction between Oxanorbornadiene and thiol or amine; Staudinger Ligation: reaction between Azide + triarylphosphine; andInverse Electron Demand Diels–Alder (IEDDA): reaction between Tetrazine + strained alkene.

[0090] In some embodiments the biopolymers are linked to form the hydrogel structure through covalent bonds (cross-links), including without limitation through bio-orthogonalATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 chemistries, such as chemistries based on strain-promoted azide-alkyne cycloaddition (SPAAC) and chemistries based on inverse electron demand Diels-Alder (IED-DA) reaction, as well as other “click”-type reactions such as thiol-ene reactions and hydrazone ligation. For such covalent chemistries the flowable biomaterial composition may be provided as two solutions that react and cross-link at the site of application under ambient conditions on a tissue surface without the need for an external stimulus such as light, for example see WO 2020 / 006255, and Madl et al., Adv. Funct. Mater.2018, 28, 1706046, each herein specifically incorporated by reference.

[0091] Other click chemistry reactions of interest include, for example, the use of copper- catalyzed azide–alkyne cycloaddition reaction (CuAAC) in applications where the toxicity of copper is not important. Alternatively the inverse–demand Diels Alder ligation pair trans– cyclooctene–tetrazine (TCO–Tz) may be used. The chemoselective TCO–Tz ligation pairs possess ultrafast kinetics (> 800 M–1s–1), selectivity, and long-term aqueous stability are advantages of TCO–Tz.

[0092] Other bio-orthogonal chemistries include, but are not limited to 1,3 dipolar cycloadditions, copper-catalyzed azide-alkyne cycloaddition reactions, Diels-Alder, inverse- electron demand Diels-Alder, Staudinger ligation, and nitrile oxide cycloaddition (see Madl / Heilshorn review in Adv Functional Materials page 4). In some cases, such as copper- catalyzed azide-alkyne cycloaddition reactions, a chelating agent may be needed to address and remove free copper ions. Other “click” type chemistries that can also be used in this invention include conjugate addition such as thiol-maleimide, thiol vinyl-sulfone, photomediated thiol-ene, hydrazone bonds, oxime ligation, and maleimide-furan Diels-Alder.

[0093] In such an embodiment, biopolymers, which may be the same or different, are modified to comprise reactive groups, where a first reactive group is an azide group and a second reactive group is a cycloalkyne group. The reacting, i.e. contacting, step results in a reaction between the azide group of the azide-modified biopolymer and the cycloalkyne group of the cycloalkyne-modified biopolymer, thereby synthetically and covalently modifying both biomolecules so that a hydrogel forms in-situ, i.e. at the site where the contacting occurs, upon the reacting step. Reactive groups of interest, include, but are not limited to, thiols, alkyne, a cyclooctyne, an azide, a phosphine, a maleimide, an alkoxy amine, an aldehyde, a thiol, a methacrylate or acrylate, and protected versions thereof, and precursors thereof.

[0094] 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.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0095] 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.

[0096] The term "transparent," as used herein, refers to at least 70%, 80, or 90% transmission of white light.

[0097] The term "DBCO," as used herein means a strained cyclooctyne molecule dibenzylcyclooctyne.

[0098] The term “BCN,” as used herein in means a strained cyclooctyne molecule, bicyclo[6.1.0]nonyne.

[0099] Polyethylene glycol chains of various lengths can be used as spacers within thewherein the first end of the polyethylene glycol chain is covalently linked on one side to a reactive group (or groups), as defined herein, including a PEG azide or a monofunctional or PEG cyclooctyne.

[0100] Using bioconjugation methodologies including bioorthogonal copper-free click chemistry methods such as the Strain Promoted Azide Alkyne Cycloaddition (SPAAC), tissue constructs may be formed in-situ, meaning at the site of application which is usually the site of injury, wound or defect, by reactions between at least polymeric "precursor" compositions and nanoclusters. Those precursor compositions are functionalized with reactive groups as described herein, which include without limitation multiple azide reactive groups in a first composition and with multiple alkyne reactive groups in a second composition. The precursor groups may be separately administered to the site of tissue injury, wound or defect. Reaction of the groups results in gelation of the combined precursor compositions to form a defined hydrogel composition at the site of injury, wound or defect. Spacers comprising polyethylene glycol (PEG) in various lengths may be used in the functionalization process. Such polymeric precursor compositions are typically flowable biomaterials.

[0101] The precursor compositions of the present invention encompass biocompatible biopolymers such as collagen that can be functionalized with reactive groups and that form hydrogels in situ upon reaction. Such in-situ gelling compositions that are functionalized with different reactive groups are applied to a site of injury, wound or damage, for example to the site of a corneal defect, and undergo a sol-gel (liquid to solid) transformation at the site of the injury, wound or defect, and so form a tissue construct upon the site of injury, wound or defect.

[0102] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, 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.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0103] Polymers contemplated for use in the instant disclosure as exemplary hydrogel- forming molecules include glycoproteins, carbohydrates, and other macromolecules, including, but not limited to, various types of collagen, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, laminin, fibronectin, extracellular matrix constituents, glycosaminoglycans, hylauronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin, polysaccharides, synthetic polyamino acids, prolamines, hydroxy methylcellulose, chitosan, combinations thereof, and other such molecules, including recombinant versions of such polymers.

[0104] Collagen, a widely used biomaterial for producing tissue scaffolds and constructs, is the major constituent of the extracellular matrix, and has been used as wound dressing, corneal shields, and engineered corneal matrix. It is well known that collagen’s molecular structure plays a crucial role in cell adhesion, migration, and differentiation.

[0105] In various embodiments of the present invention in order to demonstrate the utility of the described precursor compositions to form in-situ a tissue construct at the site of a corneal defect, bovine type-I collagen was employed as matrix due to its low immunogenicity compared to other collagen types. Collagen type I is commonly used as a cellular scaffold in three-dimensional cell culture because collagen gel matrices are more similar to the native cell environment than general two-dimensional cell culture dishes. When an acidic collagen solution is neutralized and incubated at 20 – 37 ºC, the collagen forms a gel through fibril formation. However, collagen extracted from tissue loses its original fibril density and three dimensional architecture, and as a result, neutralized non-covalently crosslinked collagen gels have low mechanical strength.

[0106] The physical properties of collagen can be modulated by crosslinking techniques that enhance mechanical strength, enzymatic degradation resistance, and transparency. For example, transparency of crosslinked biomaterials such as SPAAC-crosslinked collagen gels is an important aspect for their usefulness in corneal applications. Whereas physically collagen gels exhibit optical turbidity in proportion to the degree of randomly organized fibrillar structures in the collagen, crosslinked gels are optically clear because of the presumably reduced random organization of fibrillar structures through crosslinking.

[0107] Hyaluronic acid is another polymer of interest, which may be used alone or in combination with collagen. It is a polymer of disaccharides, themselves composed of D- glucuronic acid and N-acetyl-D-glucosamine, linked via alternating β-(1→4) and β-(1→3) glycosidic bonds. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da in vivo. The average molecular weight in human synovial fluid is 3–4 million Da, and hyaluronic acid purified from human umbilical cord is 3,140,000 Da. Hyaluronic acid is energetically stable, in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored positions, whereas the smaller hydrogens assume the less-favorable axial positions. Other macromolecules, biomolecules, or polymersATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 that can be used include but are not limited to gelatin, fibronectin, polyethylene glycol (PEG), chondroitin sulfate, other glycosaminoglycans, PLGA, PLA, polycaprolactone, dendrimers, alginate, branched polymers, and other biocompatible polymers. The same photocleavable groups can be placed on other polymer constructs such as a solid scaffolds, xerogels, aerogels, foams, electrospun fibers, polymeric microneedles, rods, pellets, emulsions, microparticle gels, or combinations thereof. Crosslinking Via Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)

[0108] In one embodiment of the invention, a bio-orthogonal approach based on bioorthogonal chemistry, that can make highly specific covalent bonds without interfering with cells and biomolecules in a living system, is used to crosslink biopolymer matrices to nanoclusters with strain-promoted azide-alkyne cycloaddition for carrying out reactions on ocular wound sites including corneal defects. Strain-promoted azide-alkyne cycloaddition (SPAAC) is a bio-orthogonal, copper-free form of click chemistry and suitable to chemically crosslink polymer or biopolymers such as collagen around cultured cells, including corneal stromal stem cells or keratocytes (in-situ encapsulation). SPAAC can be used to form covalent bonds between biomolecules in the presence of living cells.

[0109] To facilitate strain-promoted azide-alkyne cycloaddition (SPAAC) mediated crosslinking in embodiments of the present invention, biopolymer(s) and nanocluster are functionalized with either azide or dibenzocyclooctyne (DBCO) reactive groups using N- hydroxysuccinimide (NHS) coupling chemistry. A poly(ethylene glycol) (PEG) spacer may be introduced in the azide group conjugation to allow for enhanced conjugation efficiency.

[0110] SPAAC can be performed under ambient conditions in aqueous solution without the need for solvents or catalysts, produces no side reactions or free radicals or side products, and does not react with surrounding cells, proteins, or tissue. Because of these numerous advantages, SPAAC was used in the various embodiments of the present invention to crosslink collagen and encapsulate cells. METHODS

[0111] In some aspects of the invention, methods are provided for treating an injury, wound or defect that requires tissue regeneration, tissue replacement or repair, regeneration, and / or reconstruction of ocular, skin, subcutaneous tissue, nerve, muscle, bone, cartilage, vitreous, tendon, ligament, fat, retinal, conjunctival, scleral, cardiac, adrenal, and other types of tissue. In these methods, flowable biomaterials are applied to a site of injury, wound or defect where, upon crosslinking of reactive groups, a defined hydrogel structure tissue construct is formed in-situ on top of the injury, wound or defect, which serves to regenerate, reconstruct and repair the tissue injury, wound, or defect. For instance, in one embodiment of the invention, the gelATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 is applied to the peripheral part of the ocular surface in an area devoid of conjunctival tissue, and acts as a scaffold for overgrowth by surrounding conjunctiva. In other embodiments, the gel is applied in conjunction with admixed conjunctival cells to repopulate an area that is devoid of conjunctival cells. UTILITY

[0112] The injectable flowable biomaterial compositions and methods of the present invention can be applied to any clinical situation where tissue engineering, regeneration or reconstruction in a mammalian host or subject is necessary. Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The key objective in tissue engineering is the regeneration of a defective tissue through the use of materials that can integrate into the existing tissue so as to restore normal tissue function. Such injectable compositions can comprise cells that settle in the host and encourage recellularization of the wounded tissue. Furthermore, such injectable compositions can also serve as a three-dimensional tissue model for the in-vitro study of cellular responses and interplay. Application As In-Situ Forming Hydrogel Upon Ocular Defects

[0113] To address an unfilled need for effective compositions and methodologies to treat and regenerate ocular defects, including corneal defects, precursor compositions are described in various examples herein that upon crosslinking form in-situ corneal constructs on top of corneal defects. Such 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.

[0114] 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.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 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.

[0115] Such compositions may comprise functionalized biopolymers such as collagen (type I) and HA that are crosslinked in situ to a nanocluster, and where the crosslinking transforms the injectable precursor compositions into a substantially transparent hydrogel that serves as a corneal stromal scaffold, substitute or construct on top of a corneal or stromal defect, wound or wounded area to enhance the regenerative capacity of the cornea to restore viable corneal tissue.

[0116] Such polymer-based precursor compositions, when functionalized with azide-alkyne crosslinking agents and which can optionally contain a therapeutic factors, such as growth factors, a suspension of corneal keratocytes to aid in the reepithelization of the wounded corneal area, etc. are consecutively applied as flowable precursor compositions to a wounded corneal area, and then gelated on the spot (in situ) by SPAAC crosslinking to produce an in situ-forming corneal stromal scaffold which is kept in place on top of the wound site. The in situ-formed scaffold mimics the thickness and smooth, continuous surface of the cornea.

[0117] The in situ forming gels when applied to a wound site can further contain and encapsulate other elements, including but not limited to cells (to act as a vehicle for cell transplantation), the secretions of cells, biomolecules, growth factors, drugs, fibers (such as electrospun fibers) that provide additional mechanical reinforcement. Furthermore, the gels can be formed outside of the body and then processed further (e.g. via vitrification), and then placed on or within a wound and secured either by the application of additional in situ forming gel, another adhesive material (such as tissue glue or sealant), sutures, or some combination of these. In-situ molding

[0118] In embodiments of the present invention, the crosslinked gel can be applied with or without cells, and with or without an overlying contact lens (hard or soft lens) which can be used as an in situ mold to create the desired contour and curvature of the crosslinked gel on the ocular surface. This in situ molding process may be important for bestowing the desired refractive power to the surface of the cornea, since the air-cornea interface is responsible for most of the refractive power of the eye. By providing a smooth, transparent, and properly curved surface to the central cornea, the gel can restore vision to patients whose vision was severely compromised by a central defect or ulcer. Furthermore, the gel can be applied to any part of the cornea (central, paracentral, or peripheral cornea), and can be used to encapsulate stromal cells, epithelial cells, limbal cells, or combinations thereof. In other embodiments, the eyelids can be sutured shut (i.e. tarsorraphy can be placed) completely or partially to create aATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 protective environment for the eye after the gel is placed. This can be done with or without a contact lens in place over the cornea and applied gel. KITS

[0119] The present invention also provides kits comprising separate containers holding compositions comprising polymers, such as collagen, hyaluronic acid, etc., that are functionalized with azide groups, and polymers that are functionalized with alkyne groups, and optionally with spacer arm(s) bridging the azide or alkyne groups to the polymer, and optionally admixed with living cells or biomolecules (e.g. proteins) or pharmaceutical agents or combinations thereofto be delivered to the wounded tissue site.

[0120] 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.

[0121] 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. ADMINISTRATION

[0122] The precursor flowable biomaterial compositions of the present invention can be administered in the form of pharmaceutical compositions, comprising an isotonic excipient prepared under sufficiently sterile conditions for administration to a mammalian subject, particularly to a human being. In certain embodiments, multiple cycles of treatment may be administered by repeatedly applying the precursor compositions to the site of injury, wound or defect for a time period sufficient to effect at least a partial healing of the injury, wound or defect, or, preferably, for a time period sufficient to effect a complete healing of the injury, wound or defect. EXAMPLES

[0123] The present invention is based on the discovery that bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) crosslinking is useful in producing in situ-forming (collagen) corneal stromal substitutes and constructs, such as crosslinked collagen gels, that may find application in-vivo in treating and reconstructing a surgically incised or wounded cornea in a mammalian subject, and in-vitro in studying keratocyte-keratinocyte interactions.

[0124] 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. TheATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 examples are offered for illustrative purposes only, and are not intended to limit the scope of what the inventors regard as their invention.

[0125] 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. Example 1 In situ-forming nanocluster-crosslinked bio-orthogonal hydrogel for the regeneration of corneal defects

[0126] Corneal defects can lead to scarring and vision loss. In this study, an in situ-forming nanocluster (NC)-crosslinked bio-orthogonal hydrogel (NCColHA hydrogel) was developed for the regeneration of corneal stromal defects. In the hydrogel networks, NCs served both as crosslinkers as well as delivery vehicles for prednisolone and hepatocyte growth factor. NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties. They also exhibited excellent transparency and negligible swelling / de- swelling behavior. The hydrogel’s biocompatibility and capacity to support cell growth were assessed using cultured primary corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK14, α-SMA, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into transparent stroma.

[0127] Here, we engineered a bio-orthogonal hydrogel, named the NCColHA hydrogel, crosslinked with nanoclusters (NCs), with the aim of facilitating the regeneration of injured corneal tissue (Fig.1a). Human serum albumin was chosen as the nanocluster material due to the excellent water solubility, transparency, and drug loading capacity. The albumin NCs were fabricated using the ball-milling-assisted technique and their surface was decorated with dibenzocyclooctyne (DBCO) through EDC / NHS coupling. In addition to crosslinking, we assessed the functions of NCs as a delivery platform by employing prednisolone (PRD) and hepatocyte growth factor (HGF) as a model drug and a model growth factor, respectively. For hydrogel backbones, azide-modified hyaluronic acid (HA-N3) and collagen (Col-N3) were synthesized. Each component (i.e., NCs-DBCO, HA-N3, and Col-N3) exists as solution before being mixed, while they become rigid and transparent hydrogel within a couple of minutes. InATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 this study, we assessed the physicochemical properties, transparency, and swelling / de- swelling properties of NCColHA hydrogel. The biocompatibility and regeneration of corneal layers were evaluated in vitro and in vivo. Design and characterization

[0128] Albumin NCs were employed as a crosslinker for bio-orthogonal hydrogel because of the material’s transparency, biocompatibility, negative surface charge, and hydrophilicity, which were sufficient to maintain their solubility upon decoration with hydrophobic DBCOs. Moreover, previous research demonstrated that the NCs can encapsulate hydrophobic drugs within their structures and release the drugs slowly. We leveraged these properties to design a dual-release system for drugs and growth factors. In this research, PRD and HGF were employed as model hydrophobic drug and growth factor, respectively. PRD-embedded NCs were fabricated by the modified ball-milling technology (BMT). The surface of the NCs was decorated with DBCO via primary amine / N-Hydroxysuccinimide (NHS) reaction. When the NCs-DBCO were synthesized at a DBCO to HSA ratio of 30, the degree of substitution (DoS) was calculated as 35.5 ± 0.8 %. After quenching the residual NHS esters, the NCs were coated with HGF through charge-to-charge interactions. The fabricated NCs exhibited a flexible aggregation / dissociation pattern and increased an apparent water solubility of PRD (FIG.13). As counter parts to DBCO, azide-conjugated hyaluronic acid (HA-N3) and collagen (Col-N3) were synthesized via primary amine / NHS reaction. The degree of substitution (DoS) values for HA-N3 and Col-N3 were calculated as 57.8 ± 0.9 % and 50.9 ± 6.4 %, respectively. Hydrogels were fabricated by physically mixing the equal volumes of each component.

[0129] Hydrogels require robust mechanical strength to maintain their structure during wound healing processes. To optimize the fabrication conditions, we prepared various hydrogels by altering three different factors: the concentrations of HA-N3and Col-N3, and the molar ratio of DBCO to HSA. The hydrogels exhibited distinct rheological properties based on the conditions (Fig.1b). The storage modulus values of the hydrogels increased with the rising molar ratio of DBCO to HSA until reaching 30. As the rheological properties of the hydrogels appeared to be saturated at 40, we determined the DBCO to HSA ratio to be 30. The quantity of HA-N3 and Col-N3also influenced the mechanical properties. Storage modulus values increased as the amount of the polymers increased, but these values reached saturation at the highest HA- N3 amount. Therefore, we determined the concentrations of HA-N3 and Col-N3 as 30 mg / mL and 9 mg / mL, respectively. Finally, we prepared the optimized hydrogel (NCColHA hydrogel) by equally mixing NCs-DBCO (40 mg / mL; DBCO to HSA = 30), HA-N3 (30 mg / mL), and Col- N3 (9 mg / mL) in a 1:1:1 ratio (v / v / v). The fabricated hydrogel exhibited a G’ value of over 20,000 Pa, which was almost ten times higher than our previously developed bio-orthogonal hydrogels.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0130] The gelation of the mixture of each component solution was assessed by measuring the storage modulus and tan δ values over time (Fig.1c). Initially, the storage modulus value was less than 20, and its tan δ value was 0.86, indicating a sticky solution state and partial gelation. Interestingly, the modulus values increased significantly over time, and the tan δ value dropped to less than 0.1 within 5 min, suggesting that the mixture underwent gelation. Thus, the gelation of the mixture occurs in a time-dependent manner. The formation of chemical bonds between the conjugated azides and DBCOs were evaluated by analyzing the infrared spectra (FIG. 13). The azide-conjugated HA and Col exhibited an absorbance at approximately 2100 cm-1(N=N=N), and DBCO-conjugated NCs also displayed an absorbance in a similar range (2050 ~ 2150; C≡C). In contrast, NCColHA hydrogel displayed negligible absorbance at these ranges, which could be explained by depletion of azides and DBCO resulting from the click reaction.

[0131] The co-localization of NCs and polymers were observed using scanning electron microscope (SEM) (Fig. 1d). NCColHA hydrogel was lyophilized, and the surface of the lyophilized powder was observed to investigate the localization of NCs. Notably, the lyophilized NCColHA displayed a bundle of distinct aggregates polarized alongside the polymer branches, while these structures were not observed in a mixture of HA-N3 and Col- N3. These aggregate-bound branches were believed to result from NCs chemically bound to polymer chains, and the polar localization of NCs alongside the chains may imply the chemical reactions between the components.

[0132] Biodegradability of NCColHA hydrogel was assessed in a simulated body fluid solution containing 5 units / mL of hyaluronidase and ≥ 5 CDU / mL of collagenase (Fig. 1e). The NCColHA hydrogel maintained more than 90 % of its weight for 24 h, and 50 % for 48 h. These values were significantly greater than those of the physically mixed hydrogel (i.e., a mixture of unmodified Col, HA, and NCs). Furthermore, its structural integrity decreased when the DBCO / HSA ratio decreased to 20. These results suggest that the degree of crosslinking by NC-induced click reaction had a negative correlation with biodegradability.

[0133] The release kinetics of PRD and HGF in physiological conditions was observed for 72 h (Fig.1f). In our design, the PRD was embedded into NCs through hydrophobic interactions, and HGF was coated on the surface of NCs through charge-to-charge interactions. As predicted, both compounds were released continuously and slowly from the hydrogel, exhibiting sustained release patterns. These patterns can be attributed to the non-covalent molecular interactions including hydrophobic interactions and hydrogen bonding, as well as steric hinderances by the three-dimensional network of hydrogel. TransparencyATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0134] Hydrogels for corneal administration must be transparent to avoid interfering with clear visions. Hence, we assessed the transparency of the hydrogels by measuring the transmittance in visible light. The fabricated NCColHA hydrogel appeared highly transparent, allowing for a clear view of the background image (Fig. 2a). Transmittance values of the hydrogels with various conditions were measured in the visible light spectrum spanning 400 to 800 nm (Fig. 2b). The optimized NCColHA hydrogel exhibited transmittance values consistently exceeding 80 % across the entire wavelength range, indicating excellent transparency. Of note, transmittance slightly decreased with an increase in DBCO-to-HSA ratio as well as with higher concentrations of HA-N3 and Col-N3. Swelling / De-swelling behaviors

[0135] Swelling and de-swelling are common phenomena observed in hydrogels, but these characteristics are undesirable for hydrogels used in corneal defects. Ideally, the hydrogels should conform to the corneal defects and maintain their structural integrity and as-formed dimensions. To evaluate the swelling / de-swelling behaviors of NCColHA hydrogel, we first measured the weight change of the hydrogel after incubation in PBS (Fig.2c). Interestingly, NCColHA hydrogel exhibited minimal shrinking, with only a 7 % reduction in weight after 48 h, while the physically mixed hydrogel (i.e., a mixture of the unmodified components; HA, Col, and NCs) experienced a significant weight loss of 44.6 % over the same period. This suggests that the crosslinking by NCs improved its de-swelling behavior. Fig. 2d displays the appearance of NCColHA hydrogel before and after a 48-h incubation in PBS. The size of the hydrogel appeared unchanged after incubation, consistent with the results of the swelling ratio. To further confirm these swelling / de-swelling properties, the water content of NCColHA hydrogel was analyzed by Thermogravimetric analysis (TGA) (Fig.2e). The water content of NCColHA hydrogel was measured as 96.8 ± 0.2 %, and this value did not significantly differ after post-incubation for 72 h (97.0 ± 0.3 %), indicating negligible swelling and de-swelling properties of NCColHA hydrogel. Biocompatibility

[0136] The biocompatibility of NCColHA hydrogel was assessed by culturing the corneal epithelial cells (CECs) on the hydrogel. The NCColHA hydrogel was labeled with 6-FAM to visualize the localization of the hydrogel and cells. The 6-FAM-labeled NCColHA hydrogel was spread on cell culture dishes, and then the cells were cultured on the hydrogel (Fig.3a). After two days of incubation, we observed that the cells had proliferated well on the hydrogel, as demonstrated by the overlay of 6-FAM and DAPI images. Additionally, we evaluated the biocompatibility by observing the interface between the hydrogel-coated area and the uncoated area using optical microscopy (FIG.14). These results demonstrate that the CECsATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 can proliferate on the hydrogel, indicating excellent biocompatibility. Furthermore, we found that each component of hydrogel (i.e., Col-N3, HA-N3, and NCs-DBCO) exhibited excellent biocompatibility with CECs (FIG.15). In vitro cell proliferation

[0137] Cell proliferation following treatment with NCColHA hydrogel was evaluated using the cell counting kit-8 (CCK-8) assay (Fig.3b and 3c). NCColHA hydrogel was treated to CECs in two different ways: through pre-addition and post-addition of NCColHA hydrogels, allowing to assess the cell proliferation under different cell status. In the pre-addition study (Fig.3b), cells were initially seeded in the NCColHA hydrogel-pre-added well and incubated for two days (i.e., hydrogel application before cell adhesion). In the post-addition study (Fig.3c), hydrogels were added to one-day pre-cultured cells and incubated for two days (i.e., hydrogel application after cell adhesion). In both conditions, the cell population increased in a concentration-dependent manner. Considering each component did not affect the cell proliferation (FIG. 15), these growth patterns would be attributed to the promoted cell proliferation by HGF released from the hydrogel. Interestingly, the differences in cell viabilities between two conditions were not significant except for 1 mg / mL. These results suggest that NCColHA hydrogel promoted cell proliferation regardless of the status of cellular adhesion.

[0138] Epithelial cell proliferation on NCColHA hydrogel was evaluated by detecting live and dead cells over five days (Fig. 3d and 3e). CECs were cultured on the hydrogel-coated surface, and their populations of live and dead cells were visualized using calcein-AM (blue) and ethidium homodimer-1 (red), respectively (Fig.3d). The number of live cells significantly increased from day 1 to day 3 (5.01-fold) and from day 3 to day 5 (2.31-fold) (Fig, 3e). Meanwhile, the percentages of dead cells remained below 2 % through 5 days. Considering that the hydrogel is designed to support re-epithelialization in vivo, these results suggest the high potential of NCColHA hydrogel for corneal regeneration.

[0139] Epithelial cell migration from limbal stem cells is a crucial process for regenerating and maintaining epithelium, and HGF can facilitate the motility of epithelial cells. Hence, we assessed cell migration after incubating the cells with NCColHA hydrogels (Fig. 3f). CECs were incubated until they reached a confluency of over 80 %, and then a linear scratch was made in the middle of the cells to mimic a corneal epithelial wound. The initial scratched area was approximately 25 % in both groups, and within 24 h, it decreased to 12.5 ± 2.0 % in the untreated group. In contrast, the cells treated with NCColHA hydrogel displayed a faster closure of the scratched area within 24 h (0.6 ± 0.1 %), indicating enhanced cell proliferation and migration.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 Re-epithelialization of ex vivo rabbit cornea

[0140] Re-epithelialization in corneal defects is a pivotal process for wound closure to prevent bacterial infection, perforation, and scarring. Our hypothesis is that the NCColHA hydrogel can suturelessly fill corneal defects and that epithelial cells would proliferate on the outer surface of the hydrogel. To observe the re-epithelialization process, we used an ex vivo anterior lamellar keratoplasty (ALK) model using rabbit eyes. Fig. 4a shows keratectomy wounds treated with the NCColHA hydrogel. As shown in control group (PBS-treated group; day 5), the cornea exhibited a distinct groove caused by defects, with re-epithelialization occurring alongside the groove. In contrast, in the cornea treated with the NCColHA, the hydrogels are seen filling the defects (yellow color; tagged with 6-FAM) with epithelialization (indicated by orange arrows) observed on their surface.

[0141] The structural integrity of the regenerated epithelial layer was assessed by observing the expression patterns of epithelial biomarkers (Fig.4b). The epithelium typically consists of five to seven cell layers, with the expression of CD44 and CK14 being polarized in a basal layer. CD44, a cell surface adhesion receptor, plays a role in cell-to-cell interactions crucial for corneal re-epithelialization, providing adhesive strength to the epithelial layers. In addition to CD44, basal epithelial cells expressed cytokeratin-14 (CK-14), indicating limbal stem cell phenotype within the re-epithelialized layers. To further assess the structural integrity of the stratified layers, we examined the formation of tight junctions (stained with ZO-1) between the cells. Actin filaments within the cells were visualized using phalloidin (green) and DAPI (blue), while ZO-1 (white) delineated the tight junctions between the cells. These epithelial biomarkers clearly indicate the structural integrity of the regenerated epithelial layers and the re- epithelialization on the NCColHA hydrogel. In vivo cornea regeneration

[0142] Corneal regeneration by NCColHA hydrogel was assessed in rabbits (Fig.5). Corneal wounds were created using a 3.5-mm trephine during anterior lamellar keratoplasty (ALK), and NCColHA hydrogel was applied to the defects (FIG.16). In this study, the PBS-treated eyes were used as the control group. When treated with PBS, the re-epithelialization occurred within one week (Fig.5a), which is verified by fluorescein staining (Fig 5b). Then, the damaged stromal layers slowly recovered over 56 days. In contrast, in the NCColHA hydrogel group, the wounds were filled with the hydrogel (indicated by a red star) upon administration. Notably, the applied hydrogels maintained their structure for 14 days, and newly regenerated epithelial layers (indicated by an orange arrow) were clearly observed on the hydrogel. These results differ significantly from our previous work on a bio-orthogonally crosslinking collagen-HA gel, where treated corneas were followed for 1 week and the gels were less clearly visible on OCT imaging but were visible upon immunohistochemical analysis. This difference in appearanceATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 on OCT at 1 week may be attributed to the increased mechanical properties of the nanocluster-crosslinked hydrogel, which improves its resistance to dehydration and / or matrix turnover. Clinical evidence of re-epithelialization was confirmed using fluorescein staining (Fig. 5b). The epithelial layers of the treated corneas were found to all be completely healed by Day 7. We also monitored the transparency of the corneas each day (Fig. 5c). The eyes of PBS group had some scars on day 14 and 28 and disappeared on day 56. In contrast, NCColHA hydrogel group exhibited negligible opacities over the evaluation period.

[0143] The presence and appearance of the gel was monitored using OCT through 56 days after surgery and treatment. The NCColHA hydrogel appeared mostly intact through Day 14 with a characteristic dark appearance throughout its bulk and a hyper-reflective surface, which we interpreted as the re-grown epithelium. By Day 28, the dark band appeared thinner while the stromal bed appeared thicker with a thicker zone of hyper-reflectivity anteriorly, appearing to now occupy the posterior interface of the hydrogel. By Day 56, a thin, well-defined dark band remained, with a smooth overlying hyper-reflective band of epithelium, and a more discrete band of hyperreflectivity posteriorly, with an overall more compact and smoother contour and uniformity across all layers.

[0144] To quantitatively analyze corneal regeneration, the thickness of the treated and untreated (saline control) corneas were calculated and compared to that of normal (uninjured, untreated) corneas (Fig. 5d and FIG. 18). The thickness of the epithelium, stroma, and full thickness cornea in normal (n = 4) was calculated as 24 ± 7 µm, 383 ± 51 µm, and 412 ± 59 µm, respectively. In the PBS-treated group, the thickness of whole cornea slowly increased for 56 days, but it did not fully recover (323 ± 15 µm; n = 4) by 56 days, with a statistically significantly thicker epithelium (76 ± 19 µm) but thinner stroma (243 ± 27 µm), and overall thickness compared to normal corneas (Fig 5d). This observation is consistent with epithelial hyperplasia, which is a common observation during the regeneration after stromal injuries. In contrast, the injured cornea exhibited significant regeneration when treated with NCColHA hydrogel, resulting in epithelial (40 ± 9 µm), stroma (355 ± 29 µm), and overall cornea thickness (402 ± 40 µm; n = 6) that were not statistically significantly different from the normal corneas at 56 days. The epithelium thickness in the NCColHA hydrogel group was also slightly increased (40 ± 9 µm) but was not statistically significantly different from that of normal corneas. NCColHA hydrogel groups exhibited a robust recovery of cornea structures, with components appearing similar to those of normal corneas (Fig.5e). Immunohistochemical analyses

[0145] The treated corneas at day 56 were further evaluated by immunohistochemical analysis (Fig.6) to visualize the hydrogel and markers of corneal cell structure and function. Fig.6a shows that NCColHA hydrogels fills keratectomy wounds, and stromal cells are seenATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 growing alongside the hydrogel. Notably, F-actin was observed in the areas between the hydrogel and the epithelium, indicating potential cell-matrix mechanical interactions at the wound site. The NCColHA hydrogel-treated corneas exhibited a multilayered epithelium with normal thickness (Fig. 6b), similar to that of normal corneas (Fig.6c). In contrast, the PBS- treated cornea exhibited a significantly thicker epithelium (Fig. 6d). In addition to the hyperplasia, we analyzed alpha-smooth muscle actin (α-SMA) expression at the wounds site of the PBS-treated corneas, which is indicative of fibrotic healing (Fig.6e and 6f). In corneal wounds, activated keratocytes can differentiate into α-SMA-positive myofibroblasts, inducing scarring. Notably, the corneas treated with the NCColHA hydrogel exhibited significantly reduced the expression of α-SMA compared to the PBS treatment, suggesting a reduction in myofibroblastic activity and fibrosis, consistent with the relatively clear clinical appearance of the corneas with slit lamp photography (Fig 5c). CD44 expression further confirms the structural integrity and interaction of the regenerated epithelium over the NCColHA hydrogel (Fig.6g).

[0146] We have developed a nanocluster-crosslinked bio-orthogonal hydrogel for the regeneration of corneal defects. By utilizing DBCO-decorated NCs that react with azide- conjugated HA and Col, we achieved rapid gelation within a few minutes at the site of corneal defects. While DBCO was chemically tethered to the albumin in the NCs, the NCs themselves were composed of both albumin and prednisolone as a result of hydrophobic interactions, while HGF coated the surface of the NCs by virtue of electrostatic interactions. The fabricated NCColHA hydrogel exhibited excellent biocompatibility, transparency, and negligible swelling / de-swelling properties, creating an ideal environment for corneal regeneration. In a rabbit corneal defect model, our study demonstrated that NCColHA hydrogel maintained its structural integrity for at least 14 days, facilitating re-epithelialization on the hydrogel. Ultimately, the hydrogel was replaced by newly remodeled stromal layers, resulting in non- fibrotic remodeling of corneal stroma and stable, multi-layered epithelization resembling those of a normal cornea at 8 weeks post injury and treatment. Based on these findings, the NCColHA hydrogel developed and described herein provides an approach for the treatment of deep stromal corneal defects.

[0147] Table 1. Degree of substitution (DoS) values of the synthesized materials. The value was measured using MicroMolar Primary Amine Assay Kit (Profoldin, MA, USA), and absorbance at 285 nm for azide and 306 nm for DBCO. Materials DoS* (%)ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 HA-N3 57.8 ± 0.9 Col-N350.9 ± 6.4HSA-DBCO 35.5 ± 0.8*DoS = (DBCO-or N3-conjugated amines / total surface primary amines)*100

[0148] Materials. Human serum albumin (HSA), L-lysine, fluorescein, prednisolone (≥ 99 %) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyaluronic acid-Amine (MW 250k; DoS of 50 %) was obtained from Creative PEGWorks (Durham, NC, USA). Collagen (TeloCol-10) was provided from Advanced BioMatrix (Carlsbad, CA, USA). DBCO-sulfo-NHS ester (99 %) and Azido-PEG5-NHS ester (98 %) were purchased from BroadPharm (San Diego, CA, USA). Recombinant human hepatocyte growth factor protein (HGF; Active form) was purchased from Abcam (Cambridge, UK).

[0149] Fabrication of NCColHA hydrogel. To fabricate the bio-orthogonally-crosslinked hydrogel, we prepared azide-conjugated HA (i.e., HA-N3), azide-conjugated collagen (i.e., Col- N3), and DBCO-decorated NCs (NCs-DBCO). For the synthesis of HA-N3, the neutralized hyaluronic acid amine solution (HA-NH2; 20 mg / mL) was prepared using 10X PBS and 1N NaOH. Meanwhile, azido-PEG5-NHS ester solution was diluted with DMSO (dilution factor: 2). The azido-PEG-NHS ester solution (20.52 µL) was mixed with the HA-NH2solution (1 mL) and vortexed overnight under light protected condition. Then, the product solution was dialyzed (MWCO = 12 – 14 kDa) against distilled water (DW) for 2 days. DW was replaced two times a day for two days. The dialyzed solution was lyophilized and stored at -20 ºC until use.

[0150] Col-N3 was prepared in the way like the HA-N3. Collagen solution (9 mg / mL) was prepared by neutralizing with 10X PBS and 1N NaOH, and azido-PEG5-NHS ester was diluted ten times with DMSO. The diluted azido-PEG5-NHS ester solution (20.7 µL) was added into the neutralized collagen solution (1 mL) and rotated gently at 4ºC overnight. The product solution was dialyzed against PBS using Slide-A-Lyzer G2 Dialysis Cassettes at 4ºC overnight.

[0151] For the fabrication of DBCO-decorated NCs, albumin-based NCs were fabricated by modifying the Ball-Milling Technology. Briefly, HSA (20 mg) and prednisolone (PRD; 4 mg) were mixed, and 9.5% DMSO solution (in PBS; 4.75 µL) and stainless-steel bead (diameter: 5 mm; PGN Bearings) were added into the powder. Then, the mixture was vortex-mixed for 10 min. The suspension was lyophilized to clearly remove the DMSO. The lyophilized powder was reconstituted using DW (0.4 mL) and centrifuged at 5,000 rpm for 1 min. After that, the supernatant was filtrated using a syringe filter (0.45 µm) to eliminate the unboundATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 prednisolone. For the decoration of DBCO on the surface of NCs (DBCO / HSA ratio = 30), the DBCO-sulfo-NHS ester solution was prepared in PBS (100 mg / mL) and added (19.2 µL) into the NC suspension (40 mg / mL). The suspension was gently rotated for 2 h under light protected conditions. After incubation, L-lysine solution (140 mg / mL in PBS; 3.76 µL) was added the suspension to inactivate the residual NHS ester and gently rotated for 30 min. Finally, HGF solution (240 µg / mL in PBS; 2.5 µL) was added into the suspension to load the HGF on the surface of NCs. NCColHA hydrogel was prepared by mixing HA-N3, Col-N3, and NCs-DBCO in equivalent proportions (1:1:1, v / v / v).

[0152] Characterization. To investigate the rheological properties of NCColHA hydrogel according to different fabrication conditions, we prepared five kinds of NCs-DBCO (DBCO / HSA ratio = 0, 10, 20, 30, and 40), four kinds of HA-N3 solution (concentration of 10, 30, 50 mg / mL), and three types of Col-N3(concentration of 3, 6, and 9 mg / mL). The storage and loss modulus of each gel were measured by rheometer (ARES-G2; TA instrument, New Castle, DE, USA) at frequencies ranging from 0.1 to 10 at the oscillation strain of 1 at 37 ºC. The change of storage modulus and tan δ values over time was measured at oscillation strain of 1 % at 37 ºC. Tan δ value was defined as loss modulus (G’’) / storage modulus (G’). The infrared spectra of NCColHA hydrogel and each component were obtained by Fourier- transform infrared spectrometer (Nicolet iS50; Thermo Fisher Scientific; Waltham, MA, USA). The hydrogel was lyophilized, and the hydrogel powder was analyzed by FT-IR spectrometer. The SEM images (Thermo Fisher Scientific Apreo S LoVac Scanning Electron Microscope) were obtained by observing the surface of lyophilized hydrogel. In this observation, a mixture of HA-N3 and Col-N3 was also prepared to compare the microstructures.

[0153] Biodegradability. Biodegradability of NCColHA hydrogel was investigated against three kinds of NCColHA hydrogel according to DBCO / HSA ratio (20, 30, and 40). In this study, the unmodified hydrogel (i.e., physical mixture of HA-NH2, Collagen, and NCs) was employed as a control. The initial hydrogels were weighed (W1) and they were submerged in the mixture of 5 units / mL of hyaluronidase (Sigma-Aldrich) and 5 ≥ CDU / mL collagenase (Sigma-Aldrich). The hydrogels were incubated at 37ºC. The weight of the hydrogels (Wn) was measured at 2, 6, 24, and 48 h. The percent weight (%) of the hydrogels at those points was calculated as Wn / W1× 100.

[0154] In vitro HGF and PRD release. The release patterns of HGF and PRD from NCColHA hydrogel were evaluated in PBS or 1% BSA solution. To calculate the amount of PRD embedded in the NCs, PRD was extracted using DMSO (dilution factor: 10) and centrifuged (14,000 rpm, 5 min). The supernatant was further diluted with DW, and the absorbance at 240 nm was analyzed with the microplate reader (TECAN, Männedorf, Switzerland). The release kinetics of PRD was investigated in PBS. NCColHA hydrogel was inserted into the dialysis membrane tubing (3.5 kDa) and incubated with a gentle shaking. The release profile of HGFATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 was assessed in 1 % BSA solution. The release HGF was calculated using human HGF ELISA kit (Abcam).

[0155] Transparency. To visualize the transparency of NCColHA hydrogel, the fabricated hydrogel was put on a plastic card with lettering in the background, and photos were taken by focusing on the background letters. In addition to the visual images, transparency was quantified by measuring the transmittance of visible light spectrum from 400 nm to 800 nm. The percentage of transmittance was calculated for each hydrogel in comparison to that of PBS.

[0156] Swelling ratio. Swelling property of NCColHA hydrogel was assessed by measuring the weight of hydrogel after incubated in PBS at 37 ºC. Briefly, the weight of hydrogel was measured prior to incubation (W0), as well as at 12 h, 24 h, and 48 h post-incubation (Wn). The swelling ratio was calculated as follows: (Wn– W0) / W0× 100. The digital images of NCColHA hydrogel were also taken before and after incubation for 48 h. To compare the size difference after incubation, the hydrogel was placed on the plastic card with lettering background. To confirm the swelling property, the contents of NCColHA hydrogel were measured by Thermogravimetric Analyzer (TGA; TA instrument, DE, USA). The NCColHA hydrogel was incubated in PBS for 72 h, and its thermogravimetric pattern was subsequently compared to that of the hydrogel at the initial point (0 h).

[0157] Cell culture. Corneal epithelial cells (CEC; ATCC; CRL-11135) were cultured to test cell proliferation and cytotoxicity. Cells were cultured in keratinocyte-serum free medium supplemented with 5 ng / mL epidermal growth factor, 500 ng / mL hydrocortisone, 0.05 mg / mL bovine pituitary extract, and 5 μg / mL insulin. Cells were incubated at 37ºC in a 5% CO2 atmosphere, and the medium was refreshed every other day. The proliferated cells were passaged at 80% confluency using Trypsin-EDTA solution.

[0158] Biocompatibility. To investigate the biocompatibility of NCColHA hydrogel to the CECs, the NCColHA hydrogel was thinly spread on the ground of culture dishes, and cells (4 × 104cells) were seeded on the dishes. To visualize the hydrogel on the ground, Col-N3 was labeled with 6-FAM NHS ester (Lumiprobe, Maryland, USA). Briefly, FAM NHS ester solution (2 µL; 7.1 mg / mL in DMSO) was added to Col-N3 solution (100 µL) and incubated overnight at 4 ºC under light protected conditions. The cells were incubated for two days. After incubated, the cells were stained with DAPI solution, and their fluorescents were observed using confocal microscope (Carl Zeiss, Oberkochen, Germany). The cell morphology on the gel was also investigated using an optical microscope.

[0159] Cell proliferation study. To evaluate the cell viability of CECs when treated with NCColHA hydrogel, the cells (1 × 104cells) were seeded on 96 well culture plates and incubated for one day prior to treatment. Then, NCColHA hydrogel was treated to the cells at five different concentrations of NCColHA hydrogel (0, 0.5, 1, 5, and 10 mg / mL). The samplesATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 were prepared in two different ways. One is that the cells were cultured in NCColHA hydrogel- pre-added condition. The other is that NCColHA hydrogel was added to the pre-incubated cells. All the treated cells were incubated for two days, and their cell population was analyzed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, MD, USA). After incubating with CCK-8 solution for 2 h, the absorbance (450 nm) of the supernatant was analyzed with the microplate reader.

[0160] Cell proliferation on the NCColHA hydrogel-coated ground was assessed by measuring cell populations and the ratio of dead cells. NCColHA hydrogel was coated on the culture flask in the same way as for the biocompatibility study. Then, CECs (3.5 × 105cells) were seeded on the hydrogel and incubated for 5 days. The cell proliferation was assessed by monitoring the cell populations on day 1, 3, and 5. The live and dead cells were detected using Live / Dead Viability / Cytotoxicity Kit (Invitrogen, MA, USA). The cell number of each image was measured using ImageJ software.

[0161] Cell migration assay. To make the artificial wounds, the parafilm piece (1.6 mm x 5.0 mm) was stuck to the ground of cell culture dishes (100 mm). These dishes were sterilized using 70 % ethanol and ultraviolet (UV) light irradiation. CECs (1 × 105cells) were seeded on the dishes and incubated until the cells were fully proliferated. Then, the parafilm was removed to make the artificial wounds, and NCColHA hydrogel (100 µL) was added alongside the wounds. The wounds were monitored using an optical microscope at 0, 4, 8, 24, and 48 h.

[0162] Ex vivo wound healing study. The wound healing processes after application of hydrogel were assessed using ex vivo rabbit eye culture model that was previously established in our research group. Briefly, A lamellar keratectomy with a 3.5-mm trephine (Robbins Instruments, CA, USA) was carried out on the rabbit corneas. The defected corneas were cut off from the eyes leaving a 5 mm thick ring of sclera, removing lens and iris. The corneas were washed two times with 1% penicillin-streptomycin (PS) solution. The corneas were then mounted on agar plugs. The agar plugs were prepared by microwaving 2 g of agar in 75 mL of organ culture medium containing DMEM / F12 (Gibco, MT, USA), insulin-transferrin- selenium (0.05%), and PS (1%). The agar was solidified using PDMS mold, and the obtained rabbit cornea was placed on the agar plugs. Ater mounting the corneas, the culture medium was added until it met the sclera. After that, 6-FAM-labeled NCColHA hydrogel was applied to the defects (3 – 5 µL) to fill out the defects and harvested on day 1, 3, and 5. The PBS-treated cornea was employed as control group, and the control group was harvested just on day 5. The collected corneas were sliced for the analysis of immunohistochemistry.

[0163] Immunohistochemistry. To observe the morphologies and biomarkers at the hydrogel- treated areas, the corneas were sliced and stained using various kinds of antibodies. For ex vivo analysis, the slices were stained with DAPI (1:1000, Thermo Fisher Scientific), anti- cytokeratin 14 (anti-CK14; 1:100, Abcam), anti-CD44 (1:100, Abcam), anti-ZO-1 (1:100,ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 Invitrogen), and Alexa fluor 647-conjugated anti-phalloidin (1:400, Thermo Fisher Scientific). Subsequently, the anti-CK14, CD44, and ZO-1 antibodies were captured with Alexa fluor 555- conjugated anti-rabbit IgG (1:200, life technologies corporation, CA, USA), Alexa fluor 647- conjugated anti-rat IgG (1:200, life technologies corporation), and Alex fluor 555-conjugated anti-mouse IgG (1:200, life technologies), respectively. The fluorescent signals from each sample were detected with the confocal microscope. For in vivo analysis, anti-α-smooth muscle actin (α-SMA; 1:500, Abcam) was primarily used, and Alexa fluor 647-conjugated anti- mouse IgG (1:200, life technologies corporation) was subsequently added for a secondary staining.

[0164] Animals. New Zealand white rabbits (3.5 – 5.5 kg) were used in this study. Animal experiments were designed to conform with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Stanford University Institutional Animal Care and Use Committee (protocol #: APLAC-33413). All anesthesia procedures were supported by the veterinary service center (VSC) at Stanford University.

[0165] In vivo corneal regeneration. For all surgeries and follow-up studies, we dropped proparacaine hydrochloride ophthalmic solution (0.5 %; Bausch and Lomb, Laval, Canada) was added to the eye that would be examined. To establish corneal defect model, lamellar keratectomy was performed on one eye using a 3.5 mm trephine to create a deep circular cut (50 – 70 % depth of corneas) and a spatula to remove the collagen fibril layers. After applying the mixture of each hydrogel component (i.e., the mixture of HA-N3, Col-N3, and NCs-DBCO; 1:1:1, v / v / v) on the artificial defects, we waited for 10 min for the gelation of the applied solution. Then, a contact lens was applied to protect the hydrogel from scratching or other stresses, and tarsorrhaphy was carried out to prevent agitation by the animal and to help keep the contact lens and gel in place. As a post-operative cares, ofloxacin ophthalmic solution (0.3 %, Bausch and Lomb) was added three times a day for one week to prevent the bacterial infection, and prednisolone acetate ophthalmic suspension (1 %, Sandoz, Basel, Switzerland) was dropped three times a day for the day after surgery to relieve the initial inflammation caused by the lamellar keratectomy.

[0166] The corneal defects after application of NCColHA hydrogel were monitored by taking the optical coherence tomography (OCT; Heidelberg Engineering, Heidelberg, Germany) images and photographs. To check the re-epithelialization of the defected areas, fluorescein solution (1.5 %) was dropped and washed immediately with the balanced salt solution (Alcon, Geneva, Switzerland). The fluorescein-stained areas were detected using blue light. On day 7, the tarsorrhaphy was removed for the eye examination, and the defects were further monitored on day 14, 28, and 56. The eyes were enucleated and cryo-sectioned for immunostaining after examined on day 56. The cornea slices were stained with DAPI, phalloidin, anti-CK14 and anti-α-smooth muscle actin (anti-α-SMA; Abcam). The thickness ofATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 the epithelial and stromal layers, and whole cornea on day 56 was measured based on confocal microscopic images of the sliced sections.

[0167] Statistical analysis. All the statistical analyses were performed using Graphpad Prism software (version 8.0.2.263). For multiple comparisons, we employed one-way ANOVA followed by Tukey’s post-hoc analysis. For pairwise comparisons, the significance was determined using a two-tailed unpaired t-test. The significance threshold was set at p < 0.05; otherwise, the difference was labeled as “not significant (n.s.)”. Example 2 Photoactivated growth factor release from bio-orthogonally crosslinked hydrogels for the regeneration of corneal defects

[0168] In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs can improve re-epithelialization and stromal remodeling. However, challenges persist in controlling the release of therapeutic molecules from the formed hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel (PC- HACol hydrogel) containing growth factors tethered via photocleavable linkages is developed to accelerate corneal regeneration. Epidermal growth factor (EGF) is conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms, and the growth factor is released when exposed to mild ultraviolet (UV) light (2 – 5 mW / cm2). PC-HACol hydrogel rapidly gels within a few minutes when applied to corneal defects, with excellent transparency and biodegradability. The hydrogel under UV irradiation promotes the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization is positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea. In an in vivo rat study, PC-HACol hydrogel exposed to UV light significantly promotes re-epithelialization and remodeling of stromal layers, with expression of normal epithelial and stromal biomarkers, including minimal α-SMA and robust ALDH3A1, F-actin, and CK12. The remodeled cornea exhibits a full recovery of corneal thickness without hyperplasia of the epithelium or stromal scarring.

[0169] Recently, photo-responsive hydrogels have emerged as a promising avenue in the realm of addressing corneal diseases. These hydrogels utilize ultraviolet (UV) or visible light to induce crosslinking between polymers, thereby enhancing mechanical properties, adhesiveness, and transparency. Furthermore, the use of light is employed to exert control over the release of cargo from nanoparticle and membranes. The key advantage lies in their potential for spatio-temporal control over release: the ability to modulate properties precisely at a desired location and time. The UV light irradiation (365 nm) conditions utilized here are less than that is typical of the intensity of natural sunlight (<7 mW / cm2). Moreover, corneal cross-linking (CXL), an FDA-approved procedure for treating keratoconus, utilizes UV light inATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 conjunction with riboflavin-5-phosphate to crosslink corneal collagen. Inspired by these advantages, we have designed a photo-responsive hydrogel that releases growth factors under mild UV irradiation.

[0170] Here, we developed a photo-responsive bio-orthogonal hydrogel, termed the PC- HACol hydrogel, designed for the in situ-gelation and controlled release of EGF in corneal defects (FIG. 7A). This hydrogel was bio-orthogonally constructed through the copper-free click chemistry reaction strain-promoted azide-alkyne cycloaddition (SPAAC) between an collagen-azide (Col-N3) conjugate and a hyaluronic acid-polyethylene glycol- dibenzocyclooctyne (HA-PEG-DBCO) conjugate. EGF was first grafted to the HA-PEG-DBCO backbone via a photocleavable (PC) linker derived from o-nitrobenzene. This PC linker, sensitive to mild UV irradiation conditions (2 – 5 mW / cm2), facilitates the efficient release of EGF. Each component (i.e., HA-PEG-DBCO and Col-N3) exists as a solution before being mixed (before administration), becoming a solid and transparent hydrogel within minutes upon administration into corneal defects followed by in situ gelation. Our study extensively assessed the physicochemical properties and biodegradability of PC-HACol hydrogel. Furthermore, we evaluated cell proliferation and migration using primary corneal epithelial cells. The impact of multiple UV irradiations on re-epithelialization were investigated in ex vivo rabbit eyes. Additionally, we studied in vivo stromal regeneration and re-epithelialization using rats. This comprehensive analysis aims to validate the effectiveness and safety of our novel PC-HACol hydrogel for potential applications in corneal wound healing. Results and Discussion

[0171] Synthesis and fabrication of PC-HACol hydrogel. For the photo-responsive release of EGF from hydrogel, EGF was conjugated with photo-cleavable (PC) linkers via primary amine (-NH2) / N-hydroxysuccinimide (NHS) reactions. The one end of the linker was an NHS ester, allowing it to react with lysine (K) of human EGF. The azide (N3) moiety of the other end reacts with DBCO through strain-promoted alkyne-azide cycloaddition (SPAAC). Hyaluronic acid amine (HA-NH2; degree of substitution (DoS): 50 %) was reacted with DBCO-PEG-NHS ester (10 k) to form HA-PEG-DBCO. We used PEG as a solubilizing linker to offset the increased hydrophobicity induced by DBCO conjugation. As the counter part of HA-PEG-DBCO, azide- functionalized collagen (Col-N3) was synthesized via primary amine / NHS reactions. DoS values for HA-PEG-DBCO and Col-N3 were calculated as 3.36 ± 0.25 % (n = 3) and 58.5 ± 13.7 % (n = 3), respectively. The PC linker-conjugated EGFs were conjugated to HA-PEG- DBCO through click reaction (i.e., DBCO / N3 reaction), followed by mixed with Col-N3. The HA- PEG-DBCO reacted with Col-N3 through SPAAC reactions, and gelation of the mixture occurred within 5 min at 37 ℃ (FIG.19).ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0172] Optimization and characterization of PC-HACol hydrogel. Hydrogels for corneal wound healing require a robust mechanical strength to maintain their structures until stroma is regenerated. Specifically, based on our previous studies, hydrogels for ocular administration have a storage modulus of over 1000 Pa. We prepared four different concentrations of HA- PEG-DBCO (10, 30, 50, and 70 mg / mL1) and Col-N3(1, 3, 6, and 9 mg / mL1) (Figure 7b and 7c). In all conditions, EGF was conjugated to HA-PEG-DBCO through the PC linker, and the HA-PEG-DBCO and Col-N3were mixed at equal volumes (1:1, v / v). The storage modulus of the hydrogels increased with higher concentration of HA-PEG-DBCO and Col-N3. Notably, the hydrogel reached a storage modulus over 1000 Pa (~3100 Pa) when the concentrations of HA-PEG-DBCO and Col-N3 were 70 mg / mL1and 9 mg / mL1, respectively. Therefore, the final hydrogel (PC-HACol hydrogel) components were determined as 70 mg / mL1of HA-PEG- DBCO (with EGF conjugation) and 9 mg / mL1of Col-N3. The gelation of PC-HACol was investigated by measuring storage modulus and Tan δ values over time (Figure 7d). The storage modulus values of mixture continuously increased after mixing, while the Tan δ values decreased over time. Thus, the mixture of each component solution gels in a time-dependent manner, suggesting the properties as in situ-forming hydrogel. Given each component existed as a solution state, the gelation of the mixture was attributed to the formation of chemical reactions between the DBCO and azide (SPAAC reaction).

[0173] A desirable characteristic of hydrogels for ocular applications is transparency, ensuring they do not interfere with clear vision. To investigate the transparency of the hydrogels composed of HA-PEG-DBCO and Col-N3, we measured their transmittance of visible lights spanning from 400 nm to 800 nm (Figure 7e and 7f). All the hydrogels exhibited high transmittance, exceeding 90 % throughout the range. Thus, the hydrogels maintained their transparency after gelation. We further examined the transparency of PC-HACol hydrogel by placing it over lettering on a plastic card (Figure 7g). The background letters were clear and legible through the material, demonstrating the excellent transparency of PC-HACol hydrogel.

[0174] In addition to its appearance, we interrogated the microstructure of the lyophilized PC- HACol hydrogel using scanning electron microscopy (SEM) (Figure 7h). The hydrogels were lyophilized at two different times: immediately after mixing (i.e., before gelation) and 30 min after mixing (i.e., after gelation). In the ‘before gelation’ image, we observed long-chain polymer structures and numerous pores throughout the material. Meanwhile, the porosity of the structures is reduced after gelation, due to the enhanced crosslinking density resulting from chemical reactions.

[0175] Hydrogels for corneal defects should be degradable for new stromal regeneration after re-epithelialization. The biodegradability of the PC-HACol hydrogel was evaluated in biosimulating solutions containing 5 units / mL1of hyaluronidase, ≥ 2 CDU / mL1of collagenase or a combination of 5 units / mL1of hyaluronidase and ≥ 2 CDU / mL1of collagenase (Figure 7i).ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 The PC-HACol hydrogel was degraded both in collagenase solution and hyaluronidase solution, and their degradation rate was higher in the mixture of collagenase and hyaluronidase solution. Interestingly, the hydrogel was fully dissolvable in collagenase solution alone as well as the mixture, which may be caused by the complete degradation of collagen, leading to collapse of the hydrogel structure. Thus, PC-HACol hydrogel exhibited collagenase- and hyaluronidase-dependent degradation patterns, suggesting their biodegradability within corneal defects.

[0176] In vitro growth factor release. The release kinetics of EGF from PC-HACol hydrogel under UV irradiation was investigated in vitro (FIG.8). In this study, we prepared three kinds of hydrogels: PC-HACol hydrogel, Non-PC-HACol Hydrogel, and Physical hydrogel. In the ‘Non-PC-HACol hydrogel’, a non-photocleavable linker (Azido-PEG5-NHS ester) was used to conjugate EGF to HA-PEG-DBCO (FIG. 8a). The ‘physical hydrogel’ was composed of a physical mixture of native Col, HA-NH2, and EGF. The Non-PC-HACol and physical hydrogels were employed as control groups to compare the release patterns. According to our hypothesis, the photocleavable linker undergoes a series of radical reactions under mild UV irradiation and can be broken (FIG.8b and 20), thereby allowing free EGF to be released from the hydrogel networks. Thus, the release rate of EGF depends on both UV irradiation and the structural integrity of the hydrogel network.

[0177] FIG. 8c illustrates the release patterns of EGF from the hydrogels. In the physical hydrogel, EGF was released rapidly, exceeding 85 % of the accumulative release within 24 h. In contrast, the Non-PC-HACol hydrogel under UV irradiation exhibited only 5.70 ± 0.57 % release within 24 h and 7.99 ± 1.15 % release within 96 h, indicating a lack of responsiveness to UV irradiation. Notably, the PC-HACol hydrogel exhibited a controlled release pattern of EGF, which was dependent on UV irradiation. The PC-HACol hydrogel exhibited minimal release without irradiation (6.2 ± 1.8 % for 0 – 12 h; 2.8 ± 1.3 % for 24 – 36 h; 0.6 ± 0.8 % for 48 – 60 h; 0.6 ± 0.8 % for 72 – 84 h). However, it released a significantly larger amount of EGF under UV irradiation (18.2 ± 3.3 % for 12 – 24 h; 15.3 ± 6.7 % for 36 – 48 h; 5.9 ± 4.9 % for 60 – 72 h; 8.2 ± 1.5 % for 84 – 96 h). As the released amount would depend on the remaining amount in the hydrogels, we calculated the normalized EGF release under two different irradiation conditions (Figure 2S3). Interestingly, the released amount of EGF was larger under strong UV irradiation (5 mW / cm2for 10 min) compared to weak irradiation (2 mW / cm2for 10 min). Thus, PC-HACol hydrogel was likely to exhibit a strength-dependent EGF release patten.

[0178] To further investigate the release patterns according to irradiation conditions, we prepared hydrogels by four different irradiation times (0, 1, 5, and 10 min) at 2 mW / cm2(FIG. 8d) and strengths (0, 1, 2, and 5 mW / cm2) for 10 min (FIG.8e). Without irradiation, the PC- HACol hydrogel released 5.3 ± 0.2 % of EGF within 12 h, possibly due to unconjugated fractions during synthesis. Of note, PC-HACol hydrogel exhibited a time-dependent EGFATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 release (10.4 ± 1.2 % at 1 min; 12.4 ± 1.0 % at 5 min; 16.9 ± 1.2 % at 10 min) and a strength- dependent EGF release (14.1 ± 0.3 at 1 mW / cm2; 16.9 ± 1.2 % at 2 mW / cm2; 22.9 ± 0.8 % at 5 mW / cm2). These results suggest that EGF release can be controlled by adjusting the irradiation time and strength.

[0179] In vitro cell proliferation and migration. EGF can promote the proliferation and migration of epithelial cells at injury sites. To investigate cell proliferation, primary corneal epithelial cells (CECs) were incubated for one day, followed by treatment with PC-HACol hydrogel (FIG.9a). Before conducting the cellular studies, we assessed the relative cytotoxicity of our chosen UV light conditions to CECs (FIG.22). The hydrogels were irradiated with UV light (2 mW / cm2for 5 min) after being added to the cells. To evaluate the effects of UV light, one group was prepared by adding PC-HACol hydrogel without irradiation. Cell viability (%) was calculated by comparing the values with untreated cells. Notably, at all concentrations, cell viability was actually measured to be higher than 100 %, indicating that, at the very least, the UV irradiation conditions being tested were not toxic to the cells. Interestingly, without UV light, PC-HACol hydrogel did not increase the cell viability at 5 and 10 mg / mL1, and cell viability at 50 mg / mL1was significantly lower than that with UV light. These results suggest that the activities of EGF significantly decreases when conjugated to polymer chains.

[0180] In addition to cell viability, we investigated the rate of cell migration when treated with PC-HACol hydrogel (FIG. 9b). CECs were cultured until reaching a confluency exceeding 90 %, and then a linear scratch was created in the middle to simulate a corneal epithelial wound. In the control group (No treatment group), the scratched area decreased to 29.9 ± 12.2 % within 48 h. Surprisingly, PC-HACol without UV light (PC-HACol group) did not exhibit significant differences (26.4 ± 9.3 % within 48 h), compared to the no treatment group. In contrast, under UV light irradiation, PC-HACol hydrogel group (PC-HACol + UV) exhibited complete wound closure within 48 h. Thus, PC-HACol hydrogel needed to be irradiated by UV light to facilitate cell migration. These results suggest that EGF need to be released from polymer chains to function effectively.

[0181] Biocompatibility. The biocompatibility of the PC-HACol hydrogel was evaluated by observing cell growth on the hydrogel (FIG.9c and 9d). PC-HACol hydrogel was thinly spread on the bottom of the cell culture dishes, and CECs were cultured on the hydrogel-coated dishes. In the hydrogel-uncoated dishes (No treatment group), the cells reached a population of 179 ± 28 cells / cm2, with a dead cell population accounting for 1.4 ± 0.7 %. Similarly, cells grew on the hydrogel, exhibiting a cell population of 185 ± 45 cells / cm2with 2.2 ± 0.8 % of dead cells. Thus, PC-HACol hydrogel did not prohibit cell growth. Interestingly, under UV irradiation, PC-HACol hydrogel promoted cell growth, exhibiting a significantly increased cell population of 252 ± 57 cells / cm2with a decreased ratio of dead cells (0.5 ± 0.4 %).ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226

[0182] Ex vivo re-epithelialization in rabbit eyes. Re-epithelialization following corneal injuries is an initial and critical process for proper wound healing, serving as the eye’s an initial protective measure from infection and other deleterious exposures. This process can be promoted by growth factors. Based on the results from in vitro cellular studies, we hypothesized that freed EGF after photocleavage from the network would be more effective than covalently-bound EGF. To initially demonstrate the wound healing effects of the PC- HACol hydrogel, we used an ex vivo anterior lamellar keratoplasty (ALK) model using rabbit eyes. In this study, PC-HACol hydrogel was applied to the defected eyes and irradiated with UV light (2 mW / cm2for 5 min) either one or three times (FIG.10). The control group was not subjected to light irradiation. Re-epithelialization in defects was assessed by staining the defects with fluorescein (FIG. 10a). When the hydrogel was not irradiated, the fluorescein staining persisted on the wound for five days, while eyes subjected to one-time UV irradiation (at day 0, after hydrogel administration) tended to exhibit wound closure within 2-4 days (FIG. 10b). Interestingly, PC-HACol hydrogel with three UV irradiation exposure (at day 0, 1, and 2) exhibited faster re-epithelialization than that with only a one-time irradiation. These results suggest that photocleavage and release of EGF increases the wound healing effects of the PC-HACol hydrogel.

[0183] In vivo wound healing in rats. Corneal regeneration by PC-HACol hydrogel was assessed in rats over seven days. A corneal defect model was established using a 2.0-mm trephine during ALK. PC-HACol hydrogel was applied to the corneal defects, either with UV irradiated (2 mW / cm2for 10 min) or without. The PBS-treated group served as a control group. In FIG.11a, optical coherence tomography (OCT) images of cornea revealed that, at day 0, corneal defects of 40 – 60 % depth were created, and the applied hydrogels were observed in the defects (marked by orange arrows). One day post-surgery, PBS-treated eyes showed no noticeable stromal regeneration, while PC-HACol hydrogel group exhibited greater corneal regeneration. Under UV irradiation, the PC-HACol hydrogel group exhibited faster and more extensive regeneration than other groups. The differences in corneal regeneration in PC- HACol hydrogel groups (i.e., PC-HACol and PC-HACol+UV) were more evident in the following examination, compared to the PBS group. In addition to OCT images, we monitored the appearance of the wounded eyes using bright field images (FIG.11b and 23). All the PBS- treated eyes exhibited significant scars on the defects, suggesting severe vision impairment. Surprisingly, the PC-HACol hydrogel group exhibited scars for three days, and the scars markedly decreased from three to seven days, much improved optical clarity by day 7. Importantly, scars were not significantly observed in the UV-exposed PC-HACol group over the evaluation period, with a significantly less scar area than those in PBS group (FIG.24). Thus, the PC-HACol hydrogel prevented scarring during the healing processes, and UVATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 irradiation-induced photocleavage of the EGF within the network further fortified the anti- scarring effects of the hydrogel.

[0184] Re-epithelialization during healing was investigated by staining the defects treated with fluorescein (Figure 11c and 11d). In the PBS group, the corneal defects persisted for longer than those in the PC-HACol hydrogel group, and the fluorescein-stained area was significantly smaller in PC-HACol hydrogel group. These results were consistent with our previous reports on bio-engineered hydrogels for improving corneal regeneration. Thus, the application of the PC-HACol hydrogel promoted the wound healing processes even without UV irradiation. Notably, under UV irradiation, the PC-HACol hydrogel significantly accelerated re- epithelialization, achieving complete re-epithelialization in half of the rats within one day. The other half exhibited a fluorescein staining area of less than 5 %. These results suggest that EGF released from PC-HACol hydrogel promotes the proliferation and migration of epithelial cells, resulting in faster re-epithelialization.

[0185] To further analyze the materials’ effects on corneal regeneration, we measured the thickness of the cornea and epithelium (FIG. 11e and 11f). At the time of surgery on day 0, approximately 50 % of the corneas were removed in all groups (FIG.11e) through an anterior keratectomy. In the PBS group, the corneal thickness increased slowly and continuously for five days (50.9 ± 23.6 % on day 1; 60.0 ± 25.6 % on day 3; 60.5 ± 21.4 % on day 5) and dramatically increased from day 5 to 7 (82.9 ± 11.8 % on day 7). In contrast, the PC-HACol group exhibited markedly thicker corneal layers starting from day 1 (72.8 ± 8.5 %), and the layers were significantly thicker in the PC-HACol+UV group (95.7 ± 22.3 %). PC-HACol+UV group showed the highest corneal thickness over the evaluation period, regenerating to a thickness of 101.6 ± 3.2 % by day 7. Thus, the PC-HACol hydrogel exposed to UV light exhibited significantly greater wound healing effects both in terms of surface epithelialization and in terms of stromal regeneration. In addition to the cornea, we investigated the thickness of the epithelial layers because hyperplasia of the epithelium is a commonly observed abnormality during corneal wound healing processes. As expected, PBS-treated corneas exhibited a 1.53-fold thicker epithelium (52.0 ± 7.1 µm), compared to normal corneas (34.0 ± 1.6 µm) (Figure 11f). Meanwhile, this phenomenon was not observed in the PC-HACol (33.3 ± 4.0 µm) and the PC-HACol+UV (33.0 ± 1.4 µm) groups. Therefore, these results suggest that the PC-HACol hydrogel promotes a return to baseline corneal epithelium thickness after injury.

[0186] Immunohistochemistry of treated corneal defects. The regenerated corneas on day 7 were further investigated through immunohistochemical analysis to visualize the biomarkers (FIG. 12). FIGS. 12a – c depict the expression of alpha-smooth muscle actin (α-SMA) - a marker of fibrotic healing - in the regenerated stromal layers. During corneal regeneration, activated keratocytes can differentiate into α-SMA-expressing myofibroblasts, potentiallyATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 leading to mild-to-severe scarring. Importantly, PC-HACol hydrogel exhibited a markedly lower expression of α-SMA (FIG.12b) compared to PBS (FIG.12a), and the expression was much lower with UV irradiation (FIG. 12c). These results suggest a reduction in myofibroblastic activities and fibrosis in the regenerated cornea, consistent with the clear transparency observed in bright field images (FIG. 11b). To further investigate corneal transparency, we assessed the expression of aldehyde dehydrogenase 3A1 (ALDH3A1), a corneal crystallin and biomarker of corneal health and transparency (FIG.12d). The epithelium of PC-HACol hydrogel with UV light group expressed the ALDH3A1 evenly in all layers, whereas the PBS- treated group exhibited an uneven expression pattern in epithelial layers (FIG.25). Together with α-SMA expression, these results could explain the differences in transparency of the corneas between the groups. Stromal regeneration in the PC-HACol + UV group was observed by staining F-actin (FIG.12e). F-actin was highly observed in the stroma close to the epithelial layers, indicating cell-matrix mechanical interactions at the regenerated site. Additionally, we examined a cell differentiation marker in the regenerated epithelium (FIG.12f). The epithelial layers exhibited positive expression of cytokeratin 12 (CK 12), a cornea-specific cytokeratin, suggesting that PC-HACol hydrogel with UV light resulted normal epithelial differentiation of the regenerated layers.

[0187] We have developed an in situ-forming, bio-orthogonally crosslinked hydrogel capable of subsequent photoactivated accelerated release of EGF for corneal regeneration. The bio- orthogonally crosslinked hydrogel was fabricated through SPAAC click reactions between HA- PEG-DBCO and Col-N3, achieving rapid gelation within minutes in corneal defects. EGF was conjugated to HA-PEG-DBCO using photo-cleavable linkers, allowing the hydrogel to release EGF under mild UV conditions. The amount of released EGF depended on UV intensity and irradiation time. Upon UV irradiation, the PC-HACol hydrogel promoted the proliferation and migration of corneal epithelial cells with excellent biocompatibility. The rate of re- epithelialization in corneal defects was influenced by the frequency of UV irradiation, as shown through ex vivo rabbit corneal organ culture studies. In rats in vivo, the PC-HACol hydrogel after exposure to UV light significantly promoted re-epithelialization without hyperplasia and stromal regeneration with return to baseline corneal thickness compared to treatment conditions without UV irradiation and without any gel treatment. Our results indicate that in situ-forming, bio-orthogonally crosslinked hydrogels with controllable growth factor release may be a promising and advanced approach for the treatment of corneal defects. Table 2 Details on the released amount of EGF of Fig.8c.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 Time (h) % Release UV irradiation 0 – 2 2.8 ± 0.4 2 – 12 4.1 ± 1.8 12 – 24 18.2 ± 3.3 2 mW / cm2for 10 min 24 – 36 2.8 ± 1.3 36 – 48 15.3 ± 6.7 5 mW / cm2for 10 min 48 – 60 0.6 ± 0.8 60 – 72 5.9 ± 4.9 2 mW / cm2for 10 min 72 – 84 0.6 ± 0.8 84 – 96 8.2 ± 1.5 5 mW / cm2for 10 min Materials

[0188] Hyaluronic acid-Amine (MW 250k; DoS of 50 %) was obtained from Creative PEGWorks (Durham, NC, USA). Collagen (TeloCol-10) was purchased from Advanced BioMatrix (Carlsbad, CA, USA). DBCO-PEG-NHS ester (10 k), Azido-PEG5-NHS ester (98 %), and PC Azido-PEG3-NHS carbonate ester (95 %) were purchased from BroadPharm (San Diego, CA, USA). Recombinant Human Epidermal Growth Factor Protein (EGF, carrier-free) was purchased from R&D Systems (Minneapolis, MN, USA).

[0189] Fabrication of PC-HACol hydrogel. To fabricate the photo-cleavable and bio- orthogonal hydrogel, we prepared DBCO-PEG-conjugated HA (i.e., HA-PEG-DBCO) and azide-conjugated collagen (i.e., Col-N3). For the synthesis of HA-PEG-DBCO, DBCO-PEG- NHS ester solution (10 k; 160 µL; 100 mg / mL1in DMSO) was mixed with the hyaluronic acid amine solution (HA-NH2; 20 mg / mL1in PBS). The mixture was stirred overnight under protection from light. Then, the solution was dialyzed against water for 48 h. The water was replaced at least two times a day. The dialyzed solution was lyophilized and stored at -20 ºC until use. For the synthesis of collagen-N3(Col-N3), collagen solution (9 mg / mL1) was neutralized with 10X PBS and 1N NaOH, and azido-PEG5-NHS ester was diluted ten times with DMSO. The diluted azido-PEG5-NHS ester solution (20.7 µL; 100 mg / mL1) was mixed with the neutralized collagen solution (1 mL) and rotated gently at 4ºC overnight under protection from light. The product solution was dialyzed against 1X PBS using Slide-A-Lyzer G2 Dialysis Cassettes (Thermo Fisher Scientific; Waltham, MA, USA) at 4ºC overnight. For the preparation of photocleavable linker-conjugated EGF, PC azido-PEGF3-NHS ester (10 mg / mL1) was prepared in the mixture of PBS and DMSO (1:1, v / v) under protection from light. The solution (0.5 µL) was added to the EGF solution (5 µL; 500 µg / mL1in PBS). The mixture was incubated for 1 h at room temperature under protection from light. After incubation, theATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 mixture (4.4 µL) was added to the HA-PEG-DBCO solution (70 mg / mL1in PBS; 100 µL) for 1 h at room temperature under protection from light. PC-HACol hydrogel was prepared by mixing the solution with Col-N3 (100 µL; 9 mg / mL1). DoS values of DBCO-PEG-HA and Col-N3 were calculated by measuring the absorbance at 306 nm and 285 nm, respectively. The DoS value is defined as DBCO-or N3-conjugated amines / total surface primary amines*100.

[0190] In this study, we prepared two kinds of control hydrogels: Physical hydrogel and Non- PC hydrogel. Physical hydrogel was prepared by physically mixing native collagen (i.e., unmodified collagen), hyaluronic acid amine, and EGF. In this hydrogel, EGF was physically dispersed in hydrogel networks. Non-PC hydrogel was prepared by using azido-PEG5-NHS ester instead of PC Azido-PEG3-NHS carbonate. The fabrication processes were the same with PC-HACol hydrogel.

[0191] Characterization. To investigate the rheological properties of PC-HACol hydrogel at different fabrication conditions, we prepared four kinds of HA-PEG-DBCO solution (concentration of 10, 30, 50, and 70 mg / mL1) and four kinds of Col-N3 solution (concentration of 1, 3, 6, and 9 mg / mL1). The storage and loss modulus of each condition were measured by rheometer (ARES-G2; TA instrument, New Castle, DE, USA) at varying frequencies ranging from 0.1 to 10 at the fixed oscillation strain of 1 at 37 ºC. Changes of storage modulus and tan δ values over time were measured for 900 s at oscillation strain of 1 % at 37 ºC. Tan δ value was defined as the ratio of loss modulus (G’’) / storage modulus (G’). Digital image of PC-HACol hydrogel was taken after gelation on the background with letters. Transmittance of PC-HACol hydrogel in visible light spectrum was measured in the range from 400 nm to 800 nm. The percentage of transmittance was calculated based on the values of PBS. SEM images (Thermo Fisher Scientific Apreo S LoVac Scanning Electron Microscope) were obtained to observe the surface morphology of lyophilized hydrogel. The samples were prepared by lyophilizing the hydrogel mixture immediately after mixed and after incubated for 30 min. Biodegradability of PC-HACol hydrogel was investigated under bio-simulating conditions. The hydrogels were pre-incubated in PBS for one day. The pre-incubated hydrogels were weighed (W1) and submerged in the 5 units / mL1of hyaluronidase (Sigma-Aldrich) solution or 5 ≥ CDU / mL1collagenase (Sigma-Aldrich) solution or the mixture of 5 units / mL1of hyaluronidase and 5 ≥ CDU / mL1collagenase. The hydrogels were incubated at 37ºC. As pre-determined timepoints (2, 6, 24, and 48 h), the weight of hydrogels (Wn) was measured after removing enzyme solution. The percentage weight (%) of the hydrogels at those points was calculated as Wn / W1 × 100.

[0192] In vitro EGF release. EGF release kinetics from the fabricated hydrogel was investigated in vitro. PC-HACol was inserted into the dialysis bag (MWCO: 12 – 14 kDa), and the bag was submerged in 1% BSA solution. UV light (365 nm) was irradiated at 2 mW / cm2for 10 min at 12 and 60 h. At 36 and 84 h, the strength of UV light was increased (5 mW / cm2ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 for 10 min). The release medium (20 µL) was collected to analyze the amount of released EGF. At pre-determined timepoints (2, 12, 24, 36, 48, 60, 72, 84, and 96 h). For the physical hydrogel, the release medium was collected at 2, 4, 8, 12, 24, 48, 72, and 96 h. The release medium was replenished with fresh medium after collection. The collected medium was analyzed using EGF ELISA kit (Abcam, Cambridge, UK). In addition to the release kinetics, the effects of irradiation time and strength were assessed. For the effects of irradiation time, PC-HACol hydrogels were irradiated for 0, 1, 5, or 10 min at 2 mW / cm2. For the effects of irradiation strength, PC-HACol hydrogels were irradiated at 0, 1, 2, or 5 mW / cm2for 10 min.

[0193] Cell culture. Corneal epithelial cells (CECs; ATCC; CRL-11135) were cultured to assess the cell proliferation, migration, and biocompatibility. CECs were cultured in keratinocyte-serum free medium supplemented with hydrocortisone (500 ng / mL1), epidermal growth factor (5 ng / mL1), bovine pituitary extract (0.05 mg / mL1), and insulin (5 μg / mL1). Cells were incubated at 37 ºC in a 5% CO2atmosphere, and the medium was refreshed every other day. The proliferated cells were sub-cultured at 80% confluency using Trypsin-EDTA solution.

[0194] Cell proliferation. CECs (1 × 104cells) were cultured on 96-well culture plates one day before treatment. Then, PC-HACol hydrogel was added to the cells at four different concentrations of PC-HACol (0, 5, 10, and 50 mg / mL1). All the treated cells were incubated for two days, and their cell number was calculated using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, MD, USA). After incubation with CCK-8 solution for 2 h, the absorbance (450 nm) of the supernatant was analyzed with the microplate reader. The cell viability (%) values were calculated as (absorbance at each concentration / absorbance at 0 mg / mL1) × 100.

[0195] Cell migration. The UV-sterilized parafilm piece (1.6 mm x 5.0 mm) was attached to the ground of cell culture dishes (100 mm) to mimic the artificial scratches. These dishes were sterilized using 70 % ethanol and ultraviolet UV light irradiation. CECs (3 × 105cells) were seeded on the dishes and incubated until the cell population reached a confluency of more than 90 %. Then, the parafilm was removed from the ground to make the scratches, and PC- HACol hydrogel (60 µL) was added alongside the wounds. The dishes were irradiated by UV light (5 mW / cm2, 10 min). In this study, PC-HACol hydrogel without UV light was employed to compare the effect of UV light. The cell migration in the wounds was monitored using an optical microscope at 0, 6, 24, and 48 h. The wounded area was measured using ImageJ software.

[0196] Biocompatibility. The biocompatibility of PC-HACol hydrogel was evaluated by analyzing cell proliferation on the PC-HACol hydrogel-coated ground. PC-HACol hydrogel was thinly spread on the culture dish (100 mm), and CECs (3.5 × 105cells) were seeded on dish. The cells were incubated for one day, and the dish was irradiated by UV light (2 mW / cm2for 5 min). The cells were incubated one more day, and live and dead cells were monitored using the Live / Dead Viability / Cytotoxicity Kit (Invitrogen, MA, USA). The cell populations of eachATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 image were measured using ImageJ software. To visualize the cells on the hydrogel, PC- HACol hydrogel was labeled with 6-FAM NHS ester (Lumiprobe, Maryland, USA). The 6-FAM- labeled PC-HACol hydrogel-coated dishes were prepared in the same way, and the cells were cultured for two days. The images were taken using confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0197] Ex vivo re-epithelialization of rabbit eye. Re-epithelialization of defected corneas was investigated using rabbit eyes. Ex vivo rabbit eye culture model was previously established in our research group. Briefly, anterior lamellar keratectomy (ALK) with a 3.5 mm trephine (Robbins Instruments, CA, USA) was performed on rabbit corneas. The corneas were cut off from the eyeballs leaving a 5-mm thick sclera, and lens and iris were removed from the corneas. The prepared corneas were washed two times with 1% penicillin-streptomycin (PS) solution and mounted on agar plugs. The agar plugs were prepared by microwaving agar powder (2 g) in organ culture medium (75 mL) containing DMEM / F12 (Gibco, MT, USA), insulin-transferrin-selenium (0.05%), and PS (1%). The agar suspension was solidified in PDMS mold for 30 min, and the prepared rabbit corneas were placed on the agar plugs to maintain the shapes of corneas. Ater mounting the corneas, the culture medium was added until it met the sclera. After that, PC-HACol hydrogels were applied to the defects (3 – 5 µL) to fill out the defects. After gelation, the hydrogels were irradiated by UV light (2 mW / cm2for 5 min). In this study, PC-HACol hydrogels were irradiated with three different conditions: no UV light, UV light one time on day 0, and UV light three times on day 0, 1, and 2. To monitor the re-epithelialization, the corneas were stained with fluorescein solution (1.5 %) and washed with balanced salt solution (BSS, Alcon, Geneva, Switzerland). The fluorescein-stained photos were obtained under blue light irradiation. The fluorescein-stained area (%) was calculated using imageJ software.

[0198] Animals. Brown rats (Rattus norvegicus; 150 – 200 g) were used in this study. Animal experiments were designed to conform with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Stanford University Institutional Animal Care and Use Committee (protocol #: APLAC-32765). For anesthesia, the mixture of ketamine, xylazine, and water (2:1:3, v / v / v) was injected into muscle (intramuscular injection) as a dose of 1.68 mL / kg1. For the follow-up studies, rats were injected with half-dose of the mixture, and low flow of isoflurane gas was used.

[0199] In vivo corneal regeneration. For all surgeries and follow-up studies, proparacaine hydrochloride ophthalmic solution (0.5 %; Bausch and Lomb, Laval, Canada) was dropped into the eyes prior to examination. To establish corneal defect model, ALK was performed on one eye of each rat using a 2.0-mm trephine to create a deep circular cut (40 - 60 % depth of corneas) and a spatula to eliminate the collagen layers of stroma. PC-HACol hydrogel (2 – 3 µL) was treated into the defects. After gelation, the treated hydrogel was irradiated by UV lightATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 (2 mW / cm2, 10 min). As a post-operative cares, ofloxacin ophthalmic solution (0.3 %, Bausch and Lomb) was added to the eyes to prevent bacterial infections. The defects after application of PC-HACol hydrogel were examined by optical coherence tomography (OCT; Heidelberg Engineering, Heidelberg, Germany) images and digital photographs. To assess the wound closure of the defected areas, fluorescein solution (1.5 %) was dropped into the defects and washed gently with the BSS. Fluorescein-stained areas were visualized under blue light irradiation. The percentage of fluorescein-stained area was calculated using ImageJ software. The eyes were examined on day 0, 1, 3, 5, and 7, and the eyes were dissected and cryo- sectioned for immunostaining on day 7. The cornea slices were stained with DAPI, phalloidin, anti-CK12 (Abcam) and anti-α-smooth muscle actin (anti-α-SMA; Abcam). The thickness of the epithelium and whole cornea on day 7 was measured based on confocal microscopic images of the sliced sections and OCT, respectively.

[0200] Immunohistochemistry. The biomarkers were analyzed at the regenerated corneas. The treated corneas were sliced and stained with various epithelial and stromal markers. The slices were stained with DAPI (1:1000, Thermo Fisher Scientific), Alexa fluor 488-conjugated anti-cytokeratin 12 (anti-CK12; 1:100, Abcam), anti-α-smooth muscle actin (α-SMA; 1:500, Abcam), anti-ALDH3A1 (anti-ALDH3A1; 1:500, Abcam), and Alexa fluor 555-conjugated anti- phalloidin (1:400, Thermo Fisher Scientific). Subsequently, the anti-α-SMA and anti-ALDH3A1 were captured with Alexa fluor 488-conjugated anti-mouse IgG (1:200, life technologies corporation, CA, USA) and Alexa fluor 555-conjugated anti-rabbit IgG (1:200, life technologies corporation), respectively. The fluorescent signals were detected with the confocal microscope (Zeiss, Jena, Germany).

[0201] Statistical analyses. All the statistical analyses were carried out using Graphpad Prism software (version 8.0.2.263). We employed one-way ANOVA followed by Tukey’s post-hoc analysis for multiple comparisons. For pairwise comparisons, the significance was determined using a two-tailed unpaired t-test. The significance threshold between groups was set at p < 0.05; otherwise, labeled as “not significant (n.s.)”. References

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Claims

ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 What is claimed is:

1. A composition for use as an in-situ-forming hydrogel in treating or reconstructing a surgically incised or wounded area in a mammalian subject in need thereof, comprising an in situ formed polymer network of one or more polymers cross-linked through nanoclusters.

2. The composition of claim 1, wherein the nanoclusters comprise albumin.

3. The composition of claim 1 or claim 2, wherein the nanoclusters further comprise a therapeutic agent.

4. The composition of any of the preceding claims, wherein the nanocluster is crosslinked to the one or more polymers by a strain-promoted alkyne azide cycloadditions (SPAAC) reaction.

5. The composition of any of the preceding claims, wherein the nanocluster is derivatized with a SPAAC reactant at a degree of substitution from about 25 to 50.

6. A composition for use as an in-situ-forming hydrogel in treating or reconstructing a surgically incised or wounded area in a mammalian subject in need thereof, comprising an in situ formed polymer network of one or more cross-linked polymers; and one or more therapeutic factors linked to the hydrogel by a photocleavable linker that released the factor when exposed to light at a wavelength of from 300 to 450 nm.

7. The composition of claim 6, wherein the therapeutic factor is a growth factor.

8. The composition of claim 6 or claim 7, comprising two or more two or more different linkers, where the linkers may cleave at different wavelengths.

9. The composition of claim 8, wherein release of the therapeutic agent(s) from the different linkers is sequential, staggered, or simultaneous.

10. The composition of any of the preceding claims wherein the the polymer is collagen, hyaluronic acid, derivatives thereof, or a combination thereof.

11. The composition of any of the preceding claims, wherein the polymer is derivatized with a click chemistry reactant at a degree of substitution of from about 25 to 75.ATTORNEY DOCKET: STAN-2207WO CLIENT REFERENCE:S24-226 12. The composition of any of the preceding claims, wherein the hydrogel is formed by equally mixing albumin nanoclusters, hyaluronic acid and collagen in a 1:1:1 ratio (v / v / v).

13. The composition of any of the preceding claims, wherein the water content of the hydrogel is up to 80% weight / volume, up to 81%, up to 82%, up to 83%, up to 84%, up to 85%, up to 86%, up to 87%, up to 88%, up to 89%, up to 90%, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%.

14. The composition of any of the preceding claims, wherein the ratio of collagen to HA weight / weight is from about 1:25; 1:20; 1:15; 1:10: 1:5; 1:

1.

15. The composition of any of the preceding claims, wherein the hydrogel is applied to a tissue defect in a flowable liquid state and allowed to form a smooth contour on its surface as it gels.

16. The composition of claim 14, wherein said tissue defect is in an ocular tissue such as the cornea, sclera, or nerve.

17. The composition of any of the preceding claims, wherein the polymer comprises a therapeutic agent linked through a photocleavable linker.

18. The composition of claim 15, wherein the photocleavable linker is o-nitrobenzyl based.

19. The composition of claim 15 or claim 16, wherein the photocleavable linker releases at from 300 to 450 nm light.

20. The composition of any of claims 15-17, wherein the therapeutic agent is a growth factor.

21. A method of treating or reconstructing a surgically incised or wounded corneal area in a mammalian subject in need thereof, comprising administering a composition of any of claims 1-19.

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