WO2024258996A1 - Ocular drug delivery - Google Patents

Abstract

Embodiments of the disclosure encompass ocular therapeutic devices, including those that are 3D printed. In specific embodiments, the disclosure concerns 3D bioprinted drug deliver for ocular medical conditions of any kind. In particular embodiments, the disclosure concerns 3D bioprinted antifibrotic drug delivery with a 3D scaffold for the treatment of medical conditions associated with the cornea, such as corneal fibrosis.

Description

OCULAR DRUG DELIVERY

[0001] The present invention claims priority to U.S. Provisional Application Serial No. 63/508,612 filed on June 16, 2023, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under Grant EY026950 awarded by the National Institutes of Health. The government has certain rights in the invention.

I. Technical Field

[0003] This disclosure relates at least to the fields of biology, ophthalmology, cell biology, molecular biology, devices, and medicine.

II. Background

[0004] An optically transparent and clear cornea is essential for vision [1], Injuries to eyes including corneal perforations, deep corneal abrasions, chemical burns, and persistent ulceration are the major causes of destructive corneal scarring that result in vision loss [2], Each year approximately 2.4 million eye injuries occur in the United States with more than 20% of these injuries resulting in vision loss [3], Corneal scars block the light transmission and impair the visual function [4,5], The scarred and damaged corneas need to be surgically replaced with corneal transplants (penetrating keratoplasty) to restore vision [6], However, a short supply of donor corneas limits the number of corneal transplant procedures. Furthermore, postoperative complications and graft rejection reduce the functional survival time of the corneal allografts [6], Alternatives to corneal transplantation, such as stem cell therapy, amniotic membrane therapy, decellularized extracellular matrix, cell-free collagen scaffolds, and biomimetic extracellular matrix hydrogels were developed with limited success [7-12], On the other hand, current ocular pharmacotherapies are focused on the rapid healing of corneal wounds, however scarring associated with corneal wound healing is often overlooked [13], For example, eye drop formulations of anti-inflammatory corticosteroid drugs such as Dexamethasone, Prednisolone, or FluoromethoIone are commonly used for treating corneal inflammation and wounds, however they are ineffective in preventing corneal scarring and delay the wound healing process itself [14-16], Current shortage of the donor corneas, ineffectiveness of the therapeutic interventions, and the lack of effective pharmacological treatments emphasize the need for the development of novel therapeutics for the attenuation of corneal scarring and preventing vision loss. Despite considerable progress made in the understanding of corneal wound healing mechanisms, effective antifibrotic drugs are still not available to minimize corneal fibrosis.

BRIEF SUMMARY

[0005] Embodiments of the disclosure include compositions, methods, and kits that are utilized for treatment of one or more ocular medical conditions. In specific embodiments, the ocular medical condition(s) may be treated topically. In specific embodiments, the ocular medical condition(s) may be treated on the surface of the eyeball. One or more eyes in a mammal may be affected.

[0006] In particular embodiments, one or more therapeutic agents that will treat the one or more ocular medical conditions is provided to the eye with a device. The device may comprise the one or more therapeutic agents on part or all of its surface and/or within the structure(s) of the device itself. In specific embodiments, the device is utilized as a scaffold for delivery of the one or more therapeutic agents. In particular embodiments, the device is 3D-printed which may allow for control of structure of the device.

[0007] In certain embodiments, the present disclosure concerns the fabrication of an antifibrotic drug-loaded membrane (referred to herein as theramem) and its efficacy on scarless corneal wound healing (FIG. 1). In particular embodiments, an antifibrotic Nintedanib (NIN)- loaded theramem (NIN-theramem) was fabricated by 3D-bioprinting strategy. NIN is a broad tyrosine kinase inhibitor with known modulatory activity on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) receptors. NIN inhibits fibroblast proliferation and the myofibroblast (MFB) trans-differentiation from multiple resident fibroblast populations. NIN has been approved by the FDA for the treatment of idiopathic pulmonary fibrosis (IPF) and systemic sclerosis associated interstitial lung disease. The NIN-theramem efficacy was evaluated on enabling the drug molecular transport into the corneal tissue and producing scarless corneal wound healing in a mouse alkali induced ocular burn (OB) model.

[0008] Embodiments of the disclosure include a device, comprising 3D-printed material comprising one or more ocular therapeutic agents. In some cases, the device comprises a membrane. In particular embodiments, the ocular therapeutic agent treats or prevents corneal fibrosis. The device may be or may comprise a wafer, a nanowafer, a scaffold, a film, or a combination thereof. In specific embodiments, the device comprises a configuration of filaments that may comprise the one or more ocular therapeutic agents on their surface, within the filaments, or both. In some cases, the configuration of filaments is random, whereas in other cases the configuration of filaments is ordered. In some embodiments, the device comprises both random and ordered filaments. The interior of the filaments may be hollow or solid. In specific embodiments, the interior of the filaments comprises two or more coaxial layers. In some embodiments, the ordered configuration of filaments comprises multiple intersections of fibers, each intersection comprising an angle between about 1-179°. The ordered configuration of filaments comprises multiple intersections of fibers having 90° angles, in some embodiments. The device may or may not comprise apertures. In embodiments wherein the device comprises one or multiple apertures, the multiple apertures may or may not be substantially the same size. The shape of multiple apertures may generally be a polygon (e.g., a square, a rectangle, a diamond, a hexagon, etc.), or a circle. In some embodiments, the device comprises a circular shape comprising a diameter between about 1.0 mm and 15.0 mm. In some embodiments, the device comprises a polygonal shape comprising a width or diameter between about 1.0 mm and 15.0 mm. In some embodiments, the device is comprised in a liquid, such as a pharmaceutically acceptable solution. The liquid may comprise one or more ocular therapeutic agents, and the one or more ocular therapeutic agents in the liquid may be the same as the one or more ocular therapeutic agents of the device. In certain cases, one or more ocular therapeutic agents of the liquid is not the same one or more ocular therapeutic agents of the device. In some embodiments, the therapeutic agent comprises a tyrosine kinase inhibitor. In some embodiments, the device comprises between about 1 pg to 12 pg of the tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is nintedanib. In some embodiments, the device comprises between about 1 pg to 12 pg of nintedanib. In particular embodiments, the material of the device comprises a polymer having a charge that will affect release kinetics of the one or more therapeutic agents and/or polymer(s) from the device. The material of the device may comprise a polymer having a charge that will induce a synergistic effect on release kinetics of the one or more therapeutic agents and/or polymer(s) from the device. In some embodiments, the release kinetics comprise release of the ocular therapeutic agent at an average rate of about 50 ng to 1000 ng per hour, or any range derivable thereof.

[0009] Embodiments of the disclosure include methods of treating an ocular medical condition in an individual, comprising the step of applying any device encompassed herein to at least one eye of the individual. The ocular medical condition may affect one eye or both eyes. The ocular medical condition may affect the cornea. The ocular medical condition may or may not be corneal fibrosis, trauma, a genetic defect of the individual, a burn, corneal neovascularization, pterygium, meibomian gland dysfunction, an allergy, conjunctivitis, bacterial conjunctivitis, viral conjunctivitis, asthenopia, corneal injury, chemical exposure, infectious keratitis, bacterial keratitis, viral keratitis, ulcers, alkali burn, keratoconus, Fuchs' endothelial dystrophy, bullous keratopathy, ocular inflammation, ocular pain, dry eye, ocular infection, need for regenerative healing of the eye, corneal perforation, deep corneal abrasion, eye surgery, or a combination thereof. In a specific embodiment, the device comprises one or more antifibrotic agents. The therapeutic agent may or may not be nintedanib, pirfenidone, nilotinib, dexamethasone, or a combination thereof. In some embodiments, the device is comprised in a liquid, such as a pharmaceutically acceptable solution.

[0010] In certain embodiments, the device is applied to the eye of the individual prior to onset of the ocular medical condition. The device may be applied to the eye of the individual following onset of the ocular medical condition. In some cases, the device is applied to the eye of the individual prior to onset of scarring of the cornea. In some embodiments, the device may be applied to an individual at risk for having scarring of the cornea. In some embodiments, the device is applied once to the affected eye or eyes, or it may be applied more than once to the affected eye or eyes, such as daily, weekly, multiple times during a week, or monthly. In specific embodiments, the individual is a mammal, such as a human. In specific embodiments, the method further comprises the step of producing the device, such as by 3D printing of at least part of the device, including producing coaxial layers of filaments of the device. In specific cases, the 3D printed material of the device comprises one or more therapeutic agents. The device may or may not comprise one or more nerve regenerating factors and one or more neurotrophic factors. The therapeutic agent may be leteprinim. In specific embodiments, the device comprises one or more of each of an analgesic, anesthetic, antibiotic, antiviral, and/or nerve-blocking agent. In specific embodiments, the analgesic is pregabalin, gabapentin, bupivacaine, proparacaine, lidocaine, oxybuprocaine, tetracaine, or a combination thereof. In some embodiments, the device comprises antibiotics. In some embodiments, the antibiotics comprise bacteriostatic or bactericidal antibiotics. In some embodiments, the device comprises an antiviral. In some embodiments, the antiviral comprises a protease inhibitor, reverse transcriptase inhibitor, integrase inhibitor, viral entry inhibitor, and/or maturation inhibitors.

[0011] Embodiments of the disclosure include methods of producing any device encompassed herein, comprising the step of 3D printing a water-soluble polymer, optionally with one or more therapeutic agents mixed therein. The polymer may be polyvinyl alcohol (PVA). The polymer may be mucoadhesive. The polymer may be dissolvable on the eye.

[0012] Embodiments include kits comprising any device encompassed herein, wherein said device is housed in a suitable container. The kit may or may not comprise one or more ocular therapeutic agents. In specific embodiments, the kit further comprises one or more ocular therapeutic agents housed separately from the device.

[0013] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIGS. 1A-1E. NIN-theramem. FIG. 1A shows a bright field image of an NIN- theramem; FIG. IB shows a fluorescence image of an NIN-theramem; and FIG. 1C shows an NIN-theramem applied on a mouse eye. FIG. ID shows an XRD analysis of an NIN-Theramem. Scale bar: 1000 pm. FIG. IE shows examples of BioCAD designs and projections of a 3D- bioprinted device, e.g., a theramem. [0016] FIGS. 2A-2I. NIN-theramem safety on the ocular surface. FIGS. 2A-2B show results of an evaluation of NIN-theramem toxicity and maximum tolerated drug dose in human corneal epithelial cells and human corneal fibroblasts in vitro (for each plot, bars from left to right represent untreated (UT), NIN-theramem 3 pg, NIN-theramem 6 pg, NIN-theramem 9 pg, and NIN- theramem 12 pg, respectively). Whole eye images and H&E-stained corneal sections of healthy eyes (FIGS. 2C-2F), alternate day (q.o.d.) NIN-theramem treated (FIGS. 2D-2G); and once a day (q.d.) NIN-theramem treated (FIGS. 2E-2H) for 14-days. FIG. 21 shows a plot depicting the corneal thickness. *P<0.05 and ***<0.001

[0017] FIGS. 3A-3G. NIN-theramem was more effective in enhancing the NIN drug diffusion into the cornea compared to the topical NIN-eyedrops. NIN eyedrop instilled on the mouse eye: Fluorescence image (FIGS. 3A-3B), and Negative ion mode DESI-MS image of m/'z 538.25 corresponding to NIN (FIG. 3C); and NIN-theramem applied on the mouse eye: Fluorescence image (FIGS. 3D-3E), and Negative ion mode DESI image of m/z 538.25 corresponding to NIN (FIG. 3F). FIG. 3G shows NIN concentration in the corneal tissue determined by liquid chromatograph mass spectrometry (n=5 corneas/timepoint); for each time-point, the left bar represents the group treated with NIN-eyedrops and the right bar represents the group treated with NIN-theramem.

[0018] FIGS. 4A-4R. NIN-theramem promotes scarless corneal wound healing in ocular alkali burn (OB)-induced mouse model. Representative bright field and fluorescence images of Balanced Saline Solution (BSS) eyedrops (FIGS. 4A-4D), NIN-eyedrops (FIGS. 4E-4H), and NIN-theramem treated mouse eyes (FIGS. 4I-4L). H&E-stained sections of healthy cornea (FIG. 4M); OB corneas treated with BSS eyedrops (control) (FIG. 4N); OB corneas treated with NIN- eyedrops (FIG. 40); and OB corneas treated with NIN-theramems (FIG. 4P). FIG. 4Q shows a plot depicting the corneal thickness (n=3). NIN-theramem treatment promotes the recovery of corneal sensitivity measured by Cochet-Bonnett esthesiometry (n=3) (FIG. 4R).

[0019] FIGS. 5A-5F. NIN-theramem modulates corneal fibrosis in alkali induced ocular burn mouse model. Mouse corneal sections stained for fibronectin (FIGS. 5A-5C); and Collagen 1 (FIGS. 5D-5F).

[0020] FIGS. 6A-6F. NIN-theramem treatment promotes corneal wound healing. Corneal sections stained with Ki67 for dividing epithelial cells (white, FIGS. 6A-6C) and keratin-13 (white, FIGS. 6D-6F). Healthy corneas (FIG. 6A and FIG. 6D); BSS-ey edrops treated corneas (control) (FIG. 6B and FIG. 6E); and NIN-theramem treated OB corneas (FIG. 6C and FIG. 6F). Scale bar: 50pm

[0021] FIGS. 7A-7H. NFS! regulated corneal keratocyte to myofibroblast differentiation. In vitro NIN treatment inhibited the human corneal keratocyte (hCK) transformation to corneal myofibroblasts: Untreated hCK (control) (FIG. 7A); hCK treated with TGF-0 (FIG. 7B); and hCK treated with TGF-P and NIN (FIG. 7C). Mouse corneal sections stained with a-SMA for fibroblast and myofibroblast density: Healthy (FIG. 7D); PBS treated (control) (FIG. 7E); and NIN-theramem treated corneas (FIG. 7F). FIG. 7G shows quantitative flow cytometry analysis demonstrating the NIN-theramem ability to attenuate the fibroblast to myofibroblast differentiation (for each plot, left bar: healthy; middle bar: BSS; right bar: NOIN-theramem). Scale bar: 100 pm. Mean differences of the groups were considered significant at **P < 0.01.

[0022] FIG. 8. NIN-theramem downregulate the proangiogenic and profibrotic molecular mediators. *P < 0.05, **P < 0.01, ***P<0.001. NIN-theramem treatment downregulates the TGF- P expression level compared to the BSS treated mouse OB corneas (control) (FIG. 8A; for each plot, left bar: BSS (ctrl); right bar: NIN-theramem). Ctrl: control.

[0023] FIGS. 9A-9H. NIN-theramem treatment reversed the established corneal scars. Bright field and fluorescence images of BSS treated (FIGS. 9A-9D), and NIN-theramem treated OB mouse eyes (FIGS. 9E-9H).

[0024] FIGS. 10A-10F. Coaxially bioprinted ocular drug delivery 3D-scaffold. FIG. 10A shows a fluorescence image of an NIN-scaffold. FIG. 10B shows a confocal image of a tubular coaxial filament showing the outer shear and inner core. FIG. 10C shows a 3D-scaffold applied on a mouse eye. FIG. 10D shows a coaxially 3D-printed scaffold. FIG. 10E shows a microfilament structure of the 3D-scaffold under high magnification. FIG. 10F shows a 3D- printed scaffold on a fingertip. Scalebar 8 mm.

[0025] FIG. 11. 3D-scaffold drug delivery of NIN was more effective in enhancing the drug diffusion into the cornea compared to the topical NIN eyedrops. Bars represent the amount of drug released at the respective timepoint. Lines represent the cumulative amount of drug released over time. The darker portion of each bar represents NIN-drops while the lighter portion of each bar represents NIN-scaffold.

[0026] FIGS. 12A-12G. NIN-scaffold safety on the ocular surface. Whole eye and H&E- stained corneal sections of Healthy eyes (FIGS. 12A-12B); eyes treated with NIN-scaffold on alternate days (q.o.d.) for 14 days (FIGS. 12C-12D); and eyes treated daily (q.d.) with NTN- scaffold for 14 days (FIGS. 12E-12F). FIG. 12G shows a plot depicting the corneal thickness. [0027] FIG. 13. A plot depicting corneal thickness (for each plot, left bar: healthy cornea; middle bar: NIN-drops; and right bar: NIN-scaffold).

[0028] FIGS. 14A-14F NIN-scaffold modulated corneal fibrosis in alkali-induced ocular burned mouse eyes. COL1A1 and Fibronectin-stained mouse corneal sections of healthy (FIG. 14A and FIG, 14D); BSS-ey edrops treated (control) (FIG. 14B and FIG. 14E); and NIN-scaffold treated (FIG. 14C and FIG. 14F). Scale bar: 50 pm.

[0029] FIG. 15 Flow cytometry analysis of corneal myofibroblast densities in the corneal tissue. For each plot, left bar: healthy; middle bar: BSS (control); and right bar: NIN-scaffold. [0030] FIGS. 16A-16K. NIN-theramem treatment aided in reversing established corneal scar. FIGS. 16A-16D show BSS treated OB mouse eyes. FIGs. 16E-16H show NIN-theramem treated OB mouse eyes. FIGS. 16I-16K show representative H&E images of harvested mouse cornea at day 21 : Healthy (FIG. 161), BSS Treated (FIG. 16J), and NIN-theramem Treated (FIG. 16K). Scale bar: 50pm

DETAILED DESCRIPTION

[0031] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0032] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0033] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.

[0034] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. [0035] The terms “3D-printing” and “3D-bioprinting” may be used interchangeably throughout the specification and refer to a method of forming a three-dimensional (3D) structure by laminating materials, such as powders, liquids, or polymers, layer-by-layer as to form the 3D structure.

[0036] The terms “wafer” or “nanowafer” refer to structures containing reservoirs, e.g., nanosized reservoirs, for improved delivery of a substance, such as a drug.

[0037] The term “membrane” refers to a thin film. A membrane may be prepared by 3D bioprinting, casting, or any other method commonly known in the art.

[0038] The term “scaffold” refers to a hollow or solid 3D-printed core-shell polymer for deliver substances, such as drugs.

[0039] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0040] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

* * * *

[0041] Approximately 12 million people 40 years and over in the United States have vision impairment, including 1 million who are blind, 3 million who have vision impairment after correction, and 8 million who have vision impairment due to uncorrected refractive error (Vision Health Initiative (VHI); Centers for Disease Control). The World Health Organization estimates that corneal opacity accounts for over 4% of blindness worldwide, leading to around 1.5-2 million cases yearly (McKay et al., Eye volume 34, pages 271-278 (2020)). Corneal fibrosis develops in response to injury, infection, postsurgical complications, or underlying systemic disease that disrupts the tissue's homeostasis, leading to irregular extracellular matrix deposition within the stroma.

[0042] To date, the only effective treatment for vision loss due to corneal scarring is corneal transplantation. However, acute complications from the transplant include wound leakage, raised intra-ocular pressure, bleeding, and infection (Maghsoudlou P, Sood G, Akhondi H. Cornea Transplantation. [Updated 2022 Jul 25], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 Jan). Late complications, occurring months to years postoperatively, include graft rejection, corneal swelling, cataracts, astigmatism, and recurrence of the original disease. Overall, the 5-year and 15-year graft survival rates are approximately 70% and 50%, respectively. The situation is compacted by a shortage of donor tissues, resulting in 12.7 million patients waiting for corneal implants. To improve rejection and decrease complications, researchers have investigated the use of cell-free collagen scaffolds, bioengineered constructs, stem cell therapy, and corneal epithelial cell sheets; however, none of these have entered the commercial market. The inventors provide herein a scaffold for treatment, thereby reducing the complications and failure of transplants.

[0043] The present disclosure provides herein a novel 3D-printed drug-comprising scaffold, such as to be used as an alternative to transplants (for example) for treating any ocular medical condition. Such medical conditions may affect the cornea, such as corneal fibrosis.

I. Embodiments of the Device

[0044] Embodiments of the disclosure include devices that are used for delivery of at least one therapeutic drug to the surface of an eye. In specific embodiments, the device is 3D-printed. In certain embodiments, the device is a drug-delivering wafer or nanowafer. In some embodiments, the device is a membrane. The device may be an ocular drug delivery wafer, nanowafer, or membrane that is therapeutic to treat any ocular medical condition, including at least medical diseases or injuries that affect the cornea. In specific embodiments, the device is an antifibrotic drug-loaded wafer, nanowafer, or membrane, although the membrane may comprise apertures. In specific embodiments, the device is a 3D-printed drug delivery modality.

[0045] In particular embodiments, the device will dissolve over time following placement on the eye, whereas in alternative embodiments the device is not dissolvable. In certain embodiments, the device is comprised of multiple filaments, such as in an array of filaments. The filaments may or may not be hollow. In specific cases, the device comprises a configuration of filaments. In any case, the filaments may comprise the one or more ocular therapeutic agents on their surface, within the filaments, or both. The device may have a particular configuration of filaments, and the configuration may be determined by the need of the individual receiving the device. In some cases, the configuration of filaments is random, whereas in other cases, the configuration of filaments is ordered. The configuration of filaments may be such that there are apertures in the device, and the apertures may be of any shape, including a polygon (e.g., a square, a rectangle, a diamond, a hexagon, etc.), a circle, or there may be no particular or repeating shape to the aperture. Multiple apertures of the device may or may not be of the same shape and size. In specific cases, an ordered configuration of filaments comprises multiple intersections of fibers, wherein the intersections have 90° angles. In certain embodiments, multiple filaments may be perpendicular to other multiple filaments. In such cases, the filaments may be perpendicular with respect to an edge of the device, and in other cases the filaments may be non-perpendicular with respect to an edge of the device. In specific cases, an ordered configuration of filaments comprises multiple intersections of fibers, wherein the intersections have an acute angle, e.g., 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 11°, 12°, 13°, 14°, 15°, 16°, 17°, 18°, 19°, 20°, 21°, 22°, 23°, 24°, 25°, 26°, 27°, 28°,

29°, 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°,

50°, 51°, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°, 61°, 62°, 63°, 64°, 65°, 66°, 67°, 68°, 69°, 70°,

specific cases, an ordered configuration of filaments comprises multiple intersections of fibers, wherein the intersections have an obtuse angle, e.g., 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 111°, 112°, 113°, 114°, 115°,

116°, 117°, 118°, 119°, 120°, 121°, 122°, 123°, 124°, 125°, 126°, 127°, 128°, 129°, 130°, 131°,

132°, 133°, 134°, 135°, 136°, 137°, 138°, 139°, 140°, 141°, 142°, 143°, 144°, 145°, 146°, 147°,

148°, 149°, 150°, 151 °, 152°, 153°, 154°, 155°, 156°, 157°, 158°, 159°, 160°, 161°, 162°, 163°,

164°, 165°, 166°, 167°, 168°, 169°, 170°, 171°, 172°, 173°, 174°, 175°, 176°, 177°, 178°, or 179°. In specific cases, an ordered configuration of filaments comprises multiple intersections of fibers, wherein the intersections comprise acute and oblique angles.

[0046] In certain embodiments, the device is fabricated by 3D printing of tubular filaments, and a drug-polymer matrix comprises its inner core. In specific embodiments, the polymer matrix may comprise one or more drugs, one or more biologies, one or more polymeric particles, one of more biologically-derived polymers, one or more synthetic polymers, one or more drug diffusion (penetration) enhancers, and/or one or more drug loaded polymer micro/nanoparticles.

[0047] In specific embodiments, the device is configured and/or comprised of suitable material(s) to allow for the device to adhere to an ocular surface. In particular embodiments, there is a 3D-bioprinted device with controlled drug release attributes. In specific embodiments, the device is configured and/or is comprised of suitable material(s) to allow for elution of one or more therapeutic agents over a duration of time, such as over 1-24 hours, 1-7 days, 1-28 days, 1-4 weeks, and so forth. In specific embodiments, the duration of time is 1-28, 1-25, 1-20, 1-14, 1-10, 1-7, 2- 28, 2-25, 2-20, 2-14, 2-10, 2-7, 4-28, 4-25, 4-20, 4-14, 4-10, 4-7, 7-28, 7-25, 7-20, 7-14, 7-10, 10- 28, 10-25, 10-20, 10-14, 14-28, 14-25, 14-20, 20-28, 20-25, or 25-28 days. In specific embodiments, one or more therapeutic agents elutes from a suitable material in 1-4 hours. In specific embodiments, the drug is released from the device as the device itself dissolves. In some embodiments, the drug molecules are homogeneously dispersed within the device, including in the matrix of the device in some cases, although alternative embodiments they are not.

[0048] The material of the 3D-printed device may be fabricated with any material suitable for therapeutic use, but in specific embodiments the device is made with a poly(vinyl alcohol) (PVA) solution, Polyvinyl pyrrolidone (PVP), methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, polyacrylic acid, poly(lactic-co-glycolic) acid (PLGA), poly lactic acid (PLA), poly glycolic acid (PGA), hyaluronic acid, gelatin, polyethylene glycol (PEG), dextran, any polysaccharide, Calcium Stearate, polyvinylidene fluoride (PVDF), polyhydroxyethyl methacrylate (pHEMA), silicone, silicone hydrogels (e.g., galyfilcon, senofdcon, comfilcon, enfilcon), Arginine derivatives, chitosan derivatives, cyclodextrins, carboxymethylcellulose, polycaprolactone, alginate, carrageenan, chondroitin derivatives, dextran sulfate, or a combination thereof. The device may be 3D-printed in multiple layers. In some embodiments, the device is 3D-printed and has the ability to enable drug molecular transport into the cornea. In some embodiments, the device may be made via molding, casting, or laser sintering.

[0049] In some embodiments, the device may have any shape, including but not limited to a polygon or a circle. In some embodiments, the diameter of the device may be between about 1 mm to about 15 mm. In some embodiments, the diameter of the device may be 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5.0 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm, 6.0 mm, 6.1 mm, 6.2 mm, 6.3 mm, 6.4 mm, 6.5 mm, 6.6 mm, 6.7 mm, 6.8 mm, 6.9 mm, 7.0 mm, 7.1 mm, 7.2 mm, 7.3 mm, 7.4 mm, 7.5 mm, 7.6 mm, 7.7 mm, 7.8 mm, 7.9 mm, 8.0 mm, 8.1 mm, 8.2 mm, 8.3 mm, 8.4 mm, 8.5 mm, 8.6 mm, 8.7 mm, 8.8 mm, 8.9 mm, 9.0 mm, 9.1 mm, 9.2 mm, 9.3 mm, 9.4 mm, 9.5 mm, 9.6 mm, 9.7 mm, 9.8 mm, 9.9 mm, 10.0 mm, 10.1 mm, 10.2 mm, 10.3 mm, 10.4 mm, 10.5 mm, 10.6 mm, 10.7 mm, 10.8 mm, 10.9 mm,

11.0 mm, 11.1 mm, 11.2 mm, 11.3 mm, 11.4 mm, 11.5 mm, 11.6 mm, 11.7 mm, 11.8 mm, 11.9 mm, 12.0 mm, 12.1 mm, 12.2 mm, 12.3 mm, 12.4 mm, 12.5 mm, 12.6 mm, 12.7 mm, 12.8 mm,

12.9 mm, 13.0 mm, 13.1 mm, 13.2 mm, 13.3 mm, 13.4 mm, 13.5 mm, 13.6 mm, 13.7 mm, 13.8 mm, 13.9 mm, 14.0 mm, 14.1 mm, 14.2 mm, 14.3 mm, 14.4 mm, 14.5 mm, 14.6 mm, 14.7 mm,

14.8 mm, 14.9 mm, or 15.0 mm. In some embodiments, the diameter of the device is exactly or about 2.0 mm. In some embodiments, the thickness of the device may range between about 60 gm to about 100 gm. In some embodiments, the thickness of the device may be 60 gm, 61 gm, 62 gm, 63 gm, 64 gm, 65 gm, 66 gm, 67 gm, 68 gm, 69 gm, 70 gm, 71 gm, 72 gm, 73 gm, 74 gm, 75 gm, 76 gm, 77 gm, 78 gm, 79 gm, 80 gm, 81 gm, 82 gm, 83 gm, 84 gm, 85 gm, 86 gm, 87 gm, 88 gm, 89 gm, 90 gm, 91 gm, 92 gm, 93 gm, 94 gm, 95 gm, 96 gm, 97 gm, 98 gm, 99 gm, or 100 gm. In some embodiments, the device has a thickness of exactly or about 80 gm.

[0050] In some embodiments the device is flat. In some embodiments, the device is curved. In some embodiments, the device is concave. In some embodiments, the device has a base curve between about 8.0 mm to about 10.0 mm. In some embodiments, the base curve may be 8.0 mm,

8.1 mm, 8.2 mm, 8.3 mm, 8.4 mm, 8.5 mm, 8.6 mm, 8.7 mm, 8.8 mm, 8.9 mm, 9.0 mm, 9.1 mm,

9.2 mm, 9.3 mm, 9.4 mm, 9.5 mm, 9.6 mm, 9.7 mm, 9.8 mm, 9.9 mm, or 10.0 mm.

[0051] In certain embodiments, the device is housed in a liquid, such as an aqueous liquid that is sterile.

II. Embodiments of the Therapeutic Agent(s)

[0052] In particular embodiments, the device comprises at least one therapeutic agent of any kind. The therapeutic agent or agents may be therapeutic or preventative for an ocular medical condition, including one that stems from disease, genetics, or injury. The therapeutic agent may comprise one or more of a small molecule, a protein, nucleic acid, an antibody, a cell (including engineered cells), an enzyme, and so forth. The therapeutic agent may be an antifibrotic agent, an anti-inflammatory agent, an analgesic, an antibiotic, etc. In specific embodiments, the therapeutic agent is a tyrosine kinase inhibitor, such as a VEGFR-2 tyrosine kinase inhibitor. In some cases, the therapeutic agent is a corticosteroid (Dexamethasone, Prednisolone, and/or FluoromethoIone). The therapeutic agent may be nintedanib, pirfenidone, nilotinib, dexamethasone, or a combination thereof, in specific embodiments. In some cases, the therapeutic agent is mitomycin C In certain embodiments, the therapeutic agent inhibits fibroblast differentiation into myofibroblasts.

[0053] The skilled artisan recognizes that the amount of therapeutic agent associated with the device will be an amount that is not cytotoxic or have deleterious effects for the individual receiving the device. In certain embodiments, a particular amount of therapeutic agent is utilized, including no more than a certain level, and depending on the therapeutic agent.

[0054] In specific embodiments, one or more of the following therapeutic agents may be utilized with the device for any indication: Acetazolamide for glaucoma, Acetylcysteine for dry eyes, Aciclovir eye ointment, Antazoline and xylometazoline eye drops, Apraclonidine eye drops, Atropine eye drops, Azelastine eye drops for allergies, Azithromycin eye drops, Betamethasone eye drops, Betaxolol eye drops for glaucoma, Bimatoprost eye drops, Brimonidine eye drops for glaucoma, Brinzolamide eye drops for glaucoma, Bromfenac eye drops, Carbomer liquid eye gels, Carmellose sodium for dry eyes, Carteolol eye drops for glaucoma, Chloramphenicol for eye infections, Ciprofloxacin eye preparations, Cyclopentolate eye drops, Dexamethasone eye drops for inflammation, Diclofenac eye drops, Dorzolamide eye drops for glaucoma, Emedastine eye drops for hay fever, Epinastine eye drops for hay fever, FluoromethoIone eye drops for inflammation, Flurbiprofen eye drops, Fusidic acid eye drops, Ganciclovir eye gel, Gentamicin eye drops, Homatropine eye drops, Hypromellose eye drops, Ketorolac eye drops, Ketotifen eye drops for hay fever, Latanoprost eye drops, Levobunolol eye drops for glaucoma, Levofloxacin eye drops, Lodoxamide eye drops, Loteprednol eye drops for inflammation, Moxifloxacin for eye infections, Nedocromil sodium eye drops, Nepafenac eye drops, Ofloxacin eye drops, Olopatadine eye drops for hay fever, Paraffin-based eye ointments for dry eyes, Pilocarpine eye drops for acute glaucoma, Pilocarpine tablets, Polyvinyl alcohol for dry eyes, Prednisolone eye drops for inflammation, Rimexolone eye drops for inflammation, Sodium cromoglicate eye drops, Sodium hyaluronate for dry eyes, Soybean oil eye drops for dry eyes, Tafluprost eye drops, Timolol eye drops for glaucoma, Tobramycin for eye infections, Travoprost eye drops, and Tropicamide eye drops. In addition to this, or as alternative(s), the therapeutic agent may comprise (1) Corticosteroids, such as dexamethasone, prednisolone, fluormethoIone, and/or loteprednol etabonate; (2) Non-steroidal anti-inflammatory drugs (NSAIDs), such as Ketorolac tromethamine, bromfenac, nepafenac, flurbiprofen, and/or diclofenac; (3) Anti-infection therapeutic agents, such as fungal -amphotericin B, fluconazol, voriconazole, and/or natamycine; (4) anti-bacterial therapeutic agents, such as ciprofloxacin, ofloxacin, moxifloxacin, polymyxin b/trimethoprim, levofloxacin, gatifloxacin, azithromycin, bacitracin, and/or erythromycin; anti-viral therapeutic agents, such as ganciclovir, trifluridine, and/or acyclovir; (4) biologies, such as anti-VEGF, DNA, and/or RNA; (5) one or more pupil dilators; (6) therapeutic agents for Glaucoma, such as brimonidine, lopidine, timolol, betimol, and/or betaxolol; (7) antihistamines, such as bepotastine, emedastine, epinastine, lodoxamide, nedocromil, and/or pemirolast; (8) Anesthetics, such as proparacaine, lidocaine, and/or chloroprocaine; (9) vitamins, such as Vitamin B and/or Vitamin C; and/or (10) Phenylephrin, tropicamide, and/or cyclosporine. In some embodiments, the therapeutic agent may comprise bacteriostatic antibiotics (e.g., glycylcyclines, tetracyclines, lincosamides, macrolides, oxazolidinones, and sulfoamides) or bactericidal antibiotics (e.g., aminoglycosides, beta-lactams, fluoroquinolones, glycopeptides, cyclic lipopeptides, and nitroimidazoles). In some embodiments, the therapeutic agent comprises an antiviral, such as a protease inhibitor (e g., telapivir, atazanavir, darunavir, simeprevir, lopinavir, ritonavir, boceprevir, ritonavir, indinavir, nelfinavir, fosamprenavir, saquinavir, and tipranavir), which may target enzymes involved in viral replication and/or assembly; reverse transcriptase inhibitors, including nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) (e.g., nevirapine, penciclovir, tenofovir disoproxil, zidovudine, foscarnet, efavirenz, stavudine, delavirdine, lamivudine, and adefovir dipivoxil), which may block viral replication; integrase inhibitors (e.g., dolutegravir, raltegravir, cabotegravir, and elvitegravir), which may block integration of the viral genome into the host’s genome; viral entry inhibitors, which may interfere with binding, fusion, and/or entry of a virus into a host cell; and/or maturation inhibitors, which may inhibit viral assembly and/or packaging. [0055] In some embodiments, the device comprises between about 0.001 to about 500 pg, or any range deliverable therein, of the therapeutic agent. In some embodiments, the device comprises 0.001 pg, 0.01 pg, 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, 2.0 pg, 3.0 pg, 4.0 pg, 5.0 pg, 6.0 pg, 7.0 pg, 8.0 pg, 9.0 pg, 10.0 pg, 11.0 pg, 12.0 pg, 13.0 pg, 14.0 pg, 15.0 pg, 16.0 pg, 17.0 pg, 18.0 pg, 19.0 pg, 20.0 pg, 21.0 pg, 22.0 pg, 23.0 pg, 24.0 pg,

25.0 pg, 26.0 pg, 27.0 pg, 28.0 pg, 29.0 pg, 30.0 pg, 31.0 pg, 32.0 pg, 33.0 pg, 34.0 pg, 35.0 pg,

36.0 pg, 37.0 pg, 38.0 pg, 39.0 pg, 40.0 pg, 41.0 pg, 42.0 pg, 43.0 pg, 44.0 pg, 45.0 pg, 46.0 pg,

47.0 pg, 48.0 pg, 49.0 pg, 50.0 pg, 60.0 pg, 70.0 pg, 80.0 pg, 90.0 pg, 100.0 pg, 110.0 pg, 120.0 pg, 130.0 pg, 140.0 pg, 150.0 pg, 160.0 pg, 170.0 pg, 180.0 pg, 190.0 pg, 200.0 pg, 210.0 pg, 220.0 ^g, 230.0 gg, 240.0 gg, 250.0 gg, 260.0 pg, 270.0 gg, 280.0 gg, 290.0 gg, 300.0 gg, 310.0 gg, 320.0 gg, 330.0 gg, 340.0 gg, 350.0 gg, 360.0 gg, 370.0 gg, 380.0 gg, 390.0 gg, 400.0 gg, 410.0 gg, 420.0 gg, 430.0 gg, 440.0 gg, 450.0 gg, 460.0 gg, 470.0 gg, 480.0 gg, 490.0 gg, 500.0 gg, or any range deliverable therein, of the therapeutic agent. In some embodiments, the device comprises between about or exactly 1.0 gg to about or exactly 12.0 gg of the therapeutic agent. In some embodiments, the device comprises about or exactly 3.0 pg of the therapeutic agent.

III. Embodiments of Methods of Use and Treatment of Ocular Medical Conditions

[0056] Embodiments of the disclosure concern methods of use of a drug delivery device for any ocular medical condition, including for the treatment or prevention of any ocular medical condition. In specific embodiments, the ocular medical condition directly or indirectly affects the cornea. The ocular medical condition may be the result of disease, genetic defects, or injury, as examples.

[0057] The device of the disclosure is well-suited to overcome ocular surface barriers (OSB) because the device adheres to the wet surface of the eye and is stable. The device avoids rapid clearance, such as with eye drops, allowing for enhanced bioavailability of the drug to the eye. As the device remains on the eye, over time the device dissolves, allowing for continual exposure of the cornea to the drug. In some embodiments, there are methods that enable drug molecular transport into corneal tissue. Methods and compositions allow for the ability to surmount ocular surface barriers and enable the drug diffusion into the cornea, thus enhancing the drug bioavailability, compared to other methods in the art.

[0058] Examples of ocular medical conditions include at least glaucoma, corneal fibrosis, corneal injury (such as from impact with a foreign object), chemical exposure, infectious keratitis, ulcers, corneal neovascularization, alkali burn, meibomian gland dysfunction, pterygium, keratoconus, Fuchs' endothelial dystrophy, bullous keratopathy, ocular inflammation, ocular pain, dry eye, ocular infections (viral, bacterial, fungal, etc ), need for regenerative healing of the eye, corneal perforations, deep corneal abrasions, chemical burns, eye surgery, and persistent ulceration and so forth.

[0059] Embodiments of the disclosure include methods of treating or preventing corneal scarring and/or opacification that can result in significant and irreversible vision loss and blindness. In specific embodiments, any of the methods reduce enlargement or swelling of the stroma and epithelium. In some embodiments, an individual avoids a corneal transplant by utilizing the methods encompassed herein. In specific embodiments, any of the methods encompassed herein may delay the onset of corneal scarring and/or opacification and/or reduce the severity of corneal scarring and/or opacification.

[0060] Embodiments of the disclosure include treatment or prevention of an ocular inflammation-related disease. In specific embodiments, there are methods of treating or preventing cornea neovascularization, including reducing the risk of cornea neovascularization. Certain methods include treating corneal burn, such as corneal alkali burn. In some embodiments, there are methods of treating or preventing meibomian gland dysfunction. In certain cases, there are methods of treating or prevention of pterygium. Methods for inhibiting corneal fibrosis are encompassed herein. In certain aspects, methods include those that promote scarless corneal wound healing.

[0061] Individuals subject to methods and compositions encompassed herein may be at risk for scarring in the eye for any reason. In specific embodiments, the methods and compositions prevent corneal scarring and allow for regression of already-established corneal scars. The device allows for promotion of regenerative repair of corneal wounds or diseased tissue.

[0062] Embodiments of the disclosure include methods of reducing the risk for having corneal scars as a result of any cause. In specific embodiments, there are methods of reducing the severity of a corneal scar. In some embodiments, there are methods of reducing the size of a corneal scar. A corneal scar may be smaller in size with the treatment in comparison to not having the treatment. [0063] Embodiments of the disclosure include methods of avoiding partial or full vision loss for any reason, including related to the cornea. Specific cases include methods of avoiding the risk for or avoiding the need for a corneal transplant. Particular embodiments include methods of minimizing corneal fibrosis.

[0064] In some embodiments, there are methods of reducing the amount of corneal keratocyte to myofibroblast transformation or reducing its risk. In specific embodiments, there are methods of enhancing the efficacy in preventing scarring of a cornea, such as a diseased cornea or an injured cornea. In certain embodiments, there are methods of having or enhancing scarless corneal wound healing.

[0065] In certain embodiments, methods of the disclosure result in, or enhance the ability for, corneas to remain clear with no (or reduced) haze and/or opacification (upon onset or risk for an ocular medical condition) compared to when the methods are not employed. In particular aspects, the methods of the disclosure allow for uniform epithelial layer formation upon healing from an ocular medical condition. Methods and compositions of the disclosure allow for corneal re- epithelialization, including without a risk for scarring.

[0066] The 3D-printed drug delivery device of the present disclosure is configured to allow the drug delivery to overcome the ocular surface barrier and facilitate drug diffusion into the cornea. The disclosed ocular drug delivery device enhances the drug bioavailability in the corneal tissue compared to conventional eye drop formulations that do not remain on the surface of the eye for sufficient time to allow sufficient healing, including to avoid scar prevention. In specific embodiments, the 3D-scaffold upon application on the eye will release the drug for an extended period of time, thus increases the drug residence time on the ocular surface and promotes drug transport through the ocular surface barriers into the corneal tissue to enhance the drug bioavailability and efficacy.

[0067] The device may be employed prior to, during, and/or following onset of any ocular medical condition. For example only, in cases wherein an individual is subject to injury, the device may be used following the injury, and in cases wherein an individual is subject to a scheduled surgery, the device may be used prior to, during, and/or following the surgery. In cases wherein an individual is at risk for an ocular medical condition, the device may be utilized at least prior to its onset.

[0068] In particular embodiments, the device improves the drug bioavailability to a part of an eye. The device may be applied topically, in specific embodiments, and the application may or may not be applied to the surface of the eye in a pharmaceutically acceptable liquid. In some embodiments, the device is applied in an eye drop(s). The device may also be applied similar to that of applying a contact to the eyeball.

[0069] Any of the methods encompassed herein may utilize one or more of the following steps (or similar steps): treating one or more ocular medical conditions; preventing one or more ocular medical conditions; preparing a device; 3D-printing a device; 3D-printing a device comprising one or more therapeutic agents; identifying an ocular medical condition; applying a device to the surface of an eye; applying a dissolvable device to the surface of an eye; and so forth IV. Embodiments of Production of the Device

[0070] Embodiments of the disclosure include methods of producing a 3D-printed drug delivery device for treatment or prevention of at least one ocular medical condition. In certain embodiments, the production method fabricates a drug delivery device comprising a structure that comprises one or more therapeutic agents on the surface of the structure, within the structure itself, or both. In specific embodiments, the therapeutic agent is within the structure material as it is being 3D printed. In alternative cases, the therapeutic agent is applied to the device following its printing.

[0071] In particular embodiments, the selection of the material for the 3D printing is done for the purpose of allowing the structure of the device to be dissolvable once placed onto the surface of the eye. In specific aspects, the selection of the configuration of the structure of the device is such that the device applies a sufficient amount of the therapeutic agent(s) to the surface of the eye, including over time, such as including over the duration of time that the device structure dissolves. In specific embodiments, the 3D printing of the device produces an array of filaments for the device, and in some cases the pattern of the array of filaments facilitates release of the therapeutic agent. In particular embodiments the 3D printing produces an array of filaments comprising multiple apertures produced as the filaments are printed. The apertures may be of any size or shape, including a polygon (e.g., a square, rectangle, triangle, diamond, etc.), a circle, or there may be randomly shaped apertures. The interior of the filaments may be solid or hollow.

[0072] In particular embodiments, the production methods generate an antifibrotic drug delivery 3D-scaffold, such as for scarless corneal wound healing. In particular embodiments, the antifibrotic 3D-scaffolds are fabricated by coaxially 3D-bioprinting the filaments that, in specific embodiments, enables the fabrication of 3D-scaffolds comprising arrays of drug-encapsulated filaments (tubular, in some aspects) with programmable drug release kinetics.

[0073] The filaments may be monolithic or coaxial, in certain embodiments, which further enables the tunability/programmability of the drug release kinetics of the device. Currently devices lack the tunability/programmability of their drug release kinetics. In particular embodiments, the filament orientation affects the release kinetics. In particular embodiments, the filament diameter and/or the number of coaxial layers affects the release kinetics.

[0074] In certain embodiments, the width of the filament is about, or is substantially exactly, 50-200 pm. In some embodiments, the width of the filament is about, or substantially exactly, 50- 200, 50-175, 50-150, 50-100, 50-75, 75-200, 75-175, 75-150, 75-100, 100-200, 100-175, 100-150, 150-200, 150-175, or 175-200 pm in width. In specific embodiments, the width of the filament is about, or is substantially exactly, 50, 75, 100, 125, 150, 175, or 200 pm in width. In some embodiments, the filaments have 1 or multiple layers, including 3-5, 3-4, or 4-5 layers. The number of layers may be 1, 2, 3, 4, or 5, or more. The layers may be configured coaxially.

Examples

[0075] The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the embodiments of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

ANTIFIBROTIC DRUG DELIVERY THERAMEM DIRECTED SCARLESS CORNEAL WOUND HEALING

[0076] Eye injuries leading to corneal scarring are a major cause of vision loss and a significant disabling concern among the patient population. Currently, eye injuries are treated with eyedrop formulations of anti-inflammatory and antibiotics drugs with limited success in preventing scarring. In the absence of effective pharmacotherapies, injured patients need corneal transplants to restore vision. The ineffectiveness of current pharmacotherapies and the scarcity of donor corneas emphasize the critical unmet need to develop efficacious antifibrotic therapeutics to prevent corneal scarring and obviate the need for a corneal transplant. In this Example, there is described the development of an innovative ocular drug delivery system delivering antifibrotic drug, nintedanib (NIN) to prevent corneal scarring (as one example only). The inventors fabricated nintedanib-loaded theramem (NIN-theramem) by a 3D-bioprinting strategy. The NIN-theramem rapidly adhered to the wet ocular surface and released NIN for longer than the eyedrop. In alkali- induced ocular burn mouse corneas, it was shown that NIN-theramem treatment enhanced corneal healing and prevented scarring by reducing corneal keratocyte to myofibroblast transformation. This demonstrated the development of a 3D-bioprinted NIN-theramem with controlled drug release attributes and enhanced efficacy in preventing scarring of the injured corneas.

Antifibrotic NIN-theramem fabrication and characterization

[0077] The antifibrotic NIN-theramems were fabricated using a poly(vinyl alcohol) (PVA) solution by 3D-bioprinting strategy. PVA is a water-soluble, mucoadhesive polymer and its aqueous solutions are commonly used in ophthalmic formulations, particularly as artificial tears [28,29], The inventors first prepared a PVA solution (10%, 5mL) and dissolved (20mg) NIN, which was used for layer-by-layer printing. It was determined that a 2 mm circular NIN-theramem contained 3 pg by HPLC analysis. The printed theramem comprised an array of filaments. The NIN-theramems thus fabricated were characterized by optical microscopy, which demonstrated a uniform array of microfilaments in the theramem (FIGS. 1A and IB). X-ray diffraction analysis revealed that the drug molecules are homogeneously dispersed in the PVA matrix of the theramem (FIG. ID)

Theramem was safe on the corneal cell lines in vitro.

[0078] The inventors evaluated the antifibrotic theramem safety in vitro with human corneal epithelial (hCEC) and human corneal fibroblast (hCF) cells. Treatment with a single NIN- theramem containing 3 pg of NIN or two NIN-theramems (6 pg of NIN) did not elicit a toxic effect. However, treatment with three and four theramems corresponding to a higher drug concentration (9 and 12 pg of NIN) caused a decrease in the number of live cells. The cytotoxic effect was more pronounced in hCF cell cultures. Up to 6 pg of NIN treatment had negligible effect on live hCF cells, however a higher NIN concentration (>9 pg) produced cytotoxic effect and an approximately 50% decrease in live hCF cells was observed with 12 pg of NIN treatment, indicating that the hCF cells can safely tolerate up to 6 pg of NIN (FIGS. 2A and 2B).

NIN-theramem was safe on the ocular surface.

[0079] The in vivo safety of the NIN-theramems was evaluated on healthy mouse eyes. The NIN-theramems were applied on the corneas of healthy mice once-a-day (q.d.; n=3 mice) and on every alternate day (q.o.d; n=3 mice) for 14-days. Gross examination indicated the absence of corneal haze, redness, or swelling in q.o.d and q.d. treated eyes compared to the healthy eyes (FIGS. 2C-2E). Histological analysis was performed of the H&E-stained corneal eye sections which also revealed no change in the corneal thickness or integrity. These studies confirmed that both q.d. and q.o.d. NIN-thereamem treatments are safe on the ocular surface (FIGS. 2F-2H and 21).

Theramem enabled drug molecular transport into the cornea.

[0080] Theramems, when applied on the healthy mouse eyes, readily adhered to the wet mucosal surface of the eye and started to disintegrate (FIG. 3). NIN being a green-fluorescent drug, both NIN-theramem dissolution and drug molecular transport into the cornea were monitored by fluorescence time-lapse microscopy. This study revealed that the NIN-theramem was present on the ocular surface for several hours during its dissolution (FIG. 3). The eyes were green fluorescent for 3 h, indicating the presence of NIN-theramem in the cornea (FIG. 3). In comparison, topically instilled NIN-eyedrop formulations were rapidly cleared from the ocular surface and the corneas did not exhibit a measurable green fluorescence, while the eye lids were green fluorescent indicating the rapid clearance of NIN from the ocular surface within 10 minutes (data not included).

[0081] To further confirm the theramem ability to enable drug molecular transport into the cornea, the inventors performed Desorption electrospray ionization mass spectrometry (DESI-MS) imaging that allowed for the detection of NIN directly from samples and further provided visualization of its spatial distribution within the cornea [30,31], The inventors applied either NIN- theramem or NIN-eyedrop on the healthy mouse corneas then corneas were separated 3 hours postapplication. The DESI-MS images showed a high relative abundance of NIN, detected in the negative ion mode at m/z 538.25, localized to the center of theramem applied corneas (FIG. 3F) compared to eye drop instilled corneas (FIG. 3C). These results further established the theramem ability to enable the drug molecular transport into the cornea.

[0082] The inventors further evaluated NIN-theramem driven drug pharmacokinetics in the corneas of healthy mice. In this study, 24 wildtype mice were randomly selected and divided into 6 groups (4 mice/group), and a single NIN-theramem containing 3 pg of NIN was applied on the right eye of each mouse. A group of 4 mice were used for each timepoint. The corneas were collected at 1-, 2-, 3-, 6-, 12-, and 24-hours after the NIN-theramem application and the NIN concentration was quantified by liquid chromatograph mass spectrometry (LCMS). After 1 h, there was 502.7 ng of NIN present in the corneal tissue and the drug concentration progressively decreased with time, and the NIN concentration in the corneal tissue at 3-, 6-, 12- and 24-hour time points was 342, 206.5, 32.2, and 54 ng, respectively (FIG. 3G). In comparison, mouse eyes treated with NIN-ey edrops of same drug concentration (3 pg/3 pl) contained 226 ng in the first hour and 81 ng in the second hour, and no drug was detected after 2 hrs. This study demonstrated the NIN-theramem’s ability to surmount the ocular surface barriers and enable the drug diffusion into the cornea thus enhancing the drug bioavailability compared to the eye drop treatment ().

NIN-theramem promoted scarless corneal wound healing.

[0083] The efficacy of the NIN-theramems in an alkali induced severe ocular burn (OB) mouse model [17-19] was evaluated. NIN-theramems were applied soon after the OB creation and the treatment continued once every alternate day for 14 days. Bright field microscopy revealed that on the 14^th day, the NIN-theramem treated corneas were clear with no haze or opacification (FIGS. 4I-4L). On the other hand, the balanced saline solution (BSS) treated corneas (control) (FIGS. 4A-4D) and the NIN eyedrop treated corneas (FIG. 4E-4H) exhibited persistent haze even after 14 days of treatment compared to the NIN-theramem treatment, thus demonstrating an enhanced efficacy of the NIN-theramem treatment. To evaluate the effect of NIN-theramem treatment on corneal surface re-epithelialization, a fluorescein staining study was performed on the mouse corneas. The fluorescein staining of the NIN-theramem treated corneas did not exhibit green fluorescence by the 14^th day (FIG. 4L), confirming the complete corneal epithelial closure, while the NIN eyedrops treated (FIG. 4H), and the BSS treated corneas exhibited green fluorescence (FIG. 4D), indicating an incomplete corneal epithelial closure and a rough surface of the injured cornea. The H&E-stained corneal sections revealed that the NIN-theramem treated corneas completely healed with a uniform epithelial layer formation (FIG. 4P) compared to the NIN eyedrop treated sections that contained several voids in the cornea indicating an incomplete epithelium formation (FIG. 40). The BSS treated corneal sections have swollen stroma (edema) and irregular corneal epithelium (FIG. 4N) compared to the NIN-theramem treated eyes. Thickness of the NIN-theramem treated corneas was close to the healthy corneas (FIG. 4Q). NIN-theramem treatment improved the recovery of corneal sensitivity.

[0084] Corneal sensitivity was measured using a Cochet-Bonnet aesthesiometer using a 12/100 nylon thread [32], Pressure response was tested twice with two observers confirming the occurrence of a blink. The filament pressure is inversely proportional to the sensitivity and a lower filament pressure indicates an increased corneal sensitivity. The NIN-theramem treated eyes required a lower filament pressure close to that of the healthy mice to elicit a blink response compared to the BSS and NTN-eyedrops treated mice (FIG. 4R).

NIN-theramem controlled corneal fibrosis.

[0085] To ascertain the efficacy of the NIN-theramem treatment on modulating the corneal fibrotic process, the inventors stained the corneal sections for COL1A1 and fibronectin content. In the NIN-theramem treated corneal sections, a reduction in the fibronectin content (FIGS. 5A- 5C) and COL1 Al (FIGS. 5D-5F) was observed. On the other hand, BSS treated corneal sections showed an increase in collagen I and fibronectin compared to healthy corneal sections. These results confirmed the antifibrotic effect of the NIN-theramem.

NIN-theramem promoted corneal re-epithelialization.

[0086] The corneal cross sections were stained with Ki67 antibody and imaged to assess the dividing corneal epithelial cells. This study revealed that in the NIN-theramem treated sections an increased number dividing epithelial cells were observed, compared to the BSS treated corneal sections (FIGS. 6A-6C). The corneal cross sections also indicated a uniform layer of keratin 13 formation (FIGS. 6D-6F). These results demonstrated corneal re-epithelialization and a normal wound healing process with NIN-theramem treatment.

NIN regulated corneal keratocyte to myofibroblast differentiation.

[0087] To determine the effect of NIN on regulating the TGF-P induced corneal keratocyte (CKC) to myofibroblast (MFB) differentiation, the inventors treated CKC with TGF-P (lOng/ml) followed by NIN (lOpg) for 72 hours. The TGF-P+NIN treated CKCs did not exhibit a-SMA antibody staining compared to the only TGF-P treated CKCs (control), indicating that NIN attenuates the TGF-P induced CKC to MFB differentiation (FIG. 7A-C). The inventors further characterized the CKC to MFB differentiation in vivo in a mouse OB model. To evaluate the antifibrotic efficacy of the NIN-theramem, the OB mice were treated with NIN-theramems on alternate days for 14 days. The corneal sections were subjected to a-SMA antibody staining (green fluorescent) to differentiate the MFB from FB. The NIN-theramem treated corneas have fewer MFBs compared to the BSS treated corneas (control) (FIGS. 7D-F). The MFB density in the NIN- theramem treated sections was comparable to the healthy corneas. To further evaluate the effect of NIN on the fibroblast density in the cornea, a flow cytometry analysis of the corneal tissue was performed. The OB mice were treated with NIN-theramems every alternate day for 10 days. At the end of this period, the corneas were collected, cells were isolated and subjected to flow cytometry analysis. The corneas treated with BSS eyedrops were used as controls. In this study, we quantified 3 subsets of fibroblasts: a-SMA positive myofibroblasts associated with corneal haze and scar formation, S100 positive fibroblasts that promote angiogenesis, and SCA-1 positive fibroblasts associated with progenitor mesenchymal stromal cells [9], During the corneal wound healing, the u-SMA-expressing myofibroblasts are the key contributors to scar formation [33-36], This study revealed that the NIN-theramem treated corneas exhibited a decrease in the myofibroblast density compared to the BSS drops treated group (FIG. 7G). In the NIN-theramem treated eyes, the fibroblast and myofibroblast densities were close to that of the healthy corneas. Furthermore, qPCR analysis of the corneas revealed decreased expression levels of TGF- compared to the BSS eyedrops only treated corneas (FIG. 8). Taken together, these results confirmed that NIN-theramem attenuated corneal scarring during the wound healing process by regulating the TGF-0 secretion, which manifested in a reduced number of corneal myofibroblasts.

Theramem regulated corneal scarring at the molecular level.

[0088] To determine if NIN is preventing corneal scarring by down regulating the proangiogenic and profibrotic mediators, q-PCR analysis of the theramem treated corneas in OB mouse model was performed. BSS-eyedrops were used to treat OB mouse corneas as control. This study revealed that NIN-theramem treatment downregulated the expression levels of proangiogenic VEGFR1, VEGFR2, PDGFR-A, and PDGFR-B, and profibrotic TGF- , COL1A1, CTGF, and ACTA2 (FIG. 8). Down regulation of these proangiogenic genes was also effective in modulating corneal neovascularization in the wounded corneas [40], Taken together, these results indicate that NIN-theramem effectively downregulated the proangiogenic and profibrotic mediators without affecting the overall corneal wound healing process. Theramem efficacy on regressing the established corneal scars.

[0089] In one embodiment, a patient will be treated several days after the corneal injury, and often the drug treatment will begin only after the visual diagnosis of haze formation or opacification. In a preliminary study designed to replicate the clinical conditions, the inventors created OB model in two groups of mice (n=5). The first group was treated daily with BSS eyedrops for 24 days. The second group was treated with BSS eyedrops for the first 7-days followed by NIN-theramem treatment (q.o.d.) from 8^th- to 21 -days. The BSS eyedrops treated mice exhibited a persistent corneal haze even after complete corneal healing (after 24 days) (FIGS. 9A- 9D). The NIN-theramem treated mice exhibited clear and smooth corneas devoid of any haze or scar, thus establishing the NIN-theramem efficacy on regressing the corneal scars and forming clear and transparent corneas.

[0090] Theramem reversed scarring

[0091] OB were generated as previously described and OB eyes were treated with BSS or NIN-theramem on post-OB day 7 and continued for 14 days. The progress of corneal healing and wound closure was monitored by fluorescein staining (FIGs. 16A-H) and corneal epithelium morphology was assessed by H&E staining (FIG. 16I-K). NIN-theramem treated corneas showed continuous corneal epithelium comparable to Healthy (FIG. 16I-K). The BSS treated cornea shows irregular epithelial formation along with severe edema to the stroma (FIG. 16I-K).

Significance of Certain Embodiments

[0092] Evolutionarily, the body’s first and immediate response to injury is to rapidly seal the wound by excessive secretion of extracellular matrix (ECM) materials and prevent fluid loss and infection [37], In the case of eye injuries, such as deep corneal abrasions, perforations, and chemical burns, the excessive ECM secretion causes destructive corneal fibrosis and scarring resulting in vision loss [38], The corneal wound healing response involves a sequence of events that usually contribute to the spontaneous resolution of opacity during the wound healing process leading to the return of a clear cornea with normal function [33-36], However, an abnormal wound healing response results in a persistent corneal scar with a loss of corneal transparency [33], In an injured cornea, the quiescent corneal keratocytes (CK) in the stroma transform into myofibroblasts (MFB) to repair the damage by secreting extracellular matrix (ECM) [39], Myofibroblast differentiation has been identified as the critical factor that leads to corneal fibrosis [33], CK are relatively quiescent stromal cells that function to maintain collagen and other extracellular matrix components [34], Corneal injury triggers a complex stromal response leading to the development of mature myofibroblasts (MFB) that express vimentin and alpha smooth muscle actin (aSMA) [33-36], MFBs are intrinsically opaque due to limited corneal crystallin production and secrete disorganized extracellular matrix that manifests as corneal haze [40],

[0093] Corneal fibrosis is characterized by a high density of type I collagen-producing aSMA- positive MFBs and an excessive secretion of ECM proteins at the wound site [33], These underlying events result in fibrosis and scar formation. Hence, an effective approach to modulate corneal fibrosis is to target the trans-differentiation pathway responsible for the conversion of quiescent CK or fibroblasts to activated, profibrotic MFBs [34], Drugs capable of blocking the fibroblast conversion to activated MFBs could prevent the progression of corneal fibrosis [41], Moreover, pharmacologic agents capable of inhibiting the formation of MFBs in the presence of transforming growth factor-fBl (TGF-pi) without directly inhibiting TGF-pi signaling itself, may have an advantage over direct suppression of TGF-pi, which has the potential to exacerbate immune responses [41], Hence, modulating the CK differentiation into MFBs with pharmacologic agents that do not suppress TGF-pi expression could be an effective strategy to prevent corneal fibrosis and obviate the need for corneal transplant.

[0094] Recently, an antifibrotic drug Nintedanib (NIN) has been approved by the FDA for the treatment of idiopathic pulmonary fibrosis (IFF) [26,27], NIN is a broad tyrosine kinase inhibitor with known activity on proangiogenic platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) receptors and profibrotic fibronectin and collagen-lal secretion and TGF-P induced myofibroblast differentiation [22-24], Considering the fact that CK to MFB differentiation in the injured cornea leads to corneal fibrosis and scarring [34], in this study, the repurposing of NIN as a topical formulation was examined for the treatment of corneal fibrosis. A 3D-bioprinting strategy was employed to fabricate NIN-theramems with a water-soluble polymer, polyvinyl alcohol (PVA) as a delivery system to increase the drug residence time on the cornea and demonstrated its efficacy in promoting scarless corneal wound healing in a mouse ocular burn (OB) induced model. Because the PVA polymer is mucoadhesive, the NIN-theramem rapidly adheres to the wet mucosal surface of the eye. The mucoadhesive hydrogel forming PVA polymer in the theramem enables its rapid wetting and adhesion to the ocular surface without being displaced due to constant blinking and minimizes foreign body sensation in the eye. The antifibrotic drug loaded theramems can function as a drug delivery system and as an ocular surface lubricant. The NIN-theramem can be applied on the ocular surface with a fingertip like a contact lens. The studies have confirmed that when NIN-theramems applied on the eyes of healthy mice, released the drug for an extended time (up to 24 hours), increased the drug residence time on the ocular surface and promoted the drug diffusion into the corneal tissue. A longer drug residence time on the ocular surface provides sufficient time for the drug molecules to diffuse through the ocular surface barriers (OSB) such as tear-film mucus barrier, epithelial tight junctions and diffuse into the corneal tissue, thus improving the drug bioavailability. Further, NIN- theramems when applied on the eyes of alkali induced severe OB mouse model have demonstrated a reduction in the MFB density in the injured corneas compared to the NIN-eyedrop and BSS treatment groups, as confirmed by the fluorescence imaging, q-PCR, and flowcytometry analysis, respectively. Taken together, these results confirm that NIN-theramem treatment is highly effective in attenuating corneal fibrosis in the injured corneas and do not interfere with the normal corneal healing process.

[0095] In summary, the inventors have demonstrated the development of an innovative 3D- bioprinted antifibrotic drug delivery NIN-theramem and established the repurposing potential of NIN for the scarless healing of corneal injuries. The development of antifibrotic NIN-theramems is a major advancement in the treatment of corneal fibrosis and fulfils a hitherto unmet clinical need. The ocular drug delivery theramem is broadly applicable, as it can be loaded with other antiinflammatory and/or antibiotic drugs for the treatment of corneal inflammation, infections, and dry eye.

Experimental Section

[0096] Materials: The chemicals used in this study were purchased from Sigma-Aldrich Co., (St Louis, MO), Nintedanib Ethanesulfonate Salt was obtained from LC Laboratories (Woburn, MA). Antibodies were obtained from Invitrogen, Thermo Fisher Scientific, USA unless noted otherwise. PCR reagents were procured from Applied Biosystems, Thermo Fisher Scientific, USA. [0097] Polymer-drug solution for 3D-bioprinting: 10% PVA solution was synthesized by dissolving 20g PVA in 200ml water and stirred continuously on a magnetic hot plate until fully dissolved. 5ml PVA solution was transferred to a 10 ml glass vial and 20 mg NIN was added to this and vortexed to form a homogenous solution. This PVA-NTN solution was used as the bioink for 3D-printing.

[0098] Fabrication of NIN -theramem: Theramem was fabricated by layer-by-layer 3D- bioprinting (3DDiscovery; regenHU, Switzerland) of a homogenous solution of PVA and NIN in water using a pressure-gradient printhead. The spacing and thickness of the theramem can be tuned by adjusting printing parameters like array design, needle gauge, line spacing and number of layers printed. Here, a 27-gauge needle with an inner diameter of 0.2 mm was used to continuously extrude the polymer drug solution onto the glass slide at a needle pressure of 0.025 MPa and collector velocity of 12 mms'¹. After printing, the theramem was left on the stage for 60 mins to dry. All of the fabrication process was conducted inside a biosafety hood under sterile conditions. [0099] Structure analysis: Crystalline structure analysis of the NIN-theramem was done using Rigaku SmartLab X-ray diffractometer (XRD) system with CuKa radiation at a scan rate of 1.5° min'¹ and a sampling of 0.5°. The analysis was done using PDXL software.

[0100] HPLC analysis. The total drug in NIN-theramem was determined by dissolving an accurately weighed NIN-theramem in 200 pL of water and 800 pL of the mobile phase to precipitate the polymer. The solution was filtered through a 0.2 pm syringe filter then analyzed by the UV-high-performance liquid chromatography equipped with autosampler, in line degasser, and column oven set at room temperature (Shimadzu Prominence, Japan). The inventors used a Kinetex 5uXB-C18 100A (150mm x 4.6mm) column from Phenomenex. The mobile phase for NIN was a mixture of water (50%; pH 3) and acetonitrile (50%). The injection volume was 10 pL and the flow rate was 1 mL/min.

[0101] Evaluation of Nin-theramem toxicity in vitro: Potential cell toxicity of the NIN- theramem was studied using human corneal epithelial cells (hCEC) and human corneal fibroblast (hCF) cells. NIN-theramems were cut into 2 mm diameter and placed in each well of a flat bottom well plate to test the cell toxicity effect of the 3D-bioprinted Theramems. The cell lines were incubated with 1, 2, 3 and 4 NIN-theramems with 3pg, 6pg, 9pg and 12pg drug respectively. The hCEC and hCF cells were added to the wells with or without the theramems at a seeding density of 10 X 10³ cells per well. The cells were kept in an incubation chamber at 37°C and 5% CO2 throughout the study. After 48 hours the cell viability was measured against cells without treatment by MTT assay using a plate reader (CLARIOstar BMG LABTECH GmbH, Germany). [0102] Evaluation of NIN-Theramem ability to regulate corneal scarring in vitro: The ability of NIN-theramem to regulate corneal scarring was evaluated by determining the effect of NIN on modulating TGF-0 induced transition of corneal keratocyte (CKC) to myofibroblasts. The CKCs were seeded at a density of 15,000 cells per well in complete growth medium for 24 hours. The cells were then treated with lOng/ml TGF-01 followed by NIN-theramem for 72 hours. After 72 hours the cells were fixed in 4% PF A, blocked, permeabilized and incubated with oc-SMA primary antibody at 4°C overnight. This was followed by incubation with fluorescent secondary antibody protected from light for 40 mins at room temperature. The slides were mounted with Fluoromount G and sealed with glass cover slip before imaging using Nikon AR confocal microscope (Nikon Instruments, Melville, NY, USA). The cells treated with TGF-01 + NIN- Theramem was compared against cells treated with only TGF-01 and untreated controls.

[0103] Evaluation of Theramem Safety in vivo: The safety of the NIN-Theramem treatment on the cornea was evaluated by applying the theramem on the healthy cornea of C57BL/6 mice daily (n=3) and every alternate day (n=3) for 14 days. Corneal epithelial barrier was monitored daily for scarring or redness. At the end of the study period, the mice were humanely euthanized, and the eyes were harvested for histology to confirm the corneal integrity.

[0104] Evaluation of drug transport to the cornea: The theramem dissolution and drug molecule transport was tracked using fluorescent time lapse imaging and Desorption Electrospray Ionization (DESI) mass spectrometry imaging on the corneas until the drug is completely dispersed.

[0105] For fluorescent imaging, the NIN-theramems were placed on the corneas of healthy C57BL/6 mice (6-8 weeks old). Nintedanib is a green-fluorescent drug, and the efficiency of transport of this drug molecule by the theramem was evaluated by fluorescent imaging every hour for 3 hours using SMZ imaging system.

[0106] For DESI-MS imaging, the corneas were harvested after 3 hours of treatment with either NIN eyedrop or NIN-theramem. The fresh corneas were flat-mounted on glass slide for analysis. DESI-MS analyses were performed using a DESI-XS source paired with a Xevo G2-XS Q-TOF mass spectrometer (Waters Corporation) operated in sensitivity mode with a mass range m z 100 to 1500. DESI-MS imaging of cornea samples was performed at a spatial resolution of 100 pm in the positive and negative ion modes using a spray solvent of MeOH at a flow rate of 3 pL/min. DESI-MS ion images were constructed using HDI (Waters Corporation) software. Ions were identified based on high mass accuracy measurements.

[0107] NIN-Theramem pharmacokinetic study on the corneal surface: Pharmacokinetic analysis for NIN-theramem was done at 1-, 2-, 3-, 6-, 12-, and 24-hours after the NIN-theramem application on the cornea. For this study, 24 wildtype mice were randomly divided into 6 groups with 4 mice in each group for each timepoint and a NIN-theramem containing 3 pg of NIN was applied on the right eye of each mouse. At each time point the mice were euthanized and the corneas were collected. The NIN concentration on the harvested corneas were quantified using liquid chromatograph mass spectrometry (LC-MS). Here, the eyes treated with NIN eye drops were used as control.

[0108] Creation of alkali ocular burn model and NIN-theramem application: Female C57BL/6 mice 6-8 weeks (The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized via intraperitoneal injection of ketamine/xylazine, followed by the topical anesthesia of the right eye using proparacaine hydrochloride eye drops (5 uL, 0.5%, topical anesthetic). Each mouse was given Buprenorphine SR and Meloxicam as analgesia one hour before the burn. Whatman filter paper 2 mm in diameter was soaked with 1 N NaOH solution and placed on the right cornea of the mice for 20 seconds. After 20 secs the eyes were flushed three times with 20 ml sterile BSS solution. 24 hours after burning, 2 mm diameter NIN-theramem was placed gently on the mouse cornea with forceps under anesthesia followed by the addition of 5 uL drop of BSS to facilitate theramem adhesion and dissolution. This treatment was continued every alternate day for 14 days. [0109] The progress of corneal healing and wound closure was monitored by fluorescein staining. 1 uL of fluorescein (0.1%) was applied on the injured corneas for 1 min and rinsed off with 1 ml BSS. Corneal images were recorded every alternate day using a stereoscopic zoom microscope (SMZ1500, Nikon Instruments, Melville, NY, USA).

[0110] Corneal Sensitivity Measurement: The inventors evaluated the corneal sensitivity to tactile sensation by gently applying pressure using a Cochet-Bonnet aesthesiometer (nylon thread, 12/100 mm, Luneau Ophthalmologies, France). This measurement was performed at least three times by two independent observers. The length of the filament is inversely proportional to the pressure applied.

[0111] Histology: Flash-frozen eye tissue samples were sectioned at 10 um thickness using a cryostat (LEICA CM 1850, Leica Biosystems, Buffalo Grove, IL, USA) at -20 °C and collected on a glass microscope slide. The tissue-mounted glass slides were stained with hematoxylin for 4 mins and eosin for 1 minute, followed by dehydration and cleaning in ethanol and xylene. The slides were mounted using toluene-based synthetic resin mounting medium (e.g., Permount™ mounting solution) and sealed with a cover slip. The sections were imaged and analyzed using Nikon eclipse TE2000-U microscope (Nikon Instruments, Melville, NY, USA). The corneal thickness of the OB theramem treated mice were calculated from the H&E images using NIKON AR software and compared with a healthy and BSS eye drop treated group.

[0112] Immunofluorescence: Tissues sectioned at 10 um thickness were used for all the immunofluorescence study. Sections were fixed with 4% PFA, permeabilized, blocked, and incubated with the following primary antibodies: Ki67, keratin-13, ot-SMA, fibronectin and COL1A1 at 4°C, followed by fluorescent secondary antibody staining in a dark chamber for 40 mins at room temperature. The tissues were then mounted using a water soluble, non-fluorescing mounting medium (e.g., Fluoromount G™) and sealed using a glass cover slip. The tissues were imaged and analyzed using Nikon AR confocal microscope.

[0113] Flow cytometry: After separating the corneas, corneal cells were isolated followed by digestion for 2 h at 37°C. The obtained single-cell suspension was stained for viability using blue live/dead fixable cell stain kit. The cells were then fixed and permeabilized overnight at 4 °C (Fixation/Permeabilization, Affymetrix, Santa Clara, USA). On the next day, cells were stained with anti-oc-SMA, SI 00, SCA1 (Invitrogen, Thermo Sciences). A BD LSRII cytometer was used for data acquisition and data was analyzed using BD Diva Software (BD Pharmingen) and FlowJo software.

[0114] Quantification of profibrotic and proangiogenic cytokines by qPCR: The study subjects were euthanized at the end of the study period and their eyes were harvested and enucleated. Corneas were excised and dissected from the surrounding conjunctiva, lens, and uvea. TRIzol reagent was used to extract RNA from the corneal tissue samples and stored at -80°C. Genomic DNA was removed from the samples using TURBO DNA-free kit. RNA was quantified and quality assessed using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First strand cDNA was synthesized from 1.0 ug of RNA with Ready-To- Go-You-Prime-First-Strand Beads and random hexamers (Applied Biosystems, Thermo Fisher Scientific, USA). Equal amounts of synthesized cDNA were used to measure specific gene expression by RT-PCR using a TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific, USA) for specific primers: VEGFR1, VEGFR2, PDGFR-A, PDGFR-B, TGF-p, COL1 Al, CTGF, and ACTA2 from Applied Biosystems on Quantstudio 5 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, USA). GAPDH is used as an internal reference and the results were analyzed by the comparative threshold cycle method with a target change of 2-A CT _{resujts were t}h_en normalized by the CT value of GAPDH and the levels of relative expressions in the untreated, healthy group was used as the calibrator.

EXAMPLE 2

PRODUCTION OF EMBODIMENTS OF THE DEVICE

[0115] Embodiments of the disclosure encompass development of an ocular drug delivery 3D- scaffold by coaxial 3D-bioprinting strategy. In specific embodiments, this strategy enables the fabrication of 3D-scaffolds containing tubular filaments with an inner core composed of drug- polymer matrix and an outer sheath of mucoadhesive polymer. In particular embodiments, the 3D- scaffold releases the drug in a tightly controlled fashion and enhances the drug molecular diffusion into the cornea. By optimizing the drug-polymer ratio, polymer molecular weight, the outer sheath thickness and diameter of the tubular filaments, its drug release kinetics can be programmed. By optimizing the wettability and lubricity of the 3D-scaffold, its ocular comfort and compliance is improved. This strategy enables the incorporation of biologies into the 3D-scaffold without compromising their stability and biological activity.

[0116] Fabrication of 3D-bioprinted NIN-scaffold: In a preliminary study, 3D-scaffolds were fabricated by coaxial bioprinting strategy, which enables the fabrication of tubular filaments with inner core of drug-polymer matrix and an outer sheath of mucoadhesive polymer, unlike the monolithic fibers printed by current 3D-printers. The inventors designed a square patterned 3D- scaffold using BioCAD software for co-axial bioprinting with a water soluble mucoadhesive polyvinyl alcohol (PVA) polymer. The drug and fluorescent dye loaded 3D-scaffolds were fabricated using a RegenHU 3D-Discovery bioprinter. The inner core of the filament was loaded with red fluorescent dye and PVA while the outer sheath with green-fluorescent dye mixed PVA to demonstrate the ability to coaxially bioprint arrays of tubular filaments with two different fluorescent dyes (FIGS. 10A and 10B). The microfilaments in the 3D-scaffold enable rapid wetting and adhesion to the ocular surface without being displaced because of constant blinking and minimizes foreign body sensation in the eye (FIGS. 10D-10F). The controlled release of drug molecules from the NIN-PVA microfilaments of the 3D-scaffold increases the drug residence time on the corneal surface. A longer residence time on the corneal surface provides sufficient time for the drug molecules to diffuse into the corneal through the ocular surface barriers. A controlled release 3D-scaffold increases the drug bioavailability in the cornea. During the course of the drug release, the 3D-scaffold slowly dissolves and disappears. Formouse studies, 3D-scaffolds of 2 mm diameter and 80 pm thickness were fabricated. The 3D-scaffold adheres to the ocular surface without being displaced because of constant blinking and minimizes foreign body sensation in the eye (FIG. 10C)

[0117] NIN-scaffold enhanced the drug transport into the cornea: A Nintedanib (NIN) loaded 3D-scaffold was fabricated by coaxial 3D-bioprinting strategy in which the outer sheath of the tubular filament was made of PVA sheath, and the inner core is formed of NIN-PVA matrix (2: 10 w/w). The NIN loaded 3D-scaffolds (NIN-scaffolds) were used to evaluate the NIN-scaffold efficacy in promoting the drug transport into the cornea. The inventors randomly selected 24 wildtype mice and divided them into 6 groups (4 mice/group) and a single NIN-scaffold containing 3 pg of NIN was applied on the right eye of each mouse. A group of 4 mice were used for each timepoint. The corneas were collected at 1-, 2-, 3-, 6-, 12-, and 24-hours after the NIN-scaffold application and the NIN concentration was quantified by mass spectrometry. After 1 h, there was 502.7 ng of NIN present in the corneal tissue and the drug concentration progressively decreased with time and 54 ng of the drug was present after 24h. (FIG. 11). In comparison, the mouse eyes treated with NIN-eyedrops of same drug concentration (3 pg/3 pL) contained less amount of drug (226 ng) in the first hour and no drug was detected after 12 h. This study demonstrated that NIN- scaffold surmounts the ocular surface barriers and enables the drug diffusion into the cornea compared to the eye drop treatment (FIG. 11).

[0118] NIN-scaffold was safe on the ocular surface: The inventors applied the NIN scaffolds (containing 3 pg of NIN/scaffold) on the corneas of healthy mice once-a-day (q.d.) and on-every- alternate day (q.o.d.) for 14-days. Eye examination indicated the absence of corneal haze or swelling while the H&E-stained eye sections revealed no change in the corneal thickness, thus confirming safety of NIN-scaffolds on the ocular surface (FIG. 12).

[0119] NIN-scaffold promoted scarless corneal wound healing. In this study the inventors evaluated the efficacy of the NIN-scaffolds in alkali induced severe ocular burn (OB) mouse model. Considering the presence of drug in the corneal tissue even after 24 hours (FIG. 12), an alternate day NIN-wafer treatment regimen was designed to minimize the drug overdosing. The NIN wafers were applied soon after the OB creation and the treatment continued once every alternate day for 14 days. The eyes were monitored on alternate days by bright field microscopy during the course of the NIN-scaffold treatment. On the 14th day, the NIN-wafer treated corneas were clear with no haze or opacification (FIG. 4I-4L). On the other hand, the balanced saline solution (BSS) treated corneas (control) (FIGS. 4A-4D) and the NIN eyedrop treated corneas (FIGS. 4E-4H), both exhibited persistent haze and opacification even after 14 days of treatment compared to the alternate day NIN-wafer treatment, thus demonstrating an enhanced efficacy of the NIN-scaffold treatment. To further evaluate the effect of NIN-wafer treatment on corneal surface re-epithelialization, a fluorescein staining study was performed on the mouse corneas. The fluorescein staining of the NIN-scaffold treated corneas did not exhibit green fluorescence by the 14th day (FIG. 4L), confirming the complete corneal epithelial closure and the formation of a smooth and clear corneal epithelium. After 14-days of treatment, the NIN drops treated eyes exhibited slight green fluorescence in the center of the cornea (FIG. 4H), indicating an incomplete epithelial healing of the corneal injury, while the BSS treated corneas exhibited bright green fluorescence (FIG. 4D), indicating an incomplete corneal epithelial closure and a rough surface of the injured cornea. The H&E-stained corneal sections revealed that the NIN-scaffold treated corneas completely healed with a uniform epithelial layer formation (FIG. 4P) compared to the NIN eyedrop treated corneal sections that contained several voids in the cornea indicating an incomplete epithelium formation (FIG. 40). The BSS treated corneal sections have swollen stroma (edema) and irregular corneal epithelium (FIG. 4N) compared to the healthy and NIN- wafer treated eyes (FIGS. 4M and 4P). Thickness of the NIN-scaffold treated corneas was close to the healthy corneas compared to the NIN-eyedrops treated corneas (FIG. 13).

[0120] NIN-scaffold treatment improved the recovery of corneal sensitivity: Corneal sensitivity was measured using a Cochet-Bonnet aesthesiometer using a 12/100 nylon thread [42], The threshold pressure response (g/mm²) for the central cornea was obtained by starting at a pressure too low for detection and then systematically increasing the pressure by shortening the filament length until a blink was observed. Pressure response was tested twice with two observers confirming the occurrence of a blink. The filament pressure is inversely proportional to the sensitivity and a lower filament pressure indicates an increased corneal sensitivity. The NIN- scaffold treated eyes required a lower filament pressure close to that of the healthy mice to elicit a blink response compared to the BSS and NIN-eyedrops treated mice (FIG. 4R).

[0121] Elucidated the mechanism of drug action on myofibroblast-mediated corneal fibrosis: During the corneal wound repair process, keratocytes migrate to the injury site and a subset of these keratocytes in the stroma will differentiate into fibroblast (FB) and myofibroblasts (MFB) by the activation of TGF-P and PDGF secreted by the epithelial cells. The MFB produce ECM at the wound site to repair the damaged cornea. After the wound repair, the stromal MFB undergo apoptosis and the keratocytes populate the stroma to enable the structural and functional recovery of the cornea. However, a sustained secretion of TGF-P at the wound site transform the quiescent stromal cells to MFB and prevents the repopulation of keratocytes in the stroma. The MFB continue to secrete an excessive disorganized ECM that result in a permanent corneal scarring. In this embodiment, the inventors deciphered the mechanism through which NIN acts as an antifibrotic drug, i.e., if the antifibrotic effect of NIN is by modulating the persistent secretion of TGF-P and the MFB differentiation at the wound site or directly targeting the MFB and trigger their apoptosis. Studies were performed to elucidate the antifibrotic mechanism of NIN.

[0122] NIN regulated in vitro FB to MFB differentiation: To determine the effect of NIN on regulating the TGF-P induced FB to MFB transformation, keratocytes were treated with TGF- P (10 ng/ml) followed by NIN (10 pg) for 72 hours. The TGF-P+NIN treated keratocytes did not exhibit a-SMA antibody staining compared to the only TGF-P treated keratocytes (control) indicating that NIN plays a key role in attenuating the TGF-P induced keratocyte transformation to MFB (FIGS. 7A-7C).

[0123] NIN-scaffold modulated the in vivo MFB differentiation: To evaluate the antifibrotic efficacy of the NIN-scaffold, the OB mice were treated with NIN-scaffolds on alternated days for 14 days. The corneal sections were subjected to green fluorescent a-SMA antibody staining to differentiate the MFB from FB. The NIN-wafer treated corneas had fewer MFBs compared to the BSS treated corneas (control). The MFB density in the NIN-scaffold treated sections was close to the healthy corneas (FIGS. 7D-7F). NIN-scaffold treatment also reduced the fibrotic COL1 Al and fibronectin content in the corneal sections, further confirming its antifibrotic effect (FIGS. 14A-14F). These results demonstrated the efficacy of the NIN-scaffold on reducing the MFB density in the corneas compared to the BSS treated corneas (control). [0124] NIN-scaffold promoted corneal re-epithelialization: The Ki67 stained NIN-scaffold treated corneal cross sections contained an increased number of dividing epithelial cells compared to the BSS treated corneal sections (FIGS. 6A-6C). The corneal cross sections stained for Keratin 13 indicated a uniform layer of keratin 13 formation (FIGS. 6D-6F). Together, these results demonstrated the corneal re-epithelialization and a normal wound healing process with NIN- scaffold treatment.

[0125] NIN-scaffold regulated profibrotic mediator expression: q-PCR analysis revealed that the expression levels of profibrotic factors COL1 Al, CTGF, and ACTA2 were down regulated in the NIN-scaffold treated corneas compared to the BSS treated control group, indicating that NIN-scaffold operates by downregulating the expression levels of TGF-0 and profibrotic factors (FIG. 8)

[0126] 3D-scaffold delivery of NIN attenuated corneal myofibroblast differentiation in OB mouse cornea: To evaluate the effect of NIN on the myofibroblast density in the cornea, the NIN-scaffold was tested in alkali induced severe OB mouse model. After the creation of OB, the mice were subjected q.o.d. treatment of NIN-scaffolds for 10 days. At the end of this period, the corneas were collected and stained with u-SMA for myofibroblasts and subjected to flowcytometry analysis. The OB group corneas treated with PBS eyedrops were used as controls. In this study, 3 subsets of fibroblasts were quantified: a-SMA positive myofibroblasts associated with haze and scar formation, S100 positive fibroblasts that promote angiogenesis, and Sca-1 positive fibroblasts associated with progenitor mesenchymal stromal cells. During the corneal wound healing, the o- SMA-expressing myofibroblasts are the key contributors to scar formation. This study revealed that, the NIN-scaffold treated corneas exhibited a decrease in the myofibroblast density compared to the OB group (FIG. 15).

[0127] 3D-scaffold efficacy on regressing the established corneal scars: In some embodiments, the patient is treated several days after the corneal injury, and often the drug treatment begins only after the visual diagnosis of haze formation or opacification. In a study designed to replicate the clinical conditions, the inventors created optical burns in two groups of mice (n=5). The first group was daily treated with BSS eyedrops for 24-days. The second group was treated with BSS eyedrops for the first 7-days followed by NIN-scaffold treatment (q.o.d.) from 8^th- to 21^st-days. The BSS eyedrops treated mice exhibited a persistent corneal haze even after complete corneal healing (after 24 days) (FIGS. 9A-9D). The NIN-scaffold treated mice exhibited clear and smooth corneas devoid of any haze or scar.

[0128] In summary, the data demonstrated the capability to fabricate coaxially 3D-bioprinted NIN-scaffolds with controlled drug release attributes and enhanced efficacy in preventing corneal fibrosis and restoration of corneal sensitivity towards a regenerative wound repair process.

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* * *

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS;

1. A device, comprising 3D-printed material comprising one or more ocular therapeutic agents.

2. The device of claim 1, wherein the device comprises a membrane.

3. The device of claim 1 or 2, wherein the ocular therapeutic agent treats or prevents corneal fibrosis.

4. The device of any one of claims 1-3, wherein the device is a nanowafer, a scaffold, or both.

5. The device of any one of claims 1-4, wherein the device comprises a configuration of filaments.

6. The device of claim 5, wherein the filaments comprise the one or more ocular therapeutic agents on their surface, within the filaments, or both.

7. The device of claim 5 or 6, wherein the configuration of filaments is random.

8. The device of claim 5 or 6, wherein the configuration of filaments is ordered.

9. The device of any one of claims 5-8, wherein the interior of the filaments is hollow.

10. The device of any one of claims 5-8, wherein the interior of the filaments is solid.

11. The device of any one of claims 5-10, wherein the interior of the filaments comprises coaxial layers.

12. The device of claim 11, wherein the coaxial filaments comprise an inner core comprising a drug-polymer matrix and the outer core comprising a mucoadhesive polymer.

13. The device of claim 8, wherein the ordered configuration of filaments comprises multiple intersections of fibers, each intersection comprising an angle between about 1-179°.

14. The device of claim 13, wherein the intersections comprise an angle of exactly or about 90°.

15. The device of claim 8, wherein the ordered configuration of filaments comprises multiple intersections of fibers having acute, oblique, or a combination of acute and oblique angles.

16. The device of any one of claims 1-4, wherein the device comprises no apertures.

17. The device of any one of claims 1-15, wherein the device comprises one or multiple apertures.

18. The device of claim 17, wherein the multiple apertures are substantially the same size.

19. The device of claim 17 or 18, wherein the shape of multiple apertures is generally a polygon, or a circle.

20. The device of anyone of claims 1-19, wherein the device comprises a circular shape comprising a diameter between about 1.0 mm and 15.0 mm.

21. The device of anyone of claims 1-20, wherein the device comprises a thickness of about 60-100 pm.

22. The device of claim 21, wherein the device comprises a thickness of about 80 pm.

23. The device of any one of claims 1-22, wherein the device is comprised in a liquid.

24. The device of claim 23, wherein the liquid comprises a pharmaceutically acceptable solution.

25. The device of claim 23 or 24, wherein the liquid comprises one or more ocular therapeutic agents.

26. The device of claim 25, wherein the one or more ocular therapeutic agents in the liquid is the same as the one or more ocular therapeutic agents of the device.

27. The device of claim 25, wherein the one or more ocular therapeutic agents of the liquid is not the same one or more ocular therapeutic agents of the device.

28. The device of any one of claims 1-27, wherein the material comprises a polymer having a charge that will affect release kinetics of the one or more therapeutic agents and/or polymer(s) from the device.

29. The device of claim 28, wherein the material comprises a polymer having a charge that will induce a synergistic effect on release kinetics of the one or more therapeutic agents and/or polymer(s) from the device.

30. The device of claims 28 or 29, wherein the release kinetics comprise release of the ocular therapeutic agents over a period of about 24 hours.

31. The device of any one of claims 28-30, wherein the release kinetics comprise release of the ocular therapeutic agents over a period of 1 hour to 4 hours.

32. The device of claims 30 or 31, wherein the release kinetics comprise release of the ocular therapeutic agent at an average rate of about 50 ng to 1000 ng per hour.

33. The device of any one of claims 1-32, wherein the one or more ocular therapeutic agents comprises pirfenidone, nilotinib, dexamethasone, a tyrosine kinase inhibitor, or a combination thereof.

34. The device of claim 33, wherein the tyrosine kinase inhibitor comprises nintedanib.

35. The device of claim 34, wherein the device comprises between about 1 pg to 12 pg of nintedanib.

36. The device of claim 35, wherein the device comprises exactly or about 3 pg of nintedanib.

37. A method of treating an ocular medical condition in an individual, comprising the step of applying the device of any one of claims 1-36 to at least one eye of the individual.

38. The method of claim 37, wherein the ocular medical condition affects one eye.

39. The method of claim 37, wherein the ocular medical condition affects both eyes.

40. The method of any one of claims 37-39, wherein the ocular medical condition affects the cornea.

41. The method of any one of claims 37-40, wherein the ocular medical condition is corneal fibrosis, trauma, a genetic defect of the individual, a burn, corneal neovascularization, pterygium, meibomian gland dysfunction, asthenopia, corneal injury, chemical exposure, infectious keratitis, ulcers, alkali burn, keratoconus, Fuchs' endothelial dystrophy, bullous keratopathy, ocular inflammation, ocular pain, dry eye, ocular infection, need for regenerative healing of the eye, corneal perforation, deep corneal abrasion, eye surgery, or a combination thereof.

42. The method of any one of claims 37-41, wherein the device comprises an antifibrotic agent.

43. The method of any one of claims 37-41, wherein the therapeutic agent is nintedanib.

44. The method of any one of claims 37-43, wherein the device is comprised in a liquid.

45. The method of claim 44, wherein the liquid comprises a pharmaceutically acceptable solution.

46. The method of any one of claims 37-45, wherein the device is applied to the eye of the individual prior to onset of the ocular medical condition.

47. The method of any one of claims 37-45, wherein the device is applied to the eye of the individual following onset of the ocular medical condition.

48. The method of any one of claims 37-45, wherein the device is applied to the eye of the individual prior to onset of scarring of the cornea.

49. The method of any one of claims 37-45, wherein the device is applied to the eye of the individual following the onset of scarring of the cornea.

50. The method of any one of claims 37-49, wherein the device is applied once to the affected eye or eyes.

51. The method of any one of claims 37-49, wherein the device is applied more than once to the affected eye or eyes.

52. The method of any one of claims 37-51, wherein the device is applied daily, every other day, weekly, multiple times during a week, or monthly.

53. The method of any one of claims 37-52, wherein the individual is a human.

54. The method of any one of claims 37-53, further comprising the step of producing the device.

55. The method of claim 54, wherein the producing step comprises 3D printing of at least part of the device.

56. The method of claim 55, wherein the 3D printed material of the device comprises one or more therapeutic agents.

57. The method of any one of claims 37-56, wherein the device comprises one or more nerve regenerating factors and one or more neurotrophic factors.

58. The method of any one of claims 37-56, wherein the therapeutic agent is leteprinim.

59. The method of any one of claims 37-56, wherein the device comprises an analgesic, anesthetic, antibiotic, antiviral, and/or nerve-blocking agent.

60. The method of claim 58, wherein the analgesic is pregabalin, gabapentin, bupivacaine, proparacaine, lidocaine, oxybuprocaine, tetracaine, or a combination thereof.

61. The method of any one of claims 58-60, wherein the antibiotic comprises a bacteriostatic and/or bactericidal antibiotic.

62. The method of any one of claims 58-61, wherein the antiviral comprises a protease inhibitor, reverse transcriptase inhibitor, integrase inhibitor, viral entry inhibitor, and/or maturation inhibitors.

63. A method of producing the device of any one of claims 1-36, comprising the step of 3D printing a water-soluble polymer, optionally with one or more therapeutic agents mixed therein.

64. The method of claim 63, wherein the polymer is polyvinyl alcohol (PVA).

65. The method of claim 63 or 64, wherein the polymer is mucoadhesive.

66. The method of any one of claims 63-65, wherein the polymer is dissolvable on the eye.

67. A kit comprising the device of any one of claims 1-36, wherein said device is housed in a suitable container.

68. The kit of claim 67, wherein the device comprises the one or more ocular therapeutic agents.

69. The kit of claim 67, wherein the device lacks the one or more ocular therapeutic agents.

70. The kit of any one of claims 67-69, wherein the kit further comprises one or more ocular therapeutic agents housed separately from the device.

71. A method of treating corneal injury and/or corneal scarring in an individual, the method comprising the step of providing to the eye of the individual a therapeutically effective amount of nintedanib.

72. The method of claim 71, wherein nintedanib is provided as eye drops.

73. The method of claim 71, wherein nintedanib is provided using the device of any one of claims 1-36.