Peptide-modified PPCN and methods of ocular delivery and treatment of disease therewith

Abstract

Provided herein is bioactive protein / peptide-modified PPCN (e.g., A5G81, QK, VEGF-C, etc.) and methods of ocular delivery and treatment of disease (e.g., glaucoma) therewith.

Description

[0001] PEPTIDE-MODIFIED PPCN AND METHODS OF OCULAR DELIVERY AND TREATMENT OF DISEASE THEREWITH

[0002] CROSS-REFERENCE

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 659,727, filed June 13, 2024; which is incorporated by reference herein in its entirety.

[0004] FIELD

[0005] Provided herein is bioactive protein / peptide-modified PPCN (e.g., A5G81, QK, VEGF-C, etc.) and methods of ocular delivery and treatment of disease (e.g., glaucoma) therewith.

[0006] BACKGROUND

[0007] Glaucoma is the leading cause of irreversible blindness worldwide. The treatment for glaucoma is reduction of intraocular pressure (IOP) to prevent damage the lamina cribrosa of the optic nerve head and apoptitic loss of retinal ganglion cells and their axons. Many patients with glaucoma require surgery to achieve adequate IOP reduction. The most effective surgical approach for advanced glaucoma is trabeculectomy which has been in use since 1968. In this procedure, a guarded fistula between the anterior chamber of the eye and the subconjunctival space provides for an alternative drainage route for aqueous humor, thereby reducing IOP. Since the 1990s, the adjunctive use of the anti-fibrotic agent, Mitomycin-C (MMC) at the time of trabeculectomy has resulted in an improvement in the surgical success rates. Yet, failure of the surgery, most commonly due to subconjunctival fibrosis of the bleb, the conjunctival and subconjunctival tissue where aqueous humor accumulates, occurs in 35% of eyes at 5 years.

[0008] A glaucoma surgery known as microinvasive glaucoma surgery (MIGS) is now the most frequently performed incisional glaucoma surgeries in the US. Bleb-forming MIGS procedures are the more effective than those that merely allow for trabecular bypass. The XEN Gel Stent (Allergan) is available in the US and PreserFlo (Santen Pharmaceuticals) is available outside the US. These tubular microstent devices also provide direct communication between the anterior chamber and the subconjunctival space. Similar to trabeculectomy, despite the adjunctive use of MMC, these surgeries are prone to failure due to fibrosis in the subconjunctival space. A major unmet need exists for methods to maintain long-term TOP control after blebforming incisional glaucoma surgery.

[0009] SUMMARY

[0010] Provided herein is bioactive protein / peptide-modified PPCN (e.g., A5G81, QK, VEGF-C, etc.) and methods of ocular delivery and treatment of disease (e.g., glaucoma) therewith.

[0011] In some embodiments, provided herein are treatment strategies to facilitate the formation of lymphatic vessels in the region of a post-surgical bleb to improve long-term bleb survival and thereby improve the long-term success rates of glaucoma surgeries.

[0012] In some embodiments, provided herein are compositions (e.g., for use in the methods of treatment herein) comprising bioactive protein / peptide-modified PPCN. In some embodiments, the bioactive protein / peptide is laminin-derived peptide or a VEGF-derived peptide. In some embodiments, the bioactive protein / peptide is selected from A5G81, QK, VEGF, VEGF-c, SPARC113-130, VEGF-Mimic Pl, PR1P, ANGPTL4-mimetic Peptide, FGF2-Mimetic Peptide, 4N1K Peptide, RGD Peptide, IKVAV, YIGSR, Substance P, AcSDKP, or variants thereof. In some embodiments, the bioactive protein / peptide has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges of values therebetween) sequence identity to one of SEQ ID NOS: 1-14. In some embodiments, the bioactive protein / peptide has 100% sequence identity to one of SEQ ID NOS: 1-14. In some embodiments, the bioactive protein / peptide is covalently linked to PPCN. In some embodiments, the bioactive protein / peptide is covalently linked to PPCN by carbodiimide chemistry. In some embodiments, the bioactive protein / peptide is covalently linked to PPCN by click chemistry. In some embodiments, the bioactive protein / peptide is encapsulated within a matrix or hydrogel of PPCN. In some embodiments, the bioactive protein / peptide capable of being eluting from the PPCN into an aqueous or biological environment. In some embodiments, compositions further comprise lymphatic endothelial cells. In some embodiments, compositions comprise multiple (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, or more, or ranges therebetween) different bioactive proteins / peptides. In some embodiments, compositions comprise one or more bioactive proteins / peptides conjugated to the PPCN and one or more unconjugated bioactive proteins / peptides encapsulated within a matric or hydrogel of PPCN.

[0013] In some embodiments, provided herein are methods comprising administering a composition described herein (e.g., PPCN conjugated to and / or encapsulating a bioactive peptide) to a subject. In some embodiments, the subject suffers from glaucoma. In some embodiments, the subject has undergone glaucoma surgery. In some embodiments, the subject is in an acute recovery phase, subacute phase, or chronic post-surgical phase. In some embodiments the glaucoma surgery is selected from trabeculectomy, glaucoma drainage device (GDD) implantation, minimally invasive glaucoma surgery (MIGS), selective laser trabeculoplasty (SLT), and diode cyclophotocoagulation. In some embodiments, the composition is coadministered with one or more antibiotics anti-inflammatory agents, anti-fibrotic agents, pressure-lowering medications immunomodulatory agents, neuroprotective compounds, and / or antioxidant supplements.

[0014] BREIF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1. Evaluation of treatment efficiency of PPCN in rabbit trabeculectomy model, (a) H&E staining of rabbit eye after treatment with MMC and PPCN. (b) IOP at different time point after treatment with MMC and PPCN. (c) Ratio of collagen and non-collagen protein after treatment with MMC and PPCN.

[0016] Figure 2. Evaluation of tibialis anterior (TA) muscles after cell delivery in mice with hindlimb ischemia, (a) Cell morphology on the surface of peptide-modified PPCN hydrogel, (b) Representative images of Masson’s Trichrome staining for TA muscles in mice after treatment with cells delivered in PPCN, PPCN-A5G81 and PPCN-QK. (c) Percentage of fibrosis after various treatments, (d) Representative immunohistochemical staining of CD31 at the interface of PPCN-peptide hydrogels.

[0017] Figure 3. Cumulative release profile of VEGF-c encapsulated in PPCN hydrogel. After gelation, the PPCN-VEGF-c were submerged in PBS and incubated at 37 degree C. The supernatants were collected at different timepoints, and the VEGF-c concentrations were analyzed by ELISA.

[0018] Figure 4. Cell Proliferation Study with PPCN-VEGF-c Release Media. LECs are treated with media released from PPCN-VEGF-c with different loading concentration. The media were replenished every day for 72 hours before quantifying proliferation using Picogreen dsDNA analysis. **p<0.01, ***p<0.001.

[0019] Figure 5. Images and Quantification of LEC Tubular Network in Tube Formation Assay Treated with PPCN-VEGF-c Release Media. *P < 0.05. DEFINITIONS

[0020] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0022] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0023] As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.

[0024] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.

[0025] As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be by parenteral administration (e.g., orally, intraocularly, intravenously, subcutaneously, etc.).

[0026] As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0027] As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the coadministration of two or more agents or therapies is concurrent (e.g., in a single formulation / compo sition or in separate formulations / compositions). In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co- administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0028] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0029] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect against a particular disease, disorder, or condition. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptom attributable to the disease.

[0030] As used herein, the term “glaucoma surgery” refers to any surgical intervention intended to reduce intraocular pressure or improve aqueous humor outflow, including but not limited to trabeculectomy, glaucoma drainage device (GDD) implantation, minimally invasive glaucoma surgery (MIGS) (e.g., canaloplasty, goniotomy, trabecular bypass stents (e.g., iStent, Hydrus), ab intemo trabeculectomy, etc.) laser procedures (e.g., selective laser trabeculoplasty (SLT), diode cyclophotocoagulation, etc.), etc.

[0031] As used herein, the term “post-surgical period” refers to the time following surgical intervention, including the acute recovery phase (0-2 weeks), subacute phase (2-6 weeks), and chronic post-surgical phase (>6 weeks).

[0032] As used herein, the term “polymer” refers to a chain of repeating structural units (e.g., citric acid, aliphatic diol, amino acids, etc.) or “monomers”, typically of large molecular mass. Examples of polymers include homopolymers (single type of monomer subunits), copolymers (two types of monomer subunits), and heteropolymers (e.g., three or more types of monomer subunits). As used herein, the term “oligomer” refers to a polymer of only a few monomer units (e.g., 2, 3, 4, 5, or more) up to about 50 monomer units, for example a dimer, trimer, tetramer, pentamer, hexamer... decamer, etc.

[0033] As used herein, the term “linear polymer" refers to a polymer in which the molecules form long chains without branches or crosslinked structures.

[0034] As used herein, the term “branched polymer” refers to a polymer comprising a polymer backbone with one or more additional monomers, or chains of monomers, extending from polymer backbone. The degree of interconnectedness of the “branches” is insufficient to render the polymer insoluble.

[0035] As used herein, the terms “pre-polymer” refers to linear or branched polymers (e.g., soluble, not significantly crosslinked) that have the capacity to be crosslinked under appropriate conditions, but which have not yet been subjected to the appropriate conditions.

[0036] As used herein, the term “crosslinked polymer” refers to a polymer with a significant degree of interconnectedness between multiple polymer strands, the result of which is an insoluble polymer network (e.g., thermoset elastomer). For example, multiple polymer stands may be crosslinked to each other at points within their structures, not limited to the ends of the polymer chains.

[0037] As used herein, the term “hydrogel” refers to a three-dimensional (3D) crosslinked network of hydrophilic polymers that swells, rather than being dissolved, in water.

[0038] As used herein, the term “thermoresponsive” refers to materials that exhibit altered physical characteristics at different temperature ranges. Particularly relevant herein are “phasetransitioning thermoresponsive materials.” Phase-transitioning thermoresponsive materials are soluble or in a liquid state at a first temperature range (e.g., below 26°C) and insoluble or in a solid state at a second temperature range (e.g., 30-45°C). A non-limiting example of a phasetransitioning thermoresponsive polymer is PPCN.

[0039] As used herein, the term “composite” refers to a material comprising two or more molecular, polymeric, and / or supramolecular constituents that are miscible with one another, and may form a single homogeneous material. While covalent connections between the constituent components may be present, they are not required to form or maintain the composite or its homogeneity; rather, non-covalent and / or mechanical / physical interactions and associations are responsible for stabilizing the composite.

[0040] As used herein, the term “biocompatible” refers to materials and agents that are not toxic to cells or organisms. In some embodiments, a substance is considered to be "biocompatible" if its addition to cells in vitro results in less than or equal to approximately 10% cell death, usually less than 5%, more usually less than 1%, and preferably less than 0.1%.

[0041] The term “biodegradable,” as used to describe the polymers, hydrogels, composites, and / or wound dressings herein, refers to compositions that are degraded or otherwise “broken down” under exposure to physiological conditions. In some embodiments, a biodegradable substance is a broken down by cellular machinery, enzymatic degradation, chemical processes, hydrolysis, etc. In some embodiments, a biodegradeable composition comprises hydrolyzable ester linkages.

[0042] As used herein, the phrase “physiological conditions” relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the intracellular and extracellular fluids of tissues. For most tissues, the physiological pH ranges from about 7.0 to 7.4. As used herein, the term “amino acid” refers to natural amino acids, unnatural amino acids, protcinogcnic amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.

[0043] The term “proteinogenic amino acids” refers to the 20 amino acids coded for in the human genetic code, and includes alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Vai or V). Selenocysteine and pyrrolysine may also be considered proteinogenic amino acids

[0044] The term “non-proteinogenic amino acid” refers to an amino acid that is not naturally- encoded or found in the genetic code of any organism, and is not incorporated biosynthetically into proteins during translation. Non-proteinogenic amino acids may be “unnatural amino acids” (amino acids that do not occur in nature) or “naturally-occurring non-proteinogenic amino acids” (e.g., norvaline, ornithine, homocysteine, etc.). Examples of non-proteinogenic amino acids include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2- aminopimelic acid, tertiary -butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2’- diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine , N-alkylglycine including N-methylglycine, N- methylisoleucine, N-alkylpentylglycine including N-methylpentylglycine. N-methylvaline, naphthylalaninc, norvalinc, norlcucinc (“Norlcu”), octylglycinc, ornithine, pcntylglycinc, pipecolic acid, thioproline, homolysine, and homoarginine. Non-proteinogenic also include D- amino acid forms of any of the amino acids herein, as well as non-alpha amino acid forms of any of the amino acids herein (beta-amino acids, gamma-amino acids, delta-amino acids, etc.), all of which are in the scope herein and may be included in peptides herein.

[0045] The term “amino acid analog” refers to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain functional group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S- (carboxy methyl) -cy s teine sulfone .

[0046] As used herein, the term “peptide” refers a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 30 amino acids or fewer in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and / or modified amino acids. A peptide may be a subsequence of naturally occurring protein or a non-natural (synthetic) sequence.

[0047] As used herein, the term “mutant peptide” refers to a variant of a peptide having a distinct amino acid sequence from the most common variant occurring in nature, referred to as the “wildtype” sequence. A mutant peptide may be a subsequence of a mutant protein or polypeptide (e.g., a subsequence of a naturally-occurring protein that is not the most common sequence in nature), or may be a peptide that is not a subsequence of a naturally occurring protein or polypeptide. For example, a “mutant laminin-based peptide” may be a subsequence of a mutant version of native laminin or may be distinct sequence not found in naturally-occurring laminin proteins.

[0048] As used herein, the term “synthetic peptide” refers to a peptide having a distinct amino acid sequence from those found in natural peptides and / or proteins. A synthetic peptide is not a subsequence of a naturally occurring protein, either the wild-type (i.e., most abundant) or mutant versions thereof. For example, a “synthetic laminin peptide” is not a subsequence of a naturally occurring laminin. A “synthetic peptide,” as used herein, may be produced or synthesized by any suitable method (e.g., recombinant expression, chemical synthesis, enzymatic synthesis, etc.).

[0049] The terms “peptide mimetic” or “peptidomimetic” refer to a peptide-like molecule that emulates a sequence derived from a protein or peptide (e.g., A5G81). A peptide mimetic or peptidomimetic may contain amino acids and / or non-amino acid components. Examples of peptidomimitecs include chemically modified peptides, peptoids (side chains are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons), P-peptides (amino group bonded to the P carbon rather than the a carbon), etc. As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another:

[0050] 1) Alanine (A) and Glycine (G);

[0051] 2) Aspartic acid (D) and Glutamic acid (E);

[0052] 3) Asparagine (N) and Glutamine (Q);

[0053] 4) Arginine (R) and Lysine (K);

[0054] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V);

[0055] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W);

[0056] 7) Serine (S) and Threonine (T); and

[0057] 8) Cysteine (C) and Methionine (M).

[0058] Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi -conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0059] In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0060] “Non-conservative substitutions” involve the exchange of a member of one class for a member from another class.

[0061] As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) differ only by conservative and / or semiconservative amino acid substitutions. The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

[0062] As used herein, the term “substantially all,” “substantially complete” and similar terms refer to greater than 99%; and the terms “substantially none,” “substantially free of,” and similar terms refer to less than 1 %. Such terms may be appended to some embodiments herein to indicate that somewhat less than 100% or just above 0% may be encompassed.

[0063] The term “about” allows for a degree of variability in a value or range. As used herein, the term “about” refers to values within 10% of the recited value or range (e.g., about 50 is the equivalent of 45-55). Such a term may be appended to some embodiments herein to indicate that certain values need not be precise.

[0064] DETAILED DESCRIPTION

[0065] Provided herein is bioactive protein / peptide-modified PPCN (e.g., A5G81, QK, VEGF-C, etc.) and methods of ocular delivery and treatment of disease (e.g., glaucoma) therewith. In some embodiments, provided herein are compositions and methods for post-surgical treatment for ocular pressure regulation and tissue healing following ocular surgery (e.g., glaucoma surgery).

[0066] Lymphatic drainage plays a prominent role in resorption of fluid from the subconjunctival space after bleb-forming glaucoma surgery. Bleb-forming surgery does not fail due to occlusion of the fistula in the case of trabeculectomy or blockage of the microstent in the anterior chamber or along its length in the case of bleb-forming MIGS. The facilitation of the formation of lymphatic vessels in the region of a post-surgical bleb may improve long-term bleb survival and thereby improve the long-term success rates of glaucoma surgeries, including trabeculectomy and bleb-forming MIGS.

[0067] Provided herein is a thermo-responsive citrate-based hydrogel, poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN), endowed with antioxidant and anti-inflammatory properties for use in mitigating fibrosis. PPCN’s ocular biocompatibility was affirmed in a rabbit model of trabeculectomy, where it demonstrated similar efficacy compared to MMC in lowering IOP and preventing fibrosis (Figure 1). PPCN was further enhanced with bioactive peptides, transforming it into a dynamic cell-delivery system that modulates cellular activity to bolster cell survival and functionality. Experiments conducted during development of embodiments herein revealed that endothelial cells (ECs) conveyed via PPCN modified with A5G81 and QK peptides resulted in diminished fibrosis and augmented angiogenesis (Figures 2b-d). Moreover, ECs interacting with PPCN-A5G81 and PPCN-QK showed a surge in tubulogenesis (Figure 2a). It is contemplated that the newly formed vasculature at the interface between biomaterial and tissue is serves as channels, enhancing fluid permeability (Figure 2d). In some embodiments, provided herein are cell delivery strategies with a novel peptide- modificd thcrmorcsponsivc citrate -based hydrogel (PPCN-pcptidc) that provide a long-term solution to prevent fibrosis and maintain effective IOP control, e.g., after trabeculectomy. Embodiments herein improve the quality of life for glaucoma patients by reducing the need for repeated surgeries and preserving vision.

[0068] In some embodiments, provided herein are peptide hydrogels us based on the thermoresponsive biodegradable polymer poly(polyethyleneglycol co-citric acid-co-N isopropylacrylamide) (PPCN). This material undergoes a rapid and reversible phase transition from liquid to solid at physiologically relevant temperatures to form a hydrogel with excellent water retention properties, and provides antioxidant activity including iron chelation, free radical scavenging and inhibition of lipid peroxidation. Such properties make PPCN an attractive material for use with peptides therapeticus.

[0069] In some embodiments, PPCN (e.g., hydrogel) is functionalized (e.g., via covalent or non- covalent linkage) with a bioactive peptide (e.g., A5G81, QK, VEGF, VEGF-c, SPARC113-13O, VEGF-Mimic Pl, PR1P, ANGPTL4-mimetic Peptide, FGF2-Mimetic Peptide, 4N1K Peptide, RGD Peptide, IKVAV, YIGSR, Substance P, AcSDKP, variants thereof, etc.). Peptide conjugation to PPCN may be achieved through any suitable mechanism, such as click chemistry with a bifunctional linker (e.g., BMPH). In other embodiments, a bioactive peptide (e.g., A5G81, QK, VEGF, VEGF-c, SPARC113-130, VEGF-Mimic Pl, PR1P, ANGPTL4-mimetic Peptide, FGF2- Mimetic Peptide, 4N1K Peptide, RGD Peptide, IKVAV, YIGSR, Substance P, AcSDKP, variants thereof, etc.) is encapsulated within a PPCN matrix or hydrogel, without being covalently and / or non-covalently linked to the PPCN (although some attractive non-covalent forces may exist between the two).

[0070] In some embodiments, materials and composites herein comprise polymers of citric acid, polyethylene glycol, and glycerol 1,3- diglycerolate diacrylate. In some embodiments, citric acid, polyethylene glycol, and glycerol 1,3- diglycerolate diacrylate are polymerized to form a polymer (e.g., pre-polymer) of poly(polyethyleneglycol citrate) acrylate (PPCac). In some embodiments, materials and composites comprise polymers of citric acid, polyethylene glycol, glycerol 1,3- diglycerolate diacrylate, and N- isopropylacrylamide (NIPAAm). In some embodiments, PPCac and NIPAAm are reacted together to produce a poly(polyethyleneglycol citrate co N-isopropylacrylamide (PPCN) polymer. In some embodiments, PPCN is provided as a material.

[0071] In some embodiments, polymers herein (e.g., PPCN or another polymer) comprise at least 0.1% citric acid monomers

[0072] (e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, > 60%, >70%, >80%, >90%, >95%, >98%, >99%). In some embodiments, polymers herein comprise less than 99% citric acid monomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%, <4%, <3%, <2%, <1%, <0.5%,). In some embodiments, polymers comprise about 99%, about 98%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% citric acid monomers.

[0073] In some embodiments, polymers herein (e.g., PPCN or another polymer) comprise at least 0.1% polyethylene glycol monomers

[0074] (e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, > 60%, >70%, >80%, >90%, >95%, >98%, >99%). In some embodiments, polymers herein comprise less than 99% polyethylene glycol monomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%, <4%, <3%, <2%, <1%, <0.5%,). In some embodiments, polymers comprise about 99%, about 98%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% polyethylene glycol monomers.

[0075] In some embodiments, polymers herein (e.g., PPCN or another polymer) comprise at least 0.1% glycerol 1,3- diglycerolate diacrylate monomers

[0076] (e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, > 60%, >70%, >80%, >90%, >95%, >98%, >99%). In some embodiments, polymers herein comprise less than 99% glycerol 1,3- diglycerolate diacrylate monomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%, <4%, <3%, <2%, <1%, <0.5%,). In some embodiments, polymers comprise about 99%, about 98%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% glycerol 1,3- diglycerolate diacrylate monomers. In some embodiments, polymers herein (e.g., PPCN or another polymer) comprise at least 0.1% N-isopropylacrylamidc monomers

[0077] (e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, > 60%, >70%, >80%, >90%, >95%, >98%, >99%). In some embodiments, compositions herein comprise less than 99% N-isopropylacrylamide monomers (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%, <4%, <3%, <2%, <1%, <0.5%,).

[0078] In some embodiments, polymers comprise about 99%, about 98%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% N-isopropylacrylamide monomers.

[0079] In some embodiments, provided herein are poly(polyethyleneglycol citrate co N- isopropylacrylamide (PPCN)) with one or more bioactive peptides. In some embodiments, a composition herein comprises at least 0.1% PPCN (e.g., >0.1%, >0.2%, >0.5%, >1%, >2%, >3%, >4%, >5%, >10%, >20%, >30%, >40%, >50%, > 60%, >70%, >80%, >90%, >95%, >98%, >99%). In some embodiments, a composition herein comprises less than 99% PPCN (e.g., <99%, <98%, <95%, <90%, <80%, <70%, <60%, <50%, <40%, <30%, <20%, <10%, <5%, <4%, <3%, <2%, <1%, <0.5%,). In some embodiments, a composition herein comprises PPCN in an amount of about 99%, about 98%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or ranges therein. The aforementioned percentages may be wt% or molar %.

[0080] In some embodiments, synthesis of the polymers, hydrogels, networks, etc. described herein are produced by combination of the component molecules (e.g., citric acid, polyethylene glycol and glycerol 1,3-diglycerolate diacrylate; PPCac and NIPAAm, etc.) under the appropriate conditions (e.g., temperature, pressure, pH, etc.). In some embodiments, reaction, crosslinking, polymerization, etc. occurs upon combination of the components under appropriate conditions in the absence of any additional enzyme or chemical catalysts. In some embodiments, a radical initiator (e.g., AIBN) is used to induce a reaction or polymerization.

[0081] In some embodiments, components (e.g., citric acid, polyethylene glycol and glycerol 1 ,3-diglycerolate diacrylate; etc.) are heated to at least 100°C (e.g., 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, or more). In some embodiments, components (e.g., PPCac and NIPAAm, etc.) are heated to at least 40°C (e.g., 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, or more). In some embodiments, components are reacted at a temperature not exceeding 250°C (e.g., <240°C, <220°C, <200°C, <180°C, <160°C, or less).

[0082] In some embodiments, components (e.g., citric acid, polyethylene glycol and glycerol 1,3-diglycerolate diacrylate; PPCac and NIPAAm, etc.) are reacted for at least 1 minute (e.g., >1 minute, >2 minutes, >3 minutes, >4 minutes, >5 minutes, >10 minutes, >20 minutes, >30 minutes, >45 minutes, >1 hour, >2 hours, >3 hours, >4 hours, >12 hours, >24 hours, >48 hours, >72 hours, or more).

[0083] In some embodiments, citric acid, polyethylene glycol and glycerol 1,3-diglycerolate diacrylate are reacted at a ratio of 5:9:1, 5:8:2, 5:7:3, 5:6,4, 5:5:5, 4:9:2, 3:9:3, 2:9:4, 1:9:5, 6:8:1, 7:7:1, 8:6:1, 9:5:1, 10:4:1, 11:3:1, 12:2:1, 13:1:1, 4:10:1, 3:11:1, 2:12:1, 1:13:1, or any other suitable ratios thereof or rages there between. In some embodiments, PPCac and NIPAAm are reacted at a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4: 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any other suitable ratios thereof or rages there between.

[0084] In some embodiments, materials provided herein comprise a peptide component (e.g., conjugated to a polymer component) that is one or more of: anti-inflammatory, anti-angiogenic, anti-proliferative, pro-regenerative, wound-modulating, intraocular pressure-modulating, antimicrobial, anti-infective, immune-modulatory, tolerance -promoting, anti-oxidant, neuroprotective, etc. Exemplary peptides that may find use in embodiments herein (e.g., conjugated to or encapsulated within PPCN) include, but are not limited to A5G81, QK, VEGF, VEGF-c, SPARC 113-130, VEGF-Mimic Pl, PR IP, ANGPTL4-mimetic Peptide, FGF2-Mimetic Peptide, 4N1K Peptide, RGD Peptide, IKVAV, YIGSR, Substance P, AcSDKP, variants thereof, etc.

[0085] In some embodiments, compositions and methods are provided comprising a QK peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising a QK peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to KLTWQELYQLKYKGI (SEQ ID NO: 1). QK peptide is derived from VEGF (Vascular Endothelial Growth Factor), specifically from the a-helical region of VEGF. QK peptide mimics the pro-angiogenic activity of VEGF by binding to VEGF receptors (especially VEGFR2). QK peptide promotes angiogenesis and induces endothelial cell proliferation, migration, and tubule formation. QK peptide enhances vascularization in tissue.

[0086] In some embodiments, compositions and methods are provided comprising a A5G81 peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising a A5G81 peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration postglaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to AGQWHRVSVRWG (SEQ ID NO: 2). A5G81 peptide is synthetic peptide whose sequence mimics a region of Angiopoietin- 1 (Angl). A5G81 peptide promotes vascular stabilization and anti-inflammatory effects through activation of Tie2. A5G81 peptide provides vascular protection, stabilizes blood vessels, and prevents leakage. A5G81 peptide reduces endothelial cell activation and inflammation.

[0087] In some embodiments, compositions and methods are provided comprising a SPARC- derived peptide (SPARCI 13-130) or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising a SPARC-derived peptide (SPARCI 13-130) or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to FKLGHPDKLKDLPGGG (SEQ ID NO: 3). SPARC-derived peptide (SPARC 113- 130) promotes angiogenesis, enhances endothelial cell migration.

[0088] In some embodiments, compositions and methods are provided comprising VEGF-Mimic Pl or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising VEGF-Mimic Pl or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to CGGNQNSR (SEQ ID NO: 4). VEGF- Mimic Pl mimics VEGF, binds VEGFR and induces angiogenesis.

[0089] In some embodiments, compositions and methods are provided comprising PR1P or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising PR IP or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to MLGKLRSGY (SEQ ID NO: 5). PR1P binds VEGF, stabilizes it, and extends VEGF bioactivity in tissue.

[0090] In some embodiments, compositions and methods are provided comprising ANGPTL4- mimetic peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising ANGPTL4-mimetic peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to DSGLYCSST (SEQ ID NO: 6). ANGPTL4-mimetic peptide stabilizes endothelium and reduces vascular leakage.

[0091] In some embodiments, compositions and methods are provided comprising FGF2- mimetic peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising FGF2-mimetic peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to CNLMYKKPKL (SEQ ID NO: 7). FGF2-mimetic peptide binds FGF receptors, mimics FGF2 activity for cell proliferation and angiogenesis.

[0092] In some embodiments, compositions and methods are provided comprising 4N1K peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising 4N1K peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to KRFYVVMWKK (SEQ ID NO: 8). 4N1K peptide regulates angiogenesis and inflammation; can be pro- or anti- angiogenic depending on context.

[0093] In some embodiments, compositions and methods are provided comprising RGD peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising RGD peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. Tn some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to Arg-Gly-Asp (SEQ ID NO: 9) (linear’ or cyclic variants like c(RGDfK) (SEQ ID NO: 10)). RGD binds integrins (avP3, a5pi) on endothelial and other cells, and promotes cell adhesion, migration, and angiogenesis.

[0094] In some embodiments, compositions and methods are provided comprising IKVAV peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising IKVAV peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration postglaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to IKVAV (SEQ ID NO: 11).

[0095] In some embodiments, compositions and methods are provided comprising YIGSR peptide or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising YIGSR peptide or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration postglaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to YIGSR (SEQ ID NO: 12).

[0096] In some embodiments, compositions and methods are provided comprising Substance P or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising Substance P or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to RPKPQQFFGLM (SEQ ID NO: 13).

[0097] In some embodiments, compositions and methods are provided comprising AcSDKP or variants thereof conjugated to PPCN. In some embodiments, compositions and methods are provided comprising AcSDKP or variants thereof encapsulated with a PPCN matrix or hydrogel. In some embodiments, compositions herein (e.g., for administration post-glaucoma surgery) comprise a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges or values therebetween) sequence identity to Ac-Ser- Asp-Lys-Pro (SEQ ID NO: 14).

[0098] In some embodiments, the peptides described herein are covalently conjugated to PPCN, e.g., for use methods of enhancing post-glaucoma surgery and / or healing / maintenance thereof. In some embodiments, peptides are synthesized with a terminal cysteine residue (e.g., not present the peptide sequences) for conjugation to a PPCN via the hctcro-bifuncitonal cross linker N-[P- maleimidopropionic acid] hydrazide (BMPH). In some embodiments, cysteine-terminated peptides may be conjugated to polymers (e.g., displaying appropriate functional groups) directly or via other linkers. In some embodiments, unmodified (e.g., without terminal cysteines) peptides are conjugated to PPCN (e.g., directly or via a linker) using an amino acid reside already present in the peptide sequence. In some embodiments, peptides are modified (e.g., chemically, by amino acid substitution (e.g., with a natural (e.g., cysteine, lysine, etc.) or unnatural amino acid), by addition of a terminal amino acid (e.g., a natural (e.g., cysteine, lysine, etc.) or unnatural amino acid), etc.) for conjugation (e.g., directly or via a chemical linker) to a polymer.

[0099] Conjugation via a terminal cysteine and BMPH provides suitable conjugation chemistry for use in the examples herein. However, embodiments within the scope herein are not so limited.

[0100] In some embodiments, peptides and PPCN are conjugated by click chemisty, employing Diels-Alder reactions, thiol-yne reactions, and azide-alkyne reactions, thiol / maleimide, thiol / haloacetyl (e.g., iodoacetyl, etc.), azide / phosphine (Staudinger ligation), thiol / pyridyl disulfide (e.g. pyridyldithiol, etc.), sulphonyl azides / thio acids, etc. In some embodiments, a conjugation of peptides and PPCN comprises a reaction pair selected from: maleimide / thiol, succimidylester (NHS ester) / amine, azide, carboxy / EDC (l-Ethyl-3-[3- dimethylaminopropyl]carbodiimide Hydrochloride ) / amine, amine / Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate) / thiol, and amine / BMPH(N-[P-Maleimidopropionic acid]hydrazide) / thiol. Other conjugation pairs, reaction chemistries, etc. are within the scope herein.

[0101] In certain embodiments, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) are administered through various local or periocular delivery routes depending on the type of glaucoma surgery performed and the desired therapeutic profile. Local routes include topical ocular administration via eye drops, which may be formulated for daily use or timed-release. In other embodiments, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) are delivered by subconjunctival injection either intraoperatively or postoperatively, providing localized depot delivery near the surgical site. Alternatively, intracameral (within the anterior chamber) or intravitreal (within the posterior segment) injection may be employed, particularly for targeting deeper intraocular tissues. Sustained-release formulations may be delivered via implantable biodegradable devices or hydrogel depots applied to the scleral or subconjunctival surface. Periocular routes, such as sub-Tenon’s injection or retrobulbar injection, may also be suitable.

[0102] In some embodiments, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) are used in conjunction with standard post-surgical therapeutics to enhance treatment outcomes and mitigate complications. Co-administration may include antibiotics such as moxifloxacin, ciprofloxacin, or tobramycin to reduce the risk of postoperative infection. Antiinflammatory agents, including corticosteroids such as dexamethasone or NSAIDs such as ketorolac, may be administered concurrently to manage inflammation. Anti-fibrotic agents, such as mitomycin C or 5-fluorouracil, may be applied intraoperatively or postoperatively to reduce conjunctival scarring and maintain surgical patency. Additionally, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) may be combined with pressure-lowering medications such as beta-blockers, prostaglandin analogs, alpha agonists, or carbonic anhydrase inhibitors, especially in patients with persistently elevated intraocular pressure. Immunomodulatory agents, neuroprotective compounds, or antioxidant supplements may also be considered as adjunct therapies.

[0103] I In some embodiments, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) are tailored to the specific surgical modality employed in the treatment of glaucoma. Following trabeculectomy, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) are applied to the subconjunctival space to prevent fibroblast proliferation and maintain bleb function. In the context of glaucoma drainage device (GDD) implantation, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) may minimize capsular fibrosis around the implant plate and support long-term device patency. For minimally invasive glaucoma surgeries (MIGS), including trabecular meshwork bypass stents, canaloplasty, and goniotomy, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) may be used to prevent postoperative scarring in Schlemm’s canal or the trabecular meshwork, preserving the efficacy of aqueous outflow. In procedures involving laser ablation, such as diode laser cyclophotocoagulation or selective laser trabeculoplasty (SLT), In some embodiments, PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) may mitigate inflammation and promote tissue homeostasis, reducing the risk of hypotony or pain.

[0104] Provided herein are method of treating a patient following glaucoma surgery by administering a PPCN / bioactive peptide composition (e.g., conjugates, encapsulations, etc.) during the postoperative period to the affected ocular tissue. The method includes performing a glaucoma surgical procedure such as trabeculectomy, GDD implantation, MIGS, or laser-based intervention, followed by the administration of Treatment X locally to the surgical site or surrounding ocular tissues PPCN / bioactive peptide compositions (e.g., conjugates, encapsulations, etc.) may be delivered immediately at the time of surgery or during the postoperative recovery phase, either as a single dose or in a sustained-release format. The therapeutic objective is to reduce inflammation, modulate wound healing, prevent excessive fibrosis, maintain aqueous humor outflow, and preserve visual function. In some embodiments, methods includes monitoring intraocular pressure, conjunctival tissue morphology, bleb function (if present), and patient-reported outcomes. Methods may also include the co-administration of additional pharmaceutical agents selected to support the therapeutic goals of the surgical intervention and individual patient response.

[0105] EXPERIMENTAL

[0106] Synthesis and characterization of PPCN-BMPH hydrogels

[0107] PPCN (poly(polyethyleneglycol citrate-co-N isopropylacrylamide) (U.S. Pub. No. 20180085492; incorporated by reference in its entirety) was covalently linked to BMPH using standard carbodiimide chemistry. Specifically, PPCN (200 mg, 0.02 mmol) was dissolved in ultrapure water at a concentration of 50 mg / mL. N-hydroxysuccinimide (NHS, 4 mmol) and 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 2 mmol) were added to activate the carboxylic acid groups of PPCN. After a 2-hour reaction at room temperature, BMPH (60 mg, 0.2 mmol) was dissolved in 2 mL of MQ water and added to the solution to react overnight at room temperature. The resulting PPCN-BMPH product was neutralized to pH 7.4, dialyzed with 2kDa molecular weight-cutoff (MWCO) dialysis tubing for 2 days at 4 °C, and lyophilized for 2 days. For peptide conjugation, PPCN-BMPH (0.01 mmol) was mixed with peptides, including the laminin-derived peptide A5G81 and the VEGF-mimic peptide QK (0.03 mmol), in PBS to react overnight at room temperature. The PPCN-peptide was then dialyzed with 3.5kDa MWCO dialysis tubing for 2 days at 4 °C and lyophilized for 2 days.

[0108] Synthesis and characterization of PPCN-VEGF-C hydrogels

[0109] PPCN was covalently linked to BMPH using standard carbodiimide chemistry. Specifically, PPCN (200 mg, 0.02 mmol) was dissolved in ultrapure water at a concentration of 50 mg / mL. N-hydroxy succinimide (NHS, 4 mmol) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 2 mmol) were added to activate the carboxylic acid groups of PPCN. After a 2-hour reaction at room temperature, BMPH (60 mg, 0.2 mmol) was dissolved in 2 mL of MQ water and added to the solution to react overnight at room temperature. The resulting PPCN-BMPH product was neutralized to pH 7.4, dialyzed with 2kDa molecular weight-cutoff (MWCO) dialysis tubing for 2 days at 4 °C, and lyophilized for 2 days. For VEGF- C conjugation, PPCN-BMPH (0.01 mmol) was mixed with VEGF-C or VEGF-C-derived peptides (0.03 mmol), in PBS to react overnight at room temperature. The PPCN-peptide was then dialyzed with 3.5kDa MWCO dialysis tubing for 2 days at 4 °C and lyophilized for 2 days. For VEGF-C encapsulation, 100 mg / mL PPCN was mixed with 250-500 ng / mL VEGF-C.

[0110] Delivery with lymphatic endothelial cells

[0111] PPCN's ocular biocompatibility was confirmed in a rabbit model of trabeculectomy, showing similar efficacy to Mitomycin-C (MMC) in reducing IOP and preventing fibrosis (Figure 1). After the trabeculectomy surgery, 10 pL of 100 mg / mL PPCN hydrogel mixed with 100,000 lymphatic endothelial cells was administered.

[0112] Development of peptide-modified thermoresponsive citrate-based hydrogel for tubulogenesis of lymphogenic endothelial cells

[0113] Peptide-modified thermoresponsive citrate-based hydrogels are engineered, e.g., specifically targeting the tubulogenesis of lymphogenic endothelial cells. Variants of the PPCN hydrogel, e.g., PPCN-A5G81 and PPCN-QK, are synthesized and thorough characterization is conducted. The chemical structures of these materials is verified through proton magnetic resonance t1H NMR). The degree of peptide conjugation on PPCN is quantified using the 2,4,6- Trinitrobenzene Sulfonic Acid (TNBSA) assay. The mechanical properties and phase transition temperatures of the peptide-modified PPCN hydrogels is determined by rheological analysis. The potential of these biomatcrials to support tubulogcncsis is demonstrated by culturing lymphogenic endothelial cells atop the hydrogel surfaces.

[0114] Assessing cell delivery efficacy in a rabbit trabeculectomy model using PPCN-peptide hydrogels

[0115] Various formulations of PPCN hydrogels — both with and without cellular integration (e.g., PPCN alone, PPCN with cells, PPCN-A5G81, PPCN-A5G81 with cells, PPCN-QK, and PPCN-QK with cells) — are administered directly into the surgical site immediately following the procedure. MMC and a no-treatment group serve as controls for comparison. IOP is meticulously monitored on days 0, 1, 4, 8, 11, 15, 18, 22, 25, and 29. Upon reaching the 29-day mark, the tissues are harvested for detailed histological and immunohistochemical analyses to explore the extent of fibrosis and the development of lymphogenic structures.

[0116] REFERENCES

[0117] Tham, Y. C. et al. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology 121, 2081-2090, doi:10.1016 / j.ophtha.2014.05.013 (2014).

[0118] Kang, J. M. & Tanna, A. P. Glaucoma. Med Clin North Am 105, 493-510, doi:10.1016 / j.mcna.2021.01.004 (2021).

[0119] Wilkins, M., Indar, A. & Wormaid, R. Intra-operative mitomycin C for glaucoma surgery. Cochrane Database Syst Rev 2005, Cd002897, doi:10.1002 / 14651858. CD002897.pub2 (2005).

[0120] Gedde, S. J. et al. Treatment Outcomes in the Primary Tube Versus Trabeculectomy Study after 5 Years of Follow-up. Ophthalmology 129, 1344-1356, doi:10.1016 / j.ophtha.2022.07.003 (2022).

[0121] Boland, M. V, Corcoran, K. J. & Lee, A. Y. Changes in Performance of Glaucoma Surgeries 1994 through 2017 Based on Claims and Payment Data for United States Medicare Beneficiaries. Ophthalmol Glaucoma 4, 463-471, doi:10.1016 / j.ogla.2021.01.004 (2021).

[0122] Torbey, J. et al. XEN 45 Gel Stent Implantation in Open Angle Glaucoma: 5-Year Results of a Prospective Study. J Glaucoma 32, 909-917, doi:10.1097 / ijg.0000000000002302 (2023).

[0123] Lee, J. Y. et al. Structural Confirmation of Lymphatic Outflow from Subconjunctival Blebs of Live Human Subjects. Ophthalmol Sci 1, doi:10.1016 / j.xops.202L 100080 (2021).

Claims

CLAIMS1. A composition comprising bioactive protein / peptide-modified PPCN.

2. The composition of claim 1, wherein the bioactive protein / peptide is laminin-derived peptide or a VEGF-derived peptide.

3. The composition of claim 1 or 2, wherein the bioactive protein / peptide is selected from A5G81, QK, VEGF, VEGF-c, SPARC113-130, VEGF-Mimic Pl, PR1P, ANGPTL4-mimetic Peptide, FGF2-Mimetic Peptide, 4N1K Peptide, RGD Peptide, IKVAV, YIGSR, Substance P, AcSDKP, or variants thereof.

4. The composition of claim 3, wherein the bioactive protein / peptide has at least 70% sequence identity to one of SEQ ID NOS: 1-14.

5. The composition of claim 4, wherein the bioactive protein / peptide has 100% sequence identity to one of SEQ ID NOS: 1-14.

6. The composition of one of claims 1-5, wherein the bioactive protein / peptide is covalently linked to PPCN.

7. The composition of claim 6, wherein bioactive protein / peptide is covalently linked to PPCN by carbodiimide chemistry.

8. The composition of one of claims 1-5, wherein the bioactive protein / peptide is encapsulated within a matrix or hydrogel of PPCN.

9. The composition of claim 8, wherein the bioactive protein / peptide capable of being eluting from the PPCN into an aqueous or biological environment.

10. The composition of one of claims 1-9, further comprising lymphatic endothelial cells.

11. The composition of one of claims 1-10, comprising multiple different bioactive proteins / peptides.

12. The composition of claim 11, comprising bioactive proteins / peptides conjugated to the PPCN and unconjugated bioactive proteins / peptides encapsulated within a matric or hydrogel of PPCN.

13. A method comprising administering a composition of one of claims 1-12 to a subject.

14. The method of claim 13, wherein the subject suffers from glaucoma.

15. The method of claim 14, wherein the subject has undergone glaucoma surgery.

16. The method of claim 15, wherein the subject is in an acute recovery phase, subacute phase, or chronic post-surgical phase.

17. The method of claim 15, wherein the glaucoma surgery is selected from trabeculectomy, glaucoma drainage device (GDD) implantation, minimally invasive glaucoma surgery (MIGS), selective laser trabeculoplasty (SLT), and diode cyclophotocoagulation.

18. The method of one of claims 13-17, wherein the composition is co-administered with one or more antibiotics anti-inflammatory agents, anti-fibrotic agents, pressure-lowering medications immunomodulatory agents, neuroprotective compounds, and / or antioxidant supplements.