A pedf-7mer polypeptide hydrogel, and preparation method and application thereof

Abstract

The application provides a PEDF-7mer polypeptide hydrogel and a preparation method and application thereof, and belongs to the technical field of ophthalmic hydrogels. The raw material of the hydrogel comprises a maleimide-modified poloxamer F127, a thiolated hyaluronic acid and a thiol-modified PEDF-7mer polypeptide, a double covalent crosslinking network is constructed through thiol-maleimide click chemistry, the PEDF-7mer polypeptide is covalently grafted on the gel skeleton, and the problems of drug burst release and easy degradation can be solved. The hydrogel can form covalent combination with corneal surface proteins, improve the wet-state adhesion, and prolong the ocular surface retention time of the preparation; meanwhile, the system has high transparency and does not block the vision of the patient, and the clinical use comfort is improved. Animal and cell experiments prove that the hydrogel can significantly inhibit corneal neovascularization, down-regulate inflammatory factors, remove active oxygen, and reduce corneal inflammatory edema. The hydrogel can be used for treating corneal neovascularization and corneal epithelial inflammation related eye diseases.

Description

Technical Field

[0001] This invention relates to the field of ocular hydrogel technology, and more particularly to a PEDF-7mer polypeptide hydrogel, its preparation method, and its application. Background Technology

[0002] Corneal neovascularization (CNV) is a common blinding ocular surface disease, often caused by factors such as corneal acid and alkali burns, mechanical trauma, pathogenic microbial infections, and autoimmune inflammation. A normal, healthy cornea is transparent and avascular, relying on tears and aqueous humor for nutrition to maintain its light transmission function. When the corneal epithelial barrier is damaged, pro-angiogenic factors are abnormally expressed in large quantities, leading to neovascularization that invades the corneal stroma. This continuously induces corneal edema, inflammatory cell infiltration, and fibrous scar hyperplasia, resulting in permanent corneal opacity, severely impairing visual function. In advanced stages, it can directly lead to blindness, making it a pressing clinical challenge in ophthalmology.

[0003] Currently, clinical interventions for CNV are mainly divided into two categories: surgical treatment and topical eye drops. Surgical procedures such as laser photocoagulation and corneal transplantation are highly invasive and have a high risk of recurrence after surgery, and are only suitable for patients with severe and advanced CNV, thus limiting their applicability. For patients with mild to moderate CNV, topical eye drops are the preferred clinical treatment, but existing delivery systems have technical limitations that make it difficult to eradicate the CNV. Pigment epithelial-derived factor (PEDF) has multiple biological activities, including potent anti-angiogenic, antioxidant, anti-inflammatory, and corneal epithelial repair-promoting effects, making it an ideal active molecule for CNV targeted therapy. However, the full-length PEDF protein has a large molecular weight, making it difficult to penetrate the dense corneal epithelial barrier. Furthermore, the ocular surface environment is rich in various proteases, and free full-length PEDF is easily hydrolyzed, degraded, and rapidly inactivated, resulting in extremely low bioavailability of topical corneal drugs, making it impossible to directly develop conventional eye drop formulations.

[0004] Existing research confirms that the PEDF-derived 7-peptide short peptide (PEDF-7mer) retains the core pharmacological activity of PEDF, while having a smaller molecular weight and significantly better corneal tissue penetration than the full-length PEDF protein, demonstrating great potential for developing ophthalmic formulations. However, there are significant drawbacks to preparing ordinary eye drops by dissolving PEDF-7mer peptides in buffer solution: the peptides remain free on the ocular surface and are rapidly washed away by tears, resulting in a very short effective time on the cornea; ocular surface proteases can directly contact the exposed peptides, causing rapid drug degradation and a significant reduction in efficacy.

[0005] To prolong peptide retention time and improve drug stability, existing technologies often employ physical ocular hydrogels constructed with matrices such as poloxamer, sodium hyaluronate, and alginate. These hydrogels load peptide drugs through physical encapsulation. However, these gels rely solely on weak intermolecular interactions for drug encapsulation, failing to achieve covalent bonding between the drug and the carrier. This results in two major drawbacks: first, severe burst release occurs, with large amounts of peptides rapidly released and depleted within a short period, making stable and long-lasting controlled release impossible; second, the peptides are only physically embedded in the gel network, resulting in weak binding forces. Even slight tear rinsing can cause drug detachment, making it difficult to maintain a long-term effective drug concentration in the cornea. Furthermore, the peptides exposed on the outside of the gel pores are still easily degraded and inactivated by proteases.

[0006] Most commercially available ophthalmic gel matrices use unmodified poloxamer F127, relying on temperature-induced hydrophobic aggregation to achieve physical gelation. The system only exhibits dynamic physical cross-linking, lacking a stable, directional covalent network, resulting in poor gel mechanical and adhesive properties. Some composite gels simply blend hyaluronic acid and poloxamer, with no chemical cross-linking between the two components. This leads to a loose overall gel structure and uncontrollable degradation rate, making it difficult to meet the requirements for long-acting ophthalmic drug delivery. Furthermore, ordinary hyaluronic acid matrices rely solely on intermolecular hydrogen bonds for ocular surface adhesion, exhibiting weak wet adhesion and easily detaching from the corneal surface during blinking or tear flow. Simple thermosensitive poloxamer gels lack adhesive properties and cannot achieve prolonged ocular surface retention.

[0007] In summary, existing physically blended ophthalmic hydrogels cannot simultaneously solve multiple problems such as easy loss and degradation of PEDF-7mer peptides, uncontrollable release, and short residence time on the ocular surface. Clinically, there is an urgent need for a novel ophthalmic formulation that can adapt the molecular structure of PEDF-7mer to meet the needs of local ocular drug delivery, providing long-acting, highly effective, and low-frequency drug delivery for corneal neovascularization and corneal epithelial inflammation. Summary of the Invention

[0008] In view of this, the present invention provides a PEDF-7mer polypeptide hydrogel, its preparation method and application, to solve the above problems.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a PEDF-7mer polypeptide hydrogel, the raw materials of which include maleimide-modified poloxamer F127, thiolated hyaluronic acid, and thiolated PEDF-7mer polypeptide; the hydrogel forms a bivalent cross-linking system through a thio-maleimide click chemistry reaction: maleimide-modified poloxamer F127 is covalently coupled with thiolated hyaluronic acid and thiolated PEDF-7mer polypeptide through thioether bonds.

[0011] This invention also provides a method for preparing the PEDF-7mer polypeptide hydrogel, comprising the following steps:

[0012] (1) Dissolve thiolized hyaluronic acid in PBS buffer, stir for 10-15 min to obtain thiolized hyaluronic acid solution, add thiol-modified PEDF-7mer peptide to obtain thiolized hyaluronic acid peptide solution;

[0013] (2) Dissolve maleimide-modified Prönnick F127 in PBS buffer, and let the maleimide-modified Prönnick F127 solution and the thiolized hyaluronic acid polypeptide solution prepared in step (1) stand at 36~38℃ for 13~17min respectively. Then mix the two solutions and react at 36~38℃ for 45~55s to obtain PEDF-7mer polypeptide hydrogel.

[0014] In step (2), the mass fraction of the gel precursor in the mixed system is 10~20wt%.

[0015] Preferably, the concentration of the thiolized hyaluronic acid polypeptide solution is 28-32 wt%, and the concentration of the maleimide-modified Pranic F127 solution is 28-32 wt%.

[0016] Preferably, the mass ratio of thiolated hyaluronic acid, maleimide-modified Pranic F127, and thiolated modified PEDF-7mer peptide is 0.8~1.2:1.8~2.2:0.1.

[0017] Preferably, the method for preparing the thiolized hyaluronic acid includes the following steps:

[0018] (1) Dissolve hyaluronic acid in ultrapure water, adjust the pH to 5.0~5.5, and activate the carboxyl groups with EDC / NHS;

[0019] (2) Add cystamine dihydrochloride for amide coupling and graft disulfide side chains;

[0020] (3) Add excess dithiothreitol to the reaction system in step (2) to reduce and break the disulfide bonds, releasing free thiol groups;

[0021] (4) Purify and freeze dry to obtain thiolized hyaluronic acid powder.

[0022] Preferably, the preparation method of the maleimide-modified poloxamer F127 includes the following steps:

[0023] (1) 6-maleimide hexanoic acid is chlorinated with thionyl chloride to obtain 6-maleimide hexanoyl chloride;

[0024] (2) The dried maleimide-modified poloxamer F127 was dissolved in anhydrous dichloromethane and esterified with 6-maleimide hexanoyl chloride under the catalysis of triethylamine.

[0025] (3) Concentrate the reaction system of step (2), add it dropwise to pre-cooled n-hexane, collect the precipitate, and remove impurities to obtain the final product.

[0026] Preferably, the thiol-modified PEDF-7mer polypeptide is prepared using the Fmoc solid-phase polypeptide synthesis method, by introducing a cysteine ​​residue carrying a free thiol group at the C-terminus of the polypeptide.

[0027] The present invention also provides the application of the PEDF-7mer polypeptide hydrogel in at least one of the following:

[0028] (1) Application in the preparation of ophthalmic drugs that inhibit corneal neovascularization;

[0029] (2) Application in the preparation of ophthalmic drugs that inhibit corneal epithelial cell inflammation.

[0030] By adopting the above technical solution, the present invention has the following beneficial effects:

[0031] This invention uses maleimide-modified poloxamer (PF127-MAL), thiolated hyaluronic acid (HA-SH), and thiolated modified PEDF-7mer peptide as raw materials, and achieves two key "exclusive bindings" through a thio-maleimide click chemistry reaction:

[0032] 1. Covalent coupling of drug and carrier: The terminal thiol group of PEDF-7mer peptide can undergo a click reaction with the maleimide group of maleimide-modified Pluronic F127 (PF127-MAL) to generate a stable thioether bond, which allows the peptide drug to be grafted onto the gel backbone at specific sites, which is different from the traditional physical embedding drug delivery method.

[0033] 2. Covalent cross-linking of the gel skeleton: PF127-MAL and HA-SH are also bonded to each other through a thiol-maleimide click reaction to construct a structurally stable three-dimensional cross-linked network, thereby achieving precise control of gel degradation rate and drug release behavior.

[0034] 3. This invention simultaneously introduces the PF127-MAL thermosensitive drug-carrying module and the thiolated hyaluronic acid (HA-SH) adhesion module. HA-SH can form covalent bonds with corneal surface proteins, greatly improving wet adhesion and prolonging the retention time of the formulation on the ocular surface. At the same time, the system has high transparency, does not obstruct the patient's vision, and improves the comfort of clinical use.

[0035] 4. This invention integrates a covalent drug-loaded unit with a wet adhesive matrix into a single in-situ thermosensitive eye drop. The low-temperature liquid state facilitates eye drops, and the transparent three-dimensional gel is rapidly formed at body temperature. A single dose can maintain an effective drug concentration in the cornea for a long time, reducing the frequency of administration, improving patient compliance, and providing a safe, efficient, and clinically applicable new formulation for the local treatment of corneal neovascularization. Attached Figure Description

[0036] Figure 1 The gelation time of PEDF-7mer peptide hydrogels when the total concentration of gel precursors is 10 wt%, 15 wt%, and 20 wt%.

[0037] Figure 2 The transparency of PEDF-7mer peptide hydrogels was observed when the total concentration of the gel precursor was 10 wt%, 15 wt%, and 20 wt%.

[0038] Figure 3 Electron microscopy images of blank gels with total precursor concentrations of 10 wt%, 15 wt%, and 20 wt%, and peptide hydrogels loaded with PEDF-7mer at a total precursor concentration of 15 wt%.

[0039] Figure 4 The ocular surface adhesion of PEDF-7mer peptide hydrogels was determined when the total concentration of gel precursors was 10 wt%, 15 wt%, and 20 wt%.

[0040] Figure 5 Image showing blood vessel length in mice under different treatments.

[0041] Figure 6 The image shows corneal thickness and inflammation in mice under different treatments.

[0042] Figure 7 This image shows the corneal epithelial healing process in mice treated with different methods.

[0043] Figure 8 The image shows the qPCR quantification results of macrophage M1 / M2 polarization genes in an LPS-induced inflammation model.

[0044] Figure 9 The results of intracellular reactive oxygen species (ROS) fluorescence staining and quantitative statistical graphs of fluorescence intensity in each group after H2O2-induced oxidative damage are shown.

[0045] Figure 10 The images show the staining and cell viability of corneal epithelial cells in each group after H2O2-induced oxidative damage.

[0046] Figure 11 The figure shows the results of the scratch assay to detect the inhibitory effect of 15wt% PEDF-7mer on VEGF-induced cell migration.

[0047] Figure 12 The results of the Transwell migration assay show that 15 wt% PEDF-7mer inhibits VEGF-induced cell migration.

[0048] Figure 13 A schematic diagram illustrating how PEDF-7mer hydrogel alleviates corneal neovascularization and ocular inflammation. Detailed Implementation

[0049] Terminology Notes

[0050] CNV: Corneal Neovascularization;

[0051] PEDF: Pigment Epithelium-Derived Factor;

[0052] PEDF-7mer: A short, active heptapeptide derived from PEDF, corresponding to the amino acid sites Asp64–Ser70 (7 consecutive amino acids of aspartic acid-serine). It retains complete biological activity in inhibiting VEGF and blocking angiogenesis. It is the shortest known functional short peptide fragment of PEDF and was purchased from Shaanxi Kairui Aosheng Biotechnology Co., Ltd.

[0053] PF127-MAL: Maleimide-modified Ploxam F127 (maleimide-modified poloxamer F127), is a temperature-sensitive carrier specifically designed for PEDF-7mer coupling in this invention;

[0054] HA-SH: Thioyl hyaluronic acid, which is the adhesion matrix prepared in this invention for the PEDF-7mer delivery system;

[0055] VEGF: Vascular Endothelial Growth Factor;

[0056] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0057] NHS: N-hydroxysuccinimide;

[0058] DTT: Dithiothreitol;

[0059] PBS: Phosphate-buffered saline;

[0060] LPS: Lipopolysaccharide;

[0061] ROS: Reactive oxygen species;

[0062] TNF-α: Tumor necrosis factor-α;

[0063] IL-1β: Interleukin-1β;

[0064] HUVEC: Human umbilical vein endothelial cells.

[0065] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] 1. Preparation of thiolated hyaluronic acid (HA-SH)

[0068] (1) Hyaluronic acid dissolution and carboxyl activation treatment

[0069] Accurately weigh 1.5 g of hyaluronic acid (HA) powder and add it to 150 mL of ultrapure water to prepare an HA aqueous solution. Stir magnetically at a constant speed for 5 h at room temperature in the dark until the powder is completely dissolved, resulting in a homogeneous, transparent, viscous solution. After complete HA dissolution, adjust the pH of the system to 5.2 with dilute hydrochloric acid / sodium hydroxide solution to provide reaction conditions for EDC / NHS. Accurately weigh 0.910 g of EDC and 0.683 g of NHS according to the HA carboxyl group molar ratio, and add them sequentially to the solution. Activate the solution by stirring at room temperature in the dark for 90 min. EDC can activate the HA carboxyl groups to generate an active intermediate, while NHS can stabilize the intermediate, prevent its hydrolysis and inactivation, and improve the carboxyl group activation efficiency.

[0070] (2) Cystamine dihydrochloride amide coupling reaction

[0071] After the carboxyl group activation was completed, 2.529 g of cystamine dihydrochloride powder was accurately weighed according to the preset grafting ratio and slowly added in batches to the activated HA reaction solution. After the addition was completed, the reaction was continuously stirred magnetically at room temperature in the dark for 48 h, and the pH of the system was maintained at 5.5 throughout the process to ensure the efficient amidation reaction.

[0072] During the reaction, the NHS active ester activated on the surface of the HA molecule undergoes a nucleophilic substitution reaction with the amino group in the cystamine dihydrochloride molecule to form a stable amide bond. The cystamine structure with disulfide bond is covalently grafted onto the hyaluronic acid molecular chain to generate a hyaluronic acid intermediate containing disulfide bond, realizing the initial grafting of functional groups and laying the structural foundation for the subsequent introduction of free thiol groups.

[0073] (3) Preparation of free thiol groups by DTT reduction

[0074] After the amide coupling reaction was completed, excess dithiothreitol (DTT) was added to the above reaction system to specifically break the disulfide bonds in the intermediate molecule. The reduction reaction was carried out with continuous stirring for 24 h at room temperature in the dark.

[0075] The disulfide bonds in the cystamine structure grafted onto the HA molecule are broken through the reduction effect of DTT, stably releasing highly active free thiol groups (-SH), thus preparing crude thiolized hyaluronic acid. Excess DTT ensures complete reduction of the disulfide bonds, preventing incomplete reduction and low thiol substitution. Simultaneously, complete light protection during the process prevents premature oxidation and cross-linking of the free thiol groups, ensuring product activity.

[0076] (4) Product purification and drying

[0077] Gradient dialysis for impurity removal: After the reduction reaction is completed, the crude reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for purification using gradient dialysis. First, the solution is dialyzed with low-concentration physiological saline for 3 hours to remove residual salt ions, unreacted small molecule EDC, NHS, cystamine dihydrochloride, DTT, and other impurities. Then, ultrapure water is used for continuous dialysis for 24 hours, with the dialysate being replaced every 6 hours until the dialysate is clear and free of impurities and small molecule residues, thus thoroughly removing reaction byproducts and unreacted raw materials and improving product purity.

[0078] The HA-SH aqueous solution purified by dialysis was then rapidly dispensed into lyophilized bottles and placed in a low-temperature freeze dryer for freeze drying to obtain a fluffy white flocculent HA-SH solid powder.

[0079] Substitution degree detection and product preservation: The degree of thiol substitution of the prepared HA-SH powder was determined using the classic Ellman reagent method. The grafting rate of free thiol groups in the sample was accurately calculated by combining colorimetric reaction with ultraviolet spectrophotometry. The qualified HA-SH powder was sealed and packaged, and stored in a light-protected, low-temperature, dry environment at -20℃ for later use.

[0080] 2. Preparation of maleimide-modified Pluronic F127 (PF127-MAL)

[0081] (1) Activation by 6-maleimide hexanoic acid acylation

[0082] The carboxyl groups in the raw material are activated into highly reactive acyl chloride groups through an acyl chloride reaction, thereby enhancing the activity of subsequent esterification reactions. The steps are as follows: 6-maleimide hexanoic acid is used as the raw material, and excess thionyl chloride is added as both the acylating agent and solvent. The mixture is stirred and mixed at 25°C, then heated to 60°C and refluxed for 48 hours. After the reaction, residual thionyl chloride and byproducts are removed by rotary evaporation under reduced pressure to obtain purified 6-maleimide hexanoyl chloride, which is then sealed and protected from light for later use.

[0083] (2) Anhydrous catalytic esterification grafting reaction

[0084] Prönnik F127 (PF127) was thoroughly dried under vacuum beforehand to remove moisture. 2.1945 g of the dried PF127 was dissolved in 50 mL of anhydrous dichloromethane, and 97.1 μL of triethylamine was added as an acid-binding agent and catalyst. The mixture was stirred and mixed at room temperature. Under anhydrous, oxygen-free, and light-protected conditions, 6-maleimide hexanoyl chloride prepared in step (1) was slowly added dropwise, and the mixture was stirred at a constant temperature for 24 h to allow it to react fully. The acyl chloride in the system underwent an esterification reaction with the terminal hydroxyl group of PF127, covalently grafting the maleimide group onto the end of the PF127 molecular chain to generate the crude PF127-MAL product.

[0085] (3) Solvent precipitation purification and drying

[0086] After the reaction was completed, the reaction solution was concentrated, the crude product was dissolved in dichloromethane, and pre-cooled n-hexane was added dropwise for reverse precipitation. The target product was then fully analyzed. The precipitate was collected, and the precipitation and purification were repeated several times to improve the purity. Finally, the product was dried under vacuum to constant weight to remove residual organic solvents, yielding a pure white PF127-MAL solid product.

[0087] (4) Characterization of product structure and substitution rate

[0088] The structure of the dried final product was characterized: the absorption peaks of the characteristic functional groups of maleimide were detected by Fourier transform infrared spectroscopy (FTIR) to verify the successful grafting of the groups; the characteristic hydrogen integral ratio was analyzed by nuclear magnetic resonance spectroscopy (H NMR) to calculate the degree of substitution and grafting efficiency of the maleimide groups, confirming the successful modification of the product.

[0089] 3. Preparation of thiol-modified PEDF-7mer peptides

[0090] Thiol-modified PEDF-7mer peptides were prepared using the standard Fmoc solid-phase peptide synthesis process. By specifically introducing cysteine ​​residues at the C-terminus of the peptide to carry free active thiol groups, high-purity target peptides were obtained after purification and structural identification. The specific steps are as follows:

[0091] (1) Solid-phase polypeptide synthesis

[0092] A suitable solid-phase resin was selected as the synthesis carrier, and a series of Fmoc-protected amino acids were used as raw materials. Following the predetermined amino acid sequence of the PEDF-7mer peptide, deprotection, amino acid coupling, and end-capping cycling reactions were performed sequentially. During the synthesis process, a cysteine ​​residue (Cys) was introduced at the C-terminus of the peptide, utilizing its active side-chain thiol group to achieve thiol functionalization of the peptide. After all sequences were assembled, the resin was cleaved using a lysis buffer, and the protecting groups of the amino acid side chains were simultaneously removed to obtain the crude thiol-modified PEDF-7mer peptide.

[0093] (2) High performance liquid chromatography (HPLC) purification

[0094] After dissolving the crude peptide, gradient elution purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Based on the difference in retention time between the target peptide and impurities, incompletely coupled short peptides, residual amino acids, and cleavage impurities were separated and removed. The target main peak component was collected to improve the peptide purity.

[0095] (3) Mass spectrometry identification and finished product preparation

[0096] The purified peptide sample was freeze-concentrated, and its molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The theoretical and measured molecular weights were compared to verify the correctness of the peptide sequence and structure. Finally, a purified functionalized PEDF-7mer peptide with free thiol groups was obtained and stored in a sealed container at low temperature, protected from light, for later use.

[0097] Example 2. Preparation of PEDF-7mer ophthalmic composite hydrogel (15 wt%)

[0098] 1. Pre-loaded drug reaction

[0099] Accurately weigh 100 mg of thiolized hyaluronic acid (HA-SH), add 1 mL of 0.02 mol / L, pH 7.4 PBS buffer (pre-added with 0.013 mmol triethylamine), stir thoroughly to dissolve, and activate at room temperature for 15 min; then weigh 1 mg of thiol-modified PEDF-7mer peptide, add it to the above HA-SH solution and mix well to obtain HA-SH peptide solution for later use.

[0100] 2. In-situ hydrogel molding

[0101] Accurately weigh 200 mg of maleimide-modified Pranic F127 (PF127-MAL) and dissolve it in 1 mL of 0.02 mol / L, pH 7.4 PBS buffer. Place the PF127-MAL solution and the HA-SH peptide solution obtained in step 2 together at 37°C and let them stand for 15 min. Then quickly mix the two liquids and keep them at 37°C for about 50 s. The system undergoes a thiol-maleimide click reaction and forms a hydrogel in situ.

[0102] After the hydrogel was filtered through a 0.22 μm sterile filter membrane, it was dispensed into eye drop bottles and stored at room temperature in the dark to obtain PEDF-7mer polypeptide hydrogel.

[0103] The concentration of the hydrogel = (mass of thiolated hyaluronic acid + mass of maleimide-based Pranic) / total mass of the precursor solution × 100%.

[0104] Example 2: Preparation of PEDF-7mer ophthalmic composite hydrogel (10 wt%)

[0105] 1. Pre-loaded drug reaction

[0106] Accurately weigh 66.7 mg of thiolized hyaluronic acid (HA-SH), add 1 mL of 0.02 mol / L, pH 7.4 PBS buffer (the buffer is pre-added with 0.0087 mmol triethylamine, and the amount of catalyst is reduced proportionally according to the 10 wt% system), stir thoroughly to dissolve, and activate at room temperature for 15 min; then weigh 1 mg of thiol-modified PEDF-7mer peptide, add it to the above HA-SH solution and mix well to obtain the HA-SH peptide solution for later use.

[0107] 2. In-situ hydrogel molding

[0108] Accurately weigh 133.3 mg of maleimide-modified Pluronic F127 (PF127-MAL) and dissolve it in 1 mL of 0.02 mol / L, pH 7.4 PBS buffer. Place the PF127-MAL solution and the HA-SH peptide solution obtained in step 1 together at 37°C and let them stand for 15 min. Then, quickly mix the two liquids and keep them at 37°C for about 120 s. The system undergoes a thiol-maleimide click reaction and forms a 10 wt% hydrogel in situ.

[0109] The hydrogel was filtered through a 0.22μm sterile filter membrane and then dispensed into eye drop bottles and stored at room temperature away from light, resulting in 10wt% PEDF-7mer polypeptide hydrogel.

[0110] Example 3. Preparation of PEDF-7mer ophthalmic composite hydrogel (20 wt%)

[0111] 1. Pre-loaded drug reaction

[0112] Accurately weigh 133.3 mg of thiolized hyaluronic acid (HA-SH), add 1 mL of 0.02 mol / L, pH 7.4 PBS buffer (pre-added with 0.017 mmol triethylamine), stir thoroughly to dissolve, and activate at room temperature for 15 min; then weigh 1 mg of thiol-modified PEDF-7mer peptide, add it to the above HA-SH solution and mix well to obtain HA-SH peptide solution for later use.

[0113] 2. In-situ hydrogel molding

[0114] Accurately weigh 266.7 mg of maleimide-modified Pluronic F127 (PF127-MAL) and dissolve it in 1 mL of 0.02 mol / L, pH 7.4 PBS buffer. Place the PF127-MAL solution and the HA-SH peptide solution together at 37°C and let them stand for 15 min. Then, quickly mix the two liquids and keep them at 37°C for about 30 s. The system undergoes a thiol-maleimide click reaction, forming a 20 wt% hydrogel in situ.

[0115] After the hydrogel was filtered through a 0.22 μm sterile filter membrane, it was dispensed into eye drop bottles and stored at room temperature in the dark to obtain 20 wt% PEDF-7mer polypeptide hydrogel.

[0116] Experimental Example 1. Physicochemical Properties Characterization of Hydrogel Eye Drops

[0117] 1. Transparency Evaluation

[0118] The PEDF-7mer polypeptide hydrogels prepared in Examples 1, 2, and 3 were dropped onto a white substrate printed with the black letter "A," forming a gel film. Transmittance photographs of the gels were taken under natural light to visually compare the transmittance properties of the different PEDF-7mer polypeptide hydrogels, observe the clarity of the underlying letter, and evaluate the gel transparency. The results are as follows: Figure 2 As shown in the figure. The results showed that the 10 wt% and 15 wt% hydrogels were clear and transparent, with the letter "A" at the bottom clearly visible, meeting the light transmittance requirements of ophthalmic formulations; the 20 wt% gel system was turbid, and its light transmittance was significantly reduced.

[0119] 2. Characterization of micropore structure by scanning electron microscopy (SEM)

[0120] Complete hydrogels from Examples 1, 2, and 3 were rapidly frozen in liquid nitrogen and then freeze-dried under vacuum to remove internal moisture while preserving the three-dimensional porous framework structure. Three groups of complete PEDF-7mer peptide hydrogel samples (10 wt%, 15 wt%, and 20 wt%) were taken respectively, and regular gel pieces of approximately 5 mm × 5 mm × 3 mm were cut. The gel samples were then rapidly immersed in liquid nitrogen for catalytic freezing to fix the internal three-dimensional porous network structure and prevent ice crystals from damaging the pore morphology.

[0121] After freezing, the samples were quickly transferred to a vacuum freeze dryer for lyophilization to completely remove internal moisture from the gel and preserve the original cross-linked porous framework to the greatest extent possible. Once lyophilization was complete, the samples were removed and gently broken along the gel cross-section. A fresh, flat, uncompressed cross-section was selected as the observation surface. The samples were then firmly adhered to the SEM sample stage using conductive adhesive, with the cross-section facing upwards. The sample surface was then coated with gold for conductivity (5–10 nm thickness). The prepared samples were then placed in the scanning electron microscope sample chamber. Appropriate observation voltage and magnification were set to observe the three-dimensional porous microstructure of each group of hydrogels. Cross-sectional pore structure photographs were taken to compare the differences in pore size, pore distribution, and cross-linking density of hydrogels under different precursor concentrations.

[0122] SEM results ( Figure 3 The results showed that all three blank gels possessed a continuous three-dimensional porous network. With increasing polymer concentration, the pore walls thickened, the pore size gradually decreased, and the cross-linked network became denser. In the 15 wt% system, after the introduction of PEDF-7mer peptides, the original porous framework remained intact, and the peptides were uniformly dispersed within the gel pores without significant aggregation or pore blockage. Overall, the hydrogel with a 15 wt% precursor concentration exhibits both excellent light transmittance and a suitable porous structure, making it suitable for ophthalmic functional carrier applications.

[0123] Experimental Example 2. Animal Verification Experiment on Ocular Surface Retention and In vivo Sustained-Release Performance

[0124] Using healthy C57 mice as experimental subjects, three groups of PEDF-7mer drug-loaded thermosensitive hydrogel eye drops with total precursor concentrations of 10wt%, 15wt%, and 20wt% were applied topically to the corneal surface of the mice. Anterior segment OCT was used to dynamically observe the gel retention status at different time points, recording the integrity of gel coverage and retention thickness at 0h and 2h. Simultaneously, the drug retention content in the corneal tissue was measured. The adhesion, retention, and long-acting sustained-release capabilities of different concentration formulations were compared and evaluated. The results are as follows: Figure 4 As shown.

[0125] The results showed that the 10wt% gel system had insufficient mechanical strength and was easily washed away by tear flushing. The amount of gel residue on the ocular surface decreased significantly 2 hours after administration, and it could not achieve long-term retention. The 20wt% gel system had too high cross-linking density and too fast gel formation rate. The gel texture was too hard and could not adhere evenly to the corneal surface. It was prone to edge lifting and detachment, and had poor light transmittance. The 15wt% gel system had a suitable gel formation rate and could form a uniform and stable gel coating layer on the corneal surface. The gel structure was intact and there was no significant loss 2 hours after administration, providing a good structural basis for long-term ocular treatment.

[0126] Experimental Example 3. Intervention Experiment of Mouse Corneal Alkali Burn Neovascularization Model and Verification of Corneal Tissue Pathology and Mechanism of Action

[0127] Healthy SPF-grade C57 mice were selected to establish a corneal alkali burn-induced neovascularization (CNV) animal model. After successful modeling, the mice were randomly divided into four groups: normal control group, PBS group, 15wt% blank hydrogel group (Gel), and 15wt% PEDF-7mer (GP7) drug-loaded hydrogel experimental group. Each group received topical eye drops three times daily, with each drop being 5 μL, for 14 consecutive days. During the treatment period, corneal morphology was observed periodically using a slit-lamp microscope, and photographs were taken to record and quantify the corneal neovascularization coverage area and corneal opacity score to evaluate the in vivo therapeutic effect of the formulation. The results are as follows: Figure 5 As shown.

[0128] The results showed that the PBS control group mice had extensive corneal neovascularization, with disordered and dense blood vessels, severe corneal edema and opacity, and extremely poor corneal damage repair. The 15wt% blank hydrogel group could only improve ocular surface moisture and had a slight anti-inflammatory and neovascularization inhibitory effect. The 15wt% PEDF-7mer drug-loaded hydrogel experimental group could significantly reduce the corneal neovascularization coverage area, effectively alleviate corneal edema and opacity symptoms, promote corneal damage repair, and the in vivo treatment effect was significantly better than the other groups.

[0129] After 14 days of animal drug intervention, C57 mice in each group were sacrificed and corneal tissue was isolated. HE staining was used to observe the number of inflammatory cells infiltrating the corneal tissue and the degree of tissue damage in each group. The results are as follows: Figure 6 and Figure 7 As shown.

[0130] Experimental results: The blank control group and PBS group showed extensive infiltration, high density of neovascularization, and abnormally high expression of pro-angiogenic and inflammatory factors; the 15wt% blank gel group had no significant inhibitory effect on inflammation and angiogenesis; the 15wt% PEDF-7mer drug-loaded gel experimental group significantly reduced corneal inflammatory cell infiltration, reduced corneal neovascularization density, effectively blocked the vicious cycle of corneal pathology, and reduced the probability of lesion recurrence.

[0131] Experiment Example 4. In vitro cell antioxidant and anti-inflammatory activity verification experiment

[0132] 1. Preparation of hydrogel extract:

[0133] (1) Gel sample preparation and sterilization

[0134] Control group: 15 wt% blank hyaluronic acid gel (Gel);

[0135] Experimental group: 15 wt% PEDF-7mer peptide-loaded hyaluronic acid gel (GP7).

[0136] Cut the molded gel into round pieces / small blocks with a diameter of 8-10 mm and a thickness of 1-2 mm, and sterilize them by UV irradiation for 30 minutes.

[0137] (2) Pre-swelling and cleaning

[0138] Swelling system: sterile PBS buffer (pH 7.4);

[0139] Swelling conditions: Soak at 4℃ or room temperature for 8 hours until the gel reaches swelling equilibrium;

[0140] Cleaning procedure: After swelling is complete, rinse repeatedly with sterile PBS 3-5 times to fully wash away free hyaluronic acid and EDC / NHS activator residue;

[0141] (3) Extraction

[0142] Add 0.2 g of pretreated gel to 1 mL of complete culture medium and extract in a sealed cell culture incubator at 37℃ and 5% CO2 for 24 h. Remove the extract mixture, centrifuge at 3000 rpm for 5 min, collect the supernatant, filter through a 0.22 μm sterile membrane, and adjust the pH of the filtrate to 7.2-7.4. The resulting liquid is the 100% gel extract stock solution.

[0143] Extraction medium formulation: basal culture medium + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin bispecific antibody.

[0144] 2. Anti-inflammatory activity test

[0145] The experiment included a model control group, a modeling group, a 15wt% blank gel (Gel) group, and a 15wt% PEDF-7mer drug-loaded gel (GP7) group.

[0146] Blank control group: cultured in normal culture medium for 12 h;

[0147] Except for the blank control group, the modeling group, the 15wt% blank gel (Gel) group, and the 15wt% PEDF-7mer drug-loaded gel (GP7) group were first stimulated with 1μg / mLLPS for 12h to construct a macrophage M1 pro-inflammatory polarization model, and then the following treatments were performed:

[0148] Model group: 1 μg / mL LPS medium, 12 h (containing LPS throughout);

[0149] Blank gel extract group (Gel group): Pretreated with 1 μg / mL LPS for 12 h, then replaced with fresh culture medium containing 1 μg / mL LPS + 100% blank gel extract, and cultured until the endpoint;

[0150] GP7 drug-loaded gel extract group: pretreated with 1 μg / mL LPS for 12 h, then replaced with fresh culture medium containing 1 μg / mL LPS + 100% GP7 extract, and cultured until the endpoint.

[0151] 3. In vitro cellular antioxidant experiments

[0152] The experiment included a model control group, a modeling group, a 15wt% blank gel (Gel) group, and a 15wt% PEDF-7mer drug-loaded gel (GP7) group.

[0153] Blank control group: cultured in conventional complete culture medium without the addition of H2O2 or gel extract until the experimental endpoint.

[0154] Except for the blank control group, the modeling group, the 15wt% blank gel (Gel) group, and the 15wt% PEDF-7mer drug-loaded gel (GP7) group were first subjected to 200μM H2O2 to damage human corneal epithelial cells for 2h to construct an oxidative stress cell model, and then the following treatments were performed:

[0155] Modeling concentration: 200 μM H2O2, applied throughout the entire process; Intervention reagent: 100% gel extract;

[0156] Model group: Cultured in a medium containing 200 μM H2O2 without the intervention of extract solution until the experimental endpoint.

[0157] Blank gel extract group (Gel): The culture medium containing 200 μM H2O2 + 100% blank gel extract was added directly and cultured until the experimental endpoint.

[0158] PEDF-7mer drug-loaded gel extract group (GP7): The medium containing 200 μM H2O2 + 100% GP7 extract was directly added and cultured until the experimental endpoint.

[0159] After each intervention, intracellular ROS levels were measured. Quantitative real-time PCR was used to detect the mRNA and protein expression levels of pro-inflammatory markers iNOS and CD86, and anti-inflammatory repair markers CD206 and Arg1. The in vitro antioxidant and anti-inflammatory capabilities of each formulation were evaluated. Macrophage polarization inflammation assay results are as follows: Figure 8 As shown, the results of the oxidative damage experiment are as follows: Figure 9 and Figure 10 As shown.

[0160] Figure 8 This indicates that the blank gel provides only weak protection to cells; the GP7 peptide-loaded gel can significantly scavenge reactive oxygen species, improve cell survival rate, and has outstanding antioxidant and anti-cell damage effects. Figure 8The results showed that the blank gel had no significant regulatory effect; GP7 could significantly downregulate the M1 pro-inflammatory markers iNOS and CD86, upregulate the M2 anti-inflammatory repair markers CD206 and Arg1, inhibit the macrophage pro-inflammatory phenotype, and improve the local inflammatory microenvironment. Figure 9 and Figure 10 This indicates that GP7 peptide-loaded gel can effectively scavenge reactive oxygen species generated by oxidative stress, alleviate hydrogen peroxide-induced cellular oxidative damage, significantly improve the survival rate of damaged cells, and possess excellent antioxidant and cell-protective activities.

[0161] Experimental Example 5. In vitro anti-angiogenic activity verification experiment

[0162] Human umbilical vein endothelial cells (HUVECs) were used as experimental cells to construct cell scratch migration model and Transwell invasion model, respectively. At the same time, control group, VEGF (vascular endothelial growth factor) group, VEGF+Gel group, and VEGF+GP7 group were set up.

[0163] The blank control group was cultured in a standard complete culture medium without pretreatment, VEGF, or extract, and cultured throughout the entire process.

[0164] The VEGF group was first treated with VEGF-containing medium for 12 hours, and then replaced with new VEGF-containing medium. No gel extraction solution was added during the entire process.

[0165] VEGF+Gel group: Cells were pretreated with VEGF for 12 hours. After the pretreatment, the culture medium was replaced with VEGF containing 20 ng / mL VEGF + 100% blank gel extract and cultured for another 12 hours.

[0166] VEGF+GP7 group: Cells were first treated with VEGF for 12 hours. After the pretreatment, the culture medium was replaced with VEGF+100%GP7 drug-loaded gel extract containing 20 ng / mL VEGF+100%GP7 and cultured for another 12 hours.

[0167] The in vitro anti-angiogenic ability of the formulation was evaluated by quantitatively detecting cell migration rate and cell invasion number. The results are as follows: Figure 9 and Figure 10 As shown.

[0168] Experimental results: The VEGF+Gel group had limited inhibitory effects on endothelial cell migration, invasion, and lumen formation, and the duration of action was short. Compared with the VEGF and VEGF+Gel groups, the VEGF+GP7 group could significantly inhibit endothelial cell migration and invasion, and could inhibit angiogenesis in a long-term and stable manner, exhibiting excellent anti-corneal angiogenesis activity.

[0169] As can be seen from the above embodiments, the present invention provides a PEDF-7mer polypeptide hydrogel, its preparation method and application. The hydrogel prepared by the present invention can prolong the ocular surface retention time, significantly inhibit corneal neovascularization, downregulate inflammatory factors, and can be used for the treatment of corneal neovascularization and corneal epithelial inflammation-related eye diseases.

[0170] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PEDF-7mer polypeptide hydrogel, characterized in that, The raw materials include maleimide-modified poloxamer F127, thiolated hyaluronic acid, and thiolated modified PEDF-7mer peptide; the hydrogel forms a bivalent cross-linking system through a thio-maleimide click chemistry reaction: maleimide-modified poloxamer F127 is covalently coupled with thiolated hyaluronic acid and thiolated modified PEDF-7mer peptide by thioether bonds.

2. The method for preparing the PEDF-7mer polypeptide hydrogel according to claim 1, characterized in that, Includes the following steps: (1) Dissolve thiolized hyaluronic acid in PBS buffer, stir for 10-15 min to obtain thiolized hyaluronic acid solution, add thiol-modified PEDF-7mer peptide to obtain thiolized hyaluronic acid peptide solution; (2) Dissolve maleimide-modified Prönnick F127 in PBS buffer, and let the maleimide-modified Prönnick F127 solution and the thiolized hyaluronic acid polypeptide solution prepared in step (1) stand at 36~38℃ for 13~17min respectively. Then mix the two solutions and react at 36~38℃ for 45~55s to obtain PEDF-7mer polypeptide hydrogel. In step (2), the mass fraction of the gel precursor in the mixed system is 10~20wt%.

3. The preparation method according to claim 2, characterized in that, The concentration of the thiolized hyaluronic acid peptide solution is 28-32 wt%, and the concentration of the maleimide-modified Pluronic F127 solution is 28-32 wt%.

4. The preparation method according to claim 2, characterized in that, The mass ratio of thiolated hyaluronic acid, maleimide-modified Pranic F127, and thiolated modified PEDF-7mer peptide was 0.8~1.2:1.8~2.2:0.

1.

5. The preparation method according to claim 2, characterized in that, The method for preparing the thiolized hyaluronic acid includes the following steps: (1) Dissolve hyaluronic acid in ultrapure water, adjust the pH to 5.0~5.5, and activate the carboxyl groups with EDC / NHS; (2) Add cystamine dihydrochloride for amide coupling and graft disulfide side chains; (3) Add excess dithiothreitol to the reaction system in step (2) to reduce and break the disulfide bonds, releasing free thiol groups; (4) Purify and freeze dry to obtain thiolized hyaluronic acid powder.

6. The preparation method according to claim 2, characterized in that, The preparation method of the maleimide-modified poloxamer F127 includes the following steps: (1) 6-maleimide hexanoic acid is chlorinated with thionyl chloride to obtain 6-maleimide hexanoyl chloride; (2) The dried maleimide-modified poloxamer F127 was dissolved in anhydrous dichloromethane and esterified with 6-maleimide hexanoyl chloride under the catalysis of triethylamine. (3) Concentrate the reaction system of step (2), add it dropwise to pre-cooled n-hexane, collect the precipitate, and remove impurities to obtain the final product.

7. The preparation method according to claim 2, characterized in that, The thiol-modified PEDF-7mer peptide was prepared using the Fmoc solid-phase peptide synthesis method, by introducing a cysteine ​​residue carrying a free thiol group at the C-terminus of the peptide.

8. The use of the PEDF-7mer polypeptide hydrogel according to claim 1 in at least one of the following: (1) Application in the preparation of ophthalmic drugs that inhibit corneal neovascularization; (2) Application in the preparation of ophthalmic drugs that inhibit corneal epithelial cell inflammation.

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