Double-layer hydrogel for scar-free corneal injury repair and preparation method thereof

Abstract

The invention relates to double-layer hydrogel for scar-free corneal injury repair and a preparation method of the double-layer hydrogel. The preparation method comprises the following steps: (1) synthesizing oxidized polysaccharide and a sulfhydrylated polymer; the method comprises the following steps: oxidizing polysaccharide by taking NaIO4 as an oxidizing agent to obtain oxidized polysaccharide; wherein the oxidized polysaccharide is negatively charged; the preparation method comprises the following steps: modifying an amino-containing antibacterial polymer by using cysteine to obtain a sulfhydrylated polymer; (2) preparing bottom layer hydrogel; amino of the multi-arm polyethylene glycol and oxidized polysaccharide are subjected to a Schiff base reaction, and amino of the multi-arm polyethylene glycol and a multi-arm polyethylene glycol derivative are subjected to a cross-linking reaction, so that bottom-layer hydrogel is formed; wherein the multi-arm polyethylene glycol derivative has a group crosslinked with an amino group; (3) preparing double-layer hydrogel; and spraying the sulfhydrylation polymer on the bottom layer hydrogel, and carrying out UV illumination to obtain the double-layer hydrogel. According to the double-layer hydrogel disclosed by the invention, the purposes of resisting bacterial infection, regulating cascade immunoreaction, reducing scar formation and recovering cornea transparency are achieved through the cooperation of structural design, a biological mechanism and a time sequence.

Description

Technical Field

[0001] The present invention belongs to the technical field of ophthalmic biomaterials, and particularly relates to a double-layer hydrogel for scarless corneal damage repair and a preparation method thereof. Background Art

[0002] The cornea, the outermost layer of the eye, protects the inner tissues of the eye while also playing a key role in vision formation by refracting light. However, scarring caused by corneal damage is a serious threat to vision health. According to statistics, corneal scarring is the main cause of blindness. Among existing treatment options, corneal transplantation remains the most effective clinical means of restoring vision. However, its widespread application faces multiple constraints: a severe shortage of donor corneas, high surgical and treatment costs, and a lack of specialized equipment. These practical problems highlight the urgency and importance of developing alternative materials for corneal repair.

[0003] In recent years, hydrogels with excellent adjustability, transparency, and biocompatibility have become ideal candidates for corneal donors. However, conventional hydrogels without active ingredients only provide wound filling functions and are unable to intervene in the progression of corneal fibrosis, ultimately leading to unavoidable scarring. Fundamentally, this involves a complex corneal injury and repair cascade: after injury, IL-1α and TNF-α secreted by damaged epithelial cells induce apoptosis of quiescent corneal stromal cells or activate them into fibroblasts; subsequently, TGF-β and PDGF-BB drive fibroblast proliferation, chemotaxis, and differentiation into myofibroblasts, leading to disordered collagen deposition. Furthermore, bacterial keratitis, the most common early complication of corneal trauma, secretes a large number of inflammatory factors from microorganisms and corneal epithelial cells, which not only hinder wound healing and aggravate scarring, but can also lead to the serious consequence of corneal perforation.

[0004] To overcome this dilemma, innovative developments in hydrogel technology have injected new vitality into corneal scar treatment. In particular, functional hydrogels incorporating stem cells, exosomes, or growth factors have demonstrated significant therapeutic potential. However, these materials still have significant limitations: the isolation of stem cells and exosomes is complex and costly, and their long-term safety in vivo remains controversial. More importantly, existing technologies have yet to adequately address infection prevention and control after corneal transplantation. Therefore, the development of novel bioactive hydrogels that are independent of stem cells and exosomes has become a research priority. These materials are expected to achieve ideal therapeutic effects through a dual mechanism of action: first, preventing early infection; second, regulating the later wound healing cascade and synergistically inhibiting the fibrotic process, thereby effectively reducing scarring and opening up new avenues for the treatment of corneal injuries.

[0005] In summary, in the field of human corneal regeneration and repair, there is an urgent need to develop new hydrogel materials with good biological activity, which can effectively prevent infection and inhibit scar formation. Summary of the Invention

[0006] Based on the above-mentioned shortcomings and deficiencies in the prior art, one of the objects of the present invention is to at least solve one or more of the above-mentioned problems in the prior art. In other words, one of the objects of the present invention is to provide a double-layer hydrogel for scarless corneal damage repair and a preparation method thereof that meets one or more of the above-mentioned needs.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A method for preparing a double-layer hydrogel for scarless corneal damage repair comprises the following steps:

[0009] (1) Synthesis of oxidized polysaccharides and thiolated polymers;

[0010] Using NaIO4 as an oxidant, the polysaccharide is oxidized to obtain oxidized polysaccharide; wherein the oxidized polysaccharide has a negative charge;

[0011] The antibacterial polymer with amino group was modified by cysteine ​​to obtain thiol polymer;

[0012] (2) Preparation of bottom layer hydrogel;

[0013] The multi-arm polyethylene glycol amino groups react with the oxidized polysaccharide through Schiff base reaction and with the multi-arm polyethylene glycol derivative through cross-linking reaction to form a bottom layer hydrogel; wherein the multi-arm polyethylene glycol derivative has a group that cross-links with the amino group;

[0014] (3) Preparation of double-layer hydrogel;

[0015] The thiol-modified polymer is sprayed on the bottom hydrogel and irradiated with UV light to obtain a double-layer hydrogel.

[0016] The thiol polymer solution of the present invention is evenly covered on the surface of the bottom hydrogel by spraying. Then, under UV light conditions, the thiol groups in the thiol polymer undergo self-crosslinking reaction to form stable disulfide bonds, thereby constructing a dense network structure of the upper layer; at the same time, the aldehyde groups of the unreacted oxidized polysaccharide in the bottom hydrogel react with the thiol groups and amino groups in the thiol polymer respectively to form a double-layer hydrogel.

[0017] As a preferred embodiment, the synthesis process of the oxidized polysaccharide in step (1) includes:

[0018] In a dark environment, negatively charged polysaccharides and NaIO4 were dissolved in ultrapure water, stirred, and then anhydrous ethanol was added to inactivate unreacted NaIO4. The resulting solution was dialyzed, and the dialyzed purified solution was freeze-dried to obtain oxidized polysaccharides.

[0019] The ratio of polysaccharide, NaIO4, ultrapure water and anhydrous ethanol is (0.2-0.8) g: (0.05-0.4) g: (40-80) mL: (20-50) mL.

[0020] As a preferred embodiment, the negatively charged polysaccharide is heparin, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, alginic acid, fucoidan sulfate, carrageenan, pectin or xanthan gum.

[0021] As a preferred embodiment, the synthesis process of the thiol polymer in step (1) includes:

[0022] The antibacterial polymer with amino groups is dissolved in MES buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added, and cysteine ​​is added after stirring to obtain a mixed solution; the mixed solution is stirred in the dark for 12 to 24 hours, and the resulting solution is dialyzed against a HCl aqueous solution with a pH of 3 to 5, and then freeze-dried to obtain a thiol-modified polymer;

[0023] Wherein, the antibacterial polymer with amino group is ε-polylysine, α-polylysine or chitosan.

[0024] As a preferred embodiment, the ratio of the antibacterial polymer with amino group, EDC, NHS and cysteine ​​is (0.5-3) g: (0.5-1.5) g: (0.25-0.8) g: (0.2-5) g.

[0025] As a preferred solution, the step (2) specifically includes the following process:

[0026] Multi-arm polyethylene glycol amino, oxidized polysaccharide and multi-arm polyethylene glycol derivative were dissolved in PBS buffer respectively, mixed and vortexed to obtain a bottom layer hydrogel.

[0027] As a preferred embodiment, the molar ratio of the amino group in the multi-arm polyethylene glycol amino group, the aldehyde group of the oxidized polysaccharide, and the group cross-linked with the amino group in the multi-arm polyethylene glycol derivative is 10: (2 to 10): (1 to 9).

[0028] As a preferred embodiment, the multi-arm polyethylene glycol amino group is a four-arm polyethylene glycol amino group, a six-arm polyethylene glycol amino group, an eight-arm polyethylene glycol amino group, a sixteen-arm polyethylene glycol amino group or a thirty-two-arm polyethylene glycol amino group;

[0029] The multi-arm polyethylene glycol derivative is a four-arm polyethylene glycol derivative, a six-arm polyethylene glycol derivative, an eight-arm polyethylene glycol derivative, a sixteen-arm polyethylene glycol derivative or a thirty-two-arm polyethylene glycol derivative; wherein the group cross-linked with the amino group in the multi-arm polyethylene glycol derivative is a succinimidyl carbonate, an aldehyde group, a carboxyl group or a succinimide group.

[0030] As a preferred solution, the solid content of the bottom layer hydrogel is 3 to 10 wt%.

[0031] As a preferred solution, step (3) specifically includes the following process:

[0032] Thiolated polylysine was dissolved in PBS solution and sprayed on the bottom hydrogel with an intensity of 15 mW·cm -2 The UV light exposure time is 0.5 to 1 min.

[0033] The present invention also provides a double-layer hydrogel for scarless corneal damage repair prepared by the preparation method described in any of the above schemes.

[0034] The present invention also provides the use of the double-layer hydrogel described in the above scheme as a bioengineering material for treating corneal damage.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention designs a double-layer hydrogel with synergistic anti-fibrosis function. The bottom layer is formed by covalent bonding of multi-arm polyethylene glycol amino groups with oxidized polysaccharides and multi-arm polyethylene glycol derivatives. The negatively charged polysaccharides electrostatically adsorb cytokines, downregulate the wound healing cascade reaction to inhibit corneal fibrosis; the upper layer is constructed by self-crosslinking of thiol polymers, which effectively resists bacterial infection, reduces the secretion of inflammatory factors, and accelerates wound healing; the bottom layer and the upper layer are tightly chemically bonded through the reaction of the aldehyde groups of the oxidized polysaccharides with the thiol and amino groups of the thiol polymers to form thioether bonds and amide bonds, thereby enhancing mechanical stability and promoting functional synergy: the upper layer provides early antibacterial protection and reduces the generation of inflammatory factors, while the bottom layer adsorbs cytokines in the later stage to regulate fibrosis. The two synergistically accelerate tissue repair through structure, biological mechanism and time sequence, providing an efficient solution for the treatment of corneal injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the optical property characterization results of the bottom layer hydrogels of various component ratios in Example 1 of the present invention;

[0038] Figure 2 These are the characterization results of cytokine adsorption by the heparin-containing and heparin-free bottom hydrogels of Example 1 of the present invention, with A and B being the test results of the adsorption capacity of different bottom hydrogels for IL-1α and TGF-β1, respectively;

[0039] Figure 3 Figure 1 is the antibacterial performance of the double-layer hydrogels with different EPL-SH concentrations in Example 1 of the present invention. Figure A is a statistical analysis of the diameters of the inhibition rings against MRSA, and Figure B is a statistical analysis of the diameters of the inhibition rings against Pseudomonas aeruginosa.

[0040] Figure 4 These are the mechanical property characterization results of the bottom layer hydrogel and the double layer hydrogel of Example 1 of the present invention;

[0041] Figure 5 These are the test results of the cytokine adsorption capacity of the bottom layer and double-layer hydrogels of Example 1 of the present invention, A and B are the test results of the adsorption capacity of the bottom layer and double-layer hydrogels for IL-1α and TGF-β1, respectively;

[0042] Figure 6 This is an analysis of the in vivo antibacterial effect of the bottom layer hydrogel and the double layer hydrogel of Example 1 of the present invention;

[0043] Figure 7 This is an analysis of the anti-fibrosis effect of the bottom layer hydrogel and the double layer hydrogel of Example 1 of the present invention in vivo. DETAILED DESCRIPTION

[0044] The double-layer hydrogel for scarless corneal damage repair and its preparation method of the present invention are described in detail below.

[0045] The method for preparing the double-layer hydrogel for scarless corneal damage repair of the present invention comprises the following steps:

[0046] (1) Synthesis of oxidized polysaccharides and thiolated polymers;

[0047] a. Synthesis of oxidized polysaccharides;

[0048] Using NaIO4 as an oxidant, the polysaccharide is oxidized to obtain oxidized polysaccharide; wherein the oxidized polysaccharide has a negative charge;

[0049] Specifically, the synthesis process of the above-mentioned oxidized polysaccharide includes:

[0050] In a dark environment, negatively charged polysaccharides and NaIO4 were dissolved in ultrapure water, stirred, and then anhydrous ethanol was added to inactivate unreacted NaIO4. The resulting solution was dialyzed, and the dialyzed purified solution was freeze-dried to obtain oxidized polysaccharides.

[0051] The ratio of the above-mentioned polysaccharide, NaIO4, ultrapure water and anhydrous ethanol is (0.2-0.8) g: (0.05-0.4) g: (40-80) mL: (20-50) mL, which can be selected according to actual application requirements;

[0052] The negatively charged polysaccharide is selected from heparin, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, alginic acid, fucoidan sulfate, carrageenan, pectin, and xanthan gum, and can be selected according to actual application requirements.

[0053] b. Synthesis of thiol-polymers;

[0054] The antibacterial polymer with amino group was modified by cysteine ​​to obtain thiol polymer;

[0055] Specifically, the synthesis process of the above-mentioned thiol polymer includes:

[0056] The antibacterial polymer with amino groups is dissolved in MES buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added, and cysteine ​​is added after stirring to obtain a mixed solution; the mixed solution is stirred in the dark for 12 to 24 hours, and the resulting solution is dialyzed against a HCl aqueous solution with a pH of 3 to 5, and then freeze-dried to obtain a thiol-modified polymer;

[0057] The ratio of the above-mentioned antibacterial polymer with amino group, EDC, NHS and cysteine ​​is (0.5-3) g: (0.5-1.3) g: (0.25-0.79) g: (0.2-5) g, which can be selected according to actual application requirements;

[0058] Wherein, the antibacterial polymer with amino group is ε-polylysine, α-polylysine or chitosan.

[0059] (2) Preparation of bottom layer hydrogel;

[0060] The multi-arm polyethylene glycol amino groups reacted with oxidized polysaccharides through Schiff base reaction and with multi-arm polyethylene glycol derivatives through cross-linking reaction to form a bottom layer hydrogel;

[0061] Among them, the multi-arm polyethylene glycol derivative has groups cross-linked with amino groups and thiol groups;

[0062] The specific preparation process of the above-mentioned bottom layer hydrogel includes:

[0063] Multi-arm polyethylene glycol amino, oxidized polysaccharide and multi-arm polyethylene glycol derivative were dissolved in PBS buffer respectively, mixed and vortexed to obtain a bottom layer hydrogel.

[0064] The molar ratio of the amino groups in the multi-arm polyethylene glycol amino group, the aldehyde groups in the oxidized polysaccharide, and the groups cross-linked with the amino groups in the multi-arm polyethylene glycol derivative is 10:(2-10):(1-9), which can be selected according to actual application requirements to ensure that there are excess unreacted aldehyde groups of the oxidized polysaccharide, so that the bottom layer and the upper layer can react with the sulfhydryl and amino groups of the sulfhydrated polylysine through the reaction of the aldehyde groups of the oxidized polysaccharide and the sulfhydryl groups to form sulfide bonds and amide bonds, thereby achieving tight chemical bonding.

[0065] The multi-arm polyethylene glycol amino group is selected from four-arm polyethylene glycol amino group, six-arm polyethylene glycol amino group, eight-arm polyethylene glycol amino group, sixteen-arm polyethylene glycol amino group, and thirty-two-arm polyethylene glycol amino group; the specific selection can be made according to actual application requirements;

[0066] The above-mentioned multi-arm polyethylene glycol derivative is selected from a four-arm polyethylene glycol derivative, a six-arm polyethylene glycol derivative, an eight-arm polyethylene glycol derivative, a sixteen-arm polyethylene glycol derivative or a thirty-two-arm polyethylene glycol derivative; wherein the group cross-linked with the amino group in the multi-arm polyethylene glycol derivative is succinimidyl carbonate, aldehyde group, carboxyl group or succinimide group; the specific selection can be made according to actual application requirements.

[0067] The solid content of the bottom layer hydrogel is 3-10 wt%.

[0068] (3) Preparation of double-layer hydrogel;

[0069] The thiol-modified polymer is sprayed on the bottom hydrogel and irradiated with UV light to obtain a double-layer hydrogel.

[0070] The specific preparation process of the above-mentioned double-layer hydrogel includes:

[0071] The thiol-modified polymer was dissolved in PBS solution and sprayed on the bottom hydrogel with an intensity of 15 mW·cm -2 The UV light exposure time is 0.5 to 1 min.

[0072] The thiol polymer solution of the present invention is evenly covered on the surface of the bottom hydrogel by spraying. Subsequently, under UV light conditions, the thiol groups in the thiol polymer undergo self-crosslinking reaction to form stable disulfide bonds, thereby constructing a dense network structure of the upper layer; at the same time, the unreacted multi-arm polyethylene glycol derivatives in the bottom hydrogel react with the thiol groups and amino groups in the thiol polymer respectively to form a double-layer hydrogel, including the bottom hydrogel for adsorbing cytokines and the top hydrogel with antibacterial effect, which serves as a bioengineering material for treating corneal injuries and is used for scarless corneal injury repair.

[0073] The double-layer hydrogel for scarless corneal damage repair and its preparation method of the present invention are further explained below through specific examples.

[0074] Example 1:

[0075] The method for preparing the double-layer hydrogel of this embodiment comprises the following steps:

[0076] (1) Synthesis of oxidized heparin Hep-CHO;

[0077] 50 mg of NaIO4 was dissolved in 2 mL of ultrapure ddH2O, and 500 mg of heparin was dissolved in 50 mL of ddH2O. The mixture was stirred at room temperature for 2 h. After the reaction, 20 mL of anhydrous ethanol was added to inactivate unreacted NaIO4 for 30 min. The resulting solution was then dialyzed against ddH2O (MWCO = 3500 Da) for 3 days. Finally, the purified solution was lyophilized to obtain oxidized heparin Hep-CHO.

[0078] (2) Preparation of bottom layer hydrogel;

[0079] 4-Arm-PEG-NH2, 4-Arm-PEG-SC, and Hep-CHO were dissolved in PBS buffer to obtain solutions with concentrations of 100 mg / mL, 100 mg / mL, and 10 mg / mL, respectively. After mixing, the mixture was vortexed to obtain a bottom hydrogel.

[0080] Among them, the molar ratios of the amino group in 4-Arm-PEG-NH2, the succinimidyl carbonate SC group cross-linked with the amino group in 4-Arm-PEG-SC, and the aldehyde group of Hep-CHO are 10:5:6, 10:5:7, 10:5:8 and 10:5:9, respectively, and the solid content of the bottom hydrogel is 5 wt%, so as to obtain four types of heparin-containing bottom hydrogels, respectively. 5 P 10 P5H6, 5 P 10 P5H7, 5 P 10 P5H8 and 5 P 10 P5H9, as an example, 5 P 10 P5H6 indicates that the solid content of the bottom hydrogel is 5 wt %, and the molar ratio of the amino group in 4-Arm-PEG-NH2, the succinimidyl carbonate SC group cross-linked with the amino group in 4-Arm-PEG-SC, and the aldehyde group of Hep-CHO is 10:5:6.

[0081] The optical properties of the above-mentioned heparin-containing bottom layer hydrogel were tested as follows:

[0082] 100 μL of heparin-containing bottom hydrogel was prepared in a 96-well plate, and the absorbance of each well in the wavelength range of 400 to 800 nm was measured by a microplate reader (M200 PRO NanoQuant, TECAN). Figure 1 As shown, the transmittances of the four heparin-containing bottom layer hydrogels are all maintained above 90%, indicating that the bottom layer hydrogels have excellent light transmittance.

[0083] For comparison, a hydrogel without heparin was prepared. The specific preparation process differed from the above-mentioned preparation process of the hydrogel with heparin in that oxidized heparin Hep-CHO was not added. Specifically, the process included:

[0084] 4-Arm-PEG-NH2 and 4-Arm-PEG-SC were dissolved in PBS buffer to obtain solutions with concentrations of 100 mg / mL and 100 mg / mL, respectively. After mixing, the solutions were vortexed to obtain a heparin-free bottom hydrogel.

[0085] The molar ratio of the amino groups in 4-Arm-PEG-NH2 and the succinimidyl carbonate SC groups cross-linked with the amino groups in 4-Arm-PEG-SC is 1:1, the solid content of the heparin-free bottom layer hydrogel is 5 wt%, and the heparin-free bottom layer hydrogel is named PP.

[0086] The cytokine adsorption performance of the above-mentioned different bottom hydrogels was characterized as follows:

[0087] Human corneal epithelial cells (HCECs) were cultured in 10 cm culture dishes. When the cells reached a density of approximately 70%, star-shaped scratches were made on the bottom of the culture dish to allow the damaged corneal epithelial cells to secrete cytokines. After 24 hours, the culture medium was collected and centrifuged for later use. 100 μL of bottom hydrogel was prepared in a 12-well plate. The previously collected culture medium and hydrogel were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cytokine concentration was measured by ELISA. The results are shown in Figure 2. Figure 2 As shown in A, the control group is a group without hydrogel, and its IL-1α concentration is as high as 137.9 pg / mL; the IL-1α concentration of the PP hydrogel without heparin is 62.4±7.2 pg / mL; 5 P 10 The IL-1α concentration in the P5H6 hydrogel group was 19.1 ± 4.1 pg / mL; 5 P 10 The IL-1α concentration in the P5H8 hydrogel group was further reduced to 11.1 ± 1.3 pg / mL; Figure 2 As shown in B, the concentrations of TGF-β1 in each group were 728.7±20pg / mL in the control group and 125.7±18.1pg / mL in the PP group. 5 P 10 P5H6 hydrogel 62.7±8.3pg / mL, 5 P 10 The P5H8 hydrogel group had a cytokine adsorption capacity of 30.3 ± 4.2 pg / mL. This confirms that the ability of the bottom hydrogel to adsorb cytokines comes from heparin, and as the heparin content increases, the adsorption of cytokines becomes stronger.

[0088] (3) Synthesis of thiolated polylysine EPL-SH;

[0089] 1.0 g of EPL was dissolved in 0.1 M MES buffer (pH adjusted to 5.0 with 1 M HCl) to obtain an EPL solution. 1.3 g of EDC and 0.79 g of NHS were then added to the EPL solution and stirred at room temperature for 30 min to activate the carboxyl groups of EPL. Subsequently, 0.88 g of L-Cys was dissolved in 40 mL of 0.1 M MES buffer and mixed with the above solution. The mixed solution was stirred at 30°C in the dark for 1 day. Finally, the reacted solution was dialyzed (MWCO = 1000 Da) against aqueous HCl (pH = 3) for 3 days and lyophilized to obtain cysteine-modified EPL, namely EPL-SH.

[0090] (4) Preparation of double-layer hydrogel EPPH;

[0091] EPL-SH was dissolved in PBS buffer to form solutions with concentrations of 20 mg / mL, 45 mg / mL, 75 mg / mL, and 100 mg / mL, which were sprayed onto the heparin-containing bottom hydrogel PPH and irradiated with UV light to form a double-layer hydrogel: E 20 PPH, E 45 PPH, E 75 PPH and E 100 PPH.

[0092] The following is an in vitro antibacterial test of the double-layer hydrogels with different concentrations of EPL-SH, as follows:

[0093] In the in vitro antibacterial experiment, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) were used as the main research objects. A sterile coating stick was used to spread 100 μL of diluted MRSA or Pseudomonas aeruginosa suspension (5×10 3 CFU / mL) was evenly spread on the agar plate. Under sterile conditions, the hydrogel was prepared into a disc with a diameter of 5 mm and a height of 1 mm, and one side of the thiol-modified polylysine EPL-SH was attached to the agar plate. It was then placed in a 37°C constant temperature incubator and incubated for 24 hours. Finally, the diameter of the inhibition zone was measured using a ruler to evaluate its antibacterial properties. The results are shown in Figure 2. Figure 3 As shown in A, with the increase of EPL-SH concentration, the antibacterial performance of the double-layer hydrogel increased. For MRSA, EPL-SH 20 PPH,E 45 PPH,E 75 PPH and E 100The diameters of the inhibition zone in the PPH group were 15.5±3.9, 21.3±1.3, 22.3±0.5 and 24.3±1 mm, respectively. However, in the analysis of the inhibition zone diameter of MRSA, E 45 PPH and E 75 PPH did not show statistical differences; Figure 3 As shown in B, for PA, E 20 PPH,E 45 PPH,E 75 PPH and E 100 The diameters of the inhibition zone in the PPH group were 19.8±0.5, 21.3±0.5, 24.3±1 and 25.5±0.6 mm, respectively. However, in the analysis of the inhibition zone diameter of Pseudomonas aeruginosa, E 75 PPH and E 100 PPH did not show statistical difference.

[0094] The mechanical properties of the bottom layer hydrogel and the double layer hydrogel are characterized as follows:

[0095] Will 5 P 10 PPH, a double-layer hydrogel prepared with P5H8 as the bottom layer, was prepared into a 20 mm diameter, 2 mm height disc and tested on a rheometer (HAAKE MARS 60, Thermo Fisher Scientific). Frequency-dependent rheological measurements were performed in the range of 1 rad / s to 100 rad / s and at a strain of 1%. The results are shown in Figure 2. Figure 4 As shown, G′ represents the storage modulus, and G″ represents the loss modulus. When the concentration of EPL-SH is 20 mg / mL, the double-layer hydrogel E 20 The storage modulus of PPH (797±32Pa) is higher than that of PPH (601±64Pa). This is because the aldehyde groups of the unreacted oxidized polysaccharide in the bottom hydrogel react with the sulfhydryl and amino groups in the sulfhydrylated polylysine, further strengthening the chemical bond between the bottom layer and the upper layer, making E 20 PPH exhibits slightly enhanced mechanical properties. However, as the EPL-SH concentration increases, the storage modulus of the bilayer hydrogel decreases, though it remains slightly stronger than PPH. This is because the dynamic disulfide-crosslinked polylysine network in the upper layer mimics the flexibility of the corneal epithelium, influencing the overall mechanical properties of the hydrogel. This dual crosslinking strategy not only achieves a tight connection between the two hydrogel layers but also imparts excellent overall mechanical properties to the material.

[0096] The performance of the double-layer hydrogel in adsorbing cytokines is characterized as follows:

[0097] Human corneal epithelial cells (HCECs) were cultured in a 10 cm culture dish. When the cells reached a density of about 70%, star-shaped scratches were made on the bottom of the culture dish to allow the damaged corneal epithelial cells to secrete cytokines. After 24 hours, the culture medium was collected and centrifuged for later use. 100 μL of the culture medium was prepared in a 12-well plate. 5 P 10 The culture medium collected previously and the hydrogel were incubated in a 37°C, 5% CO2 incubator for 24 h, and the cytokine concentrations were determined by ELISA. Figure 5 A and Figure 5 As shown in B, the concentrations of IL-1α in each group were 135.4±2.4 pg / mL in the control group, 5 P 10 P5H8 hydrogel 11±1.2 pg / mL, E 45 The concentrations of TGF-β1 in the PPH hydrogel group were 7.6±1pg / mL; the concentrations of TGF-β1 in each group were 728.7±20pg / mL in the control group, 5 P 10 P5H8 hydrogel 62.7±8.3pg / mL, E 45 The PPH hydrogel group had a concentration of 53.2±3.7 pg / mL. The above results confirmed that the addition of the top hydrogel did not affect the cytokine adsorption performance of the bottom layer, but instead improved the efficiency of cytokine absorption.

[0098] The following is a test of the in vivo antibacterial and anti-fibrosis synergistic effect of the double-layer hydrogel, as follows:

[0099] (a) Before surgery, the experimental animals were anesthetized by intramuscular injection and oxybuprocaine hydrochloride eye drops were used. Lamellar keratectomy was performed in the center of the rabbit cornea using a 4 mm trephine drill. 3 μL of hydrogel was filled in the wound surface, and then 10 μL of 10% hydrogel was dripped on the surface of the hydrogel. 3 The experimental animals were divided into a normal group (normal), an injury group (no bacterial solution was added and the defect was not filled), a PPH group (PPH-) with bacterial solution but no antibacterial eye drops, a PPH group (PPH+) with bacterial solution and antibacterial eye drops, and an EPPH group (EPPH) with bacterial solution but no eye drops.

[0100] (b) To evaluate the antibacterial effect, corneas from the PPH-, PPH+, and EPPH groups were collected on day 3. The corneal homogenate was plated on agar plates and cultured at 37°C for 24 h before colony counts. Figure 6 As shown, the bacterial colonies in the PPH+ and EPPH groups were significantly reduced compared with the PPH- group; the results showed that EPPH hydrogel had significant antibacterial properties;

[0101] (c) If Figure 7 As shown, the first column of each group represents the slit lamp bright field image; the second column represents the corneal status under the cobalt blue light of the slit lamp, and the area stained with sodium fluorescein represents the corneal defect; the cornea of ​​the normal group was transparent and the surface was smooth; a small amount of corneal fibrosis was visible in the injury group under the slit lamp bright field on the 7th day, and the fibrosis gradually worsened and eventually formed a scar by the 90th day; when the bacterial keratitis model after corneal injury was established, the PPH- group developed eyeball adhesive secretions, conjunctival congestion, and anterior chamber purulent exudate on the 3rd day; while the PPH+ and EPPH groups did not develop infection, which confirmed that the EPPH group can reduce the postoperative care burden and reduce the risk of infection; and the slit lamp images from the 7th to the 90th day showed that the corneal epithelial defect in the EPPH group was repaired and the corneal transparency was restored, because the double-layer hydrogel effectively reduced the occurrence of fibrosis and infection, accelerated wound healing and corneal function reconstruction.

[0102] The Chinese names of the raw materials in the above examples are as follows:

[0103] 4-Arm-PEG-NH2: four-arm polyethylene glycol amino group;

[0104] 4-Arm-PEG-SC: four-arm polyethylene glycol succinimidyl carbonate;

[0105] EPL: ε-polylysine;

[0106] NaIO4: sodium periodate;

[0107] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

[0108] NHS: N-hydroxysuccinimide;

[0109] L-Cys: L-cysteine;

[0110] MES: 2-(N-morpholino)ethanesulfonic acid.

[0111] Given the numerous embodiments of the present invention, the raw materials and amounts involved can be selected within a limited range according to actual needs. The experimental data for each embodiment is voluminous and it is not suitable to list and explain them one by one here. However, the content required for verification and the final conclusions obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be described one by one here.

[0112] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a double-layer hydrogel for scarless corneal damage repair, characterized in that: The following steps are involved: (1) Synthesis of oxidized polysaccharides and thiolated polymers; Using NaIO4 as an oxidant, the polysaccharide is oxidized to obtain an oxidized polysaccharide having an aldehyde group; wherein the oxidized polysaccharide has a negative charge; The antibacterial polymer with amino group was modified by cysteine ​​to obtain thiol polymer; (2) Preparation of bottom layer hydrogel; The multi-arm polyethylene glycol amino groups react with the oxidized polysaccharide through Schiff base reaction and with the multi-arm polyethylene glycol derivative through cross-linking reaction to form a bottom layer hydrogel; wherein the multi-arm polyethylene glycol derivative has a group that cross-links with the amino group; (3) Preparation of double-layer hydrogel; The thiol-modified polymer is sprayed on the bottom hydrogel and irradiated with UV light to obtain a double-layer hydrogel.

2. The preparation method according to claim 1, characterized in that The synthesis process of the oxidized polysaccharide in step (1) comprises: In a dark environment, negatively charged polysaccharides and NaIO4 were dissolved in ultrapure water, stirred, and then anhydrous ethanol was added to inactivate unreacted NaIO4. The resulting solution was dialyzed, and the dialyzed purified solution was freeze-dried to obtain oxidized polysaccharides. The ratio of polysaccharide, NaIO4, ultrapure water and anhydrous ethanol is (0.2-0.8) g: (0.05-0.4) g: (40-80) mL: (20-50) mL.

3. The preparation method according to claim 2, characterized in that The negatively charged polysaccharide is heparin, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, alginic acid, fucoidan sulfate, carrageenan, pectin or xanthan gelatin.

4. The preparation method according to claim 1, characterized in that The synthesis process of the thiol polymer in step (1) comprises: The antibacterial polymer with amino groups is dissolved in MES buffer, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added, and cysteine ​​is added after stirring to obtain a mixed solution; the mixed solution is stirred in the dark for 12 to 24 hours, and the resulting solution is dialyzed against a HCl aqueous solution with a pH of 3 to 5, and then freeze-dried to obtain a thiol-modified polymer; Wherein, the antibacterial polymer with amino group is ε-polylysine, α-polylysine or chitosan.

5. The preparation method according to claim 4, characterized in that The ratio of the antibacterial polymer with amino group, EDC, NHS and cysteine ​​is (0.5-3) g: (0.5-1.5) g: (0.25-0.8) g: (0.2-5) g.

6. The preparation method according to claim 1, characterized in that The step (2) specifically includes the following process: Multi-arm polyethylene glycol amino, oxidized polysaccharide and multi-arm polyethylene glycol derivative were dissolved in PBS buffer respectively, mixed and vortexed to obtain a bottom layer hydrogel.

7. The preparation method according to claim 6, characterized in that The molar ratio of the amino group in the multi-arm polyethylene glycol amino group, the aldehyde group in the oxidized polysaccharide, and the group cross-linked with the amino group in the multi-arm polyethylene glycol derivative is 10: (2-10): (1-9).

8. The preparation method according to claim 7, characterized in that The multi-arm polyethylene glycol amino group is a four-arm polyethylene glycol amino group, a six-arm polyethylene glycol amino group, an eight-arm polyethylene glycol amino group, a sixteen-arm polyethylene glycol amino group or a thirty-two-arm polyethylene glycol amino group; The multi-arm polyethylene glycol derivative is a four-arm polyethylene glycol derivative, a six-arm polyethylene glycol derivative, an eight-arm polyethylene glycol derivative, a sixteen-arm polyethylene glycol derivative or a thirty-two-arm polyethylene glycol derivative; wherein the group cross-linked with the amino group in the multi-arm polyethylene glycol derivative is a succinimidyl carbonate, an aldehyde group, a carboxyl group or a succinimide group.

9. The preparation method according to claim 1, characterized in that The solid content of the bottom layer hydrogel is 3-10 wt%.

10. A double-layer hydrogel for scarless corneal damage repair obtained by the preparation method according to any one of claims 1 to 9.

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