An artificial cornea that can release nitric oxide under light control

Abstract

The present invention relates to an artificial cornea capable of light-controlled release of nitric oxide, and belongs to the field of medical devices. A carboxymethyl chitosan and sodium alginate gel coating is prepared on the surface of the artificial cornea, and the coating contains liposome nanoparticles encapsulating NO donors, and the NO donors can release NO in response to light, and play the role of regulating intraocular pressure, antibacterial and anti-inflammatory. At the same time, EDC and NHS are used to connect metal porphyrins as a peroxynitrite scavenging layer to remove highly toxic peroxynitrite produced by the reaction of NO with endogenous superoxide anions, and prevent the damage of peroxynitrite caused by NO release. The artificial cornea can reduce complications such as glaucoma, infection and inflammation caused by artificial corneal transplantation.

Description

Technical Field

[0001] The present invention relates to the field of medical devices, and in particular to an artificial cornea capable of light-controlled release of NO. Background Art

[0002] Artificial cornea is a medical device used to replace diseased or damaged cornea, and has important therapeutic value for improving vision loss and eye diseases. However, the current artificial cornea still has certain limitations in terms of biocompatibility, functional recovery and long-term stability. Artificial cornea transplantation is prone to cause complications such as glaucoma, infection and endophthalmitis. Studies have shown that more than 10% of patients need glaucoma surgery after artificial cornea transplantation, and local anti-glaucoma drugs are less effective in artificial cornea transplantation. Infection and endophthalmitis can be treated by topical drops of antibiotics and other drugs, but long-term use of antibiotics is prone to cause drug resistance, thereby reducing the antibacterial effect. Therefore, the development of an artificial cornea that can reduce complications such as glaucoma and infectious endophthalmitis is a hot topic in current research.

[0003] Nitric oxide (NO) is a gas molecule with a wide range of biological activities, including anti-inflammatory, anti-proliferative, and angiogenesis-promoting effects. In the field of ophthalmology, NO has been shown to have a positive effect on corneal repair and regeneration. Nitric oxide reduces intraocular pressure by affecting the fluid circulation in the anterior chamber of the eye and regulating the tension of the ciliary muscle. It can promote the expansion of capillaries in the anterior chamber of the eye, thereby increasing fluid circulation and flow and reducing intraocular pressure. In addition, nitric oxide can also reduce intraocular pressure by affecting the relaxation of the ciliary muscle. In addition, studies have shown that nitric oxide has excellent anti-inflammatory and antibacterial activities. Therefore, introducing NO into the design of artificial corneas is expected to improve the biocompatibility and functional recovery effects of artificial corneas.

[0004] Existing studies have applied NO to the treatment of glaucoma, but they are all limited to local administration. In addition, the half-life of NO is very short, the duration of action is short, and high concentrations of NO will also produce excessive peroxynitrite, thereby causing cytotoxicity.

[0005] However, there is no report on an artificial cornea capable of light-controlled release of NO. Therefore, the present invention proposes an artificial cornea capable of light-controlled release of nitric oxide, aiming to achieve release of NO through light-controlled technology, thereby improving the therapeutic effect of the artificial cornea. Summary of the invention

[0006] According to the defects of existing artificial cornea applications, the present invention provides an artificial cornea that can release nitric oxide under light control. The artificial cornea includes an artificial cornea, a peroxynitrite scavenging layer located on the artificial cornea, and a NO donor layer located on the outermost layer of the artificial cornea; the NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate, which can trigger the release of NO in response to light; the peroxynitrite scavenging layer contains a peroxynitrite scavenger, which is incubated with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide and connected to the artificial cornea, and can scaveng peroxynitrite generated by NO release when NO is excessive;

[0007] Furthermore, the artificial cornea is prepared from a transparent material and surface activated by (3-aminopropyl)triethoxysilane;

[0008] Furthermore, the NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate, the mass ratio of carboxymethyl chitosan to sodium alginate in the gel coating is 1-1.2:0.8-1, and the solution pH is 7.0-7.6;

[0009] Furthermore, the NO donor is encapsulated in the liposome nanoparticles in the NO donor layer, and the concentration ratio of the NO donor to the liposome nanoparticles is 1:20-50;

[0010] Furthermore, the NO donor in the NO donor layer is photoresponsive, and may be a combination of one or more of N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine and S-nitrosothiol, which can trigger the release of NO in response to light;

[0011] Preferably, the NO donor is S-nitrosothiol;

[0012] Further, the peroxynitrite scavenging layer comprises a peroxynitrite scavenger, which may be one of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin iron(III), 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrin iron(III) chloride, 5,10,15,20-tetrakis(4-benzoic acid)porphyrin iron(III), benzenesulfonyl manganese porphyrin, manganese(III)-tetrakis(4-N-methylpyridyl)porphyrin, meso-tetrakis(4-carboxyphenyl)porphyrin manganese(III) chloride, and heme;

[0013] Preferably, the peroxynitrite scavenger is meso-tetrakis(4-carboxyphenyl)porphyrin manganese(III) chloride;

[0014] Further, the peroxynitrite scavenger in the peroxynitrite scavenging layer is connected by incubation with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride and n-hydroxysuccinimide;

[0015] Further, the peroxynitrite scavenger is dissolved in a solution of maleinethanesulfonic acid and sodium chloride;

[0016] Furthermore, the peroxynitrite scavenger connection concentration in the peroxynitrite scavenging layer is 1.3-1.7 mMoL / mL.

[0017] The present invention provides an artificial cornea capable of light-controlled release of nitric oxide, wherein the controlled release of NO can be achieved through illumination, and the NO donor can release NO in response to illumination, thereby regulating intraocular pressure, and having antibacterial and anti-inflammatory effects, and at the same time, the peroxide scavenging layer can effectively prevent the hazards caused by highly toxic peroxynitrite. The preparation method of the present invention is simple and feasible, and the application is convenient and flexible, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the result diagram of artificial cornea cell vitality;

[0019] Figure 2 This is the result graph of NO release from artificial cornea in vitro;

[0020] Figure 3 This is the result graph of the in vitro antibacterial rate of artificial cornea;

[0021] Figure 4 This is a graph of intraocular pressure results of artificial cornea in glaucoma animal model. DETAILED DESCRIPTION

[0022] The present invention introduces an artificial cornea capable of light-controlled release of nitric oxide, and the technical scheme is as follows:

[0023] 1. An artificial cornea comprises an artificial cornea, a peroxynitrite scavenging layer located on the artificial cornea, and an NO donor layer located on the outermost layer of the artificial cornea; the NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate, and can trigger the release of NO in response to light; the peroxynitrite scavenging layer comprises a peroxynitrite scavenger, which is connected to the artificial cornea after being incubated with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide, and can scaveng peroxynitrite generated by NO release when NO is excessive;

[0024] 2. The artificial cornea is made of transparent material and surface activated by (3-aminopropyl) triethoxysilane;

[0025] 3. The NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate, the mass ratio of carboxymethyl chitosan to sodium alginate in the gel coating is 1-1.2:0.8-1, and the solution pH is 7.0-7.6;

[0026] 4. The NO donor layer contains NO donor encapsulated in the liposome nanoparticles, and the concentration ratio of NO donor to liposome nanoparticles is 1:20-50;

[0027] 5. The NO donor in the NO donor layer is photoresponsive, and is a combination of one or more of N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine and S-nitrosothiol, and can trigger the release of NO in response to light;

[0028] 6. The peroxynitrite scavenging layer comprises a peroxynitrite scavenger, which is one of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin iron(III), 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrin iron(III) chloride, 5,10,15,20-tetrakis(4-benzoic acid)porphyrin iron(III), benzenesulfonyl manganese porphyrin, manganese(III)-tetrakis(4-N-methylpyridyl)porphyrin, meso-tetrakis(4-carboxyphenyl)porphyrin manganese(III) chloride, and heme;

[0029] 7. The peroxynitrite scavenger in the peroxynitrite scavenging layer is connected by incubation with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride and n-hydroxysuccinimide;

[0030] 8. The peroxynitrite scavenger is dissolved in morpholineethanesulfonic acid and sodium chloride solution;

[0031] 9. The concentration of peroxynitrite scavenger in the peroxynitrite scavenging layer is 1.3-1.7 mMoL / mL.

[0032] Preparation of artificial cornea matrix and preparation before coating: Glass was selected as the substrate material to prepare an artificial cornea with a middle optical part of 540 μm and a sutureable hole bracket on all sides. The glass artificial cornea matrix was placed in a 60°C 2mol hydrochloric acid solution and gently stirred for 20 minutes. Take it out, wash it once with 95% ethanol, 70% ethanol, and 50% ethanol, wash it three times with ultrapure water, dry it, place the glass artificial cornea matrix in a 1% (3-aminopropyl) triethoxysilane solution, incubate it at 100°C for 30 minutes, then wash it with ultrapure water, and place it in ultrapure water for use.

[0033] Example 1

[0034] A 1.5 mMol / mL aqueous solution of meso-tetra(4-carboxyphenyl)porphyrin manganese chloride (III) (MnTBAP) was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, and 2.2 mMol EDC and 5 mM NHS were added. The pH was adjusted to 6.0. After 30 minutes, it was soaked in 2-mercaptoethanol for 3 minutes, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, soaked in glycine for 5 minutes, and finally thoroughly washed with ultrapure water and placed in ultrapure water. 10 mg / mL of S-nitrosothiol (RSNO) was encapsulated with 0.2 mg / mL of the amphiphilic copolymer DSPE-PEG2000 to prepare NO donor nanoparticles. A 3% carboxymethyl chitosan solution and a sodium alginate solution were prepared using a 3% acetic acid solution, respectively. The two solutions were then mixed in a 1:1 ratio, the pH was adjusted to 7.4, incubated at 60°C for 1 hour, and then placed at 4°C for 5 hours, during which time NO donor nanoparticles were added in a 2:1 ratio. The artificial cornea was placed in the hydrogel solution, taken out and leveled, and placed at room temperature for 3 days until a gel coating was produced.

[0035] Example 2

[0036] A 1.3 mMol / mL MnTBAP aqueous solution was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, and 1.9 mMol EDC and 4.3 mM NHS were added. The pH was adjusted to 4.5. After 30 minutes, it was soaked in 2-mercaptoethanol for 3 minutes, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, soaked in glycine for 5 minutes, and finally thoroughly washed with ultrapure water and placed in ultrapure water. 4 mg / mL RSNO was encapsulated with 0.1 mg / mL of the amphiphilic copolymer DSPE-PEG2000 to prepare NO donor nanoparticles. Use 2% acetic acid solution to prepare 2% carboxymethyl chitosan solution and sodium alginate solution, respectively. Then, mix the two solutions at a ratio of 1:0.8, adjust the pH to 7.0, incubate at 60 °C for 1 hour, and then place at 4 °C for 5 hours. During this period, NO donor nanoparticles are added at a ratio of 2:1. The artificial cornea is placed in the hydrogel solution, taken out and leveled, and placed at room temperature for 3 days until a gel coating is produced.

[0037] Example 3

[0038] A 1.7 mMol / mL MnTBAP aqueous solution was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, 2.2 mMol EDC and 5 mM NHS were added, and the pH was adjusted to 7.2. After 30 minutes, it was soaked in 2-mercaptoethanol for 3 minutes, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, soaked in glycine for 5 minutes, and finally thoroughly washed with ultrapure water and placed in ultrapure water. 20 mg / mL RSNO was encapsulated with 0.4 mg / mL of the amphiphilic copolymer DSPE-PEG2000 to prepare NO donor nanoparticles. Use 1% acetic acid solution to prepare 1% carboxymethyl chitosan solution and sodium alginate solution, respectively. Then, mix the two solutions at a ratio of 1:1.2, adjust the pH to 7.6, incubate at 60°C for 1 hour, and then place at 4°C for 5 hours. During this period, NO donor nanoparticles are added at a ratio of 2:1. The artificial cornea is placed in the hydrogel solution, taken out and leveled, and placed at room temperature for 3 days until a gel coating is produced.

[0039] Comparative Example 1

[0040] A 1.5 mMol / mL MnTBAP aqueous solution was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, and finally thoroughly washed with ultrapure water and placed in ultrapure water. 10 mg / mL RSNO was encapsulated with 0.2 mg / mL amphiphilic copolymer DSPE-PEG2000 to prepare NO donor nanoparticles. A 2% carboxymethyl chitosan solution and a sodium alginate solution were prepared with 2% acetic acid solution, and then the two solutions were mixed 1:1, the pH was adjusted to 7.4, incubated at 60 degrees Celsius for 1 hour, and then placed at 4°C for 5 hours. During this period, NO donor nanoparticles were added at a ratio of 2:1, and the artificial cornea was placed in the hydrogel solution, taken out and flattened, and placed at room temperature for 3 days until the gel coating was produced.

[0041] Comparative Example 2

[0042] A 1.5 mMol / mL MnTBAP aqueous solution was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, and 2.2 mMol EDC and 5 mM NHS were added. The pH was adjusted to 6.0. After 30 minutes, it was soaked in 2-mercaptoethanol for 3 minutes, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, soaked in glycine for 5 minutes, and finally thoroughly washed with ultrapure water and placed in ultrapure water. A 2% carboxymethyl chitosan solution and a sodium alginate solution were prepared with a 2% acetic acid solution, and then the two solutions were mixed 1:1, the pH was adjusted to 7.4, incubated at 60 degrees Celsius for 1 hour, and then placed at 4°C for 5 hours. During this period, 10 mg / mL RSNO was added, and the artificial cornea was placed in the hydrogel solution, taken out and flattened, and placed at room temperature for 3 days until the gel coating was produced.

[0043] Comparative Example 3

[0044] A 1.5 mMol / mL MnTBAP aqueous solution was prepared with 0.1 M morpholineethanesulfonic acid and 0.3 M sodium chloride, and the pre-treated glass-based artificial cornea was placed in it and incubated at room temperature for 23 hours. After incubation, it was washed with ultrapure water and 95% ethanol in turn, and finally thoroughly washed with ultrapure water and placed in ultrapure water. A 2% carboxymethyl chitosan solution and a sodium alginate solution were prepared with a 2% acetic acid solution, respectively, and then the two solutions were mixed 1:1, the pH was adjusted to 7.4, incubated at 60 degrees Celsius for 1 hour, and then placed at 4°C for 5 hours, during which 10 mg / mL RSNO was added, and the artificial cornea was placed in the hydrogel solution, taken out and flattened, and placed at room temperature for 3 days until the gel coating was produced.

[0045] Comparative Example 4

[0046] 10 mg / mL RSNO was encapsulated with 0.2 mg / mL amphiphilic copolymer DSPE-PEG2000 to prepare NO donor nanoparticles. 2% carboxymethyl chitosan solution and sodium alginate solution were prepared with 2% acetic acid solution, and then the two solutions were mixed at a ratio of 1:1, the pH was adjusted to 7.4, incubated at 60 degrees Celsius for 1 hour, and then placed at 4 degrees Celsius for 5 hours, during which NO donor nanoparticles were added at a ratio of 2:1, and the artificial cornea was placed in the hydrogel solution, taken out and leveled, and placed at room temperature for 3 days until the gel coating was produced.

[0047] Verification Example

[0048] In order to illustrate the technical solution and effect of an artificial cornea that can release nitric oxide under light control, the above case was experimentally verified.

[0049] Cytotoxicity test: The artificial cornea was extracted at 37°C for 24 hours according to the cytotoxicity test method. The extract was not protected from light during the extraction process, and then the extract was used for cell culture to detect cytotoxicity.

[0050] In vitro release test plan: without modifying porphyrin, 5 artificial corneas were immersed in PBS and continuously illuminated. The amount of NO released was detected using a commercial Griess detection kit, and the results at 0min, 10min, 20min, 30min, 1h, 1.5h, and 2h were recorded and organized.

[0051] In vitro antibacterial test: 0.1mL of Escherichia coli suspension (1×106cfu / mL) was dropped on the sample surface for co-culture. Subsequently, the mixed solution of artificial cornea and bacteria was placed at 37°C for 30 minutes, during which the mixed solution was irradiated with simulated fluorescent light. Then 50μL of the diluted bacterial solution was evenly inoculated on LB agar, and after incubation at 37°C, the bacterial colonies on the plate were counted, and the antibacterial rate was calculated through negative control.

[0052] Animal experiment plan: Male New Zealand white rabbits were selected and glaucoma models were established, with 5 rabbits in each group. The intraocular pressure of the glaucoma rabbits was measured. According to the experimental specifications, one artificial cornea was implanted in each rabbit. The intraocular pressure was tested immediately after surgery, and then 10 minutes of light was exposed every day. The intraocular pressure was measured 12 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 30 days, and 3 months after surgery to observe the changes in intraocular pressure.

[0053] The specific cell viability test results are as follows Figure 1 As shown, since the released porphyrin can remove the peroxynitrite produced by the release of nitric oxide, Example 1, Example 2, and Comparative Example 1 all have excellent cell viability. In Example 3, due to the high content of nitric oxide donor, the released NO increases, so that the peroxynitrite cannot be effectively and timely removed, so the cell viability is reduced, but still higher than 80%. Comparative Example 2 is due to the instantaneous release of nitric oxide and the slow release of porphyrin, so that the peroxynitrite produced in a short time cannot be removed, so the cell viability is relatively low. Comparative Example 4 does not contain porphyrin, so the peroxynitrite cannot be removed, so the cell viability is the lowest.

[0054] The specific in vitro release test results are as follows Figure 2 As shown, since the NO donor in Example 1 was encapsulated by liposomes and gel coating, and the free incubation in Comparative Example 2, NO in Comparative Example 2 was released in large quantities within 30 minutes, while the release amount in Example 1 was linearly related to time and was slowly released along with the degradation of the gel layer.

[0055] Specific in vitro antibacterial test results are as follows Figure 3 As shown in the figure, the results show that the increase of NO makes the artificial cornea have a certain antibacterial effect, and the addition of porphyrin reduces the antibacterial effect of NO to a certain extent. At the same time, the antibacterial effect of NO and porphyrin incubation through chemical bonds and gel coating is better than that of free incubation.

[0056] Specific animal test results such as Figure 4 As shown, the results show that the intraocular pressure can be reduced after artificial cornea transplantation. At the same time, the addition of NO can promote the reduction of intraocular pressure. The intraocular pressure reduction caused by the addition of free NO in the comparative example is instantaneous, while the slow release of NO in the embodiment has a more lasting effect of reducing intraocular pressure. In addition, there is no increase in intraocular pressure after surgery in the embodiment.

Claims

1. An artificial cornea capable of light-controlled release of nitric oxide, characterized in that: The invention comprises an artificial cornea, a peroxynitrite scavenging layer located on the artificial cornea and an NO donor layer located on the outermost layer of the artificial cornea; the NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate, and can trigger the release of NO in response to light; the peroxynitrite scavenging layer contains a peroxynitrite scavenger, which is connected to the artificial cornea after being incubated with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide, and can scaveng peroxynitrite generated by NO release when NO is excessive.

2. An artificial cornea capable of light-controlled release of nitric oxide according to claim 1, characterized in that: The artificial cornea is made of transparent material and surface activated by (3-aminopropyl) triethoxysilane.

3. The artificial cornea capable of light-controlled release of nitric oxide according to claim 1, characterized in that: The NO donor layer is a gel coating prepared from liposome nanoparticles, carboxymethyl chitosan and sodium alginate. The mass ratio of carboxymethyl chitosan to sodium alginate in the gel coating is 1-1.2:0.8-1, and the solution pH is 7.0-7.

6.

4. The artificial cornea capable of light-controlled release of nitric oxide according to claim 1, characterized in that: The NO donor is encapsulated in the liposome nanoparticles in the NO donor layer, and the concentration ratio of the NO donor to the liposome nanoparticles is 1:20-50.

5. An artificial cornea capable of light-controlled release of nitric oxide according to claim 1 or 4, characterized in that: The NO donor in the NO donor layer is photoresponsive, and is a combination of one or more of N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine and S-nitrosothiol, and can trigger the release of NO in response to light.

6. The artificial cornea capable of light-controlled release of nitric oxide according to claim 1, characterized in that: The peroxynitrite scavenging layer comprises a peroxynitrite scavenger, which is one of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin iron(III), 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrin iron(III) chloride, 5,10,15,20-tetrakis(4-benzoic acid)porphyrin iron(III), benzenesulfonyl manganese porphyrin, manganese(III)-tetrakis(4-N-methylpyridyl)porphyrin, meso-tetrakis(4-carboxyphenyl)porphyrin manganese(III) chloride, and heme.

7. An artificial cornea capable of light-controlled release of nitric oxide according to claim 1 or 6, characterized in that: The peroxynitrite scavenger in the peroxynitrite scavenging layer was linked by incubation with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

8. The artificial cornea capable of light-controlled release of nitric oxide according to claim 6, characterized in that: The peroxynitrite scavenger is dissolved in a solution of maleic acid and sodium chloride.

9. The artificial cornea capable of light-controlled release of nitric oxide according to claim 7, characterized in that: The peroxynitrite scavenger concentration in the peroxynitrite scavenging layer is 1.3-1.7 mMoL / mL.

Images