A coating-modified ophthalmic material and its preparation method

Abstract

The present invention discloses a coating-modified ophthalmic material with improved biocompatibility and its preparation method, belonging to the field of biomedical materials. The material preparation mainly includes the following steps: (1) treating the ophthalmic material with 3,4-dihydroxybenzaldehyde to introduce aldehyde groups thereto; (2) reacting the aldehyde-group-containing ophthalmic material in step (1) with recombinant human type I collagen or human amniotic membrane extract to obtain the coating-modified ophthalmic material. While meeting the mechanical and optical property requirements of the ophthalmic material, this material significantly reduces the adhesion of proteins and macrophages, improves biocompatibility, and potentially can extend the service life of the ophthalmic material, having broad application prospects in ophthalmic implant materials.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to an ophthalmic material with a coating modification for improving biocompatibility and a preparation method thereof. Background Art

[0002] Currently, ophthalmic materials with great clinical application demands include intraocular lens materials, artificial corneal endothelial materials, and scleral lens materials.

[0003] ICL (Implantable Collamer Lens) is called an implantable contact lens. The principle is to implant it long-term between the iris and the natural lens of the human eye. Through the coordination of the ICL and the natural lens, the external light entering the eye is focused on the retina, achieving the effect of clear vision. This surgery is also called posterior chamber intraocular lens implantation. As an alternative to glasses and contact lenses, refractive surgery has developed over the years, with continuous innovation in surgical methods. The optimization of surgical methods is reflected in many aspects such as safety, convenience, accuracy, correction range, and postoperative recovery. The mainstream myopic refractive surgeries mainly include corneal refractive surgery and lens implantation surgery. The principle of corneal refractive surgery is the "subtraction principle" of removing part of the corneal tissue. The lens implantation surgery is the "addition principle" of putting ophthalmic materials without damaging the corneal tissue. Clinically, a very large number of moderate and high myopia patients cannot accept excimer laser treatment due to reasons such as too thin corneal tissue, and the invention of ICL solves this clinical need. Different from laser and other surgeries that cut corneal tissue, the ICL intraocular lens implantation surgery does not require cutting the cornea. The lens is implanted into the eye through a minimally invasive surgery, without damaging the cornea, achieving the purpose of long-term correction of refractive errors, and providing a new option for the treatment of high myopia.

[0004] Staar Surgical is the global leader in ICL. Its products include implantable lenses and a delivery system for delivering the lenses into the eye. Among them, the implantable lenses mainly include: 1) the Visian ICL lens series for myopia correction refractive surgery; 2) the ICL lens series for hyperopia and astigmatism correction refractive surgery; 3) the IOL series of ophthalmic materials for cataract surgery. In addition to STAAR's ICL in China, there is only one posterior chamber implantable lens, the Yijing PRL under Aijinglun, in the same category.

[0005] An artificial corneal endothelium is a thin film implant attached to the posterior surface of the cornea, which can treat corneal edema caused by endothelial dysfunction and may become a substitute for the donor cornea in endothelial keratoplasty. The artificial endothelium is placed in the central position, covering about 40% of the posterior surface of the cornea. The remaining uncovered outer edge surface provides an appropriate amount of water filtration to ensure the nutrition of the covered tissue. A new balance is created to reduce edema and maintain corneal function and health.

[0006] A scleral lens is a lens located on the sclera. There is a space between the lens and the cornea, and it does not directly contact the cornea. When designing the scleral lens, it bulges in the middle to form a vault, and the vault ensures the gap between the lens and the cornea. Before wearing, the lens needs to be filled with fluid, which can be antiseptic saline or physiological saline. The scleral lens consists of three parts: a tactile part that contacts the sclera, a dome that ensures the gap between the lens and the cornea, and an optical part of the lens. The lens only contacts the sclera. By filling it with liquid (physiological saline, tears), the uneven corneal surface becomes "flat" again, thereby improving vision.

[0007] Existing ophthalmic materials include intraocular lens materials (such as 2-hydroxyethyl methacrylate), artificial corneal endothelial materials (such as hydrophilic acrylic acid), and scleral lens materials (such as fluorosiloxane acrylate). Their disadvantage is that they will adhere and contaminate with proteins and cells in the local intraocular environment, resulting in a short service life of the ophthalmic materials. Summary of the Invention

[0008] In view of the above problems existing in the prior art, the present invention provides a coating-modified ophthalmic material with improved biocompatibility and its preparation method. While meeting the mechanical and optical performance requirements of the ophthalmic material, the modified material significantly reduces the adhesion of proteins and macrophages, improves biocompatibility, and potentially can extend the service life of the ophthalmic material.

[0009] The present invention is achieved through the following technical solutions:

[0010] A coating modification method for an ophthalmic material, which can significantly improve the biocompatibility of the material, mainly includes the following steps:

[0011] (1) Treat the ophthalmic material with 3,4-dihydroxybenzaldehyde to introduce aldehyde groups;

[0012] (2) React the aldehyde-grouped ophthalmic material in step (1) with recombinant human type I collagen or human amniotic membrane extract,

[0013] to obtain the coating-modified ophthalmic material.

[0014] As an optional method, in the above preparation method, the ophthalmic material includes intraocular lens materials, artificial corneal endothelial materials, and scleral lens materials.

[0015] As an optional method, in the above preparation method, the core sequence of the recombinant human type I collagen is GPPGPPGKNGDDGEAGKPGRPGERGPPEE, and it has a uniform positive and negative charge distribution. The recombinant human type I collagen in the present invention is designed by computer-aided design, and its secondary structure angle is greater than that of the native type I collagen with a sequence identity of 100%.

[0016] As an alternative, in the above preparation method, the human amniotic membrane extract is a product with a particle size less than 1 μm obtained by using fresh human amniotic membrane as the raw material, through decellularization, fragmentation by a mechanical crusher, decomposition by biological enzymes and strong alkali, freeze-drying, and sieving through a stainless-steel standard sieve.

[0017] As an alternative, in the above preparation method, in step (1), the mass concentration of 3,4-dihydroxybenzaldehyde is 1% - 10%, the solvent is an ethanol / water mixed solution with a volume ratio of 1:1, and the reaction is carried out at 37°C for 24 hours.

[0018] As an alternative, in the above preparation method, in step (2), the mass concentration of recombinant human type I collagen or human amniotic membrane extract is 1% - 10%, and the reaction is carried out at 37°C for 24 hours.

[0019] As an alternative, in the above preparation method, the human amniotic membrane extract is a product with a particle size less than 1 μm obtained by using fresh human amniotic membrane as the raw material, through decellularization, fragmentation by a mechanical crusher, decomposition by biological enzymes and strong alkali, freeze-drying, and sieving through a stainless-steel standard sieve.

[0020] As an alternative, in the above preparation method, step (2) includes:

[0021] Step S1: Obtain and amplify plasmid DNA; specifically, use competent cells and Escherichia coli to amplify plasmid DNA capable of expressing the target sequence.

[0022] Step S2: Extract plasmid DNA; specifically, use a commercial plasmid extraction kit to extract and purify the amplified plasmid.

[0023] Step S3: Induce the expression of the target polypeptide; specifically, transfer the recombinant plasmid extracted in step S2, and verify the transformants by colony PCR.

[0024] Step S4: Purify the target polypeptide.

[0025] The present invention also provides an ophthalmic material prepared by the above method, and the ophthalmic material has better biocompatibility and a longer service life compared with traditional ophthalmic materials.

[0026] The present invention also provides the use of a human amniotic membrane extract or recombinant human type I collagen with a core sequence of GPPGPPGKNGDDGEAGKPGRPGERGPPEE in the preparation of ophthalmic materials.

[0027] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0028] Advantages of the present invention:

[0029] In the present invention, ophthalmic materials are treated with 3,4-dihydroxybenzaldehyde. The catechol group in 3,4-dihydroxybenzaldehyde has a broad-spectrum adhesion property similar to that of mussels and can effectively adhere to ophthalmic materials. On the other hand, the aldehyde group in 3,4-dihydroxybenzaldehyde can be introduced onto the ophthalmic materials simultaneously. By reacting the aldehyde group with the amino group in recombinant human type I collagen or human amniotic membrane extract, a chemically bonded coated modified ophthalmic material is obtained.

[0030] The recombinant human type I collagen in the present invention is obtained by biosynthetic means after screening and has a sequence with a degree of overlap of more than 90% with a partial sequence of human type I collagen, and has low immunogenicity; similarly, the raw material of human amniotic membrane extract is taken from the human body and also has low immunogenicity.

[0031] The recombinant human type I collagen in the present invention has a uniform positive and negative charge distribution, can significantly reduce the adhesion and contamination of proteins and cells, improve the biocompatibility of the material, and potentially can extend the service life of ophthalmic materials. Description of the drawings

[0032] Figure 1 It is the test result of the solid surface potential.

[0033] Figure 2 It is the test result of immunogenicity.

[0034] Figure 3 It is the test result of protein adhesion.

[0035] Figure 4 It is the test result of macrophage adhesion.

[0036] Figure 5 It is the test result of mechanical properties.

[0037] Figure 6 It is the test result of light transmittance. Detailed implementation manners

[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation manners. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments.

[0039] Example 1

[0040] (1) Constructing recombinant human type I collagen by biosynthetic method

[0041] Step S1: Obtaining plasmid DNA and amplification

[0042] The core polypeptide sequence GPPGPPGKNGDDGEAGKPGRPGERGPPEE was designed independently. The plasmid DNA lyophilized powder capable of expressing the above core sequence was customized from a professional biological company. Then, the plasmid DNA lyophilized powder expressing the core sequence was resuspended, and the competent cells were melted on ice. 2 μL of the plasmid was added to 100 μL of the melted competent cells. Then, the plasmid-competent cell complex was successively treated with ice bath for 30 min, heat shock at 42 °C for 45 s, and ice bath for 2 min. Subsequently, the complex was added to the liquid medium without antibiotics and placed on a shaker at 37 °C and cultured at a rotation speed of 200 rmp for 60 min. Then, it was taken out, centrifuged at a rate of 5000 rmp for 5 min, the supernatant was discarded, 60 μL of the liquid medium containing antibiotics was added, and after resuspension, it was dropped on the antibiotic-containing plate and spread for overnight culture. In a 20 mL test tube, the Escherichia coli carrying the required plasmid was inoculated into 5 mL of the medium and cultured with shaking at 37 °C for 12 - 16 h.

[0043] Step S2: Extract plasmid DNA

[0044] Using the E.Z.N.A.TM Plasmid Mini Kit I small extraction kit from Omega Company, the bacterial liquid containing plasmid DNA obtained after amplification in step S1 was extracted. Specifically: Take 2 mL of the bacterial liquid, centrifuge at 6000×g for 2 min at room temperature, discard the supernatant, add 250 μL of Solution I (containing RNase A), and vortex with a vortex oscillator until the bacteria are completely suspended. Add 250 μL of Solution II, gently invert the centrifuge tube 4 - 6 times to obtain a clear lysate. Add 350 μL of Solution III, gently invert several times to mix until a white flocculent precipitate appears, and centrifuge at 13000×g for 10 min at room temperature. Aspirate the supernatant after centrifugation and transfer it to a clean adsorption column assembled with a 2 mL centrifuge tube, centrifuge at 13000×g for 1 min at room temperature until the lysate completely passes through the adsorption column. Discard the filtrate, add 500 μL of Buffer HB, centrifuge at 13000×g for 1 min, and discard the filtrate. Then wash the adsorption column with 750 μL of Wash Buffer diluted with 100% ethanol, centrifuge at 13000×g for 1 min. Place the adsorption column in a clean 1.5 mL centrifuge tube, add 60 μL of sterile deionized water on the filter membrane, and centrifuge at 13000×g for 5 min. Collect the precipitate to obtain the extracted and purified plasmid.

[0045] Step S3: Induce the expression of the target polypeptide

[0046] Transfer the recombinant plasmid extracted in step S2 into Escherichia coli, and verify the transformants by colony PCR. Pick the positive clones on the plate and inoculate them into 5 mL of liquid LB medium (containing Amp resistance) for overnight liquid culture to prepare the seed solution. Then inoculate 1 mL of the seed solution into 100 mL of liquid LB medium (containing Amp resistance) and culture at 37 °C until the OD600 value reaches 2, then induce the expression of the target protein and culture overnight with shaking at 18 °C. Centrifuge the fermentation broth at 5000 r / min for 10 min at 4 °C, collect the bacterial cells, add 5 - 10 volumes of bacterial lysis buffer, and centrifuge the mixture at 20000 r / min for 20 min at 4 °C. Collect the supernatant and precipitate respectively and detect the expression of the target polypeptide by SDS-PAGE.

[0047] Step S4: Purification of the target polypeptide

[0048] Equilibrate the column with 5 mL of LS-ID buffer containing 400 mmol / L imidazole. Then wash the column with LS-ID buffer. Flow the collected cell lysate supernatant through a filter membrane and through the pre-equilibrated resin at a certain flow rate to bind the target polypeptide to the resin. Elute the column with 5 mL of rinse buffer to remove non-specifically bound polypeptides. Collect the eluate and detect it by SDS-PAGE to obtain the purified recombinant human type I collagen.

[0049] (2) Treatment of intraocular lens material with 3,4-dihydroxybenzaldehyde

[0050] Prepare a 1% (mass concentration) solution of 3,4-dihydroxybenzaldehyde in ethanol / water (volume ratio 1:1). After washing the intraocular lens material with deionized water, soak it in the 3,4-dihydroxybenzaldehyde solution and react at 37 °C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water.

[0051] (3) Preparation of coated and modified intraocular lens material

[0052] Using the recombinant human type I collagen prepared in step (1), prepare a 1% (mass concentration) aqueous solution of recombinant human type I collagen. Immerse the intraocular lens material treated with 3,4-dihydroxybenzaldehyde in step (1) into the aqueous solution of recombinant human type I collagen and react at 37 °C for 24 hours. Then, take out the ophthalmic material and wash it with deionized water to obtain the coated and modified intraocular lens material.

[0053] Example 2

[0054] The preparation method of recombinant human type I collagen is the same as that in Example 1.

[0055] (1) Treatment of intraocular lens material with 3,4-dihydroxybenzaldehyde: Prepare an ethanol / water (volume ratio 1:1) solution of 3,4-dihydroxybenzaldehyde with a mass concentration of 5%. After washing the intraocular lens material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution and react at 37 °C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water.

[0056] (2) Preparation of coated and modified intraocular lens material: Recombinant human type I collagen is prepared by a biosynthetic method, and its core sequence is GPPGPPGKNGDDGEAGKPGRPGERGPPEE. Prepare an aqueous solution of recombinant human type I collagen with a mass concentration of 5%. Immerse the intraocular lens material treated with 3,4-dihydroxybenzaldehyde in step (1) in the aqueous solution of recombinant human type I collagen and react at 37 °C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water to obtain the coated and modified intraocular lens material.

[0057] Example 3

[0058] The preparation method of recombinant human type I collagen is the same as that in Example 1.

[0059] (1) Treatment of intraocular lens material with 3,4-dihydroxybenzaldehyde: Prepare an ethanol / water (volume ratio 1:1) solution of 3,4-dihydroxybenzaldehyde with a mass concentration of 10%. After washing the intraocular lens material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution and react at 37 °C for 24 hours. Then, take out the intraocular lens and wash it with deionized water.

[0060] (2) Preparation of coated and modified intraocular lens: Recombinant human type I collagen is prepared by a biosynthetic method, and its core sequence is GPPGPPGKNGDDGEAGKPGRPGERGPPEE. Prepare an aqueous solution of recombinant human type I collagen with a mass concentration of 10%. Immerse the intraocular lens material treated with 3,4-dihydroxybenzaldehyde in step (1) in the aqueous solution of recombinant human type I collagen and react at 37 °C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water to obtain the coated and modified intraocular lens material.

[0061] Example 4

[0062] (1) Treatment of intraocular lens material with 3,4-dihydroxybenzaldehyde: Prepare an ethanol / water (volume ratio 1:1) solution of 3,4-dihydroxybenzaldehyde with a mass concentration of 10%. After washing the intraocular lens material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution and react at 37 °C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water.

[0063] (2) Preparation of human amniotic membrane extract: Using fresh human amniotic membrane as raw material, a product with a particle size less than 1 micron is obtained through decellularization, crushing by a mechanical pulverizer, decomposition by biological enzymes and strong alkali, freeze-drying, and sieving through a stainless steel standard sieve. The specific steps are as follows: Decellularize the fresh amniotic membrane with 0.1% sodium dodecyl sulfate at room temperature for 24 hours, then crush it with a mechanical pulverizer at a rotational speed of 500 rpm for 10 minutes, decompose it with 0.1% pepsin and 1 M sodium hydroxide for 24 hours, and after freeze-drying, sieve it through a stainless steel standard sieve to obtain a product with a particle size less than 1 micron.

[0064] (3) Preparation of coated and modified intraocular lens material: Prepare an aqueous solution of human amniotic membrane extract with a mass concentration of 10% in step (2), immerse the intraocular lens material treated with 3,4-dihydroxybenzaldehyde in step (1) into the aqueous solution of human amniotic membrane extract, and react at 37°C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water to obtain the coated and modified intraocular lens material.

[0065] Example 5

[0066] (1) Treatment of artificial corneal endothelial material with 3,4-dihydroxybenzaldehyde: Prepare a solution of 3,4-dihydroxybenzaldehyde in ethanol / water (volume ratio 1:1) with a mass concentration of 10%, after washing the artificial corneal endothelial material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution, and react at 37°C for 24 hours. Then, take out the artificial corneal endothelial material and wash it with deionized water.

[0067] (2) Preparation of human amniotic membrane extract: Using fresh human amniotic membrane as raw material, a product with a particle size less than 1 micron is obtained through decellularization, crushing by a mechanical pulverizer, decomposition by biological enzymes and strong alkali, freeze-drying, and sieving through a stainless steel standard sieve. The specific steps are as follows: Decellularize the fresh amniotic membrane with 0.1% sodium dodecyl sulfate at room temperature for 24 hours, then crush it with a mechanical pulverizer at a rotational speed of 500 rpm for 10 minutes, decompose it with 0.1% pepsin and 1 M sodium hydroxide for 24 hours, and after freeze-drying, sieve it through a stainless steel standard sieve to obtain a product with a particle size less than 1 micron.

[0068] (3) Preparation of coated and modified artificial corneal endothelial material: Prepare an aqueous solution of human amniotic membrane extract with a mass concentration of 10% in step (2), immerse the artificial corneal endothelial material treated with 3,4-dihydroxybenzaldehyde in step (1) into the aqueous solution of human amniotic membrane extract, and react at 37°C for 24 hours. Then, take out the artificial corneal endothelial material and wash it with deionized water to obtain the coated and modified artificial corneal endothelial material.

[0069] Example 6

[0070] (1) Treatment of scleral lens material with 3,4-dihydroxybenzaldehyde: Prepare an ethanol / water (volume ratio 1:1) solution of 3,4-dihydroxybenzaldehyde with a mass concentration of 10%. After cleaning the scleral lens material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution and react at 37°C for 24 hours. Then, take out the scleral lens material and wash it with deionized water.

[0071] (2) Preparation of human amniotic membrane extract: Use fresh human amniotic membrane as the raw material, and obtain a product with a particle size less than 1 micron through decellularization, crushing with a mechanical crusher, decomposition with biological enzymes and strong alkali, freeze-drying, and sieving through a stainless steel standard sieve. The specific steps are as follows: Decellularize fresh amniotic membrane with a 0.1% mass concentration of sodium dodecyl sulfate at room temperature for 24 hours, then crush it with a mechanical crusher at a rotation speed of 500 rpm for 10 minutes, decompose it with 0.1% mass concentration of pepsin and 1 M sodium hydroxide for 24 hours, and obtain a product with a particle size less than 1 micron after freeze-drying and sieving through a stainless steel standard sieve.

[0072] (3) Preparation of coated and modified scleral lens material: Prepare an aqueous solution of human amniotic membrane extract with a mass concentration of 10% in step (2). Immerse the scleral lens material treated with 3,4-dihydroxybenzaldehyde in step (1) in the aqueous solution of human amniotic membrane extract and react at 37°C for 24 hours. Then, take out the scleral lens material and wash it with deionized water to obtain the coated and modified scleral lens material.

[0073] Comparative Example 1

[0074] Comparative Example 1 is an untreated intraocular lens material.

[0075] Comparative Example 2

[0076] The preparation method of recombinant human type I collagen is basically the same as that in Example 1, except that the amino acid sequence is replaced with GPPGPPGKNGDDGEAGKPGRPGERGPP, and its sequence has 100% overlap with human type I collagen.

[0077] (1) Treatment of intraocular lens material with 3,4-dihydroxybenzaldehyde: Prepare an ethanol / water (volume ratio 1:1) solution of 3,4-dihydroxybenzaldehyde with a mass concentration of 10%. After cleaning the intraocular lens material with deionized water, immerse it in the 3,4-dihydroxybenzaldehyde solution and react at 37°C for 24 hours. Then, take out the intraocular lens material and wash it with deionized water.

[0078] (2) Preparation of Coated Modified Intraocular Lens Material: Recombinant human type I collagen was prepared by a biosynthetic method, and its core sequence is GPPGPPGKNGDDGEAGKPGRPGERGPP. An aqueous solution of recombinant human type I collagen with a mass concentration of 10% was prepared, and the intraocular lens material treated with 3,4-dihydroxybenzaldehyde in step (1) was immersed in the aqueous solution of recombinant human type I collagen and reacted at 37 °C for 24 hours. Then, the intraocular lens material was taken out and washed with deionized water to obtain the coated modified intraocular lens material.

[0079] Comparative Example 3

[0080] Comparative Example 3 is untreated artificial corneal endothelial material.

[0081] Comparative Example 4

[0082] Comparative Example 4 is untreated scleral lens material.

[0083] Test Example 1: Computer Simulation Test of Polypeptide Secondary Structure

[0084] This test includes the following two recombinant human type I collagen sequences: namely, GPPGPPGKNGDDGEAGKPGRPGERGPPEE in Examples 1-3 and GPPGPPGKNGDDGEAGKPGRPGERGPP in Comparative Example 2.

[0085] The computer simulation results show that the secondary structure angle of GPPGPPGKNGDDGEAGKPGRPGERGPPEE in Examples 1-3 is 175.1 degrees, and the secondary structure angle of GPPGPPGKNGDDGEAGKPGRPGERGPP in Comparative Example 2 is 172.3 degrees.

[0086] Test Example 1: Solid Surface Potential Test

[0087] This test includes the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, the intraocular lens materials modified with human amniotic extract were used in Example 4. The artificial corneal endothelial materials modified with human amniotic extract were used in Example 5. The scleral lens materials modified with human amniotic extract were used in Example 6. Comparative Example 1 is unmodified intraocular lens material, Comparative Example 2 is intraocular lens material modified with a polypeptide sequence 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 is untreated artificial corneal endothelial material. Comparative Example 4 is untreated scleral lens material.

[0088] Testing method: Cut the sample into rectangular strips of 5 cm × 1 cm (n = 6), wash the sample 3 times with deionized water and air dry naturally. Then paste it on the test sample stage with double-sided tape, and place it in a solid surface membrane potential meter to measure data at pH = 7.4.

[0089] The experimental results show that ( Figure 1 ), the modified ophthalmic materials all have a surface charge closer to neutral.

[0090] Test Example 3: Immunogenicity test

[0091] This test includes the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, the intraocular lens materials modified with human amniotic membrane extract were used in Example 4. The human amniotic membrane extract-modified artificial corneal endothelial materials were used in Example 5. The scleral lens materials modified with human amniotic membrane extract were used in Example 6. Comparative Example 1 was an unmodified intraocular lens material, Comparative Example 2 was an intraocular lens material modified with a polypeptide sequence 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 was an unmodified artificial corneal endothelial material. Comparative Example 4 was an unmodified scleral lens material.

[0092] The specific testing method is as follows: The immunogenicity of the material was characterized by immunoglobulin M (IgM) and immunoglobulin G (IgG) binding assays. Collect blood from healthy people, incubate and fluorescently label the material with anti-human IgM / IgG antibodies, and characterize the immunogenicity of the material by the fluorescence intensity of specific binding.

[0093] The experimental results show that ( Figure 2 ), the modified ophthalmic materials all have lower fluorescence intensity of specific binding of human IgM / IgG, that is, lower immunogenicity.

[0094] Test Example 4: Protein adhesion test

[0095] This test includes the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, the intraocular lens materials modified with human amniotic membrane extract were used in Example 4. The human amniotic membrane extract-modified artificial corneal endothelial materials were used in Example 5. The scleral lens materials modified with human amniotic membrane extract were used in Example 6. Comparative Example 1 was an unmodified intraocular lens material, Comparative Example 2 was an intraocular lens material modified with a polypeptide sequence 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 was an unmodified artificial corneal endothelial material. Comparative Example 4 was an unmodified scleral lens material.

[0096] The samples were cut into small round pieces with a diameter of 6 mm, washed three times with PBS, and then the samples were soaked in PBS solution overnight. The samples were incubated with a 1 mg / mL fluorescein-labeled fibronectin solution in a constant temperature oven at 37 °C for 2 h. After that, they were washed 3 times with PBS, and the fluorescence images of the adhesion proteins were taken using a laser confocal microscope. The fluorescence intensity was calculated by ImageJ software.

[0097] The experimental results showed ( Figure 3 ) that the modified ophthalmic materials all had lower protein adhesion properties.

[0098] Test Example 5: Macrophage Adhesion Test

[0099] This test included the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, the intraocular lens materials modified with human amniotic membrane extract were used in Example 4. The artificial corneal endothelial materials modified with human amniotic membrane extract were used in Example 5. The scleral lens materials modified with human amniotic membrane extract were used in Example 6. Comparative Example 1 was an unmodified intraocular lens material, and Comparative Example 2 was an intraocular lens material modified with a polypeptide sequence that was 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 was an unmodified artificial corneal endothelial material. Comparative Example 4 was an unmodified scleral lens material.

[0100] The samples were placed in 48-well plates, and stainless steel rings were placed on top of the samples to prevent the samples from floating. 500 μL of macrophage suspension (40,000 cells / mL) was added to each well. They were incubated in a cell culture incubator for 1 day and 3 days. 48 hours after inoculation, the supernatant of the samples expected to be cultured for 3 days was replaced with complete medium. After the culture was completed, the samples were rinsed 3 times with PBS for 5 minutes each. Subsequently, 200 μL of complete medium containing 10% CCK-8 reagent was added and incubated in a cell culture incubator for 1 hour. Subsequently, the absorbance of the medium at 450 nm was measured. A high absorbance indicated a large number of viable cells.

[0101] The experimental results showed ( Figure 4 ) that the modified ophthalmic materials all had lower macrophage adhesion properties.

[0102] Test Example 6: Mechanical Test

[0103] This test includes the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, and the intraocular lens materials modified with human amniotic membrane extract were used in Example 4. The artificial corneal endothelial materials modified with human amniotic membrane extract were used in Example 5. The scleral lens materials modified with human amniotic membrane extract were used in Example 6. Comparative Example 1 was an unmodified intraocular lens material, Comparative Example 2 was an intraocular lens material modified with a polypeptide sequence that was 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 was an unmodified artificial corneal endothelial material. Comparative Example 4 was an unmodified scleral lens material.

[0104] Tensile strength mechanical test: Cut the materials into dumbbell-shaped standard specimens and test them according to GB / T1040-92. The tensile rate is 100mm / min, and the arithmetic mean of 5 specimens is taken for each data.

[0105] The experimental results show that ( Figure 5 ), compared with the unmodified ophthalmic materials of the same type, the modified ophthalmic materials have similar tensile strength.

[0106] Test Example 7: Transmittance test

[0107] This test includes the following materials: Examples 1-6 and Comparative Examples 1-4. Among them, the intraocular lens materials modified with recombinant human type I collagen were used in Examples 1-3, and the intraocular lens materials modified with human amniotic membrane extract were used in Example 4. The artificial corneal endothelial materials modified with human amniotic membrane extract were used in Example 5. The scleral lens materials modified with human amniotic membrane extract were used in Example 6. Comparative Example 1 was an unmodified intraocular lens material, Comparative Example 2 was an intraocular lens material modified with a polypeptide sequence that was 100% identical to the amino acid sequence of human type I collagen. Comparative Example 3 was an unmodified artificial corneal endothelial material. Comparative Example 4 was an unmodified scleral lens material.

[0108] Measure the transmittance of the materials with a colorimeter in the wavelength range of 400-700nm.

[0109] The experimental results show that ( Figure 6 ), compared with the unmodified ophthalmic materials of the same type, the modified ophthalmic materials have similar transmittance.

[0110] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for coating modification of an ophthalmic material, characterized in that, This method can significantly improve the biocompatibility of the material, reduce the adhesion and contamination of proteins and cells, and mainly includes the following steps: (1) Treat the ophthalmic material with 3,4-dihydroxybenzaldehyde to introduce aldehyde groups into it; (2) React the aldehyde-group-containing ophthalmic material in step (1) with recombinant human type I collagen to obtain an ophthalmic material with a modified coating. The core sequence of the recombinant human type I collagen is GPPGPPGKNGDDGEAGKPGRPGERGPPEE, which has a uniform positive and negative charge distribution.

2. The coating modification method of the ophthalmic material according to claim 1, wherein, In step (1), the mass concentration of 3,4-dihydroxybenzaldehyde is 1%-10%, the solvent is an ethanol / water mixed solution with a volume ratio of 1:1, and the reaction is carried out at 37°C for 24 hours.

3. The coating modification method of the ophthalmic material according to claim 1, wherein In step (2), the mass concentration of the recombinant human type I collagen is 1%-10%, and the reaction is carried out at 37°C for 24 hours.

4. The coating modification method of the ophthalmic material according to claim 1, characterized in that, The preparation steps of the recombinant human type I collagen include: Step S1: Obtain plasmid DNA and amplify it; Step S2: Extract plasmid DNA; Step S3: Induce the expression of the target polypeptide; Step S4: Purify the target polypeptide.

5. The coating modification method of the ophthalmic material according to claim 4, characterized in that, Step S1 is specifically to amplify the plasmid DNA capable of expressing the target sequence by using competent cells and Escherichia coli.

6. The coating modification method of the ophthalmic material according to claim 4, characterized in that, Step S3 is specifically to transfer the recombinant plasmid extracted in step S2, and verify the transformants by colony PCR.

7. An ophthalmic material prepared by the method according to any one of claims 1-6.

Images