An artificial vitreous body and a method of making the same

Abstract

The application discloses an artificial vitreous body and a preparation method thereof, and belongs to the technical field of medical materials, and comprises the following steps: preparing a buffer solution, preparing a polylysine solution, purifying the polylysine solution, preparing a crosslinking agent reaction solution, preparing a sodium hyaluronate gel, and preparing the artificial vitreous body. The application selects chlorinated DMTMM as a condensing agent, thereby not only avoiding the problem that unreacted monomers of BDDE need to be removed through a complex purification process, reducing production cost and process complexity, but also eliminating the mutation risk caused by the residual BDDE, the local inflammation caused by the non-specific reaction between the epoxy group and the intraocular protein, the immune response or the potential toxicity risk of retinal cells, and significantly improving the biological safety of the artificial vitreous body.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to an artificial vitreous body and a preparation method thereof. BACKGROUND

[0002] Natural vitreous body is a transparent gelatinous material located in the eyeball, mainly composed of water, hyaluronic acid, collagen and a small amount of glycoprotein, which bears the core physiological functions of maintaining intraocular pressure stability, supporting the integrity of retinal structure and realizing refraction transmission. When the natural vitreous body is liquefied, turbid or detached due to aging, trauma or pathological factors, an artificial vitreous body substitute needs to be implanted through surgery to restore the normal physiological structure and function of the eye. Therefore, the research and development of artificial vitreous body is a key direction in the field of medical materials for the treatment of eye diseases.

[0003] In the existing chemical cross-linking technology, 1,4-butanediol diglycidyl ether (BDDE) becomes a commonly used cross-linking agent because of its mild reaction conditions and the ability to stably connect polymer chains through ether bonds. However, its inherent chemical properties lead to significant defects in application: the unreacted monomers in BDDE need to be removed through a complex purification process, and the residual epoxy groups in BDDE may react non-specifically with intraocular proteins, causing local inflammation or immune response and potential toxicity risk to retinal cells.

[0004] At the same time, the purification process design for functional adjuvants in the existing artificial vitreous body preparation process is not perfect, which easily leads to the presence of residual free metal ions, unremoved solid particles and macromolecular polymer impurities in the system. These impurities can destroy the optical uniformity of the artificial vitreous body and significantly reduce the light transmission performance of the material. Since light transmission is a core prerequisite for the refraction transmission function of the artificial vitreous body, the existing process cannot stably guarantee the light transmission performance to meet the standard, resulting in insufficient functional matching between the artificial vitreous body and the natural vitreous body, and the inability to reliably meet the physiological needs of eye refraction transmission.

[0005] Based on this, the present application designs an artificial vitreous body and a preparation method thereof to solve the above problems. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the present application provides an artificial vitreous body, comprising the following steps:

[0007] S1. Adding 0.12-0.16 parts of sodium dihydrogen phosphate, 0.53-0.58 parts of disodium hydrogen phosphate and 9-11 parts of sodium chloride into 1000 parts of water for injection, stirring and filtering to obtain a buffer solution;

[0008] S2. Preparing a polylysine solution according to a ratio of 2.5-3.2 parts of polylysine per 1000 parts of phosphate buffer solution;

[0009] S3. Purification of polylysine solution;

[0010] 3.1 Take polylysine solution, adjust pH to 2.5-3.5 with hydrochloric acid, add aminotri methylene phosphonic acid, stir, centrifuge to collect supernatant;

[0011] 3.2 Pass the supernatant through a sterile microfiltration device, filter to collect sterile microfiltrate;

[0012] 3.3 Equilibrate the chromatography column with cation exchange resin with sterile acetic acid-sodium acetate buffer until the column efficiency is stable;

[0013] 3.4 Load the sterile microfiltrate into the chromatography column, after loading is complete, rinse the chromatography column with sterile acetic acid-sodium acetate buffer, elute with sterile acetic acid-sodium acetate buffer containing sodium chloride, collect the eluate in sections, use the color reaction of polylysine with ninhydrin reagent to determine in real time whether the eluate contains polylysine, and collect the polylysine-rich liquid;

[0014] 3.5 Use polyaspartic acid-modified Fe3O4 magnetic nanoparticles as affinity medium, suspend in sterile citric acid buffer, load into a sterile chromatography column and equilibrate to obtain an affinity chromatography column;

[0015] 3.6 Load the polylysine-rich liquid into the affinity chromatography column, rinse the column with buffer after adsorption, collect the rinse liquid; desorb with sterile Tris-HCl buffer, collect the desorption liquid to obtain high-purity polylysine liquid;

[0016] 3.7 Filter the high-purity polylysine liquid, collect sterile polylysine solution, reduce the pressure to concentrate the sterile polylysine solution to 1 / 5-1 / 4 of the original volume to obtain purified polylysine solution;

[0017] S4. Add 85-92 parts of purified polylysine solution to 1000 parts of buffer, stir to obtain a crosslinking agent reaction solution;

[0018] S5. Add 0.8-1.5 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir to obtain a sodium hyaluronate crosslinking reaction solution, and stand to obtain a sodium hyaluronate gel;

[0019] S6. Weigh 0.09-0.13 parts of DMTMM and 100 parts of buffer pre-cooled to 8-12℃, mix and filter to obtain a condensing agent reaction solution;

[0020] Pre-cool the sodium hyaluronate gel to 8-10℃, under the stirring at 150-260r / min, add 105-118 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel at a speed of 20-30 parts / h, after the dropwise addition is completed, stir for 1-2h to make it uniformly dispersed, seal and crosslink at 8-10℃ for 65-78h to obtain the gel;

[0021] S7. Put the gel into a dialysis bag, add buffer as the dialysis external solution to dialyze, squeeze and mix the dialyzed gel, cold fill and sterilize to obtain the artificial vitreous body.

[0022] Further, S1 is specifically: add 0.12-0.16 parts of sodium dihydrogen phosphate, 0.53-0.58 parts of disodium hydrogen phosphate and 9-11 parts of sodium chloride to 1000 parts of water for injection, stir at 25-30℃ and 200-320r / min for 30-40min, and filter to obtain the buffer solution.

[0023] Further, 3.1 is specifically: take the polylysine solution, adjust the pH to 2.5-3.5 with 0.1mol / L hydrochloric acid, add 0.1-0.5 parts of amino-trimethylene phosphonic acid, stir at 80-120rpm and 20-30℃ for 15-30min to complex the free metal ions in the solution, and then centrifuge at 4000-7000rpm for 15-25min to collect the supernatant.

[0024] Further, 3.4 is specifically: load the sterile microfiltrate into the chromatography column at a flow rate of 1-3BV / h, after the loading is completed, flush the chromatography column with sterile acetic acid-sodium acetate buffer at a flow rate of 2-4BV / h, use gradient elution, the eluent is sterile acetic acid-sodium acetate buffer containing 0.1-0.3mol / L sodium chloride, the concentration of sodium chloride is increased by 0.05mol / L gradient, the flow rate is 1.5-2.5BV / h, collect the eluent in sections, use the color reaction of polylysine and ninhydrin reagent to determine whether the eluent contains polylysine in real time, and collect the polylysine-rich liquid.

[0025] Further, the method for modifying Fe3O4 magnetic nanoparticles with polyaspartic acid is as follows: dissolve FeCl3 6H2O and FeSO4 7H2O in water for injection according to a molar ratio of 2:1, add polyaspartic acid at 5-10 times the total mass of the iron salt at 55-65℃ to form a complex, warm to 80-85℃, adjust the pH to 8.0-9.0 with 1mol / L sodium hydroxide, and react at 800-1000rpm for 25-30min to obtain polyaspartic acid-modified Fe3O4 magnetic nanoparticles.

[0026] Further, 3.6 specifically is: the polylysine enrichment liquid is loaded into the affinity chromatography column at a flow rate of 0.8-2 BV / h, static adsorption is carried out at 25-30 DEG C for 30-60 min, the chromatography column is washed with the buffer at a flow rate of 1.5-3 BV / h, and the washing liquid is collected;

[0027] The high-purity polylysine liquid is desorbed by using a sterile Tris-HCl buffer with a pH of 5.5-6.5 at a flow rate of 1-2 BV / h, and the desorption liquid is collected to obtain the high-purity polylysine liquid.

[0028] Further, 3.7 specifically is: the high-purity polylysine liquid is filtered through a 0.22 mu m sterile polyether sulfone microporous filter membrane, and the sterile polylysine solution is collected; the sterile polylysine solution is placed in a rotary evaporator, and is concentrated under reduced pressure to 1 / 5-1 / 4 of the original volume under the conditions of 35-45 DEG C and a vacuum degree of 0.08-0.095 MPa to obtain the purified polylysine solution.

[0029] An artificial vitreous body prepared according to the preparation method of the artificial vitreous body.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1. The present application selects 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methyl morpholine chloride (DMTMM) as a condensing agent, which not only avoids the problem of complex purification process for removing unreacted monomers of BDDE, reduces production cost and process complexity, but also eliminates the risk of mutagenesis caused by residual BDDE and the risk of local inflammation, immune response or potential toxicity of retinal cells caused by non-specific reaction of epoxy groups with intraocular proteins, significantly improving the biological safety of the artificial vitreous body.

[0032] 2. The present application can effectively remove impurities affecting light transmission and substances that may produce cytotoxicity in the system through a multi-step polylysine purification process, cooperate with post-processing, make the light transmission characteristics of the artificial vitreous body closer to the natural vitreous body, and reduce the residual toxic impurities, thereby protecting the low toxicity of the material to the intraocular cells and meeting the biological compatibility requirements of medical implant materials.

[0033] 3. The present application cooperatively designs the buffer preparation, polylysine purification and cross-linking system construction, sodium hyaluronate gel cross-linking and high-temperature sterilization processes, and the prepared artificial vitreous body is more consistent with the physiological function of the natural vitreous body in terms of viscoelasticity, transparency and biological stability, can reliably realize the core functions of maintaining intraocular pressure, supporting the retina and refractive transmission, and balances the long-term in-vivo retention ability and biological safety of the material relying on the selection of low-toxicity condensing agent and multi-step purification process, thereby solving the technical problems of insufficient matching of the performance of the artificial vitreous body with the natural vitreous body or poor biological safety in the prior art, and enhancing the reliability of clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0035] Figure 1 State diagram when the ninhydrin color developing solution and polylysine in step 3.4 have a positive reaction;

[0036] Figure 2 State diagram when the ninhydrin color developing solution and polylysine in step 3.4 have a negative reaction;

[0037] Figure 3 Actual physical diagram of the artificial vitreous body prepared in Example 1. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0039] Example 1: The present embodiment provides a preparation method of an artificial vitreous body, comprising the following steps:

[0040] S1. Preparation of buffer solution;

[0041] 0.12 parts of sodium dihydrogen phosphate, 0.53 parts of disodium hydrogen phosphate and 9 parts of sodium chloride are added into 1000 parts of water for injection, stirred at 25℃, 200r / min for 30min, and filtered to obtain the buffer solution;

[0042] S2. Preparation of polylysine solution;

[0043] The polylysine solution is prepared according to the proportion of 2.5 parts of polylysine (ε-polylysine is selected) per 1000 parts of phosphate buffer solution;

[0044] S3. Purification of polylysine solution;

[0045] 3.1 Take the polylysine solution, adjust the pH to 2.5 with 0.1 mol / L hydrochloric acid, add 0.1 part of aminotri-methylene phosphonic acid, complex the free metal ions in the solution by stirring at 80 rpm for 15 min at 20°C, and then centrifuge at 4000 rpm for 15 min, and collect the supernatant;

[0046] 3.2 Filter the supernatant through a sterile microfiltration device (filter membrane material: polyether sulfone) at 0.15 MPa and 20°C to remove the solid particles and macromolecular polymer impurities in the solution that were not completely centrifuged, and collect the sterile microfiltrate;

[0047] 3.3 Equilibrate the cation exchange resin (matrix: cross-linked agarose) chromatography column with sterile acetic acid-sodium acetate buffer at pH 3.0 until the column efficiency is stable (pH difference between effluent and sterile acetic acid-sodium acetate buffer ≤0.1);

[0048] 3.4 Load the sterile microfiltrate into the chromatography column at a flow rate of 1 BV / h, and after the loading is complete, rinse the chromatography column with sterile acetic acid-sodium acetate buffer at a flow rate of 2 BV / h, use gradient elution (eluted in 3 times), and the eluent is sterile acetic acid-sodium acetate buffer containing 0.1-0.2 mol / L sodium chloride, the concentration of sodium chloride is increased by 0.05 mol / L in gradient, the flow rate is 1.5 BV / h, collect the eluate in sections, use the color reaction of polylysine with ninhydrin reagent (blue-purple compound is generated after heating), determine in real time whether the eluate contains polylysine, and collect the polylysine-rich liquid;

[0049] The specific steps of the color reaction experiment are as follows: prepare the ninhydrin color developing solution (0.1% ninhydrin is dissolved in 50% ethanol-water mixture), during the gradient elution process, collect the eluate every 3 mL, take 1 mL of the collected liquid, add 0.5 mL of the ninhydrin color developing solution, heat in a boiling water bath for 5 min, observe the color after cooling, when the solution shows obvious blue-purple color (positive reaction, as shown in Figure 1 ), start collecting the eluate continuously; until the collected liquid shows light color after reacting with ninhydrin (close to negative, as shown in Figure 2 ), stop collecting, and the eluate collected during this period is the polylysine-rich liquid.

[0050] 3.5 Use Fe3O4 magnetic nanoparticles with a particle size of 50 nm modified by polyaspartic acid (synthesis of Fe3O4 magnetic nanoparticles with a particle size of 50 nm modified by polyaspartic acid: dissolve 2.5 g of FeCl3 6H2O and 1.5 g of FeSO4 7H2O was dissolved in water for injection at a molar ratio of 2:1, and polyaspartic acid was added at a total mass of 5 times that of the iron salt at 55°C, and the mixture was stirred to form a complex. The temperature was raised to 80°C, and the pH was adjusted to 8.0 with 1 mol / L sodium hydroxide. The reaction was carried out at 800 rpm for 25 min to obtain polyaspartic acid-modified Fe3O4 magnetic nanoparticles, which were used as an affinity medium. The nanoparticles were suspended in sterile citric acid buffer at pH 4.0, loaded into a sterile chromatography column, and equilibrated until the effluent was clear and the pH was stable. The affinity chromatography column was obtained.

[0051] 3.6 The polylysine-rich solution was loaded into the affinity chromatography column at a flow rate of 0.8 BV / h, and the column was statically adsorbed at 25°C for 30 min. The column was then washed with buffer at a flow rate of 1.5 BV / h, and the wash was collected.

[0052] The polylysine was desorbed with sterile Tris-HCl buffer at pH 5.5 at a flow rate of 1 BV / h, and the desorbed solution was collected to obtain high-purity polylysine solution.

[0053] It should be noted that the polyaspartic acid-modified Fe3O4 magnetic nanoparticles were used as an affinity medium to adsorb polylysine through electrostatic interaction. Polyaspartic acid is a negatively charged polyelectrolyte (containing a large number of carboxylate groups), while polylysine is positively charged at physiological pH. The two are combined and adsorbed through electrostatic attraction. The key to desorption is to break the electrostatic interaction between polyaspartic acid and polylysine. Tris-HCl buffer achieves desorption through pH adjustment: the isoelectric point of polylysine is about 9.0-10.0, and at pH 4.0-4.5 (loading conditions), the amino groups of polylysine are protonated (-NH3 + ), and are combined with the -COO - groups of polyaspartic acid. When the pH is raised to 5.5-6.5, the amino groups of polylysine are deprotonated, the positive charge density is reduced, and the electrostatic attraction is weakened, thereby achieving desorption.

[0054] 3.7 The high-purity polylysine solution was filtered through a 0.22 μm sterile polyether sulfone microporous filter (positive pressure filtration, pressure 0.1 MPa), and sterile polylysine solution was collected. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 5 of the original volume at 35°C and a vacuum degree of 0.08 MPa to obtain purified polylysine solution.

[0055] S4. Preparation of crosslinking agent reaction solution;

[0056] Add 85 parts of purified polylysine solution to every 1000 parts of buffer, and stir at 25°C and 150 r / min for 8 min to obtain the crosslinking agent reaction solution.

[0057] S5. Preparation of sodium hyaluronate crosslinking reaction solution;

[0058] Add 0.8 parts of sodium hyaluronate powder to 100 parts of the crosslinking agent reaction solution, stir at 25℃ and 150r / min for 8min to obtain a sodium hyaluronate crosslinking reaction solution, and stand to obtain a clear, transparent and uniform sodium hyaluronate gel;

[0059] S6. Preparation of gel;

[0060] Weigh 0.09 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and 100 parts of buffer solution pre-cooled to 8℃, and filter (through 0.22μm sterile polyether sulfone microporous filter membrane) after mixing to obtain a condensation agent reaction solution;

[0061] Pre-cool the sodium hyaluronate gel to 8℃, and add 105 parts of the condensation agent reaction solution to 1000 parts of the sodium hyaluronate gel at a speed of 20 parts / h under stirring at 150r / min, and stir for 1h after the dropwise addition is completed to make it uniformly dispersed, and then seal and crosslink at 8℃ for 65h to obtain a gel;

[0062] S7. Post-treatment;

[0063] Put the gel into a dialysis bag, the gel loading amount is not higher than 400g / m, and after uniform paving, place it in a dialysis container, add buffer solution as dialysis external liquid, add 10000 parts of dialysis external liquid per 1000 parts of gel, the temperature is 8℃, replace the dialysis liquid once a day, and the dialysis time is not less than 72h;

[0064] Put the dialyzed gel into a stainless steel extruder for extrusion and mixing, collect it into a container, seal the container, and refrigerate and fill it;

[0065] Put the gel after refrigeration and filling into a sterilization rack and sterilize it at 121℃ for 12min to obtain an artificial vitreous body.

[0066] Embodiment 2: The embodiment provides a preparation method of an artificial vitreous body, which comprises the following steps:

[0067] S1. Preparation of buffer solution;

[0068] Add 0.16 parts of sodium dihydrogen phosphate, 0.58 parts of disodium hydrogen phosphate and 11 parts of sodium chloride to 1000 parts of water for injection, stir at 30℃ and 320r / min for 40min, and filter to obtain a buffer solution;

[0069] S2. Preparation of polylysine solution;

[0070] Prepare a polylysine solution according to the proportion of 3.2 parts of polylysine (ε-polylysine is selected) per 1000 parts of phosphate buffer solution;

[0071] S3. Purification of polylysine solution;

[0072] 3.1 Take polylysine solution, adjust pH to 3.5 with 0.1 mol / L hydrochloric acid, add 0.5 parts of aminotri-methylene phosphonic acid, complex free metal ions in the solution by stirring at 120 rpm for 30 min at 30℃, then centrifuge at 7000 rpm for 25 min, collect supernatant;

[0073] 3.2 Filter the supernatant through a sterile microfiltration device (filter membrane material is polyether sulfone) at 0.3 MPa and 28℃ to remove solid particles and macromolecular polymer impurities in the solution that have not been completely centrifuged, and collect sterile microfiltrate;

[0074] 3.3 Equilibrate the chromatography column with sterile acetic acid-sodium acetate buffer at pH 3.5 using cation exchange resin (matrix is cross-linked agarose) until the column efficiency is stable (pH difference between effluent and sterile acetic acid-sodium acetate buffer is ≤0.1);

[0075] 3.4 Load the sterile microfiltrate into the chromatography column at a flow rate of 3 BV / h, and after loading is complete, rinse the chromatography column with sterile acetic acid-sodium acetate buffer at a flow rate of 4 BV / h, use gradient elution (elution in 3 times), and the eluent is sterile acetic acid-sodium acetate buffer containing 0.2-0.3 mol / L sodium chloride, the concentration of sodium chloride is increased by 0.05 mol / L gradient, the flow rate is 2.5 BV / h, collect the eluent in sections, use the color reaction of polylysine and ninhydrin reagent (blue-purple compounds are generated after heating) to determine in real time whether polylysine is contained in the eluent, and collect the polylysine enrichment solution;

[0076] 3.5 Use polyaspartic acid modified Fe3O4 magnetic nanoparticles with a particle size of 100 nm (dissolve FeCl3 6H2O and FeSO4 7H2O in water for injection at a molar ratio of 2:1, add polyaspartic acid at 10 times the total mass of iron salt at 65℃, stir to form a complex, heat to 85℃, adjust pH to 9.0 with 1 mol / L sodium hydroxide, and react at 1000 rpm for 30 min to generate polyaspartic acid modified Fe3O4 magnetic nanoparticles), suspend as an affinity medium with sterile citric acid buffer at pH 4.5, load into a sterile chromatography column and equilibrate until the effluent is clear and the pH is stable, to obtain an affinity chromatography column;

[0077] 3.6 Load the polylysine enrichment solution into the affinity chromatography column at a flow rate of 2 BV / h, and allow it to statically adsorb for 60 min at 30℃, then rinse the chromatography column with buffer at a flow rate of 3 BV / h, and collect the rinse solution;

[0078] Desorb with sterile Tris-HCl buffer solution of pH 6.5 at a flow rate of 2 BV / h, collect the desorption liquid to obtain high-purity polylysine liquid;

[0079] 3.7 The high-purity polylysine liquid is filtered through a 0.22 μm sterile polyether sulfone microporous filter (positive pressure filtration, pressure 0.2 MPa), and the sterile polylysine solution is collected. The sterile polylysine solution is placed in a rotary evaporator and concentrated under reduced pressure to 1 / 4 of the original volume at 45°C and a vacuum degree of 0.095 MPa to obtain a purified polylysine solution;

[0080] S4. Preparation of crosslinking agent reaction solution;

[0081] Add 92 parts of purified polylysine solution to every 1000 parts of buffer solution, and stir at 34°C and 200 r / min for 12 min to obtain a crosslinking agent reaction solution;

[0082] S5. Preparation of sodium hyaluronate crosslinking reaction solution;

[0083] Add 1.5 parts of sodium hyaluronate powder to 100 parts of the crosslinking agent reaction solution, and stir at 34°C and 200 r / min for 12 min to obtain a sodium hyaluronate crosslinking reaction solution. Stand to obtain a clear, transparent and uniform sodium hyaluronate gel;

[0084] S6. Preparation of gel;

[0085] Weigh 0.13 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride) and 100 parts of pre-cooled buffer solution to 12°C, and filter (through a 0.22 μm sterile polyether sulfone microporous filter) to obtain a condensation agent reaction solution;

[0086] Pre-cool the sodium hyaluronate gel to 10°C, and add 118 parts of the condensation agent reaction solution to 1000 parts of the sodium hyaluronate gel at a speed of 30 parts / h under stirring at 260 r / min. After the addition is completed, stir for 2 h to make it uniformly dispersed, and seal it at 10°C for crosslinking for 78 h to obtain a gel;

[0087] S7. Post-treatment;

[0088] The gel is loaded into a dialysis bag, and the gel loading amount is not more than 400 g / m. After being evenly laid, it is placed in a dialysis container, and buffer solution is added as dialysis external liquid. Every 1000 parts of gel is added with 10000 parts of dialysis external liquid, the temperature is 10°C, the dialysis liquid is replaced once a day, and the dialysis time is not less than 72 h;

[0089] The dialyzed gel is loaded into a stainless steel extruder for extrusion and mixing, collected into a container, sealed, and refrigerated and filled;

[0090] The refrigerated filled gel is sterilized on a sterilization rack at 121°C for 12 min to obtain an artificial vitreous body.

[0091] Embodiment 3 provides a preparation method of an artificial vitreous body, comprising the following steps:

[0092] S1. Preparation of a buffer solution;

[0093] 0.13 parts of sodium dihydrogen phosphate, 0.55 parts of disodium hydrogen phosphate and 10 parts of sodium chloride are added into 1000 parts of water for injection, stirred at 27°C and 280 r / min for 35 min, and filtered to obtain a buffer solution;

[0094] S2. Preparation of a polylysine solution;

[0095] The polylysine solution is prepared according to the proportion of 2.8 parts of polylysine (ε-polylysine is selected) per 1000 parts of phosphate buffer solution;

[0096] S3. Purification of the polylysine solution;

[0097] 3.1 The polylysine solution is taken, the pH is adjusted to 3.2 with 0.1 mol / L hydrochloric acid, 0.3 parts of aminotri-methylene phosphonic acid is added, and the solution is stirred at 25°C and 100 rpm for 26 min to complex free metal ions in the solution, and then centrifuged at 5800 rpm for 21 min to collect the supernatant;

[0098] 3.2 The supernatant is filtered through a sterile microfiltration device (the filter membrane material is polyether sulfone) at 0.26 MPa and 24°C to remove solid particles and macromolecular polymer impurities in the solution that are not completely centrifuged, and a sterile microfiltrate is collected;

[0099] 3.3 The cation exchange resin (matrix is cross-linked agarose) is used to equilibrate the chromatography column with sterile acetic acid-sodium acetate buffer solution at pH 3.2 until the column efficiency is stable (the difference between the effluent pH and the sterile acetic acid-sodium acetate buffer solution pH is ≤0.1);

[0100] 3.4 The sterile microfiltrate is loaded into the chromatography column at a flow rate of 2 BV / h, and after the loading is completed, the sterile acetic acid-sodium acetate buffer solution is used to flush the chromatography column at a flow rate of 3 BV / h, gradient elution (elution for 3 times) is adopted, the eluent is sterile acetic acid-sodium acetate buffer solution containing 0.14-0.24 mol / L sodium chloride, the concentration of sodium chloride is increased by 0.05 mol / L in gradient, the flow rate is 1.9 BV / h, and the eluent is collected in sections, the color reaction (blue-purple compound is generated after heating) of polylysine with ninhydrin reagent is used to determine whether the eluent contains polylysine in real time, and a polylysine-rich solution is collected;

[0101] 3.5 Fe3O4 magnetic nanoparticles with a particle size of 78 nm modified by polyaspartic acid (FeCl3 6H2O and FeSO4 7H2O were dissolved in water for injection at a molar ratio of 2:1, and polyaspartic acid was added at 7 times the total mass of the iron salt at 62°C, and the mixture was stirred to form a complex. The temperature was raised to 83°C, and the pH was adjusted to 8.0 with 1 mol / L sodium hydroxide. The reaction was carried out at 950 rpm for 27 min to obtain polyaspartic acid-modified Fe3O4 magnetic nanoparticles, which were used as an affinity medium. The nanoparticles were suspended in sterile citric acid buffer at pH 4.3, loaded into a sterile chromatography column, and equilibrated until the effluent was clear and the pH was stable. The affinity chromatography column was obtained.

[0102] 3.6 The polylysine-rich solution was loaded into the affinity chromatography column at a flow rate of 1.7 BV / h, and static adsorption was carried out at 28°C for 53 min. The column was washed with buffer at a flow rate of 2.2 BV / h, and the washing solution was collected.

[0103] The solution was desorbed with sterile Tris-HCl buffer at pH 6.5 at a flow rate of 2 BV / h, and the desorbed solution was collected to obtain a high-purity polylysine solution.

[0104] 3.7 The high-purity polylysine solution was filtered through a 0.22-μm sterile polyether sulfone microporous filter (positive pressure filtration at a pressure of 0.2 MPa), and sterile polylysine solution was collected. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 5 of the original volume at 42°C and a vacuum degree of 0.86 MPa. A purified polylysine solution was obtained.

[0105] S4. Preparation of a crosslinking agent reaction solution;

[0106] To 100 parts of the crosslinking agent reaction solution, 88 parts of the purified polylysine solution were added, and the mixture was stirred at 32°C and 195 r / min for 11 min to obtain a crosslinking agent reaction solution.

[0107] S5. Preparation of a sodium hyaluronate crosslinking reaction solution;

[0108] To 100 parts of the crosslinking agent reaction solution, 1.3 parts of sodium hyaluronate powder were added, and the mixture was stirred at 32°C and 170 r / min for 10 min to obtain a sodium hyaluronate crosslinking reaction solution. The solution was allowed to stand to obtain a clear, transparent, and uniform sodium hyaluronate gel.

[0109] S6. Preparation of a gel;

[0110] 0.10 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and 100 parts of a buffer solution pre-cooled to 10°C were weighed and mixed, and the mixture was filtered through a 0.22-μm sterile polyether sulfone microporous filter to obtain a condensing agent reaction solution.

[0111] The sodium hyaluronate gel is pre-cooled to 10℃, and 112 parts of the condensing agent reaction solution is added to 1000 parts of the sodium hyaluronate gel at a speed of 28 parts / h under stirring at 240 r / min. After the dropwise addition is completed, the mixture is stirred for 1.5 h to make it uniformly dispersed. The mixture is sealed and cross-linked at 9℃ for 74 h to obtain a gel;

[0112] S7. Post-treatment;

[0113] The gel is loaded into a dialysis bag, and the gel is loaded in an amount of not more than 400 g / m. After being evenly laid, the gel is placed in a dialysis container, and a buffer solution is added as an external dialysis solution. For every 1000 parts of the gel, 10000 parts of the external dialysis solution is added. The temperature is 9℃, the dialysis solution is replaced once a day, and the dialysis time is not less than 72 h;

[0114] The dialyzed gel is loaded into a stainless steel extruder for extrusion and mixing, collected into a container, sealed, and refrigerated and filled;

[0115] The gel after refrigerated filling is placed on a sterilization rack and sterilized at 121℃ for 12 min to obtain an artificial vitreous body.

[0116] Embodiment 4: The embodiment provides a preparation method of an artificial vitreous body, comprising the following steps:

[0117] S1. Preparation of a buffer solution;

[0118] 0.15 parts of sodium dihydrogen phosphate, 0.56 parts of disodium hydrogen phosphate, and 9 parts of sodium chloride are added to 1000 parts of water for injection, stirred at 28℃ and 310 r / min for 38 min, and filtered to obtain a buffer solution;

[0119] S2. Preparation of a polylysine solution;

[0120] The polylysine solution is prepared according to the proportion of 3.1 parts of polylysine (ε-polylysine is selected) per 1000 parts of the phosphate buffer solution;

[0121] S3. Purification of the polylysine solution;

[0122] 3.1 The polylysine solution is adjusted to pH 2.8 with 0.1 mol / L hydrochloric acid, and 0.4 parts of aminotri (methylenephosphonic acid) is added. The solution is stirred at 28℃ and 110 rpm for 28 min to complex the free metal ions in the solution, and then centrifuged at 6600 rpm for 23 min to collect the supernatant;

[0123] 3.2 The supernatant is filtered through a sterile microfiltration device (the filter membrane material is polyether sulfone) at 0.22 MPa and 25℃ to remove the solid particles and macromolecular polymer impurities in the solution that are not completely centrifuged, and the sterile microfiltrate is collected;

[0124] 3.3 Use cation exchange resin (matrix is cross-linked agarose) to equilibrate the chromatography column with sterile acetic acid-sodium acetate buffer at pH 3.3 until the column efficiency is stable (pH difference between effluent and sterile acetic acid-sodium acetate buffer is ≤0.1);

[0125] 3.4 Load the sterile microfiltrate into the chromatography column at a flow rate of 3 BV / h. After the loading is completed, rinse the chromatography column with sterile acetic acid-sodium acetate buffer at a flow rate of 4 BV / h. Use gradient elution (in 3 times) with eluent being sterile acetic acid-sodium acetate buffer containing 0.18-0.28 mol / L sodium chloride, and the concentration of sodium chloride is increased by 0.05 mol / L gradient, at a flow rate of 2.4 BV / h. Collect the eluent in sections. Use the color reaction of polylysine with ninhydrin reagent (blue-violet compound is generated after heating) to determine whether the eluent contains polylysine in real time. Collect the polylysine-rich liquid.

[0126] 3.5 Use polyaspartic acid-modified Fe3O4 magnetic nanoparticles (92 nm in size) (dissolve FeCl3 6H2O and FeSO4 7H2O in water for injection at a molar ratio of 2:1. At 59°C, add polyaspartic acid in an amount of 8 times the total mass of iron salt to form a complex. Increase the temperature to 81°C. Adjust the pH to 8.0 with 1 mol / L sodium hydroxide. React at 980 rpm for 29 min to generate polyaspartic acid-modified Fe3O4 magnetic nanoparticles), which is used as an affinity medium. Suspend it in sterile citric acid buffer at pH 4.4. Load it into a sterile chromatography column and equilibrate it until the effluent is clear and the pH is stable. Obtain an affinity chromatography column.

[0127] 3.6 Load the polylysine-rich liquid into the affinity chromatography column at a flow rate of 1.2 V / h. Allow static adsorption at 26°C for 55 min. Rinse the chromatography column with buffer at a flow rate of 2.8 BV / h. Collect the rinse liquid.

[0128] Desorb it with sterile Tris-HCl buffer at pH 5.8 at a flow rate of 2 BV / h. Collect the desorption liquid to obtain high-purity polylysine liquid.

[0129] 3.7 Pass the high-purity polylysine liquid through a 0.22 μm sterile polyether sulfone microporous filter (use positive pressure filtration at a pressure of 0.2 MPa). Collect the sterile polylysine solution. Place the sterile polylysine solution in a rotary evaporator. Concentrate it to 1 / 4 of the original volume under reduced pressure at 38°C and a vacuum degree of 0.92 MPa. Obtain a purified polylysine solution.

[0130] S4. Prepare a crosslinking agent reaction solution.

[0131] Add 90 parts of purified polylysine solution to 1000 parts of buffer solution, stir at 31℃, 190r / min for 10min, to obtain the crosslinking agent reaction solution;

[0132] S5. Prepare the sodium hyaluronate crosslinking reaction solution;

[0133] Add 1.2 parts of sodium hyaluronate powder to 100 parts of the crosslinking agent reaction solution, stir at 29℃, 180r / min for 11min, to obtain the sodium hyaluronate crosslinking reaction solution, and stand to obtain the clear, transparent and uniform sodium hyaluronate gel;

[0134] S6. Prepare the gel;

[0135] Weigh 0.12 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and 100 parts of buffer solution pre-cooled to 11℃, and filter (through 0.22μm sterile polyether sulfone microporous filter membrane) after mixing to obtain the condensing agent reaction solution;

[0136] Pre-cool the sodium hyaluronate gel to 11℃, and add 116 parts of the condensing agent reaction solution to 1000 parts of the sodium hyaluronate gel at a speed of 25 parts / h under stirring at 220r / min, after the dropwise addition is completed, stir for 1.5h to make it uniformly dispersed, and seal and crosslink at 11℃ for 69h to obtain the gel;

[0137] S7. Post-treatment;

[0138] Pack the gel into a dialysis bag, the gel loading amount is not higher than 400g / m, and after being evenly laid, place it in a dialysis container, add buffer solution as the dialysis external liquid, add 10000 parts of dialysis external liquid per 1000 parts of gel, the temperature is 11℃, replace the dialysis liquid once a day, and the dialysis time is not less than 72h;

[0139] Pack the dialyzed gel into a stainless steel extruder for extrusion and mixing, collect into a container, seal, and refrigerate and fill;

[0140] Place the gel after refrigerated filling on a sterilization rack and sterilize at 121℃ for 12min to obtain the artificial vitreous body.

[0141] Comparative Example 1: The difference between this comparative example and Example 3 is that this comparative example does not perform S3.3-3.4.

[0142] Comparative Example 2: The difference between this comparative example and Example 3 is that this comparative example does not perform S3.5-3.6.

[0143] Comparative Example 3: The difference between this comparative example and Example 3 is that this comparative example does not perform S3.

[0144] Experimental Example 1: The dynamic viscosity of the artificial vitreous body prepared by each embodiment and the comparative examples of the present application is between 0.02-0.1 Pa·s, which is close to that of natural vitreous body, measured according to the standard of BH GSO ISO 3105:2024.

[0145] Experimental Example 2: Light transmittance test, measured according to GB / T 2410-2008.

[0146] Experimental Example 3: Cytotoxicity test, measured according to GB / T 16886.5-2017, wherein ARPE-19 human retinal pigment epithelial cells are selected, DMEM+10% FBS+1% double antibody, 37℃, 5% CO2;

[0147] The relative survival rate of cells (reflecting cell viability, the higher the value, the lower the toxicity) of the experimental group and the control group is calculated according to the following formula:

[0148] The relative survival rate of cells (%) = [(OD value of the experimental group-OD value of the blank group) / (OD value of the negative control group-OD value of the blank group)]x100%;

[0149] The toxicity classification is shown in the following table:

[0150] Relative cell survival (RS) Toxicity classification Material evaluation ≥80% 0 grade No cytotoxicity, meets clinical requirements 60%≤ RS < 80% 1 grade Mild toxicity, further evaluation in combination with in vivo experiments 40%≤ RS < 60% 2 grade Moderate toxicity, does not meet requirements 20%≤ RS < 40% 3 grade Severe toxicity, does not meet requirements ＜20% 4 grade Serious toxicity, does not meet requirements

[0151] The experimental results are shown in the following table:

[0152] Item Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Transmittance (%) 96.7 96.9 96.5 96.1 94.1 94.2 91.4 RS (%) 95.5 95.6 95.8 95.4 93.6 94.7 93.2 Toxicity classification 0 grade 0 grade 0 grade 0 grade 0 grade 0 grade 0 grade

[0153] From the above table, it can be seen that the comparative example 1 lacks the cation exchange chromatography step, the comparative example 2 lacks the affinity chromatography step, and the comparative example 3 does not perform purification at all, resulting in relatively higher impurity residue, which in turn affects the light transmittance performance, which shows that the multi-step purification process is a key link to ensure the light transmittance of the artificial vitreous body;

[0154] The present application selects DMTMM as a condensing agent, which replaces the traditional BDDE with residual mutagenic risk and potential toxicity, and the unreacted condensing agent and small molecular impurities are removed by subsequent dialysis, combined with the above-mentioned purification steps to remove impurities that may produce cytotoxicity, reducing the adverse effects on ARPE-19 cells. The embodiments have better cytotoxicity control because these processes are completely implemented.

[0155] In addition, the comparative examples have a small amount of toxicity-related impurities due to incomplete purification steps, and the relative survival rate of cells is slightly lower, but all meet the 0-level toxicity standard, which shows that the synergistic effect of reasonable selection of condensing agent, multi-step purification and sufficient dialysis ensures the biocompatibility of the artificial vitreous body.

[0156] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of preparing an artificial vitreous body, characterized in that, Comprising the following steps: S1. Add 0.12-0.16 parts of sodium dihydrogen phosphate, 0.53-0.58 parts of disodium hydrogen phosphate and 9-11 parts of sodium chloride to 1000 parts of water for injection, stir and filter to obtain a buffer solution; S2. Prepare a polylysine solution according to the ratio of 2.5-3.2 parts of polylysine per 1000 parts of phosphate buffer solution; S3. Polylysine solution purification; 3.1 Take the polylysine solution, adjust the pH to 2.5-3.5 with hydrochloric acid, add aminotri (methylenephosphonic acid), stir and centrifuge to collect the supernatant; 3.2 Pass the supernatant through a sterile microfiltration device and filter to collect sterile microfiltrate; 3.3 Equilibrate the cation exchange resin with sterile acetic acid-sodium acetate buffer and chromatograph the column until the column efficiency is stable; 3.4 Load the sterile microfiltrate into the chromatography column, after completion of loading, rinse the chromatography column with sterile acetic acid-sodium acetate buffer, elute with sterile acetic acid-sodium acetate buffer containing sodium chloride, collect the eluate in sections, use the color reaction of polylysine with ninhydrin reagent to determine whether the eluate contains polylysine in real time, and collect the polylysine-rich liquid; 3.5 Use polyaspartic acid-modified Fe3O4 magnetic nanoparticles as affinity medium, suspend in sterile citric acid buffer, load into a sterile chromatography column and equilibrate to obtain an affinity chromatography column; 3.6 Load the polylysine-rich liquid into the affinity chromatography column, adsorb and then rinse the column with buffer to collect the rinse liquid; desorb with sterile Tris-HCl buffer to collect the desorption liquid to obtain high-purity polylysine liquid; 3.7 Filter the high-purity polylysine liquid, collect sterile polylysine solution, and concentrate the sterile polylysine solution under reduced pressure to 1 / 5-1 / 4 of the original volume to obtain purified polylysine solution; S4. Add 85-92 parts of purified polylysine solution to 1000 parts of buffer solution and stir to obtain a crosslinking agent reaction solution; S5. Add 0.8-1.5 parts of sodium hyaluronate powder to 100 parts of the crosslinking agent reaction solution and stir to obtain a sodium hyaluronate crosslinking reaction solution. Stand to obtain a sodium hyaluronate gel; S6. Weigh 0.09-0.13 parts of DMTMM and 100 parts of buffer solution pre-cooled to 8-12℃, mix and filter to obtain a condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 8-10℃, add 105-118 parts of the condensing agent reaction solution to 1000 parts of the sodium hyaluronate gel at a speed of 20-30 parts / h under stirring at 150-260 r / min, after the addition is completed, stir for 1-2 h to make it uniformly dispersed, seal and crosslink at 8-10℃ for 65-78 h to obtain a gel; S7. Load the gel into a dialysis bag, add buffer solution as dialysis external liquid for dialysis, mix the dialyzed gel by extrusion, cold fill and sterilize to obtain an artificial vitreous body.

2. The method for preparing an artificial vitreous body according to claim 1, characterized in that, S1 is specifically: add 0.12-0.16 parts of sodium dihydrogen phosphate, 0.53-0.58 parts of disodium hydrogen phosphate and 9-11 parts of sodium chloride to 1000 parts of water for injection, stir at 25-30℃, 200-320 r / min for 30-40 min, filter to obtain a buffer solution.

3. The method for preparing artificial vitreous body according to claim 1, characterized by, 3.1 specifically: taking polylysine solution, adjusting pH to 2.5-3.5 with 0.1 mol / L hydrochloric acid, adding 0.1-0.5 parts of aminotri-methylene phosphonic acid, stirring at 80-120 rpm for 15-30 min at 20-30℃ to complex free metal ions in the solution, and then centrifuging at 4000-7000 rpm for 15-25 min to collect the supernatant.

4. The method for preparing artificial vitreous body according to claim 1, characterized by, 3.4 specifically: loading the sterile microfiltrate into the chromatography column at a flow rate of 1-3 BV / h, after the loading is completed, flushing the chromatography column with sterile acetic acid-sodium acetate buffer at a flow rate of 2-4 BV / h, using gradient elution, the eluent being sterile acetic acid-sodium acetate buffer containing 0.1-0.3 mol / L sodium chloride, the concentration of sodium chloride being increased by 0.05 mol / L in gradient, the flow rate being 1.5-2.5 BV / h, collecting the eluate in sections, using the color reaction of polylysine with ninhydrin reagent to determine in real time whether the eluate contains polylysine, and collecting the polylysine-rich liquid.

5. The method of claim 1, wherein the artificial vitreous is prepared by the steps of: The method for modifying Fe3O4 magnetic nanoparticles with polyaspartic acid is as follows: FeCl3 6H2O and FeSO4 7H2O are dissolved in water for injection in a molar ratio of 2:1, polyaspartic acid is added in an amount of 5-10 times the total mass of iron salt at 55-65°C, the mixture is stirred to form a complex, the temperature is raised to 80-85°C, 1 mol / L sodium hydroxide is used to adjust the pH to 8.0-9.0, and the reaction is carried out at 800-1000 rpm for 25-30 min to obtain polyaspartic acid-modified Fe3O4 magnetic nanoparticles.

6. The method for preparing artificial vitreous body according to claim 1, characterized by, 3.6 specifically: loading the polylysine-rich liquid into the affinity chromatography column at a flow rate of 0.8-2 BV / h, static adsorption at 25-30℃ for 30-60 min, flushing the chromatography column with buffer at a flow rate of 1.5-3 BV / h, and collecting the flushing liquid; desorbing with sterile Tris-HCl buffer at pH 5.5-6.5 at a flow rate of 1-2 BV / h, and collecting the desorbed liquid to obtain high-purity polylysine liquid.

7. The method of claim 1, wherein the artificial vitreous body is prepared by the steps of: 3.7 specifically: passing the high-purity polylysine liquid through a 0.22 μm sterile polyether sulfone microporous filter, collecting the sterile polylysine solution, and placing the sterile polylysine solution in a rotary evaporator, concentrating under reduced pressure to 1 / 5-1 / 4 of the original volume at 35-45℃ and a vacuum degree of 0.08-0.095 MPa to obtain purified polylysine solution.

8. An artificial vitreous body prepared by the method of any one of claims 1-7.

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