Artificial glass body and preparation method thereof

Abstract

The invention discloses an artificial vitreous body and a preparation method thereof, and belongs to the technical field of medical materials, and the preparation method comprises the following steps: preparing a buffer solution, preparing a polylysine solution, purifying the polylysine solution, preparing a cross-linking agent reaction solution, preparing sodium hyaluronate gel, and preparing the artificial vitreous body. According to the method disclosed by the invention, DMTMM chloride is selected as a condensing agent, so that the problem that unreacted BDDE monomers need to be removed by a complex purification process is avoided, and the production cost and the process complexity are reduced; the mutagenic risk possibly caused by BDDE residues and the local inflammation, immune response or retinal cell potential toxicity risk caused by non-specific reaction of epoxy groups and intraocular protein are eliminated, and the biological safety of the artificial vitreous body is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to an artificial vitreous body and its preparation method. Background Technology

[0002] The natural vitreous body is a transparent, gel-like substance located inside the eyeball. It is mainly composed of water, hyaluronic acid, collagen, and a small amount of glycoproteins. It plays a core physiological role in maintaining stable intraocular pressure, supporting the integrity of the retinal structure, and performing refractive conduction. When the natural vitreous body liquefies, becomes cloudy, or detaches due to aging, trauma, or pathological factors, an artificial vitreous body replacement needs to be surgically implanted to restore the normal physiological structure and function of the eye. Therefore, the research and development of artificial vitreous bodies is a key direction in the field of medical materials for the treatment of eye diseases.

[0003] Among existing chemical cross-linking technologies, 1,4-butanediol diglycidyl ether (BDDE) has become a commonly used cross-linking agent due to its mild reaction conditions and ability to stably link polymer chains via ether bonds. However, its inherent chemical properties lead to significant drawbacks in its application: unreacted monomers in BDDE need to be removed through complex purification processes, and residual epoxy groups in BDDE may react nonspecifically with proteins in the eye, triggering local inflammation or immune responses and posing a potential toxic risk to retinal cells.

[0004] Meanwhile, the existing artificial vitreous preparation process has an imperfect purification process design for functional excipients, which can easily lead to residual free metal ions, unremoved solid particles, and macromolecular polymer impurities in the system. These impurities can damage the optical homogeneity of the artificial vitreous and significantly reduce the light transmittance of the material. Light transmittance is the core prerequisite for the artificial vitreous to achieve the refractive conduction function. The existing process cannot reliably guarantee that the light transmittance meets the standards, resulting in insufficient functional matching between the artificial vitreous and the natural vitreous, and failing to reliably meet the physiological needs of the eye for refractive conduction.

[0005] Based on this, the present invention designs an artificial vitreous body and its preparation method to solve the above problems. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides an artificial vitreous body, 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 at a ratio of 2.5-3.2 parts polylysine per 1000 parts phosphate buffer; S3. Purification of polylysine solution; 3.1 Take a polylysine solution, adjust the pH to 2.5-3.5 with hydrochloric acid, add aminotrimethylenephosphonic acid, stir, centrifuge and collect the supernatant; 3.2 The supernatant is passed through a sterile microfiltration device to filter and collect the sterile microfiltrate; 3.3 Equilibrate the chromatography column with cation exchange resin and sterile acetate-sodium acetate buffer until the column efficiency is stable; 3.4 Load the sterile microfiltrate onto the chromatography column. After loading, wash the chromatography column with sterile acetate-sodium acetate buffer. Elute with a gradient of sterile acetate-sodium acetate buffer containing sodium chloride. Collect the eluent in fractions. Use the colorimetric reaction of polylysine and ninhydrin reagent to determine in real time whether polylysine is present in the eluent. Collect the polylysine enrichment solution. 3.5 Fe3O4 magnetic nanoparticles modified with polyaspartic acid were used as the affinity medium, suspended in sterile citrate buffer, packed into a sterile chromatography column and equilibrated to obtain the affinity chromatography column; 3.6 Load the polylysine enrichment solution onto an affinity chromatography column. After adsorption, wash the column with buffer and collect the wash solution. Desorb the polylysine solution with sterile Tris-HCl buffer and collect the desorbed solution to obtain a high-purity polylysine solution. 3.7 Filter the high-purity polylysine solution, collect the sterile polylysine solution, and concentrate the sterile polylysine solution under reduced pressure to 1 / 5-1 / 4 of the original volume to obtain a purified polylysine solution. S4. Add 85-92 parts of purified polylysine solution to every 1000 parts of buffer solution and stir to obtain the cross-linking agent reaction solution; S5. Add 0.8-1.5 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain 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 condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 8-10℃, and add 105-118 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel while stirring at 150-260r / min at a rate of 20-30 parts / h. After the addition is complete, stir for 1-2h to disperse it evenly, and seal it at 8-10℃ for 65-78h to obtain the gel. S7. Place the gel into a dialysis bag, add buffer solution as the dialysis fluid, and perform dialysis. After dialysis, squeeze and mix the gel, then refrigerate, fill, and sterilize to obtain the artificial vitreous body.

[0007] Furthermore, S1 specifically involves 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 to 1000 parts of water for injection, stirring at 25-30℃ and 200-320 r / min for 30-40 min, and then filtering to obtain the buffer solution.

[0008] Furthermore, 3.1 specifically involves: taking a polylysine solution, adjusting the pH to 2.5-3.5 with 0.1 mol / L hydrochloric acid, adding 0.1-0.5 parts of aminotrimethylenephosphonic acid, stirring at 80-120 rpm for 15-30 min at 20-30℃ to complex the free metal ions in the solution, and then centrifuging at 4000-7000 rpm for 15-25 min to collect the supernatant.

[0009] Furthermore, step 3.4 specifically involves: loading the sterile microfiltrate onto the chromatography column at a flow rate of 1-3 BV / h; after loading, washing the column with sterile acetate-sodium acetate buffer at a flow rate of 2-4 BV / h; using gradient elution with a sterile acetate-sodium acetate buffer containing 0.1-0.3 mol / L sodium chloride, increasing the sodium chloride concentration in increments of 0.05 mol / L at a flow rate of 1.5-2.5 BV / h; collecting the eluent in fractions; and using the colorimetric reaction of polylysine with ninhydrin reagent to determine in real time whether the eluent contains polylysine, and collecting the polylysine-enriched solution.

[0010] Furthermore, the method for modifying Fe3O4 magnetic nanoparticles with polyaspartic acid is as follows: FeCl3 6H2O and FeSO4 Dissolve 7H2O in water for injection at a molar ratio of 2:1, add 5-10 times the total mass of polyaspartic acid of iron salt at 55-65℃, stir to form a complex, heat 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.

[0011] Furthermore, 3.6 specifically involves: loading the polylysine enrichment solution onto the affinity chromatography column at a flow rate of 0.8-2 BV / h, statically adsorbing at 25-30℃ for 30-60 min, washing the chromatography column with buffer at a flow rate of 1.5-3 BV / h, and collecting the wash solution; The polylysine solution was desorbed using sterile Tris-HCl buffer at pH 5.5-6.5 at a flow rate of 1-2 BV / h, and the desorbed solution was collected to obtain a high-purity polylysine solution.

[0012] Furthermore, step 3.7 specifically involves: passing high-purity polylysine solution through a 0.22μm sterile polyethersulfone microporous filter membrane to collect the sterile polylysine solution; placing the sterile polylysine solution in a rotary evaporator and concentrating it under reduced pressure to 1 / 5-1 / 4 of its original volume at 35-45℃ and a vacuum of 0.08-0.095MPa to obtain a purified polylysine solution.

[0013] An artificial vitreous body prepared according to the method described above.

[0014] Compared with the prior art, the beneficial effects of this invention are as follows: 1. This invention uses 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine (DMTMM) as a condensing agent, which not only avoids the problem of removing unreacted BDDE monomers through complex purification processes, reducing production costs and process complexity, but also eliminates the mutagenic risks that may be caused by BDDE residues and the potential toxicity risks of local inflammation, immune reactions, or retinal cells caused by non-specific reactions between epoxy groups and intraocular proteins, significantly improving the biosafety of artificial vitreous bodies.

[0015] 2. This invention utilizes a multi-step purification process for polylysine, combined with post-treatment, to effectively remove impurities that affect light transmittance and substances that may cause cytotoxicity within the system. This makes the light transmittance characteristics of the artificial vitreous body closer to those of the natural vitreous body, while reducing the residue of toxicity-related impurities, ensuring low toxicity of the material to intraocular cells, and meeting the biocompatibility requirements of medical implant materials.

[0016] 3. Through the synergistic design of processes such as buffer preparation, polylysine purification and cross-linking system construction, sodium hyaluronate gel cross-linking, and high-temperature sterilization, the artificial vitreous body prepared in this invention is more compatible with the physiological functions of the natural vitreous body in terms of viscoelasticity, transparency, and biostability. It can reliably achieve the core functions of maintaining intraocular pressure, supporting the retina, and refractive conduction. At the same time, relying on the selection of low-toxicity condensing agents and multi-step purification processes, the long-term in vivo retention capacity and biosafety of the material are balanced, solving the technical problems of insufficient matching between the performance of artificial vitreous bodies and natural vitreous bodies or poor biosafety in existing technologies, and enhancing the reliability of clinical applications. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1This is a diagram showing the state of the reaction between ninhydrin and polylysine in step 3.4. Figure 2 This is a diagram showing the state of the ninhydrin colorimetric solution during the negative reaction with polylysine in step 3.4. Figure 3 This is a photograph of the artificial vitreous body prepared in Example 1. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Example 1: This example provides a method for preparing an artificial vitreous body, including the following steps: S1. Prepare buffer solution; Add 0.12 parts of sodium dihydrogen phosphate, 0.53 parts of disodium hydrogen phosphate and 9 parts of sodium chloride to 1000 parts of water for injection, stir at 25℃ and 200 r / min for 30 min, and filter to obtain buffer solution; S2. Prepare polylysine solution; Prepare a polylysine solution by adding 2.5 parts of polylysine (preferably ε-polylysine) to every 1000 parts of phosphate buffer. S3. Purification of polylysine solution; 3.1 Take a polylysine solution, adjust the pH to 2.5 with 0.1 mol / L hydrochloric acid, add 0.1 part of aminotrimethylenephosphonic acid, stir at 80 rpm for 15 min at 20 °C to complex the free metal ions in the solution, then centrifuge at 4000 rpm for 15 min and collect the supernatant. 3.2 The supernatant was filtered through a sterile microfiltration device (the filter membrane material was polyethersulfone) at 0.15 MPa and 20 °C to remove incompletely centrifuged solid particles and macromolecular polymer impurities from the solution, and the sterile microfiltrate was collected. 3.3 Equilibrate the chromatography column with a cation exchange resin (with cross-linked agarose matrix) and a sterile acetate-sodium acetate buffer solution at pH 3.0 until the column efficiency is stable (the pH difference between the effluent and the sterile acetate-sodium acetate buffer solution is ≤0.1). 3.4 Load the sterile microfiltrate onto the chromatography column at a flow rate of 1 BV / h. After loading, wash the column with sterile acetate-sodium acetate buffer at a flow rate of 2 BV / h. Use gradient elution (elution in 3 fractions) with sterile acetate-sodium acetate buffer containing 0.1-0.2 mol / L sodium chloride. The sodium chloride concentration should be increased in a gradient of 0.05 mol / L at a flow rate of 1.5 BV / h. Collect the eluent in fractions. Use the colorimetric reaction of polylysine with ninhydrin reagent (a blue-purple compound is generated upon heating) to determine in real time whether polylysine is present in the eluent. Collect the polylysine-enriched solution. The specific steps of the colorimetric reaction experiment are as follows: Prepare the ninhydrin colorimetric solution (0.1% ninhydrin dissolved in a 50% ethanol-water mixture). During the gradient elution process, collect the eluent every 3 mL. Take 1 mL of the collected solution, add 0.5 mL of the ninhydrin colorimetric solution, heat in a boiling water bath for 5 min, and observe the color after cooling. When the solution shows a distinct blue-purple color (positive reaction, such as...), the color will develop. Figure 1 When the eluent (as shown) is in the figure, begin collecting the eluent continuously; continue until the collected eluent reacts with ninhydrin to produce a faint color (close to negative, such as...). Figure 2 When the eluent collected during the period shown in the figure is stopped, the eluent collected during this period is the polylysine enrichment solution.

[0021] 3.5 Fe3O4 magnetic nanoparticles with a particle size of 50 nm modified with polyaspartic acid (FeCl3) 6H2O and FeSO4 7H2O was dissolved in water for injection at a molar ratio of 2:1. Polyaspartic acid, five times the total mass of iron salt, was added at 55°C and 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 generate polyaspartic acid-modified Fe3O4 magnetic nanoparticles. These nanoparticles were used as the affinity medium and suspended in sterile citrate buffer at pH 4.0. The mixture was then packed into a sterile chromatography column and equilibrated until the eluent was clear and the pH was stable, thus obtaining the affinity chromatography column. 3.6 Load the polylysine enrichment solution onto the affinity chromatography column at a flow rate of 0.8 BV / h, allow it to adsorb statically at 25 °C for 30 min, and then wash the column with buffer at a flow rate of 1.5 BV / h and collect the wash solution. The polylysine solution was desorbed using sterile Tris-HCl buffer at pH 5.5 at a flow rate of 1 BV / h, and the desorbed solution was collected to obtain a high-purity polylysine solution. It should be noted that polyaspartic acid-modified Fe3O4 magnetic nanoparticles act as an affinity medium, adsorbing polylysine through electrostatic interactions. Polyaspartic acid is a negatively charged polyelectrolyte (containing a large number of carboxyl groups), while polylysine is positively charged at physiological pH. The two are adsorbed by electrostatic attraction. The key to desorption is to disrupt the electrostatic interaction between polyaspartic acid and polylysine. Tris-HCl buffer achieves desorption through pH adjustment: the isoelectric point of polylysine is approximately 9.0-10.0. At pH 4.0-4.5 (sample loading conditions), the amino group of polylysine is protonated (-NH3). + ), with polyaspartic acid -COO - Combined, when the pH rises to 5.5-6.5, the amino group of polylysine is deprotonated, the positive charge density decreases, and the electrostatic attraction weakens, thereby enabling desorption.

[0022] 3.7 The high-purity polylysine solution was passed through a 0.22 μm sterile polyethersulfone microporous membrane (using positive pressure filtration, pressure 0.1 MPa) to collect the sterile polylysine solution. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 5 of the original volume at 35℃ and a vacuum of 0.08 MPa to obtain a purified polylysine solution. S4. Prepare the crosslinking agent reaction solution; Add 85 parts of purified polylysine solution to every 1000 parts of buffer solution, and stir at 25℃ and 150r / min for 8 min to obtain the cross-linking agent reaction solution; S5. Prepare the sodium hyaluronate cross-linking reaction solution; Add 0.8 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir at 25℃ and 150r / min for 8min to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain clear, transparent and uniform sodium hyaluronate gel; S6. Prepare the gel; Weigh 0.09 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride) and 100 parts of buffer solution pre-cooled to 8°C, mix and filter (through a 0.22 μm sterile polyethersulfone microporous filter membrane) to obtain the condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 8°C. While stirring at 150 r / min, add 105 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel at a rate of 20 parts / h. After the addition is complete, stir for 1 h to disperse it evenly. Seal and crosslink at 8°C for 65 h to obtain the gel. S7. Post-processing; Pack the gel into a dialysis bag, with a gel volume not exceeding 400 g / m. Spread it evenly and place it in a dialysis container. Add buffer solution as the external dialysis solution for dialysis. Add 10,000 portions of external dialysis solution for every 1,000 portions of gel. Maintain a temperature of 8°C. Change the dialysis solution once a day. The dialysis time should not be less than 72 hours. The dialyzed gel is loaded into a stainless steel extruder and squeezed to mix. The gel is then collected into a container, sealed, and refrigerated for filling. The refrigerated and filled gel was placed on a sterilization rack and sterilized at 121°C for 12 minutes to obtain artificial vitreous body.

[0023] Example 2: This example provides a method for preparing an artificial vitreous body, including the following steps: S1. Prepare buffer solution; 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 320 r / min for 40 min, and filter to obtain buffer solution; S2. Prepare polylysine solution; Prepare a polylysine solution by adding 3.2 parts of polylysine (preferably ε-polylysine) to every 1000 parts of phosphate buffer solution; S3. Purification of polylysine solution; 3.1 Take a polylysine solution, adjust the pH to 3.5 with 0.1 mol / L hydrochloric acid, add 0.5 parts of aminotrimethylenephosphonic acid, stir at 120 rpm for 30 min at 30 °C to complex the free metal ions in the solution, then centrifuge at 7000 rpm for 25 min and collect the supernatant. 3.2 The supernatant was filtered through a sterile microfiltration device (the filter membrane material was polyethersulfone) at 0.3 MPa and 28°C to remove incompletely centrifuged solid particles and macromolecular polymer impurities from the solution, and the sterile microfiltrate was collected. 3.3 Use a cation exchange resin (with cross-linked agarose as the matrix) to equilibrate the chromatography column with sterile acetate-sodium acetate buffer at pH 3.5 until the column efficiency is stable (the difference between the pH of the effluent and the pH of the sterile acetate-sodium acetate buffer is ≤0.1). 3.4 Load the sterile microfiltrate onto the chromatography column at a flow rate of 3 BV / h. After loading, wash the column with sterile acetate-sodium acetate buffer at a flow rate of 4 BV / h. Use gradient elution (elution in 3 fractions) with sterile acetate-sodium acetate buffer containing 0.2-0.3 mol / L sodium chloride. The sodium chloride concentration should be increased in a gradient of 0.05 mol / L at a flow rate of 2.5 BV / h. Collect the eluent fractions. Use the colorimetric reaction of polylysine with ninhydrin reagent (a blue-purple compound is generated upon heating) to determine in real time whether polylysine is present in the eluent. Collect the polylysine-enriched solution. 3.5 Fe3O4 magnetic nanoparticles with a particle size of 100 nm modified with polyaspartic acid (FeCl3) 6H2O and FeSO4 7H2O was dissolved in water for injection at a molar ratio of 2:1. Polyaspartic acid, at a mass ratio of 10 times the total iron salt, was added at 65°C and stirred to form a complex. The temperature was raised to 85°C, and the pH was adjusted to 9.0 with 1 mol / L sodium hydroxide. The reaction was carried out at 1000 rpm for 30 min to generate polyaspartic acid-modified Fe3O4 magnetic nanoparticles. These nanoparticles were used as the affinity medium and suspended in sterile citrate buffer at pH 4.5. The mixture was then packed into a sterile chromatography column and equilibrated until the eluent was clear and the pH was stable, thus obtaining the affinity chromatography column. 3.6 Load the polylysine enrichment solution onto the affinity chromatography column at a flow rate of 2 BV / h, allow it to adsorb statically at 30 °C for 60 min, then wash the column with buffer at a flow rate of 3 BV / h and collect the wash solution. The polylysine solution was desorbed using 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. 3.7 The high-purity polylysine solution was passed through a 0.22 μm sterile polyethersulfone microporous membrane (using positive pressure filtration, pressure 0.2 MPa) to collect the sterile polylysine solution. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 4 of the original volume at 45℃ and a vacuum degree of 0.095 MPa to obtain a purified polylysine solution. S4. Prepare the crosslinking agent reaction solution; Add 92 parts of purified polylysine solution to every 1000 parts of buffer solution, and stir at 34℃ and 200r / min for 12min to obtain the crosslinking agent reaction solution; S5. Prepare a sodium hyaluronate cross-linking reaction solution; Add 1.5 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir at 34℃ and 200r / min for 12min to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain clear, transparent and uniform sodium hyaluronate gel. S6. Prepare the gel; Weigh 0.13 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride) and 100 parts of buffer solution pre-cooled to 12°C, mix and filter (through a 0.22 μm sterile polyethersulfone microporous filter membrane) to obtain the condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 10°C. While stirring at 260 r / min, add 118 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel at a rate of 30 parts / h. After the addition is complete, stir for 2 h to disperse it evenly. Seal and crosslink at 10°C for 78 h to obtain the gel. S7. Post-processing; Pack the gel into a dialysis bag, with a gel volume not exceeding 400 g / m. Spread it evenly and place it in a dialysis container. Add buffer solution as the dialysis fluid for dialysis. Add 10,000 parts of dialysis fluid for every 1,000 gels. Maintain a temperature of 10°C. Change the dialysis fluid once a day. The dialysis time should not be less than 72 hours. The dialyzed gel is loaded into a stainless steel extruder and squeezed to mix. The gel is then collected into a container, sealed, and refrigerated for filling. The refrigerated and filled gel was placed on a sterilization rack and sterilized at 121°C for 12 minutes to obtain artificial vitreous body.

[0024] Example 3: This example provides a method for preparing an artificial vitreous body, including the following steps: S1. Prepare buffer solution; Add 0.13 parts sodium dihydrogen phosphate, 0.55 parts disodium hydrogen phosphate and 10 parts sodium chloride to 1000 parts water for injection, stir at 27℃ and 280 r / min for 35 min, and filter to obtain buffer solution; S2. Prepare polylysine solution; Prepare a polylysine solution by adding 2.8 parts of polylysine (preferably ε-polylysine) to every 1000 parts of phosphate buffer solution. S3. Purification of polylysine solution; 3.1 Take polylysine solution, adjust the pH to 3.2 with 0.1 mol / L hydrochloric acid, add 0.3 parts of aminotrimethylenephosphonic acid, stir at 100 rpm for 26 min at 25 °C to complex the free metal ions in the solution, then centrifuge at 5800 rpm for 21 min and collect the supernatant. 3.2 The supernatant was filtered through a sterile microfiltration device (the filter membrane material was polyethersulfone) at 0.26 MPa and 24 °C to remove incompletely centrifuged solid particles and macromolecular polymer impurities from the solution, and the sterile microfiltrate was collected. 3.3 Equilibrate the chromatography column with a cation exchange resin (with cross-linked agarose matrix) and a sterile acetate-sodium acetate buffer solution at pH 3.2 until the column efficiency is stable (the pH difference between the effluent and the sterile acetate-sodium acetate buffer solution is ≤0.1). 3.4 Load the sterile microfiltrate onto the chromatography column at a flow rate of 2 BV / h. After loading, wash the column with sterile acetate-sodium acetate buffer at a flow rate of 3 BV / h. Use gradient elution (elution in 3 fractions) with sterile acetate-sodium acetate buffer containing 0.14-0.24 mol / L sodium chloride. The sodium chloride concentration should be increased in a gradient of 0.05 mol / L at a flow rate of 1.9 BV / h. Collect the eluent in fractions. Use the colorimetric reaction of polylysine with ninhydrin reagent (a blue-purple compound is generated upon heating) to determine in real time whether polylysine is present in the eluent. Collect the polylysine-enriched solution. 3.5 Fe3O4 magnetic nanoparticles with a particle size of 78 nm modified with polyaspartic acid (FeCl3) 6H2O and FeSO4 7H2O was dissolved in water for injection at a molar ratio of 2:1. Polyaspartic acid, at a mass of 7 times the total iron salt, was added at 62°C and 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 generate polyaspartic acid-modified Fe3O4 magnetic nanoparticles. These nanoparticles were used as the affinity medium and suspended in sterile citrate buffer at pH 4.3. The mixture was then packed into a sterile chromatography column and equilibrated until the eluent was clear and the pH was stable, thus obtaining the affinity chromatography column. 3.6 The polylysine enrichment solution was loaded onto the affinity chromatography column at a flow rate of 1.7 BV / h and statically adsorbed at 28 °C for 53 min. The column was then washed with buffer at a flow rate of 2.2 BV / h, and the wash solution was collected. The polylysine solution was desorbed using 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. 3.7 The high-purity polylysine solution was passed through a 0.22 μm sterile polyethersulfone microporous membrane (using positive pressure filtration, pressure 0.2 MPa) to collect the sterile polylysine solution. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 5 of the original volume at 42℃ and a vacuum of 0.86 MPa to obtain a purified polylysine solution. S4. Prepare the crosslinking agent reaction solution; Add 88 parts of purified polylysine solution to every 1000 parts of buffer solution, and stir at 32℃ and 195r / min for 11min to obtain the cross-linking agent reaction solution; S5. Prepare a sodium hyaluronate cross-linking reaction solution; Add 1.3 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir at 32℃ and 170r / min for 10min to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain clear, transparent and uniform sodium hyaluronate gel; S6. Prepare the gel; Weigh 0.10 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride) and 100 parts of buffer solution pre-cooled to 10°C, mix and filter (through a 0.22 μm sterile polyethersulfone microporous filter membrane) to obtain the condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 10°C. While stirring at 240 r / min, add 112 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel at a rate of 28 parts / h. After the addition is complete, stir for 1.5 h to disperse it evenly. Seal and crosslink at 9°C for 74 h to obtain the gel. S7. Post-processing; Pack the gel into a dialysis bag, with a gel volume not exceeding 400 g / m. Spread it evenly and place it in a dialysis container. Add buffer solution as the external dialysis solution for dialysis. Add 10,000 portions of external dialysis solution for every 1,000 portions of gel. Maintain a temperature of 9°C. Change the dialysis solution once a day. The dialysis time should not be less than 72 hours. The dialyzed gel is loaded into a stainless steel extruder and squeezed to mix. The gel is then collected into a container, sealed, and refrigerated for filling. The refrigerated and filled gel was placed on a sterilization rack and sterilized at 121°C for 12 minutes to obtain artificial vitreous body.

[0025] Example 4: This example provides a method for preparing an artificial vitreous body, including the following steps: S1. Prepare buffer solution; Add 0.15 parts sodium dihydrogen phosphate, 0.56 parts disodium hydrogen phosphate and 9 parts sodium chloride to 1000 parts water for injection, stir at 28℃ and 310 r / min for 38 min, and filter to obtain buffer solution; S2. Prepare polylysine solution; Prepare a polylysine solution by adding 3.1 parts of polylysine (preferably ε-polylysine) to every 1000 parts of phosphate buffer solution. S3. Purification of polylysine solution; 3.1 Take polylysine solution, adjust the pH to 2.8 with 0.1 mol / L hydrochloric acid, add 0.4 parts of aminotrimethylenephosphonic acid, stir at 110 rpm for 28 min at 28℃ to complex the free metal ions in the solution, then centrifuge at 6600 rpm for 23 min and collect the supernatant. 3.2 The supernatant was filtered through a sterile microfiltration device (the filter membrane material was polyethersulfone) at 0.22 MPa and 25°C to remove incompletely centrifuged solid particles and macromolecular polymer impurities from the solution, and the sterile microfiltrate was collected. 3.3 Equilibrate the chromatography column with a cation exchange resin (with cross-linked agarose matrix) and a sterile acetate-sodium acetate buffer solution at pH 3.3 until the column efficiency is stable (the pH difference between the effluent and the sterile acetate-sodium acetate buffer solution is ≤0.1). 3.4 Load the sterile microfiltrate onto the chromatography column at a flow rate of 3 BV / h. After loading, wash the column with sterile acetate-sodium acetate buffer at a flow rate of 4 BV / h. Use gradient elution (elution in 3 fractions) with sterile acetate-sodium acetate buffer containing 0.18-0.28 mol / L sodium chloride. The sodium chloride concentration should be increased in a gradient of 0.05 mol / L at a flow rate of 2.4 BV / h. Collect the eluent in fractions. Use the colorimetric reaction of polylysine with ninhydrin reagent (a blue-purple compound is generated upon heating) to determine in real time whether polylysine is present in the eluent. Collect the polylysine-enriched solution. 3.5 Fe3O4 magnetic nanoparticles with a particle size of 92 nm modified with polyaspartic acid (FeCl3) 6H2O and FeSO4 7H2O was dissolved in water for injection at a molar ratio of 2:1. Polyaspartic acid, at a mass of 8 times the total iron salt, was added at 59°C and stirred to form a complex. The temperature was raised to 81°C, and the pH was adjusted to 8.0 with 1 mol / L sodium hydroxide. The reaction was carried out at 980 rpm for 29 min to generate polyaspartic acid-modified Fe3O4 magnetic nanoparticles. These nanoparticles were used as the affinity medium and suspended in sterile citrate buffer at pH 4.4. The mixture was then packed into a sterile chromatography column and equilibrated until the eluent was clear and the pH was stable, thus obtaining the affinity chromatography column. 3.6 Load the polylysine enrichment solution onto the affinity chromatography column at a flow rate of 1.2 V / h, allow it to adsorb statically at 26 °C for 55 min, then wash the column with buffer at a flow rate of 2.8 BV / h and collect the wash solution. The polylysine solution was desorbed using sterile Tris-HCl buffer at pH 5.8 at a flow rate of 2 BV / h, and the desorbed solution was collected to obtain a high-purity polylysine solution. 3.7 The high-purity polylysine solution was passed through a 0.22 μm sterile polyethersulfone microporous membrane (using positive pressure filtration, pressure 0.2 MPa) to collect the sterile polylysine solution. The sterile polylysine solution was placed in a rotary evaporator and concentrated under reduced pressure to 1 / 4 of the original volume at 38℃ and a vacuum of 0.92 MPa to obtain a purified polylysine solution. S4. Prepare the crosslinking agent reaction solution; Add 90 parts of purified polylysine solution to every 1000 parts of buffer solution, and stir at 31℃ and 190r / min for 10min to obtain the cross-linking agent reaction solution; S5. Prepare the sodium hyaluronate cross-linking reaction solution; Add 1.2 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir at 29℃ and 180r / min for 11min to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain clear, transparent and uniform sodium hyaluronate gel; S6. Prepare the gel; Weigh 0.12 parts of DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride) and 100 parts of buffer solution pre-cooled to 11°C, mix and filter (through a 0.22 μm sterile polyethersulfone microporous filter membrane) to obtain the condensing agent reaction solution; The sodium hyaluronate gel was pre-cooled to 11°C. While stirring at 220 r / min, 116 parts of condensing agent reaction solution were added to 1000 parts of sodium hyaluronate gel at a rate of 25 parts / h. After the addition was completed, the mixture was stirred for 1.5 h to disperse it evenly. The gel was then sealed and crosslinked at 11°C for 69 h to obtain the gel. S7. Post-processing; Pack the gel into a dialysis bag, with a gel volume not exceeding 400 g / m. Spread it evenly and place it in a dialysis container. Add buffer solution as the external dialysis solution for dialysis. Add 10,000 portions of external dialysis solution for every 1,000 portions of gel. Maintain a temperature of 11°C. Change the dialysis solution once a day. The dialysis time should not be less than 72 hours. The dialyzed gel is loaded into a stainless steel extruder and squeezed to mix. It is then collected into a container, sealed, and refrigerated for filling. The refrigerated and filled gel was placed on a sterilization rack and sterilized at 121°C for 12 minutes to obtain artificial vitreous body.

[0026] Comparative Example 1: This comparative example differs from Example 3 in that S3.3-3.4 were not performed in this comparative example.

[0027] Comparative Example 2: This comparative example differs from Example 3 in that S3.5-3.6 were not performed in this comparative example.

[0028] Comparative Example 3: This comparative example differs from Example 3 in that S3 was not performed in this comparative example.

[0029] Experimental Example 1: The dynamic viscosity of the artificial vitreous bodies prepared in each embodiment and comparative example of the present invention was measured according to the standard of BH GSO ISO 3105:2024, which is between 0.02-0.1 Pa·s and similar to that of natural vitreous bodies.

[0030] Experimental Example 2: Light transmittance test, measured according to GB / T 2410-2008.

[0031] Experimental Example 3: Cytotoxicity test, conducted according to GB / T 16886.5-2017, using ARPE-19 human retinal pigment epithelial cells, DMEM + 10% FBS + 1% double antibody, at 37℃ and 5% CO2. Calculate the relative cell viability (reflecting cell viability; higher values ​​indicate lower toxicity) for the experimental and control groups using the following formula: Relative cell viability (%) = [(OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group)] × 100%; The toxicity classification is shown in the table below: Relative cell viability (RS) Toxicity classification Material Evaluation ≥80% Level 0 It has no cytotoxicity and meets clinical requirements. 60%≤RS<80% Level 1 Mild toxicity, further evaluation is needed based on in vivo experiments. 40%≤RS<60% Level 2 Moderate toxicity, does not meet requirements 20%≤RS<40% Level 3 Severely toxic, does not meet requirements ＜20% Level 4 Severe toxicity, does not meet requirements The experimental results are shown in the table below: project Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Light 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 Level 0 Level 0 Level 0 Level 0 Level 0 Level 0 Level 0 As can be seen from the table above, Comparative Example 1 of the present invention lacked the cation exchange chromatography step, Comparative Example 2 lacked the affinity chromatography step, and Comparative Example 3 did not perform purification at all, resulting in a relatively higher amount of impurities, which in turn affected the light transmittance. This shows that the multi-step purification process is the key link to ensure the light transmittance of artificial vitreous bodies. This invention uses DMTMM as a condensing agent to replace the traditional BDDE, which has residual mutagenic risks and potential toxicity. Furthermore, unreacted condensing agents and small molecule impurities are removed by dialysis, and the aforementioned purification steps remove impurities that may cause cytotoxicity, reducing adverse effects on ARPE-19 cells. Because these processes are fully implemented in the examples, cytotoxicity control is better. In addition, the comparative sample had a small amount of toxicity-related impurities remaining due to incomplete purification steps, resulting in a slightly lower relative cell survival rate, but all met the grade 0 toxicity standard. This demonstrates that the rational selection of condensing agents, the synergistic effect of multi-step purification and thorough dialysis ensured the biocompatibility of the artificial vitreous body.

[0032] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions 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 invention.

Claims

1. A method for preparing an artificial vitreous body, characterized in that, Includes 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 at a ratio of 2.5-3.2 parts polylysine per 1000 parts phosphate buffer; S3. Purification of polylysine solution; 3.1 Take a polylysine solution, adjust the pH to 2.5-3.5 with hydrochloric acid, add aminotrimethylenephosphonic acid, stir, centrifuge and collect the supernatant; 3.2 The supernatant is passed through a sterile microfiltration device to filter and collect the sterile microfiltrate; 3.3 Equilibrate the chromatography column with cation exchange resin and sterile acetate-sodium acetate buffer until the column efficiency is stable; 3.4 Load the sterile microfiltrate onto the chromatography column. After loading, wash the chromatography column with sterile acetate-sodium acetate buffer. Elute with a gradient of sterile acetate-sodium acetate buffer containing sodium chloride. Collect the eluent in fractions. Use the colorimetric reaction of polylysine and ninhydrin reagent to determine in real time whether polylysine is present in the eluent. Collect the polylysine enrichment solution. 3.5 Fe3O4 magnetic nanoparticles modified with polyaspartic acid were used as the affinity medium, suspended in sterile citrate buffer, packed into a sterile chromatography column and equilibrated to obtain the affinity chromatography column; 3.6 Load the polylysine enrichment solution onto an affinity chromatography column. After adsorption, wash the column with buffer and collect the wash solution. Desorb the polylysine solution with sterile Tris-HCl buffer and collect the desorbed solution to obtain a high-purity polylysine solution. 3.7 Filter the high-purity polylysine solution, collect the sterile polylysine solution, and concentrate the sterile polylysine solution under reduced pressure to 1 / 5-1 / 4 of the original volume to obtain a purified polylysine solution. S4. Add 85-92 parts of purified polylysine solution to every 1000 parts of buffer solution and stir to obtain the cross-linking agent reaction solution; S5. Add 0.8-1.5 parts of sodium hyaluronate powder to 100 parts of crosslinking agent reaction solution, stir to obtain sodium hyaluronate crosslinking reaction solution, and let stand to obtain 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 condensing agent reaction solution; Pre-cool the sodium hyaluronate gel to 8-10℃, and add 105-118 parts of condensing agent reaction solution to 1000 parts of sodium hyaluronate gel while stirring at 150-260r / min at a rate of 20-30 parts / h. After the addition is complete, stir for 1-2h to disperse it evenly, and seal it at 8-10℃ for 65-78h to obtain the gel. S7. Place the gel into a dialysis bag, add buffer solution as the dialysis fluid, and perform dialysis. After dialysis, squeeze and mix the gel, then refrigerate, fill, and sterilize to obtain the artificial vitreous body.

2. The method for preparing artificial vitreous body according to claim 1, characterized in that, S1 is specifically prepared by 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 to 1000 parts of water for injection, stirring at 25-30℃ and 200-320 r / min for 30-40 min, and filtering to obtain the buffer solution.

3. The method for preparing artificial vitreous body according to claim 1, characterized in that, 3.1 Specifically: Take a polylysine solution, adjust the pH to 2.5-3.5 with 0.1 mol / L hydrochloric acid, add 0.1-0.5 parts of aminotrimethylenephosphonic acid, stir at 80-120 rpm for 15-30 min at 20-30℃ to complex the free metal ions in the solution, and then centrifuge at 4000-7000 rpm for 15-25 min and collect the supernatant.

4. The method for preparing artificial vitreous body according to claim 1, characterized in that, 3.4 Specifically: The sterile microfiltrate was loaded onto the chromatography column at a flow rate of 1-3 BV / h. After loading, the column was washed with sterile acetate-sodium acetate buffer at a flow rate of 2-4 BV / h. Gradient elution was used, with the eluent being sterile acetate-sodium acetate buffer containing 0.1-0.3 mol / L sodium chloride. The sodium chloride concentration was increased in a gradient of 0.05 mol / L at a flow rate of 1.5-2.5 BV / h. The eluent was collected in fractions. The presence of polylysine in the eluent was determined in real time by the colorimetric reaction of polylysine and ninhydrin reagent. The polylysine-enriched solution was then collected.

5. The method for preparing artificial vitreous body according to claim 1, characterized in that, The method for modifying Fe3O4 magnetic nanoparticles with polyaspartic acid is as follows: FeCl3 6H2O and FeSO4 Dissolve 7H2O in water for injection at a molar ratio of 2:1, add 5-10 times the total mass of polyaspartic acid of iron salt at 55-65℃, stir to form a complex, heat 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.

6. The method for preparing artificial vitreous body according to claim 1, characterized in that, 3.6 Specifically: Load the polylysine enrichment solution onto the affinity chromatography column at a flow rate of 0.8-2 BV / h, allow it to statically adsorb at 25-30℃ for 30-60 min, wash the chromatography column with buffer at a flow rate of 1.5-3 BV / h, and collect the wash solution; The polylysine solution was desorbed using sterile Tris-HCl buffer at pH 5.5-6.5 at a flow rate of 1-2 BV / h, and the desorbed solution was collected to obtain a high-purity polylysine solution.

7. The method for preparing artificial vitreous body according to claim 1, characterized in that, 3.7 Specifically: High-purity polylysine solution is passed through a 0.22μm sterile polyethersulfone microporous filter membrane to collect sterile polylysine solution. The sterile polylysine solution is then placed in a rotary evaporator and concentrated under reduced pressure at 35-45℃ and a vacuum of 0.08-0.095MPa to 1 / 5-1 / 4 of the original volume to obtain purified polylysine solution.

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

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