Vitreous body gel as well as preparation method and application thereof

Abstract

The invention provides vitreous gel and a preparation method and application thereof, and relates to the field of medical materials.The vitreous gel is prepared from aldehyde sodium hyaluronate, aminated sodium hyaluronate and a buffer solution; the ring opening rate of the formylated sodium hyaluronate is 40%-70%, and the molecular weight of the formylated sodium hyaluronate is 10-24 kDa; the grafting rate of the aminated sodium hyaluronate is 10%-24%, and the molecular weight of the aminated sodium hyaluronate is 150-550 kDa; the mass ratio of the aldehyde sodium hyaluronate to the aminated sodium hyaluronate is (4-9): (72-88). According to the technical scheme, the molar ratio of aldehyde groups to amino groups during mixing is smaller than 1 according to the mass ratio of the aldehyde sodium hyaluronate to the aminated sodium hyaluronate, and the proportion of the aldehyde groups is reduced, so that potential damage possibly caused by the aldehyde groups to intraocular tissues is reduced, and the healing speed of the intraocular tissues is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a vitreous gel and a preparation method and application thereof. Background Art

[0002] The vitreous body is a colorless, transparent colloid in the cavity at the back of the eyeball. It fills between the lens and the retina and occupies more than 2 / 3 of the volume of the eyeball. Its main components include water, collagen, hyaluronic acid, proteoglycans, etc. The vitreous body provides proper support for the retina, protects the surrounding eye tissues, allows light to reach the receptors at the back of the eyeball, regulates the intraocular oxygen content and the shape of the eyeball, and allows the diffusion and transport of metabolites and other solutes. Vitrectomy is an important ophthalmic surgery used to treat ophthalmic diseases such as retinopathy and retinal detachment. With the development of vitreous surgery technology, its range of indications continues to expand. Vitreous cavity filling is required for vitrectomy. The filler is filled in the vitreous cavity, pressing the retina to keep it in the correct position and maintain the shape of the eyeball. Therefore, vitreous fillers have become an important factor affecting the effect of surgery.

[0003] Currently, the vitreous filling materials used in clinical practice mainly include gas, silicone oil, and perfluorocarbon liquid. These fillers are suitable for vitreous filling in different surgical situations, but there are still many complications. For example, the gas filling time is short and may cause lens opacity, perfluorocarbon liquid has high toxicity, and the commonly used silicone oil filling is prone to emulsification and has certain toxicity to eye tissues, often causing complications such as glaucoma and cataracts. In particular, increased intraocular pressure after vitreous filling is very common in clinical practice. Severe high intraocular pressure can cause ischemia and lead to vision loss, and long-term chronic persistent high intraocular pressure may also damage the optic nerve. Summary of the invention

[0004] The main purpose of the present invention is to provide a vitreous gel and a preparation method and application thereof, aiming to reduce the damage of the vitreous gel to intraocular tissues.

[0005] To achieve the above object, the present invention provides a vitreous gel, wherein the raw materials of the vitreous gel include aldehyded sodium hyaluronate, amino sodium hyaluronate and a buffer solution;

[0006] The ring opening rate of the aldehyded sodium hyaluronate is 40% to 60%, and the molecular weight is 10 to 24 kDa;

[0007] The grafting rate of the amino sodium hyaluronate is 14% to 24%, and the molecular weight is 150 to 550 kDa;

[0008] The mass ratio of the aldehyded sodium hyaluronate to the amino sodium hyaluronate is (4-7):(76-88).

[0009] In one embodiment, the buffer is a phosphate buffer, and the phosphate concentration ranges from 4 mM to 30 mM.

[0010] In one embodiment, the mass concentration of the aldehyded sodium hyaluronate in the buffer solution is 4 mg / mL to 7 mg / mL.

[0011] In one embodiment, the mass concentration of the amino sodium hyaluronate in the buffer solution is 19 mg / mL to 22 mg / mL.

[0012] The present invention also provides a method for preparing vitreous gel, comprising the following steps:

[0013] Preparation of aldehyde-modified sodium hyaluronate: mixing sodium hyaluronate solution with sodium periodate solution, reacting for 4 to 8 hours, adding ethylene glycol to terminate the reaction, ultrafiltration and freeze-drying to obtain aldehyde-modified sodium hyaluronate;

[0014] Preparation of aminated sodium hyaluronate: mixing a diamino compound solution with sodium hyaluronate, adding a catalyst, reacting for 1 to 12 hours, adding a NaOH solution to terminate the reaction, ultrafiltration and freeze-drying to obtain aminated sodium hyaluronate;

[0015] Preparation of vitreous gel: Mixing aldehyde-modified sodium hyaluronate, amino-modified sodium hyaluronate and buffer to obtain vitreous gel.

[0016] In one embodiment, in the step of preparing aldehyde-modified sodium hyaluronate, the molecular weight of sodium hyaluronate in the sodium hyaluronate solution is 300-700 kDa; and / or,

[0017] After mixing, the concentration ratio of sodium periodate to sodium hyaluronate is (1.3-1.6):1; and / or,

[0018] The reaction temperature after mixing is 45-55°C.

[0019] In one embodiment, in the step of preparing amino sodium hyaluronate, the diamino compound includes hexamethylenediamine adipate, and the catalyst includes DMTMM.

[0020] In one embodiment, in the step of preparing amino sodium hyaluronate, the molecular weight of sodium hyaluronate in the sodium hyaluronate solution is 300-500 kDa; and / or,

[0021] After mixing, the concentration ratio of hexamethylenediamine adipate to sodium hyaluronate is (2-20):1; and / or,

[0022] The reaction temperature after mixing is 55-65°C.

[0023] In one embodiment, in the step of preparing amino sodium hyaluronate, the mass concentration of NaOH in the reaction solution is 3-5 mg / mL; and / or,

[0024] The termination reaction time is 10 min to 60 min.

[0025] The present invention also provides a use of vitreous gel in an intraocular filler, wherein the vitreous gel includes the vitreous gel as described above, or the vitreous gel prepared by the vitreous gel preparation method as described above.

[0026] The technical solution of the present invention adjusts the ring-opening rate of aldehyded sodium hyaluronate and the grafting rate of amination sodium hyaluronate, and adjusts the mass ratio of aldehyded sodium hyaluronate to amination sodium hyaluronate to (4-7): (76-88), so that the molar ratio of aldehyde group to amino group when mixed is less than 1. In some embodiments of the present invention, the molar ratio of aldehyde group to amino group is 0.15-0.79, which reduces the proportion of amino group, thereby reducing the potential damage that aldehyde group may cause to intraocular tissue and accelerating the healing speed of intraocular tissue. By using aldehyded sodium hyaluronate with a molecular weight of 10-24 kDa and amination sodium hyaluronate with a molecular weight of 150-550 kDa, the re-dissolution time of aldehyded sodium hyaluronate and amination sodium hyaluronate can be shortened to meet the needs of clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is an intraocular pressure-time line graph of Example 1 and its control group in the present invention;

[0029] Figure 2 This is an optical microscope image of Example 1 and its control group after surgery in the present invention;

[0030] Figure 3 The optical coherence tomography images of Example 1 and its control group after surgery in the present invention;

[0031] Figure 4 The graphs show the effects of Example 1 and Comparative Example 1 of the present invention on the back skin of New Zealand white rabbits at different times.

[0032] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0034] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0035] Currently, the vitreous filling materials used in clinical practice mainly include gas, silicone oil, and perfluorocarbon liquid. These fillers are suitable for vitreous filling in different surgical situations, but there are still many complications. For example, the gas filling time is short and may cause lens opacity, perfluorocarbon liquid has high toxicity, and the commonly used silicone oil filling is prone to emulsification and has certain toxicity to eye tissues, often causing complications such as glaucoma and cataracts. In particular, increased intraocular pressure after vitreous filling is very common in clinical practice. Severe high intraocular pressure can cause ischemia and lead to vision loss, and long-term chronic persistent high intraocular pressure may also damage the optic nerve.

[0036] In view of this, the present invention proposes a vitreous gel, the raw materials of which include aldehyded sodium hyaluronate, amino sodium hyaluronate and buffer solution; the ring opening rate of the aldehyded sodium hyaluronate is 40% to 60%, and the molecular weight is 10 to 24 kDa; the grafting rate of the amino sodium hyaluronate is 14% to 24%, and the molecular weight is 150 to 550 kDa; the mass ratio of the aldehyded sodium hyaluronate to the amino sodium hyaluronate is (4 to 7): (76 to 88).

[0037] The technical solution of the present invention adjusts the ring-opening rate of aldehyded sodium hyaluronate and the grafting rate of amination sodium hyaluronate, and adjusts the mass ratio of aldehyded sodium hyaluronate to amination sodium hyaluronate to (4-7): (76-88), so that the molar ratio of aldehyde group to amino group when mixed is less than 1. In some embodiments of the present invention, the molar ratio of aldehyde group to amino group is 0.15-0.79, which reduces the proportion of amino group, thereby reducing the potential damage that aldehyde group may cause to intraocular tissue and accelerating the healing speed of intraocular tissue. By using aldehyded sodium hyaluronate with a molecular weight of 10-24 kDa and amination sodium hyaluronate with a molecular weight of 150-550 kDa, the re-dissolution time of aldehyded sodium hyaluronate and amination sodium hyaluronate can be shortened to meet the needs of clinical applications.

[0038] Compared with traditional intraocular fillers (air, expansive gas, silicone oil and heavy silicone oil), the vitreous gel provided by the present invention uses higher surface tension and expansion pressure to press the detached retina to reposition. The water content of the vitreous gel itself is as high as 98%, and the transmittance (>93% in the wavelength range of 400-700nm) and refractive index (1.3357-1.3370) are almost the same as those of natural vitreous, which can quickly restore vision after surgery; the high water content prevents the exchange of nutrients in the vitreous cavity from being disturbed, resulting in faster postoperative recovery and fewer complications. The vitreous gel is mainly composed of modified derivatives of sodium hyaluronate, which is one of the main components of natural vitreous. It has excellent biocompatibility in the eye and has no effect on the cornea, lens, ciliary body, and retina, effectively reducing the occurrence of complications such as postoperative cataracts, glaucoma, and retinal toxicity.

[0039] Compared with patent CN110652610B, the mixing ratio is adjusted from 1:1 to 1:4. In order to reduce the potential damage that aldehyde groups may cause to intraocular tissues, the ring opening rate of aldehyded sodium hyaluronate and the grafting rate of amino sodium hyaluronate are adjusted so that the molar ratio of aldehyde groups to amino groups when mixed is less than 1. The molar ratio of the corresponding functional groups (-CHO:-NH2) in patent CN110652610B is 0.08 to 4.6, and in the present invention it is 0.14 to 0.79.

[0040] The order of feeding is adjusted in the preparation method of the second component. In patent CN110652610B, the sodium hyaluronate solution is first activated, and then the diamino compound is added for amination reaction. In the present invention, the diamino compound is first dissolved, then the sodium hyaluronate solution is added, and finally the amination reaction is catalyzed by an activator. Such an adjustment allows the amino group on the diamino compound and the carboxyl group on the sodium hyaluronate to be fully attracted by electrostatic action, reducing the physical distance, and improving the reaction efficiency during activation. In the order of feeding in patent CN110652610B, before the diamino compound is added, the sodium hyaluronate itself is prone to self-crosslinking, and its hydroxyl and carboxyl groups have a certain probability of forming ester bonds under the action of the activator.

[0041] In one embodiment, the buffer is a phosphate buffer, and the phosphate concentration ranges from 4 mM to 30 mM.

[0042] The technical solution of the present invention adopts phosphate buffer to maintain the pH value of the solution stable, and sets the phosphate ion concentration between 4mM and 30mM, which can effectively control the pH change in the reaction system and stabilize the pH value between 6 and 9, thereby ensuring the structural stability of the aldehyded sodium hyaluronate and the amino sodium hyaluronate in the vitreous gel, so that the vitreous gel can maintain a relatively stable environment during use, ensuring the rapid healing and smoothing of the retina and pigmented cortex.

[0043] In one embodiment, the mass concentration of the aldehyded sodium hyaluronate in the buffer solution is 4 mg / mL to 7 mg / mL.

[0044] The technical solution of the present invention can ensure that the aldehyded sodium hyaluronate has good solubility in the buffer solution by setting the mass concentration of the aldehyded sodium hyaluronate between 4 mg / mL and 7 mg / mL, avoiding precipitation or aggregation caused by excessively high concentration, thereby ensuring the uniformity and stability of the solution; within this concentration range, the aldehyded sodium hyaluronate can maintain its original biocompatibility, and for cell culture, drug carriers or tissue engineering materials in biomedical applications, such a concentration can better support cell growth and tissue repair.

[0045] In one embodiment, the mass concentration of the amino sodium hyaluronate in the buffer solution is 19 mg / mL to 22 mg / mL.

[0046] The technical solution of the present invention can form a stable solution in a buffer solution by adjusting the mass concentration of amino sodium hyaluronate to 19 mg / mL to 22 mg / mL. This stability helps to maintain the structural integrity and functional properties of sodium hyaluronate and reduce performance changes caused by concentration fluctuations. Since sodium hyaluronate itself has good biocompatibility and moisturizing properties, the amino modification enhances its binding ability to cell surface receptors, thereby improving its biological activity in cell culture, tissue engineering and drug delivery systems.

[0047] The present invention also proposes a method for preparing vitreous gel, comprising the following steps: preparing aldehyde-modified sodium hyaluronate: mixing a sodium hyaluronate solution with a sodium periodate solution, reacting for 4 to 8 hours, adding ethylene glycol to terminate the reaction, ultrafiltration and freeze-drying to obtain aldehyde-modified sodium hyaluronate; preparing aminated sodium hyaluronate: mixing a diamino compound solution with sodium hyaluronate, adding a catalyst, reacting for 1 to 12 hours, adding a NaOH solution to terminate the reaction, ultrafiltration and freeze-drying to obtain aminated sodium hyaluronate; preparing vitreous gel: mixing aldehyde-modified sodium hyaluronate, aminated sodium hyaluronate and a buffer solution to obtain vitreous gel.

[0048] The technical solution of the present invention uses sodium periodate to perform oxidation treatment on sodium hyaluronate to generate aldehyded sodium hyaluronate. This process is simple and easy to control. The reaction time of 4 to 8 hours is sufficient to fully introduce aldehyde groups, thereby ensuring the chemical stability and reactivity of the product. Amination reaction is performed on sodium hyaluronate by using a diamino compound in the presence of a catalyst to introduce amino groups into the sodium hyaluronate molecular chain. This modification not only increases the functional diversity of sodium hyaluronate, but also improves its reactivity with other biological molecules. Aldehydated sodium hyaluronate and amino sodium hyaluronate are mixed in a buffer solution to form a cross-linked network through the Schiff base reaction between the aldehyde group and the amino group, thereby preparing a vitreous gel with a three-dimensional structure. This method can accurately control the cross-linking density of the gel, thereby adjusting its physical and mechanical properties. Aldehydated and amino sodium hyaluronate treated by ultrafiltration and freeze-drying has high purity and stability and is not easily affected by the external environment, so that the prepared vitreous gel can still maintain its performance during long-term storage and transportation.

[0049] In one embodiment, in the step of preparing aldehyde-modified sodium hyaluronate, the molecular weight of sodium hyaluronate in the sodium hyaluronate solution is 300-700 kDa; and / or, after mixing, the concentration ratio of sodium periodate to sodium hyaluronate is (1.3-1.6):1; and / or, the reaction temperature after mixing is 45-55°C.

[0050] The technical solution of the present invention can ensure that the obtained aldehyde-modified sodium hyaluronate has the expected physical and chemical properties by selecting sodium hyaluronate with a molecular weight in the range of 300kDa to 700kDa. Sodium hyaluronate in this molecular weight range usually has good solubility, biocompatibility and appropriate viscoelasticity, and is suitable for preparing high-performance biomedical materials; by adjusting the concentration ratio of sodium periodate to sodium hyaluronate, the degree of oxidation reaction can be effectively controlled to ensure that an appropriate amount of aldehyde groups are introduced into the sodium hyaluronate molecule without excessive degradation, which can ensure the reaction activity of the product and maintain its structural integrity; controlling the reaction temperature between 45°C and 55°C can accelerate the process of oxidation reaction, shorten the reaction time, and prevent side reactions caused by excessive temperature or thermal degradation of sodium hyaluronate, which helps to balance the relationship between reaction rate and product quality, thereby improving the overall efficiency of the reaction.

[0051] In one embodiment, in the step of preparing amino sodium hyaluronate, the diamino compound includes hexamethylenediamine adipate, and the catalyst includes DMTMM.

[0052] The technical solution of the present invention adopts hexamethylenediamine adipate as a diamino compound, which can react with the hydroxyl group in the sodium hyaluronate molecule to form a stable covalent bond connection, thereby achieving efficient amination; adopts DMTMM (4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride) as a catalyst, which can reduce the reaction activation energy, accelerate the amination reaction rate, make the reaction proceed under mild conditions, and reduce the occurrence of side reactions; by using hexamethylenediamine adipate and DMTMM for amination reaction, a sodium hyaluronate derivative with uniform amination degree and stable structure can be obtained.

[0053] In one embodiment, in the step of preparing amino sodium hyaluronate, the molecular weight of sodium hyaluronate in the sodium hyaluronate solution is 300-500 kDa; and / or, after mixing, the concentration ratio of hexamethylenediamine adipate to sodium hyaluronate is (2-20):1; and / or, the reaction temperature after mixing is 55-65°C.

[0054] The technical solution of the present invention can ensure that the prepared amino sodium hyaluronate has suitable physical and chemical properties, good solubility and biocompatibility by selecting sodium hyaluronate with a molecular weight in the range of 300 kDa to 500 kDa, and can provide sufficient reaction sites to facilitate subsequent amino modification; the concentration ratio of hexamethylenediamine adipate to sodium hyaluronate is set between (2 to 20): 1, which can effectively promote the amination reaction, ensure the introduction of an appropriate amount of amino groups on the sodium hyaluronate molecules, improve the selectivity and conversion rate of the amination reaction, and avoid unnecessary side reactions caused by excessive hexamethylenediamine adipate; the reaction temperature is controlled between 55° C. and 65° C., which can promote the chemical reaction between hexamethylenediamine adipate and sodium hyaluronate, accelerate the reaction process, shorten the reaction time, and maintain the structural integrity of the product.

[0055] In one embodiment, in the step of preparing amino sodium hyaluronate, the mass concentration of NaOH in the reaction solution is 3 to 5 mg / mL; and / or the reaction termination time is 10 min to 60 min.

[0056] The technical solution of the present invention can effectively adjust the pH value of the reaction system by setting the mass concentration of NaOH in the reaction solution to 3-5 mg / mL, thereby controlling the speed and direction of the amination reaction, inhibiting the coupling reaction between sodium hyaluronate and the diamino compound, and ensuring that the degree of amination of the product is uniform and controllable; the termination time of the reaction is set between 10 and 60 minutes, which can effectively prevent overreaction and avoid the generation of by-products.

[0057] The present invention also provides a use of vitreous gel in an intraocular filler, wherein the vitreous gel includes the vitreous gel as described above, or the vitreous gel prepared by the vitreous gel preparation method as described above.

[0058] Since the application of the vitreous gel in intraocular filling material adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0059] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0060] Example 1

[0061] This embodiment provides a vitreous gel, the preparation method of which comprises the following steps:

[0062] Preparation of aldehyde-modified sodium hyaluronate:

[0063] 1. Weigh 4.0g sodium hyaluronate and dissolve it in 370.0g injection water until it is completely dissolved at room temperature;

[0064] 2. Weigh 3.5g sodium periodate and dissolve it in 34.0g water for injection. Dissolve completely at room temperature.

[0065] 3. Heat the sodium hyaluronate solution in 1 to 45-51°C, add 3.1 g of sodium periodate solution (in 2), stir and react for 6-8 hours in the dark;

[0066] 4. Add 3.7 g of ethylene glycol to the reaction system in step 3 to terminate the reaction;

[0067] 5. Ultrafiltration of the solution in 4 was performed using a hollow fiber column (molecular weight cut-off 10 kDa) to remove small molecules and obtain a purified aldehyded sodium hyaluronic acid solution;

[0068] 6. Freeze-dry the aldehyde-modified sodium hyaluronate solution in 5 to obtain aldehyde-modified sodium hyaluronate.

[0069] Preparation of Amination Sodium Hyalate:

[0070] 1. Weigh 5.7g of adipic acid dihydrazide and dissolve it in 588.0g of injection water until it dissolves completely at room temperature;

[0071] 2. Add 2.4g sodium hyaluronate powder into 1 and dissolve completely at room temperature;

[0072] 3. Weigh 2.1g DMTMM and dissolve it in 14.1g injection water. Dissolve completely at room temperature.

[0073] 4. Heat the adipic acid dihydrazide / sodium hyaluronate solution in 2 to 59-65°C, add 13.8 g of DMTMM solution (in 3) and stir to react for 2-4 hours;

[0074] 5. Weigh 2.88g NaOH and dissolve it in 14.4g injection water. Add 13.80g NaOH solution into the reaction system in 4 and continue the reaction for 20-40min.

[0075] 6. Weigh 10.0g NaH2PO4·H2O and dissolve it in 36.0g injection water. Take 41.7g NaH2PO4 solution and add it to 5. Stop heating and continue stirring for 5-10min.

[0076] 7. Ultrafiltration of the solution in step 6 was performed using a hollow fiber column (molecular weight cut-off 80 kDa) to remove small molecules and obtain a purified amino-modified sodium hyaluronic acid solution;

[0077] 8. Freeze-dry the aminated sodium hyaluronate solution in 7 to obtain aminated sodium hyaluronate.

[0078] Reconstitution:

[0079] 1. Weigh 70 mg of aldehyded sodium hyaluronate and dissolve it in 10 mL of phosphate buffer (5 mM) to obtain solution A;

[0080] 2. Weigh 190 mg of amino sodium hyaluronate and dissolve it in 10 mL of phosphate buffer (5 mM) to obtain solution B.

[0081] use:

[0082] Draw 2 mL of solution A and 8 mL of solution B, mix them through a mixing head or a double mixing bag to form a gel.

[0083] Comparative Example 1

[0084] This comparative example provides a vitreous gel, and its preparation method is similar to that of Example 1, except for the parameters in the preparation process of the aldehyde- and amino-modified sodium hyaluronate.

[0085] Preparation of aldehyde-modified sodium hyaluronate:

[0086] 1. Weigh 4.0g sodium hyaluronate and dissolve it in 370.0g injection water until it is completely dissolved at room temperature;

[0087] 2. Weigh 3.5g sodium periodate and dissolve it in 34.0g water for injection. Dissolve completely at room temperature.

[0088] 3. Heat the sodium hyaluronate solution in 1 to 40-44°C, add 3.1 g of sodium periodate solution (in 2), stir and react for 1-2 hours in the dark;

[0089] 4. Add 3.7 g of ethylene glycol to the reaction system in step 3 to terminate the reaction;

[0090] 5. Ultrafiltration of the solution in 4 was performed using a hollow fiber column (molecular weight cut-off 10 kDa) to remove small molecules and obtain a purified aldehyded sodium hyaluronic acid solution;

[0091] 6. Freeze-dry the aldehyde-modified sodium hyaluronate solution in 5 to obtain aldehyde-modified sodium hyaluronate.

[0092] Preparation of Amination Sodium Hyalate:

[0093] 1. Weigh 5.7g of adipic acid dihydrazide and dissolve it in 588.0g of injection water until it dissolves completely at room temperature;

[0094] 2. Add 2.4g sodium hyaluronate powder into 1 and dissolve completely at room temperature;

[0095] 3. Weigh 2.1g DMTMM and dissolve it in 14.1g injection water. Dissolve completely at room temperature.

[0096] 4. Heat the adipic acid dihydrazide / sodium hyaluronate solution in 2 to 65-70°C, add 13.8 g of DMTMM solution (in 3) and stir to react for 2-4 hours;

[0097] 5. Weigh 2.88g NaOH and dissolve it in 14.4g injection water. Add 13.80g NaOH solution into the reaction system in 4 and continue the reaction for 80-100min.

[0098] 6. Weigh 10.0g NaH2PO4·H2O and dissolve it in 36.0g injection water. Take 41.7g NaH2PO4 solution and add it to 5. Stop heating and continue stirring for 5-10min.

[0099] 7. Ultrafiltration of the solution in step 6 was performed using a hollow fiber column (molecular weight cut-off 80 kDa) to remove small molecules and obtain a purified amino-modified sodium hyaluronic acid solution;

[0100] 8. Freeze-dry the aminated sodium hyaluronate solution in 7 to obtain aminated sodium hyaluronate.

[0101] Reconstitution:

[0102] 1. Weigh 70 mg of aldehyded sodium hyaluronate and dissolve it in 10 mL of phosphate buffer (5 mM) to obtain solution A;

[0103] 2. Weigh 70 mg of amino sodium hyaluronate and dissolve it in 10 mL of phosphate buffer (5 mM) to obtain solution B.

[0104] use:

[0105] Draw 5 mL of solution A and 5 mL of solution B, mix them through a mixing head or a double mixing bag to form a gel.

[0106] Performance Testing

[0107] Eighteen adult blue rabbits of both sexes were divided into three groups, each with 6 rabbits, namely, a hydrogel group, a silicone oil group, and a BSS (balanced salt solution) group. Vitrectomy was performed on the right eye of each rabbit to create a rhegmatogenous hole in the retina. Then, sterile air was filled to flatten the hole, and then two circles of laser were used around the flattened hole. Afterwards, the air in the vitreous cavity was replaced with the hydrogel, silicone oil, and BSS in Example 1, respectively.

[0108] Measure intraocular pressure before surgery and 1, 3, 7, 14, and 30 days after surgery. Figure 1 .Depend on Figure 1 It can be found that no high intraocular pressure was observed in each group after surgery. The intraocular pressure of each group was lower than normal in the first 3 days after surgery and returned to normal on the 7th day. The low intraocular pressure in the first 3 days after surgery may be due to the fact that the sclera of rabbits is thinner than that of humans, and there is still some leakage of ocular contents before the ocular surface wound is completely healed after suturing.

[0109] Fundus photography was performed before surgery and 7, 14, and 30 days after surgery to observe the transparency of the vitreous cavity and fundus conditions. Figure 2 .Depend on Figure 2 It can be seen that the retina in the BSS group detached again, while the retina in the hydrogel group and the silicone oil group was well reattached; compared with the silicone oil group, the color of the reattached retina in the hydrogel group was closer to the surrounding area, which may be due to the fact that hydrogel has better biocompatibility than silicone oil and is more conducive to the exchange of intraocular substances.

[0110] Optical coherence tomography (OCT) was performed before surgery and 14 and 30 days after surgery. Figure 3 .Depend on Figure 3 It can be found that the OCT results showed that in the BSS group, the retinal neuroepithelium and pigment epithelium were separated at the hole, while in the hydrogel group and silicone oil group, the holes were closed, the retina was flat, the retina was thinned at the hole, and the rest of the parts were normal.

[0111] Verification that reducing the aldehyde content in the gel can reduce the potential damage that the gel may cause to intraocular tissues:

[0112] The aldehyde content of the aldehyde-modified sodium hyaluronate provided in Example 1 and Example 2 is 2700 μmol / g and 980 μmol / g, respectively, and the amino content of the aminated sodium hyaluronate provided in Example 1 and Example 2 is 390 μmol / g and 870 μmol / g, respectively.

[0113] Two gels were prepared using the aldehyded sodium hyaluronate and the amino sodium hyaluronate provided in Example 1 and Example 2, respectively, and injected subcutaneously into the back of New Zealand white rabbits:

[0114] Example 1: The aldehyde-modified sodium hyaluronate solution was re-dissolved in phosphate buffer (5 mM) to a concentration of 7 mg / mL to obtain solution A1. The amino-modified sodium hyaluronate 1 solution was re-dissolved in phosphate buffer (5 mM) to a concentration of 19 mg / mL to obtain solution B1. Solution A1 and solution B1 were mixed in a volume ratio of 1:4 and injected into the subcutaneous tissue of the back of New Zealand white rabbits to form gel 1. The aldehyde concentration in the gel was 3.78 μmol / mL, the amino concentration was 5.93 μmol / mL, and the aldehyde group:amino group = 0.64:1 (molar ratio).

[0115] Comparative Example 1: The aldehyde-modified sodium hyaluronate solution 2 was re-dissolved in phosphate buffer (5mM) to a concentration of 7mg / mL to obtain solution A2. The amino-modified sodium hyaluronate solution 2 was re-dissolved in phosphate buffer (5mM) to a concentration of 7mg / mL to obtain solution B2. Solution A2 and solution B2 were mixed in a volume ratio of 1:1 and injected into the subcutaneous tissue of the back of New Zealand white rabbits to form gel 2. The aldehyde concentration in the gel was 3.43μmol / mL, the amino concentration was 3.05μmol / mL, and the aldehyde group:amino group = 1.13:1 (molar ratio).

[0116] The color was scored from light to dark (1 to 5). The lighter the color (the lower the score), the milder the inflammatory reaction of the gel under the skin. The observation period was 24h, 48h and 72h. Each New Zealand white rabbit was injected with 12 points on the back (gel 1 and gel 2 were arranged symmetrically on the left and right, 6 points each). A total of 3 New Zealand white rabbits were used. The average score of each group of gels at each time point was recorded, as shown in Table 1. The test results are shown in Table 1. Figure 4 shown.

[0117] Table 1 Evaluation of the effect of vitreous gel on inflammation of the back skin of New Zealand white rabbits

[0118] 24h 48h 72h Example 1 1 1 1 Comparative Example 1 2 2 2

[0119] According to Table 1 and Figure 4 It can be seen that the inflammatory response of the vitreous gel provided in Example 1 at 24 hours, 48 ​​hours and 72 hours is relatively mild, while the inflammatory response of the vitreous gel provided in Comparative Example 1 at 24 hours, 48 ​​hours and 72 hours is relatively obvious, indicating that the subcutaneous inflammatory response of the vitreous gel provided in Example 1 is relatively mild, the biocompatibility of the vitreous gel is good, and the damage to the intraocular tissue is relatively small.

[0120] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A vitreous gel, characterized in that: The raw materials of the vitreous gel include aldehyde-modified sodium hyaluronate, amino-modified sodium hyaluronate and buffer solution; The ring opening rate of the aldehyded sodium hyaluronate is 40% to 60%, and the molecular weight is 10 to 24 kDa; The grafting rate of the amino sodium hyaluronate is 14% to 24%, and the molecular weight is 150 to 550 kDa; The mass ratio of the aldehyded sodium hyaluronate to the amino sodium hyaluronate is (4-7):(76-88).

2. The vitreous gel according to claim 1, characterized in that The buffer is a phosphate buffer with a phosphate concentration ranging from 4 mM to 30 mM.

3. The vitreous gel according to claim 1, characterized in that The mass concentration of the aldehyded sodium hyaluronate in the buffer solution is 4 mg / mL to 7 mg / mL.

4. The vitreous gel according to claim 1, characterized in that The mass concentration of the amino sodium hyaluronate in the buffer solution is 19 mg / mL to 22 mg / mL.

5. A method for preparing vitreous gel, characterized in that: The following steps are involved: Preparation of aldehyde-modified sodium hyaluronate: mixing sodium hyaluronate solution with sodium periodate solution, reacting for 4 to 8 hours, adding ethylene glycol to terminate the reaction, ultrafiltration and freeze-drying to obtain aldehyde-modified sodium hyaluronate; Preparation of aminated sodium hyaluronate: mixing a diamino compound solution with sodium hyaluronate, adding a catalyst, reacting for 1 to 12 hours, adding a NaOH solution to terminate the reaction, ultrafiltration and freeze-drying to obtain aminated sodium hyaluronate; Preparation of vitreous gel: Mixing aldehyde-modified sodium hyaluronate, amino-modified sodium hyaluronate and buffer to obtain vitreous gel.

6. The method for preparing vitreous gel according to claim 5, characterized in that: In the step of preparing aldehyde-modified sodium hyaluronate, the molecular weight of sodium hyaluronate in the sodium hyaluronate solution is 300 to 700 kDa; and / or, After mixing, the concentration ratio of sodium periodate to sodium hyaluronate is (1.3-1.6):1; and / or, The reaction temperature after mixing is 45-55°C.

7. The method for preparing vitreous gel according to claim 5, characterized in that: In the step of preparing aminated sodium hyaluronate, the diamino compound includes hexamethylenediamine adipate, and the catalyst includes DMTMM.

8. The method for preparing vitreous gel according to claim 7, characterized in that: In the step of preparing the aminated sodium hyaluronate, the molecular weight of the sodium hyaluronate in the sodium hyaluronate solution is 300 to 500 kDa; and / or, After mixing, the concentration ratio of hexamethylenediamine adipate to sodium hyaluronate is (2-20):1; and / or, The reaction temperature after mixing is 55-65°C.

9. The method for preparing vitreous gel according to claim 5, characterized in that: In the step of preparing aminated sodium hyaluronate, the mass concentration of NaOH in the reaction solution is 3 to 5 mg / mL; and / or, The termination reaction time is 10 min to 60 min.

10. A use of vitreous gel as an intraocular filling material, characterized in that: The vitreous gel includes the vitreous gel according to any one of claims 1 to 4, or the vitreous gel prepared by the vitreous gel preparation method according to any one of claims 5 to 9.

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