WO2019210496A1 - 一种注射用水凝胶的制备方法及其应用 - Google Patents

一种注射用水凝胶的制备方法及其应用 Download PDF

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WO2019210496A1
WO2019210496A1 PCT/CN2018/085546 CN2018085546W WO2019210496A1 WO 2019210496 A1 WO2019210496 A1 WO 2019210496A1 CN 2018085546 W CN2018085546 W CN 2018085546W WO 2019210496 A1 WO2019210496 A1 WO 2019210496A1
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hydrogel
group
crosslinking
polysaccharide
preparation
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PCT/CN2018/085546
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French (fr)
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魏长征
曹彬
吴袆
王晓彤
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上海其胜生物制剂有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • the invention relates to the field of joint cavity viscoelastic supplement, in particular to a preparation method of injection hydrogel and application thereof.
  • Osteoarthritis is a very common clinical disease with an overall incidence of about 15.6%, and in a population over 60 years old, the proportion is over 50%. Osteoarthritis can cause pain and movement disorders, severely reducing the quality of life. Synovial fluid acts as a lubricant in the joints, which reduces the friction of the articular cartilage during exercise. As the age increases, the quality of the joint fluid becomes worse and worse, leading to degeneration of the cartilage, which in turn causes osteoarthritis. In order to restore and delay the progression of joint wear, a common and effective method is to add new "lubricants" to the joint cavity, especially for patients with mild to moderate osteoarthritis and young people.
  • Hyaluronic acid is the main component of synovial fluid and an ideal viscous supplement. It has been proven by many research literatures and clinical practice applications for decades. Although the injection of these hyaluronic acid products can relieve pain and restore the mobility of patients with osteoarthritis, hyaluronic acid is rapidly degraded by hyaluronidase after injection, and this effect can only last for a while. In order to maintain the lubrication for a certain period of time, patients with osteoarthritis usually inject 3-5 times per course, which increases the medical expenses, and also brings the risk of inconvenience and infection.
  • Chitosan is a chitin-deacetylated polysaccharide with a molecular structure and lubricity similar to hyaluronic acid. Since there is no specific enzyme in the human body to degrade chitosan, its half-life in vivo is much longer than that of hyaluronic acid. In addition to having a long maintenance time, chitosan also has antibacterial and hemostatic properties, and inhibits the proliferation of fibroblasts compared with hyaluronic acid. Chitosan has been shown to support the expression of extracellular matrix proteins by human chondrocytes and to reduce the production of inflammatory mediators. Therefore, chitosan is a very promising candidate for viscoelastic supplements.
  • chitosan is a semi-rigid polymer with a large number of internal/external hydrogen bonds, so it cannot be directly dissolved in pure water.
  • an acidic solution such as a 1% acetic acid solution is used to dissolve chitosan, and since the pH of these acidic solutions differs from the physiological pH, the chitosan solution cannot be directly used in the body.
  • One way to solve this problem is to introduce a carboxymethyl group into the chitosan backbone to destroy the semi-rigid structure.
  • the obtained carboxymethyl chitosan is soluble in pure water and has practical value as a viscoelastic supplement.
  • Carboxymethyl chitosan can significantly inhibit the mRNA expression of MMP-1 and MMP-3 in osteoarthritic cartilage and reduce the severity of cartilage degeneration.
  • Bai injected carboxymethyl chitosan and hyaluronic acid into the joint cavity of osteoarthritis patients respectively. The results showed that both carboxymethyl chitosan and hyaluronic acid can relieve pain and stiffness, but carboxymethyl chitosan The number of injections is less than hyaluronic acid.
  • Carboxymethyl chitosan can protect cartilage by inhibiting the apoptosis of chondrocytes by lowering the levels of nitric oxide and reactive oxygen species.
  • Hydrogels are a network of macromolecules that contain large amounts of water, and high shear rates can damage the hydrogel network.
  • minimally invasive therapies are recommended in many areas such as knee stick viscoelastic supplements. Therefore, it is equally important that the carboxymethyl chitosan hydrogel is injectable.
  • the present invention aims to provide a hydrogel having both thermal stability and injectability and its use.
  • a method of preparing a hydrogel comprising the steps of:
  • a catalyst is used in the pre-crosslinking.
  • the mass ratio of the polysaccharide to the catalyst is from 1:1 to 10:1;
  • the catalyst is selected from the group consisting of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) or 4-(4, 6-Dimethoxy-1,3,5-triazin-2-yl)triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).
  • EDC/NHS 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide
  • DTMM 4-(4, 6-Dimethoxy-1,3,5-triazin-2-yl)triazin-2-yl)-4-methylmorpholinium chloride
  • the crosslinking agent is a substance having two or more functional groups reactive with a carboxyl group and/or a hydroxyl group in the polysaccharide; the molar ratio of the carboxyl group on the polysaccharide to the functional group (amino group) of the crosslinking agent is 2 More preferably, the molar ratio of the hydroxyl group on the polysaccharide to the functional group of the crosslinking agent is from 1:1 to 30:1.
  • one of the crosslinking agent molecules may be reacted with at least two carboxyl groups or two hydroxyl groups or one carboxyl group and one hydroxyl group on the polysaccharide.
  • the pre-crosslinking reaction time is 2-96 hours, and the reaction temperature is 0-50 ° C; more preferably 0-37 ° C.
  • the ratio of addition of the polysaccharide at the start of the pre-crosslinking reaction is from 1% (w/v) to 6% (w/v).
  • the polysaccharide is selected from the group consisting of carboxymethyl chitosan, hyaluronic acid or a derivative thereof, cellulose, starch, dextran, asparagine, arginine, chondroitin sulfate, sulfuric acid skin , keratin sulfate, heparin or a derivative thereof;
  • the carboxymethyl chitosan has a molecular weight of between 100 kDa and 800 kDa, the carboxymethyl group has a DS of not less than 1.0, and the acetamido group has a DD of not more than 20%; and the hyaluronan derivative is selected from the group consisting of Sodium hyaluronate or potassium hyaluronate having a molecular weight of between 100 kDa and 3000 kDa.
  • the crosslinking agent is selected from one or more of the following: oxalyl dioxime, cystamine dihydrochloride, 1,3-diamino-2-propanol, 1, 4-diamino-2-butanone dihydrochloride, 1,11-diamino-3,6,9-trioxadecane, diamino polyethylene glycol, lysine, glutamine, day Asparagine, arginine, divinyl sulfone, 1,4-butanediol diglycidyl ether, divinyl sulfone (DVS) and 1,4-butanediol diglycidyl ether (BDDE).
  • oxalyl dioxime cystamine dihydrochloride
  • 1,3-diamino-2-propanol 1, 4-diamino-2-butanone dihydrochloride
  • 1,11-diamino-3,6,9-trioxadecane diamino poly
  • the alkaline solution is a water-soluble alkaline solution having a pH of 9.0-13.5; more preferably, the alkaline solution is an aqueous solution of NaOH or KOH.
  • the volume ratio of the crosslinking system to the pre-crosslinked system is between 2:1 and 4:1.
  • reaction time for the further crosslinking is from 2 to 24 hours, and the reaction temperature is from 0 to 50 °C.
  • the volume ratio of the organic solvent to the crosslinking system is not less than 3:1.
  • the organic solvent is a solvent that is soluble in water but insoluble in the polysaccharide; more preferably, the organic solvent is selected from the group consisting of C1-3 alcohols, acetone or chloroform; most preferably methanol or ethanol.
  • the buffer solution is selected from the group consisting of a phosphate buffer solution or a 0.9% NaCl solution.
  • the precipitate or lyophilizate is dissolved in the buffer solution at a dissolved concentration between 1% and 4% (w/v).
  • the method further includes the steps of:
  • the dialysis has a molecular weight cut off (MWCO) of not less than 6,000.
  • the dialysate used for dialysis is the same as the buffer solution in step (4); the volume ratio of the dialysate to the hydrogel is not less than 8:1.
  • the method further includes the steps of:
  • the autoclaving temperature is 120-125 ° C for 10-30 minutes.
  • a hydrogel comprising a polysaccharide and a crosslinking agent; wherein the total weight of the hydrogel comprises from 1.0 to 2.0% by weight of the polysaccharide.
  • the crosslinking agent is a substance having two or more functional groups reactive with a carboxyl group and/or a hydroxyl group in the polysaccharide.
  • the polysaccharide is selected from the group consisting of carboxymethyl chitosan, sodium hyaluronate or potassium hyaluronate; the molecular weight of the carboxymethyl chitosan is between 100 kDa and 800 kDa, carboxymethyl
  • the DS of not less than 1.0, the DD of the acetamide group is not more than 20%; the molecular weight of the sodium hyaluronate or potassium hyaluronate is between 100 kDa and 3000 kDa.
  • the crosslinking agent is selected from one or more of the following: oxalyl dioxime, cystamine dihydrochloride, 1,3-diamino-2-propanol, 1, 4-diamino-2-butanone dihydrochloride, 1,11-diamino-3,6,9-trioxadecane, diamino polyethylene glycol, lysine, glutamine, day Asparagine, arginine, divinyl sulfone, 1,4-butanediol diglycidyl ether, divinyl sulfone (DVS) and 1,4-butanediol diglycidyl ether (BDDE).
  • oxalyl dioxime cystamine dihydrochloride
  • 1,3-diamino-2-propanol 1, 4-diamino-2-butanone dihydrochloride
  • 1,11-diamino-3,6,9-trioxadecane diamino poly
  • the hydrogel further contains drugs and/or cells.
  • the drug is an anti-inflammatory or anesthetic; the cell is selected from the group consisting of a chondrocyte or a mesenchymal stem cell.
  • the anti-inflammatory agent is selected from the group consisting of triamcinolone acetonide, ibuprofen and/or diclofenac; and the anesthetic is lidocaine.
  • the hydrogel has an oriented layered structure.
  • the hydrogel is injectable and thermally stable.
  • the hydrogel is obtained by the preparation method provided by the present invention as described above.
  • a hydrogel of the invention as described above, for use in the preparation of a reagent useful for viscoelastic supplementation or as a viscoelastic supplement; Used to prepare drug delivery systems, stents that can be used for tissue engineering, or soft tissues that can be filled.
  • the viscoelastic supplement comprises a hydrogel and an anti-inflammatory agent, an anesthetic or a cell provided by the present invention as described above;
  • the anti-inflammatory agent is selected from the group consisting of triamcinolone acetonide, ibuprofen and/or Or diclofenac);
  • the anesthetic is lidocaine;
  • the cells are selected from chondrocytes or mesenchymal stem cells.
  • the present invention provides a novel carboxymethyl chitosan hydrogel having thermal stability and injectability.
  • Figure 1 shows the storage modulus (G') and loss modulus (G") of a critical carboxymethyl chitosan hydrogel before and after autoclaving in Example 8, showing the hydrogel of the present invention in a sol and a gel. There is a critical state between them; where ⁇ denotes G' and ⁇ denotes G".
  • FIG. 2 shows the storage modulus (G') and loss modulus (G") of a carboxymethyl chitosan solution used as a viscoelastic supplement.
  • G" is always greater than G' throughout the test range. Although it can lubricate the cartilage surface, it does not absorb enough vibrational energy during joint motion compared to hydrogels.
  • ⁇ represents G' and ⁇ represents G".
  • Figure 3 is a photograph of a live/dead staining of chondrocytes cultured in a carboxymethyl chitosan hydrogel in Example 15, showing that the cross-linking agent used in the present invention can increase cell adhesion.
  • Figure 4 is a SEM micrograph of a carboxymethyl chitosan hydrogel before (A) and after alkalization (B) in Example 12, showing that the alkalization process can structure the crosslinked network from a random porous The shape changes to an oriented layer.
  • the layered structure of the hydrogel contributes to thermal stability and injectability.
  • Figure 5 is The SEM micrographs show a random porous structure. In fact, most common hydrogels have this structure.
  • Figure 6 is a photomicrograph of actin fluorescent staining of neuronal cells cultured on a carboxymethyl chitosan hydrogel in Example 19, the cell orientation being dependent on the oriented layered structure of the hydrogel.
  • Figure 7 is a graph showing the dynamic viscosity (?) of a carboxymethyl chitosan hydrogel before and after autoclaving in Example 10, indicating that the hydrogel of the present invention maintains network integrity and mechanical properties during autoclaving.
  • ⁇ means before autoclaving and ⁇ means after autoclaving.
  • Figure 8 is a section II immunohistochemical staining of type II collagen after injection of a carboxymethyl chitosan viscoelastic agent and a 0.9% NaCl solution into a rabbit arthritis model, respectively, in Example 14.
  • Panel A is a viscoelastic group and Panel B is a 0.9% NaCl solution group.
  • a polysaccharide and a cross-linking agent can be obtained by a step of crosslinking, alkalization, precipitation (or freeze-drying) and re-dissolution to obtain a hydrogel which is both injectable and thermally stable. gum.
  • injectable means that the hydrogels provided herein can maintain their mechanical properties and network integrity under conditions of high shear rate, such as when injected by a syringe.
  • thermal stability means that the hydrogels provided by the present invention retain their mechanical properties and network integrity under high temperature conditions, such as autoclaving.
  • the hydrogel provided by the present invention is a gel containing water as a dispersion medium containing a polysaccharide and a crosslinking agent; and containing 1.0 to 2.0% by weight of the polysaccharide based on the total weight of the hydrogel.
  • the microstructure of the hydrogel provided by the present invention exhibits a better ordered arrangement, that is, an oriented layered structure, which is greatly different from the random porous structure of a general hydrogel.
  • the oriented layered structure ie, the ordered array of microstructures, contributes to the injectability and thermal stability of the hydrogel.
  • the properties of the hydrogel can range from liquid to solid depending on factors such as cross-linking ratio, chemical/physical bonding properties, pH, temperature, and the like. Because soft tissue consists primarily of water, hydrogels can be easily adjusted to the nature of soft tissue, such as stiffness and elasticity. In fact, the use of common rheological terms such as dynamic viscosity ( ⁇ , resistance to deformation), storage modulus (G', reflecting the elasticity of the material) and loss modulus (G), reflecting the viscosity of the material, describe the hydraulic condensation. The nature of the glue is very convenient and practical. For viscoelastic materials, the relative size of G' and G" indicates the physical state of the material.
  • G' ⁇ G indicates that the carboxymethyl chitosan hydrogel herein is in a critical condition between a fluid material and a solid-like substance over a wide frequency range. This is unusual; it should be noted that the hydrogel provided by the present invention is still in a critical state (G' ⁇ G" even after autoclaving, which means the elasticity of the hydrogel and The viscosity is not damaged by heat.
  • hydrogels having properties similar to those of joint synovial fluid should be prioritized.
  • Synovial fluid is a pseudoplastic fluid whose apparent viscosity decreases as the shear rate increases, and the hydrogel of the present invention also has such characteristics.
  • G" is always greater than G' (as shown in Figure 2).
  • G' is always equal to or greater than G" over a wide range; by adjusting the method of the invention Specific parameters, the G' and G" of the hydrogel of the present invention can be easily adjusted. It is apparent that the ⁇ of the injectable croscarmellose hydrogel is higher than the shell at the same shear rate.
  • the glycan solution which means that the hydrogel performs better during the joint movement than the solution absorbs vibration and lubricates the joint surface.
  • the carboxymethyl chitosan used in the present invention is directly prepared from chitin, and the DD (deacetylation degree) can be controlled to be 20% or less, and the DS (carboxymethyl substitution degree) is more than 1.0.
  • the chitosan derivative used in the present invention that is, carboxymethyl chitosan, has DS ⁇ 1.0 and DD ⁇ 20%, and can be abbreviated as CCT in the present invention.
  • hydrogels in the examples are made of CCT, those skilled in the art can easily select other similar materials to prepare hydrogels having similar properties according to the technical solution of the present invention. It is within the scope of the invention.
  • other polysaccharides such as hyaluronic acid, cellulose, starch, dextran, asparagine, arginine, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin and its derivatives or others have Polymers of similar structure can also be used in accordance with the procedures described herein.
  • the CCT is selected as a representative material for a better understanding of the invention and the preparation process, but does not limit the selection of other materials.
  • the preparation method of the hydrogel provided by the invention comprises the steps of:
  • the aqueous polysaccharide solution, the crosslinking agent and the catalyst are mixed to obtain a pre-crosslinking reaction system;
  • an alkaline solution is added to the pre-crosslinking reaction system for further crosslinking to obtain a crosslinking reaction system
  • an organic solvent is added to the crosslinking reaction system to obtain a precipitated product; or the crosslinking reaction system is freeze-dried to obtain a lyophilized product;
  • the precipitated product or the lyophilized product washed with the organic solvent is dissolved in a buffer solution to obtain a hydrogel
  • the hydrogel is dialyzed against a buffer solution, filtered or autoclaved to obtain a hydrogel that can be used for injection.
  • the present invention provides a technique for preparing an injectable and thermostable carboxymethyl chitosan hydrogel comprising cross-linking, alkalization, precipitation (or freeze-drying), redissolution, filtration, and autoclaving .
  • pre-crosslinking and alkalization are the key to obtaining an oriented layered structure.
  • the hydrogel should be injectable, ie its network will not rupture after injection through the needle. Therefore, on the one hand, the cross-linking reaction ratio should not be too high, because the solid-like hydrogel cannot maintain its integrity through the syringe. On the other hand, sufficient cross-linking imparts sufficient viscoelasticity to the hydrogel to absorb vibrations and lubricate the articular surface, which means that the reaction ratio between the corresponding groups should not be too low. Moreover, proper cross-linking can extend the duration of joint viscoelastic supplementation. Since most of the functional groups in CCT are a carboxyl group and a hydroxyl group, these two functional groups are selected as the reaction sites in the present invention.
  • the molar ratio of the carboxymethyl group derived from CCT to the amino group derived from the crosslinking agent is between 2:1 and 20:1. It is worth noting that the true reaction ratio is lower than the ratios listed above, as not every amino or carboxyl group can participate in the reaction.
  • the MCT employed in the present invention is between 100 and 800 kDa. A higher Mw means that the CCT molecules have a greater length and cause more entanglement in the solution, resulting in higher viscoelastic behavior.
  • a low CCT/crosslinker ratio is required for a low Mw CCT
  • a high CCT/crosslinker ratio is required for a high Mw CCT.
  • hyaluronic acid (HA) is also an important candidate for the main components of injectable hydrogels.
  • Mw varies from 100 to 3,000 kDa. Since HA has a molecular structure similar to CCT, the conditions of CCT can be referred to for the selection of the crosslinking agent and the molar ratio of the HA/crosslinking agent.
  • the choice of crosslinking agent is relatively flexible, and in theory any substance having two or more functional groups reactive with CCT can be selected as the crosslinking agent.
  • the crosslinking agent in the first step is selected from the group consisting of oxalyl dihydrazide, cystamine dihydrochloride, 1,3-diamino-2-propanol, and 1,4- Diamino-2-butanone dihydrochloride, diamino polyethylene glycol, lysine, divinyl sulfone, 4-butanediol diglycidyl ether, these are just some examples but not limited to crosslinkers choose a range.
  • cross-linking agents can impart new properties to hydrogels, such as lysine, because it is a natural amino acid, and when lysine is used as a cross-linking agent, cells are more favorable for adhesion to hydrogels (see figure 3).
  • EDC/ 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide
  • NHS 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)triazin-2-yl)-4-methylmorpholinium chloride
  • DMTMM is sensitive to pH and can catalyze the cross-linking reaction at neutral pH, which is beneficial to the integrity of CCT molecules. Despite this, in fact EDC/NHS and DMTMM are common catalysts for amidation reactions, and there is no strict limit to the choice of any catalyst.
  • the mass ratio of CCT and EDC/NHS or DMTMM may vary from 1:1 to 10:1.
  • the catalytic efficiency is proportional to temperature, which means that the higher the temperature, the faster the reaction occurs.
  • the reaction is usually carried out at 0 to 37 °C. Based on the ratio of CCT/crosslinker/catalyst and the reaction temperature, the degree of crosslinking is also proportional to the reaction time, and the reaction time is changed from 2 hours to 96 hours.
  • CCT In addition to amino and carboxyl groups, CCT also contains a large amount of hydroxyl groups.
  • divinyl sulfone (DVS) or 1,4-butanediol diglycidyl ether (BDDE) can also be used as the crosslinking agent in the above first step.
  • the molar ratio of CCT to DVS or BDDE can vary from 1:1 to 30:1.
  • CCT in the first step, is cross-linked with DVS to form an injectable hydrogel for viscoelastic supplementation, since DVS is most effective at 40-50 ° C and the reaction temperature is set at 45 ° C. Time 2-6 hours.
  • BDDE is used as a crosslinking agent to react with CCT to form an injectable hydrogel for viscoelastic replenishment, and the reaction temperature can be below 10 ° C for a period of 90-100 hours.
  • a 2-4 times alkaline solution may be added to the reaction system (pre-crosslinking reaction system) to carry out further crosslinking by pH 9.0 to 13.5 and mechanical stirring for 2 to 24 hours.
  • the alkaline solution penetrates, the crosslinked network swells, and external and internal hydrogen bonds break.
  • CCT molecules are more flexible and fluid, and more reactive sites are exposed to participate in further reactions.
  • the addition of a large amount of alkaline solution will dilute the concentration of CCT, and further crosslinking will not result in the formation of a solid-like hydrogel. It should be noted that the concentration of CCT here is quite important, and only hydrogels with certain fluidity are injectable.
  • hydrogel exhibits a random porous morphology before the addition of the alkali solution; however, the hydrogel exhibits an oriented layered structure after the addition of the alkali solution, indicating that the addition of the alkaline solution does change.
  • Hydrogel network Generally, hydrogels have a random porous structure, such as (As shown in Figure 5), the hydrogel of the present invention provides a more stable oriented layered structure that directs cell orientation and nutrient delivery when applied in vivo (as shown in Figure 6).
  • the temperature for further crosslinking in the second step above is set to not exceed 50 ° C to reduce degradation.
  • the reaction temperature is even as low as 0 °C.
  • the monomeric units of CCT and HA are linked by glycosidic linkages which are acid sensitive to base insensitivity.
  • a base such as NaOH or KOH can increase the ionic strength of the reaction system, thereby reducing the degree of ionization of CCT, thereby reducing the degradation rate of CCT.
  • a base such as NaOH or KOH
  • the CCT molecules After network expansion and further cross-linking, the CCT molecules have been gently modified by the cross-linking agent.
  • an organic solvent such as ethanol is added in the above third step. Since the crosslinked network is insoluble in ethanol and ethanol is miscible with water, the network can be precipitated from the reaction system by adding sufficient organic solvent.
  • some cross-linking agents such as lysine and catalysts such as EDC/NHS are also soluble in certain organic solvents, such as ethanol, unreacted cross-linking agents and catalysts can be passed through the precipitation step followed by multiple times with organic solvents (eg However, it is not limited to 3 times) washing and removal. In order to completely precipitate the product, an organic solvent exceeding 3 times the volume of the reaction system is added in the above third step.
  • the precipitate may be washed at least 3 times in the fourth step above using an organic solvent of 1-2 volumes in the reaction system.
  • the precipitate may optionally be dried in a vacuum oven for more than 24 hours, then stored at -20 ° C until use; or redissolved in a buffer solution to a concentration such as phosphate buffered saline (PBS) or 0.9% NaCl solution.
  • PBS phosphate buffered saline
  • ethanol as an organic solvent in the examples of the present invention does not mean that ethanol is the only choice, and any solvent which can dissolve with water and cannot dissolve with the polysaccharide in the above fourth step can be used for the crosslinked network.
  • Precipitates such as methanol, acetone and chloroform.
  • the third step is that the temperature of the reaction system (crosslinking reaction system) is lowered to -20 ° C in less than 10 minutes at the end of further crosslinking. It was then pre-frozen for 4 hours in a freeze dryer at -45 °C. After that, the vacuum pump was turned on and the temperature was started (10 ° C for 12 hours, -5 ° C for 12 hours, 0 ° C for 6 hours, 5 ° C for 4 hours, 10 ° C for 4 hours, and 20 ° C for 2 hours). The lyophilized product was stored at -20 ° C until use. Prior to redissolution, wash with 3 times of organic solvent similar to the wash pellet described above for at least 1 hour each time.
  • the semi-finished hydrogel can be obtained by dissolving the CCT precipitate in PBS or 0.9% NaCl solution by the procedure described above.
  • the hydrogel still contains residues of a crosslinking agent, a catalyst, and an organic solvent such as ethanol, a further purification procedure is required.
  • the hydrogel in the fifth step, is placed in a dialysis bag with a MWCO ⁇ 6000, and immersed in a dialysate (PBS or 0.9% NaCl solution), and the volume of the dialysate is larger than the dialysis bag. 8 times the hydrogel, and dial the dialysate once a day. In order to reduce the residue to an acceptable level, it usually takes more than 3 days of dialysis. At the end of the dialysis, the concentration of the hydrogel was measured by the method in Example 11. Additional PBS or 0.9% NaCl solution was added to the hydrogel to achieve the desired concentration.
  • the hydrogel eventually appears as a fluid-like gel suitable for injection with a syringe. Moreover, since the hydrogel has shear thinning characteristics, its viscosity will be significantly lowered upon injection, which is quite advantageous for the operation.
  • the sterilization in the fifth step above is performed prior to using the hydrogel as a viscoelastic supplement.
  • Most of the chitin or chitosan solution products currently on the market are manufactured by filtration sterilization or using sterile raw materials under aseptic conditions, because these solutions are thermally unstable and cannot be autoclaved.
  • these sterilization methods are highly dependent on the cleaning of the environment and the procedures are cumbersome.
  • the hydrogel obtained by the present invention has a slight crosslinkability and a stable oriented layer structure, and can withstand a certain process of heat treatment, so that high pressure steam sterilization can be employed here. In order to eliminate microorganisms, the sterilization process was carried out at 121 ° C for 10-30 minutes.
  • the ⁇ of the CCT hydrogel is nearly identical before and after autoclaving (shown in Figure 7), which means that the heating process is complete to the hydrogel by proper cross-linking and orientation of the layered structure. Sex has little effect, thus maintaining its mechanical properties.
  • the invention includes the preparation of an injectable and heat stable croscarmellose hydrogel and its clinical use, particularly as a viscoelastic supplement.
  • the present invention can impart a hydrogel oriented layered structure, it facilitates cell guidance and delivery of nutrients, drugs, and biologically active proteins.
  • an oriented carboxymethyl chitosan scaffold having a reinforced modulus was produced based on the procedure of the present invention.
  • the scaffold can guide the directional alignment of neuronal cells (Figure 6), which means it can be used to repair nerve damage.
  • biologically active substances such as anti-inflammatory drugs and cells are incorporated into the hydrogel to increase the efficiency of viscoelastic supplementation or for other clinical applications.
  • the resulting hydrogel can be used not only for viscoelastic supplements, but also for soft tissue filling such as face, hand and earlobe.
  • microscopically oriented hydrogel of the present invention contributes to the injectability and thermal stability of the hydrogel.
  • the present invention provides for the first time a preparation method which allows the hydrogel to have the above microstructure.
  • hydrogels provided by the present invention can be used or used to make viscoelastic supplements, which can contain drugs or cells for a wider range of uses.
  • the solution was mechanically stirred at room temperature for 1 hour.
  • 1.5% (w/v) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was added to the The solution was mechanically stirred at 25 ° C for 2 hours.
  • Two volumes of deionized water were then added to the reaction system and the pH of the system was adjusted to 12.5 by the addition of NaOH.
  • 2% (w/v) DMTMM was added to the solution.
  • Mechanical stirring was carried out at 37 ° C for 2 hours.
  • 4 volumes of a 0.9% NaCl solution was added to the reaction system, and the pH of the system was adjusted to 11.0 by the addition of KOH.
  • a 4-fold amount of ethanol was added dropwise to the reaction system, and the product was precipitated under constant stirring.
  • the precipitate was dried in a vacuum oven for 96 hours and then stored at -20 ° C until use.
  • BDDE 1,4-butanediol diglycidyl ether
  • the obtained hydrogel was then passed through a 50 ⁇ m filter membrane, placed in a glass syringe, and autoclaved at 121 ° C for 15 minutes.
  • a 2% carboxymethyl chitosan injectable hydrogel that can be used for viscoelastic supplementation has thus far been obtained.
  • the obtained hydrogel was then passed through a 50 ⁇ m filter membrane, placed in a glass syringe, and autoclaved at 121 ° C for 15 minutes.
  • a 1% carboxymethyl chitosan injectable hydrogel that can be used for viscoelastic supplementation has thus far been obtained.
  • the obtained hydrogel was then passed through a 50 ⁇ m filter membrane, placed in a glass syringe, and autoclaved at 121 ° C for 20 minutes.
  • a 1.5% carboxymethyl chitosan injectable hydrogel that can be used for viscoelastic supplementation has thus been obtained.
  • Um is the molecular weight of the monomer unit of carboxymethyl chitosan, which is 219.
  • a solution of % (w/v) was mechanically stirred at room temperature for 4 hours.
  • 1% (w/v) EDC/NHS was added to the solution and the pH was adjusted to 4.5 by the addition of HCl. It was then mechanically stirred at 25 ° C for 12 hours.
  • Four volumes of deionized water were then added to the reaction system and the pH of the system was adjusted to 10.0 by the addition of NaOH.
  • the anterior and posterior cruciate ligaments of the hind legs of Sprague Dawley rats were cut off to construct an animal model of osteoarthritis.
  • the viscoelastic supplement of Example 10 was mixed with chondrocytes (extracted from Sprague Dawley rats) and injected into the rat joint at a density of 1,000,000/mL.
  • chondrocytes extracted from Sprague Dawley rats
  • 0.5 mL of viscoelastic supplement containing chondrocytes was injected into one of the hind legs, and 0.5 mL of 0.9% NaCl solution was injected to the other hind leg as a control.
  • the observation period was 1 month and 2 months, and 10 rats were used each time.
  • methylprednisolone (2 mg/kg), azathioprine (2 mg/kg) and methylprednisolone (2 mg/kg) daily. Chondocytes were cultured on the hydrogel to examine their biocompatibility prior to in vivo studies.
  • VDS divinyl sulfone
  • phosphate buffer phosphate buffer
  • VDS divinyl sulfone
  • the vacuum pump was turned on and the temperature was started (10 ° C for 12 hours, -5 ° C for 12 hours, 0 ° C for 6 hours, 5 ° C for 4 hours, 10 ° C for 4 hours, and 20 ° C for 2 hours).
  • the lyophilized product was washed 3 times with ethanol and dissolved in a phosphate buffer to a concentration of 20 mg/mL.
  • the concentration of the hydrogel was measured according to the method in Example 11. Additional phosphate buffer was added to the hydrogel to ensure a final carboxymethyl chitosan concentration of 10 mg/mL. Finally, the obtained hydrogel was passed through a 50 ⁇ m filter membrane, poured into a glass syringe, and autoclaved at 121 ° C for 15 minutes. A 1% strength carboxymethyl chitosan injectable hydrogel for viscoelastic supplementation is now available.
  • the solution was stirred at room temperature for 4 hours.
  • 3% (w/v) DMTMM was added to the solution.
  • the reaction was carried out at 4 ° C and stirring was continued for 4 hours.
  • 2 times of deionized water was added to the reaction system and stirred uniformly, and the pH of the reaction system was adjusted to 9.0 by adding NaOH.
  • the reaction system was then placed in an ice water bath and stirred for 2 hours and then placed at 4 ° C for 24 hours.
  • Example 18 After washing the freeze-dried stent 3 times with ethanol, the stent was dried in a vacuum oven for 24 hours and then placed under glutaraldehyde vapor for 48 hours to increase the degree of crosslinking to enhance the modulus. Thereafter, the stent was dried in a vacuum oven for 72 hours to remove residual glutaraldehyde.

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Abstract

本发明公开了一种具有取向层状结构的多糖水凝胶及其制备方法。所述方法包括步骤：(1)使多糖和交联剂进行预交联，得到预交联体系；(2)使预交联体系在碱性溶液中进一步交联，得到交联体系；(3)将交联体系与有机溶剂混合得到沉淀物或将交联体系冷冻干燥得到冻干物；和(4)将沉淀物或冻干物溶解于缓冲溶液中得到水凝胶。此外，本发明还提供了这类水凝胶的一些临床应用，特别是作为关节腔粘性补充。

Description

一种注射用水凝胶的制备方法及其应用 技术领域
本发明涉及关节腔粘弹补充领域，尤其涉及一种注射用水凝胶的制备方法及其应用。
背景技术
骨关节炎是一种非常常见的临床疾病，总体发病率约为15.6％，在60岁以上人口中，比例超过50％。骨关节炎可引起疼痛和运动障碍，严重降低生活质量。滑液在关节中起着润滑作用，可以减少关节软骨在运动过程中的摩擦。随着年龄的增长，关节液的质量越来越差，导致软骨退化，进而引起骨关节炎。为了恢复和延缓关节磨损的进程，一种普遍而有效的方法是为关节腔补充新的“润滑剂”，特别是对轻度和中度骨关节炎患者以及年轻人。
透明质酸是滑液的主要成分，是一种理想的粘性补充剂，数十年来，已被许多研究文献和临床实践应用证明。尽管注射这些透明质酸产品可以缓解疼痛并恢复骨关节炎患者的活动能力，由于注射后，透明质酸迅速被透明质酸酶降解，这种效果只能持续一段时间。为了保持一定时间的润滑作用，骨关节炎患者一般会每疗程注射3-5次，增加了医疗费用，也带来不方便和感染的风险。
壳聚糖是甲壳素脱乙酰化的多糖，具有与透明质酸类似的分子结构和润滑性。由于人体内没有特定的酶来降解壳聚糖，因此其体内半衰期比透明质酸长得多。壳聚糖除具有较长的维持时间外，还具有抗菌作用和止血性能，与透明质酸相比，还能抑制成纤维细胞的增殖。壳聚糖已被证明可支持人类软骨细胞表达细胞外基质蛋白，并且可减少炎症介质的产生。因此，壳聚糖是一个非常有前景的粘弹补充剂候选材料。
然而，壳聚糖是一种半刚性聚合物，具有大量的内/外氢键，因此它不能直接溶于纯水中。通常，酸性溶液如1％乙酸溶液用于溶解壳聚糖，由于这些酸性溶液的pH值与生理pH之间有差距，壳聚糖溶液不能直接用于体内。解决这个问题的一个方法就是将羧甲基引入壳聚糖主链中，破坏半刚性结构，得到的羧甲基壳聚糖可溶于纯水中，作为粘弹补充剂使用具有实用价值。
羧甲基壳聚糖可显著抑制骨关节炎软骨中MMP-1和MMP-3的mRNA 表达，降低软骨退变的严重程度。Bai将羧甲基壳聚糖和透明质酸分别注入骨关节炎患者的关节腔中，结果表明羧甲基壳聚糖和透明质酸均能缓解疼痛和僵硬，但羧甲基壳聚糖的注射次数要少于透明质酸。羧甲基壳聚糖可以通过降低一氧化氮和活性氧的水平来抑制软骨细胞凋亡从而保护软骨。
可是，热稳定性差阻碍了羧甲基壳聚糖粘弹补充产品的上市。壳聚糖在加热过程中会逐渐降解；随着温度和加热时间的增加，对壳聚糖链的破坏会加速。作为壳聚糖的衍生形式，羧甲基壳聚糖在加热期间也降解。因此，绝大多数壳聚糖产品都是以粉末、颗粒或薄膜的形式存在的，它们在高压灭菌或辐照灭菌过程中能抵抗破坏。例如，众所周知的止血产品
Figure PCTCN2018085546-appb-000001
就是由壳聚糖颗粒组成。但是，对于粘弹补充剂而言，为了注射到关节中，只有具有流体或凝胶形式的产品才是可接受的；在这种情况下，需要解决如何保护壳聚糖产品免受终端灭菌破坏的问题。
水凝胶是一种含有大量水分的大分子网络，高剪切速率会破坏水凝胶网络。随着医疗技术的进步，许多领域例如膝关节粘弹补充推荐使用微创疗法。因此，羧甲基壳聚糖水凝胶具备可注射性同样重要。
因此，本领域，特别是在关节腔粘弹补充领域，迫切需要提供一种具有热稳定性和可注射性的新型羧甲基壳聚糖水凝胶。
发明内容
本发明旨在提供一种兼具热稳定性和可注射性的水凝胶及其应用。
在本发明的第一方面，提供了一种水凝胶的制备方法，所述方法包括步骤：
(1)使多糖和交联剂进行预交联，得到预交联体系；
(2)使预交联体系在碱性溶液中进一步交联，得到交联体系；
(3)将交联体系与有机溶剂混合得到沉淀物或将交联体系冷冻干燥得到冻干物；
(4)将沉淀物或冻干物溶解于缓冲溶液中得到水凝胶。
在另一优选例中，进行预交联时使用催化剂。
在另一优选例中，所述多糖和催化剂的质量比在1:1至10:1；
更优选地，所述催化剂选自1-乙基-3-(3'-二甲基氨基丙基)碳二亚胺/N- 羟基琥珀酰亚胺(EDC/NHS)或4-(4,6-二甲氧基-1,3,5-三嗪-2-基)三嗪-2-基)-4-甲基吗啉鎓氯化物(DMTMM)。
在另一优选例中，所述交联剂为具有两个以上与多糖中的羧基和/或羟基反应的官能团的物质；多糖上羧基与交联剂所述官能团(氨基)的摩尔比为2:1至20:1；更优选地，多糖上羟基与交联剂所述官能团的摩尔比为1：1至30：1。
在另一优选例中，1种所述交联剂分子可至少与多糖上的2个羧基或2个羟基或1个羧基和1个羟基反应。
在另一优选例中，所述预交联的反应时间为2-96小时，反应温度为0-50℃；更优选0-37℃。
在另一优选例中，所述预交联的反应开始时多糖的添加比例为1％(w/v)-6％(w/v)。
在另一优选例中，所述多糖选自羧甲基壳聚糖、透明质酸或其衍生物、纤维素、淀粉、葡聚糖、天冬酰胺、精氨酸、硫酸软骨素、硫酸皮肤素、硫酸角蛋白、肝素或其衍生物；
更优选地，所述羧甲基壳聚糖的分子量介于100kDa至800kDa之间，羧甲基的DS不低于1.0，乙酰胺基的DD不超过20％；所述透明质衍生物选自透明质酸钠或透明质酸钾，所述透明质酸钠或透明质酸钾的分子量介于100kDa至3000kDa之间。
在另一优选例中，所述交联剂选自下述的一种或一种以上：草酰二肼、胱胺二盐酸盐、1,3-二氨基-2-丙醇、1,4-二氨基-2-丁酮二盐酸盐、1,11-二氨基-3,6,9-三氧杂十一烷、二氨基聚乙二醇、赖氨酸、谷氨酰胺、天冬酰胺、精氨酸、二乙烯基砜、1,4-丁二醇二缩水甘油基醚、二乙烯基砜(DVS)和1,4-丁二醇二缩水甘油醚(BDDE)。
在另一优选例中，所述碱性溶液为pH 9.0-13.5的水溶性碱性溶液；更优选地，所述碱性溶液为NaOH或KOH的水溶液。
在另一优选例中，所述交联体系和预交联体系的体积比在2：1和4：1之间。
在另一优选例中，所述进一步交联的反应时间为2-24小时，反应温度为0-50℃。
在另一优选例中，所述有机溶剂与交联体系的体积比不小于3:1。
在另一优选例中，所述有机溶剂为可溶于水但不能溶解多糖的溶剂；更优选地，所述有机溶剂选自C 1-3的醇、丙酮或氯仿；最优选甲醇或乙醇。
在另一优选例中，所述缓冲溶液选自磷酸盐缓冲溶液或0.9％NaCl溶液。
在另一优选例中，所述沉淀物或冻干物溶解于缓冲溶液的溶解浓度在1％和4％(w/v)之间。
在另一优选例中，所述方法还包括步骤：
(5)将得到的水凝胶进行透析。
在另一优选例中，所述透析的截留分子量(MWCO)不小于6000。
在另一优选例中，透析使用的透析液与步骤(4)中的缓冲溶液相同；所述透析液与水凝胶的体积比不小于8:1。
在另一优选例中，所述方法还包括步骤：
(6)将经过透析的水凝胶进行过滤或高压灭菌。
在另一优选例中，所述高压灭菌的温度为120-125℃，时间为10-30分钟。
在本发明的第二方面，提供了一种水凝胶，所述水凝胶含有多糖和交联剂；以水凝胶的总重量计，其中含有1.0-2.0wt％多糖。
在另一优选例中，所述交联剂为具有两个以上与多糖中的羧基和/或羟基反应的官能团的物质。
在另一优选例中，所述多糖选自羧甲基壳聚糖、透明质酸钠或透明质酸钾；所述羧甲基壳聚糖的分子量介于100kDa至800kDa之间，羧甲基的DS不低于1.0，乙酰胺基的DD不超过20％；所述透明质酸钠或透明质酸钾的分子量介于100kDa至3000kDa之间。
在另一优选例中，所述交联剂选自下述的一种或一种以上：草酰二肼、胱胺二盐酸盐、1,3-二氨基-2-丙醇、1,4-二氨基-2-丁酮二盐酸盐、1,11-二氨基-3,6,9-三氧杂十一烷、二氨基聚乙二醇、赖氨酸、谷氨酰胺、天冬酰胺、精氨酸、二乙烯基砜、1,4-丁二醇二缩水甘油基醚、二乙烯基砜(DVS)和1,4-丁二醇二缩水甘油醚(BDDE)。
在另一优选例中，所述水凝胶中还含有药物和/或细胞。
在另一优选例中，所述药物为抗炎药或麻醉剂；所述细胞选自软骨细胞或间充质干细胞。
在另一优选例中，所述抗炎药选自曲安奈德、布洛芬和/或双氯芬酸)； 所述麻醉剂为利多卡因。
在另一优选例中，所述水凝胶具有取向层状结构。
在另一优选例中，所述水凝胶具有可注射性和热稳定性。
在另一优选例中，所述水凝胶通过如上所述的本发明提供的制备方法获得。
在本发明的第三方面，提供了一种如上所述的本发明提供的水凝胶的用途，所述水凝胶用于制备可用于粘弹补充的试剂或用作粘弹补充剂；或用于制备药物输送系统、可用于组织工程的支架或可填充的软组织。
在另一优选例中，所述粘弹补充剂包含如上所述的本发明提供的水凝胶和抗炎药、麻醉剂或细胞；所述抗炎药选自曲安奈德、布洛芬和/或双氯芬酸)；所述麻醉剂为利多卡因；所述细胞选自软骨细胞或间充质干细胞。
据此，本发明提供了一种具有热稳定性和可注射性的新型羧甲基壳聚糖水凝胶。
附图说明
为了更好地理解本发明及其应用，列出了来自一些优选实施例的附图。
图1显示实施例8中高压灭菌前后临界羧甲基壳聚糖水凝胶的储能模量(G')和损耗模量(G”)情况，表示本发明水凝胶在溶胶和凝胶之间处于临界状态；其中△表示G'，▽表示G”。
图2显示用作粘弹补充的羧甲基壳聚糖溶液的储能模量(G')和损耗模量(G”)情况。在整个测试范围内，G”总是大于G'。虽然它可以润滑软骨表面，但与水凝胶相比，在关节运动过程中不能吸收足够的振动能量。其中，△表示G'，▽表示G”。
图3是实施例15中软骨细胞在羧甲基壳聚糖水凝胶中培养后的活/死染色荧光染色照片，表明本发明使用的交联剂可以增加细胞的粘附。
图4是实施例12中碱化前(A)和碱化后(B)的羧甲基壳聚糖水凝胶的SEM显微照片，表明碱化过程可以将交联网络的结构从无规多孔状改变为取向层状。水凝胶的层状结构有助于热稳定性和可注射性。
图5是
Figure PCTCN2018085546-appb-000002
的SEM显微照片呈现无规多孔状结构。事实上，大 多数普通的水凝胶都具有这种结构。
图6是实施例19中在羧甲基壳聚糖水凝胶上培养的神经元细胞的肌动蛋白荧光染色显微照片，细胞取向取决于所述水凝胶的取向层状结构。
图7是实施例10中高压灭菌前后羧甲基壳聚糖水凝胶的动态粘度(η)，表明本发明的水凝胶在高压灭菌过程中能保持网络完整性和机械性能。其中□表示高压灭菌前，△表示高压灭菌后。
图8是实施例14中将羧甲基壳聚糖粘弹剂和0.9％NaCl溶液分别注射入兔关节炎模型后2个月的II型胶原免疫组化染色切片。A图是粘弹剂组，B图是0.9％NaCl溶液组。
具体实施方式
发明人经过广泛而深入的研究，发现将多糖和交联剂通过交联、碱化、沉淀(或冷冻干燥)和再溶解等步骤可以获得一种兼具可注射性和热稳定性的水凝胶。
如本发明所用，“可注射性”是指本发明提供的水凝胶可以在高剪切速率条件下(如通过注射器注射时)保持其机械性能和网络完整性。
如本发明所用，“热稳定性”是指本发明提供的水凝胶可以在高温条件下(如高温高压灭菌)保持其机械性能和网络完整性。
水凝胶
本发明提供的水凝胶是一种以水为分散介质的凝胶，含有多糖和交联剂；以水凝胶的总重量计，其中含有1.0-2.0wt％多糖。
本发明提供的水凝胶的微观结构表现出较好的有序排列，即取向层状结构，与一般水凝胶的无规多孔状结构有很大的不同。取向层状结构即有序排列微结构有助于所述水凝胶的可注射性和热稳定性。
基于交联比例、化学/物理键合性质、pH值、温度等因素，水凝胶的性质可以从液态横跨到固态。因为软组织主要由水组成，水凝胶可以根据软组织的性质轻松调整，如刚度和弹性。事实上，用普通流变学术语如动态粘度(η，抗变形能力)、储能模量(G'，反映材料的弹性)和损耗模量(G”，反映材料的粘度)来描述水凝胶的性质是非常方便和实用的。对于粘弹性材 料，G'和G”的相对大小表示材料的物理状态。例如，当G'>G”时，水凝胶表现得更像固体；而当G”>G'时，水凝胶表现得更像流体。在一个实施例中，在较宽的频率范围内，G'≈G”(如图1所示)表示这里的羧甲基壳聚糖水凝胶处于流体状物质和类固体物质之间的临界条件下，这是不同寻常的；应当注意的是，即使在高压灭菌之后，本发明提供的水凝胶仍然处于临界状态(G'≈G”)，这意味着所述水凝胶的弹性和粘性不受热损害。
为了制备适合于粘弹补充的水凝胶，应该优先考虑与关节滑液性质相似的水凝胶。滑液是一种假塑性流体，它的表观粘度随着剪切速率的增加而降低，本发明的水凝胶也具有这种特性。对于未交联的壳聚糖溶液，G”总是大于G'(如图2所示)。交联后，G'在很宽的范围内总是等于或大于G”；通过调整本发明方法的具体参数，本发明水凝胶的G'和G”可以很容易地调节。很明显，在相同的剪切速率下，可注射的交联羧甲基壳聚糖水凝胶的η高于壳聚糖溶液，这意味着水凝胶在关节运动过程中比溶液在吸收振动和润滑关节表面表现得更好。
本发明采用的羧甲基壳聚糖直接由几丁质制得，DD(脱乙酰度)可控制在20％以下，DS(羧甲基取代度)大于1.0。除另有说明外，本发明中采用的壳聚糖衍生物，即羧甲基壳聚糖，其DS≥1.0，DD≤20％，在本发明中可以简写为CCT。
尽管实施例中的大部分水凝胶都是由CCT制成的，但是本领域技术人员可以根据本发明的技术方案很容易地选择其他相似的材料来制备具有类似性能的水凝胶，这些仍然在本发明的范围之内。在实际应用中，其他多糖，如透明质酸、纤维素、淀粉、葡聚糖、天冬酰胺、精氨酸、硫酸软骨素、硫酸皮肤素、硫酸角蛋白、肝素及其衍生物或其他具有类似结构的聚合物，也可以按本发明所描述的步骤使用。选择CCT作为代表性材料是为了更好地理解本发明发明和制备工艺，但不限制选择其他材料。
制备方法
本发明提供的水凝胶的制备方法包括步骤：
第一步，将多糖水溶液、交联剂和催化剂混合，得到预交联反应体系；
第二步，在预交联反应体系中加入碱性溶液进行进一步交联，得到交联反应体系；
第三步，在交联反应体系中加入有机溶剂获得沉淀产物；或将交联反应体系进行冷冻干燥得到冻干产物；
第四步，将经有机溶剂洗涤的沉淀产物或冻干产物溶解于缓冲溶液中得到水凝胶；
第五步，水凝胶经缓冲溶液透析后进行过滤或高压灭菌，得到可用于注射的水凝胶。
本发明提供一种制备具有可注射性和热稳定性羧甲基壳聚糖水凝胶的技术，所述技术包括交联、碱化、沉淀(或冷冻干燥)、再溶解、过滤和高压灭菌。其中预交联和碱化(进一步交联)是获得取向层状结构的关键。
作为粘弹补充剂，水凝胶应该是可注射的，即通过注射针后其网络不会破裂。因此，一方面交联反应比例不宜太高，因为类固体的水凝胶在通过注射器不能维持其完整性。另一方面，充分的交联可赋予水凝胶以足够的粘弹性，使其可吸收振动并润滑关节表面，这意味着相应基团之间的反应比率不应该太低。而且，适量交联可以延长关节粘弹补充的持续时间。由于CCT中的大部分官能团是羧基和羟基，这两种官能团被选作本发明中的反应位点。根据实验，当以羧基为反应位点时，来自CCT的羧甲基与来自交联剂的氨基的摩尔比在2:1和20:1之间。值得注意的是，真实的反应比率要低于上面列出的比例，因为不是每个氨基或羧基都可以参加反应。本发明采用的CCT的Mw在100至800kDa之间。较高的Mw意味着CCT分子具有较大的长度，在溶液中会导致更多的缠结，从而具有更高的粘弹性行为。因此，当其他影响因素相同时，为了获得相似的粘弹性，对于低Mw的CCT需要低的CCT/交联剂比例，而对于高Mw的CCT需要高的CCT/交联剂比例。除CCT外，透明质酸(HA)也是可注射水凝胶的主要成分的重要候选者。对于HA，Mw是从100至3,000kDa变化的。由于HA具有与CCT相似的分子结构，对于交联剂的选择和HA/交联剂的摩尔比，可以参考CCT的条件。交联剂的选择比较灵活，理论上任何具有两个以上可与CCT反应的官能团的物质都可被选为交联剂。
在本发明的一种实施方式中，上述第一步中所述交联剂选自草酰二肼、胱胺二盐酸盐、1,3-二氨基-2-丙醇、1,4-二氨基-2-丁酮二盐酸盐、二氨基聚乙二醇、赖氨酸、二乙烯基砜、4-丁二醇二缩水甘油醚，这些仅仅是一些例子但不限制交联剂的选择范围。不同的交联剂可赋予水凝胶新的性质，例如 赖氨酸，因为它是天然氨基酸，当使用赖氨酸作为交联剂时，细胞更有利于粘附在水凝胶上(见图3)。
为了促进羧基与氨基之间的反应，上述第一步中可以使用1-乙基-3-(3'-二甲基氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)或4-(4,6-二甲氧基-1,3,5-三嗪-2-基)三嗪-2-基)-4-甲基吗啉鎓氯化物(DMTMM)作为催化剂。由于EDC/NHS在微酸性条件下(pH≈4-5)最为有效，采用该催化剂体系时应考虑在酸性条件下CCT的可能降解。DMTMM由于对pH不敏感，能催化中性pH下的交联反应，有利于CCT分子的完整性。尽管如此，事实上EDC/NHS和DMTMM都是酰胺化反应的常用催化剂，对任何一种催化剂的选择没有严格的限制。
在本发明的一种实施方式中，CCT和EDC/NHS或DMTMM的质量比可以在1：1至10：1的范围内变动。在适当的温度范围内，催化效率与温度成正比，这意味着温度越高，反应发生得越快。为了更好地控制交联度，反应通常在0-37℃下进行。基于CCT/交联剂/催化剂的比例和反应温度，交联度也与反应时间成比例，反应时间从2小时变化到96小时。
除了氨基和羧基，CCT还含有大量的羟基。从这个方面来说，上述第一步中还可以使用二乙烯基砜(DVS)或1,4-丁二醇二缩水甘油醚(BDDE)作为交联剂。CCT与DVS或BDDE的摩尔比可在1：1至30：1的范围内变动。
在一个实施例中，上述第一步中，CCT与DVS交联形成用于粘弹补充的可注射水凝胶，因为DVS在40-50℃是最有效的，反应温度设定在45℃，时间2-6小时。在另一个实施例中，使用BDDE作为交联剂与CCT反应形成可注射水凝胶用于粘弹补充，反应温度可在10℃以下，时间90-100小时。
在上述第二步中，可以将2-4倍的碱性溶液加入到反应体系(预交联反应体系)中，使pH9.0-13.5，机械搅拌2-24小时进行进一步交联。随着碱性溶液的渗透，交联网络膨胀，外部和内部氢键断裂。在这种条件下，CCT分子更加灵活和流动，更多的反应位点暴露出来参与进一步反应。此外，大量的碱性溶液的加入会稀释CCT的浓度，进一步的交联不会导致形成类似固体的水凝胶。需要指出的是，这里CCT的浓度是相当重要的，只有具有一定流动性的水凝胶是可注射的。由于本文中CCT的DS和DD分别大于1.0和小于20％，所以CCT的单体单元非常类似于HA。因此，假定CCT和HA在水溶液中具有相似的行为是合理的。羧基和乙酰氨基可在水溶液中形成氢键， 有助于分子骨架的刚性；然而，随着碱性溶液例如NaOH或KOH的加入，氢键被破坏。因此，分子骨架的刚度减弱、回转半径和流体动力学体积减小、自扩散系数和示踪扩散系数增加，这意味着分子的迁移率和渗透性增加(S Ghosh,I Khobal,D.Zanette,and W F Reed.Conformational contraction and hydrolysis of hyaluronate in sodium hydroxide solutions.Macromolecules.1993；26:4684–91.P Gribbon,BC Heng,TE Hardingham.The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.Biophysical Journal.1999；77:2201-6.I Gate,M Popa,M Rinaudo.Role of pH on hyarluronan behavior in aqueous solution.Biomacromolecuels.2005；6:61-7)。如图4所示，所述水凝胶在加入碱溶液之前呈现无规多孔形态；然而，所述水凝胶在加入碱溶液之后呈现取向层状结构，这表明碱性溶液的加入确实改变了水凝胶的网络。通常，水凝胶具有无规多孔结构，如
Figure PCTCN2018085546-appb-000003
(如图5所示)，本发明的水凝胶提供了更为稳定的取向层状结构，可以在体内应用时引导细胞的取向和营养的传递(如图6所示)。总之，碱的加入会影响多糖主链的状态，改变网络结构。由于CCT和HA都是多糖，许多因素都会影响它们的降解，如酸、碱、温度、机械力等。因此，加入碱后，应考虑对应多糖的降解。
在本发明的一种实施方式中，上述第二步中进一步交联的温度设定为不超过50℃以减少降解。在一个实施例中，反应温度甚至低至0℃。CCT和HA的单体单元通过糖苷键连接，其对酸敏感对碱不敏感。此外，加入碱(如NaOH或KOH)可以提高反应体系的离子强度，从而减少CCT的离子化程度，从而降低CCT的降解速率。总之，因为反应温度低、离子强度高，碱性溶液的加入对CCT的降解影响不大。
在网络扩展和进一步交联之后，CCT分子已被交联剂温和地修饰。为了停止反应并将交联网络与反应体系分开，上述第三步中有机溶剂例如乙醇被加入。由于交联网络不溶于乙醇，而乙醇与水互溶，可以通过加入足够的有机溶剂使网络从反应体系中沉淀出来。另外，一些交联剂如赖氨酸和催化剂如EDC/NHS也可溶于某些有机溶剂，例如乙醇，未反应的交联剂和催化剂可以通过沉淀步骤和随后用有机溶剂的多次(例如但不限于，3次)洗涤而除去。为了完全沉淀产物，上述第三步中会加入超过反应体系3倍体积的有机溶剂。
为了清除残留物，上述第四步中可使用1-2体积于反应体系的有机溶剂洗涤沉淀物至少3次。沉淀物可选择在真空干燥箱中干燥24小时以上，然后在-20℃保存备用；或者重新溶解在缓冲溶液中至一定浓度，例如磷酸盐缓冲液(PBS)或0.9％NaCl溶液。需要说明的是，本发明实施例中采用乙醇作为有机溶剂，并不意味着乙醇是唯一的选择，上述第四步中任何能与水相互溶解且不能与多糖溶解的溶剂均可用于交联网络沉淀，如甲醇、丙酮和氯仿。
除了上述基于相分离的方法之外，为了从反应体系中分离交联网络，也可以使用冷冻干燥。在本发明的一种实施方式中，上述第三步是在进一步交联结束时，反应体系(交联反应体系)的温度在不到10分钟内降至-20℃。然后在-45℃的冷冻干燥器中预冷冻4小时。之后开启真空泵，开始程序升温(-10℃12小时，-5℃12小时，0℃6小时，5℃4小时，10℃4小时，20℃持续2个小时)。冻干产物在-20℃下保存备用。在重新溶解之前，用足够的与上述洗涤沉淀物类似的有机溶剂洗涤3次，每次至少1小时。
上述第四步中，半成品水凝胶可通过如上所述的步骤将CCT沉淀物溶解在PBS或0.9％NaCl溶液中获得。
由于所述水凝胶仍含有交联剂、催化剂和有机溶剂如乙醇等残留物，需要进一步的纯化程序。在本发明的一种实施方式中，上述第五步中将所述水凝胶放入MWCO≥6000的透析袋中，浸入透析液(PBS或0.9％NaCl溶液)中，透析液体积大于透析袋中水凝胶的8倍，每天更换1次透析液。为了将残留物降至可接受的程度，通常需要3天以上的透析。在透析结束时，通过实施例11中的方法检测水凝胶的浓度。将额外的PBS或0.9％NaC1溶液加入到水凝胶中以获得期望的浓度。
由于在交联过程中精心地控制了交联度，所述水凝胶最终表现为类似流体状的凝胶，适合用注射器进行注射。而且，由于所述水凝胶具有剪切变稀特性，注射时其粘度将显著降低，这对操作是相当有利的。
在本发明的一种实施方式中，在将所述水凝胶用作粘弹补充剂之前，需先进行上述第五步中的灭菌。目前市场上的几丁质或壳聚糖溶液产品大都采用过滤灭菌或使用无菌原料在无菌条件下制成产品，因为这些溶液热不稳定，不能采用高压灭菌。但这些灭菌方法高度依赖于环境的清洁，程序繁琐。本发明得到的水凝胶具有轻微的交联性和稳定的取向层状结构，可承受一定程序的热处理，所以在此可采用高压蒸汽灭菌。为了消除微生物，灭菌过程在 121℃下进行10-30分钟。在一个实施例中，CCT水凝胶的η在高压灭菌前后(图7中显示)几乎相同，这意味着通过适当的交联和取向层状结构，加热过程对所述水凝胶的完整性几乎没有影响，从而保持其力学性能。
由于CCT和HA具有相似的单体结构，所以上述的所有反应和后处理也适用于具有相似结构的HA或其他分子。
用途
本发明包括一种可注射和热稳定的交联羧甲基壳聚糖水凝胶的制备及其临床应用，尤其用作粘弹补充剂。
由于本发明可以赋予水凝胶取向层状结构，有利于细胞的引导和营养物质、药物和生物活性蛋白质的输送。在实施例19中，基于本发明步骤制造出具有增强模量的取向羧甲基壳聚糖支架。所述支架可以引导神经元细胞的定向排列(图6)，这意味着它可能够用于修复神经损伤。
在某些实施方式中，将生物活性物质如抗炎药和细胞掺入所述水凝胶中以提高粘弹补充的效率或用于其它临床应用。
至于HA或其他具有相似结构的分子，最终得到的水凝胶不仅可用于粘弹补充剂，而且还可用于软组织填充如面部、手和耳垂。
本发明的主要优点包括：
1.本发明水凝胶微观具有取向层状结构有助于所述水凝胶的可注射性和热稳定性。
2.本发明首次提供了可令水凝胶具有上述微观结构的制备方法。
3.本发明提供的水凝胶可用作或用于制作粘弹补充剂，其中可以含有药物或细胞使用途更为广泛。
在下面的说明中将会详细阐述上述化合物、方法、药物组合物的各个具体方面、特性和优势，使本发明的内容变得十分明了。在此应理解，下述的详细说明及实例描述了具体的实施例，仅用于参考。在阅读了本发明的说明内容后，本领域的技术人员可对本发明作各种改动或修改，这些等价形势同样落于本申请所限定的范围。
实施例1
交联羧甲基壳聚糖粉末的制备
将质量比为1：2的草酰二酰肼和羧甲基壳聚糖(Mw＝800,000)溶于蒸馏水中，得到羧甲基壳聚糖浓度为2％(w/v)的溶液。然后向溶液中加入1.5％(w/v)1-乙基-3-[3-二甲基氨基丙基]碳化二亚胺盐酸盐/N-羟基琥珀酰亚胺(EDC/NHS)，加入HCl将pH调节至4.5。反应在4℃下持续搅拌72小时。然后将3倍去离子水加入到反应体系中并搅拌均匀，加入NaOH将反应体系的pH调节至13.0。然后将反应体系置于4℃环境中连续搅拌12小时。之后，在搅拌条件下将3倍于反应体系的乙醇滴加到反应体系中以沉淀产物。用乙醇洗涤沉淀3次后，将沉淀物在真空干燥箱中干燥72小时，然后在-20℃下保存备用。
实施例2
交联羧甲基壳聚糖粉末的制备
将质量比为1:1的二氨基聚乙二醇(Mw＝1,000)和羧甲基壳聚糖(Mw＝400,000)溶于蒸馏水中，得到羧甲基壳聚糖浓度为4％(w/v)的溶液，在室温下搅拌4小时。然后向溶液中加入2％(w/v)EDC/NHS，并加入HCl将pH调节至4.5。反应在冰水浴中进行，持续搅拌48小时。然后将4倍体积去离子水加入到反应体系中并均匀，加入NaOH将反应体系的pH调节至10.0。然后将反应体系置于冰水浴中持续搅拌的24小时。之后，在搅拌条件下将3倍体积的乙醇滴加到反应体系中以沉淀产物。用乙醇洗涤沉淀3次后，将沉淀物在真空干燥箱中干燥48小时，然后在-20℃下保存备用。
实施例3
交联羧甲基壳聚糖粉末的制备
将质量比为2：3的胱氨酸二盐酸盐和羧甲基壳聚糖(Mw＝600,000)溶于磷酸盐缓冲液中，得到羧甲基壳聚糖浓度为3％(w/v)的溶液，室温下机械搅拌1小时。然后将1.5％(w/v)4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基吗啉鎓氯化物(DMTMM)加入到该溶液中，并将该体系在25℃下机械搅拌2小时。之后将2倍体积去离子水加入到反应体系中，并且通过加入NaOH将体系的pH调节至12.5。在4℃下搅拌2小时后，在反应体系中加 入3倍量的乙醇，不断搅拌，使产物沉淀。用乙醇洗涤沉淀物3次后，将沉淀物在真空干燥箱中干燥24小时，然后在-20℃下保存备用。
实施例4
交联羧甲基壳聚糖粉末的制备
将质量比为1：4的赖氨酸和羧甲基壳聚糖(Mw＝200,000)溶于0.9％NaCl溶液中，得到羧甲基壳聚糖浓度为6％(w/v)的溶液，室温下机械搅拌4小时。然后将2％(w/v)DMTMM加入到溶液中。在37℃下机械搅拌2小时。之后将4倍体积的0.9％NaCl溶液加入到反应体系中，并且通过加入KOH将体系的pH调节至11.0。在37℃下搅拌12小时后，在反应体系中滴加4倍量的乙醇，在不断搅拌下沉淀产物。用乙醇洗涤沉淀物3次后，将沉淀物在真空干燥箱中干燥96小时，然后在-20℃下保存备用。
实施例5
交联羧甲基壳聚糖粉末的制备
将质量比为1：3的1,3-二氨基-2-丙醇和羧甲基壳聚糖(Mw＝300,000)溶解在磷酸盐缓冲液中，得到羧甲基壳聚糖浓度为4％(w/v)的溶液，在室温下机械搅拌2小时。然后在溶液中加入1.5％(w/v)DMTMM，在4℃下机械搅拌24小时。之后将4倍体积的磷酸盐缓冲液加入到反应体系中，并且通过加入KOH将体系的pH调节至9.0。在4℃下机械搅拌18小时后，在反应体系中加入4倍量的乙醇，不断搅拌，使产物沉淀。用乙醇洗涤沉淀3次后，将沉淀物在真空干燥箱中干燥48小时，然后在-20℃下保存备用。
实施例6
交联羧甲基壳聚糖粉末的制备
将羧甲基壳聚糖(Mw＝500,000)溶于蒸馏水中，得到3％(w/v)的溶液，在室温下机械搅拌2小时。然后向溶液中加入0.5％(w/v)二乙烯基砜(DVS)，在45℃下机械搅拌4小时。之后将4倍体积的去离子水加入到反应体系中，并将pH调节至13.5。在45℃下搅拌2小时后，在反应体系中滴加4倍量的乙醇，并持续搅拌以沉淀产物。沉淀物用乙醇洗涤4次后，在真空干燥箱中干燥72小时，然后在-20℃下保存备用。
实施例7
交联羧甲基壳聚糖粉末的制备
将羧甲基壳聚糖(Mw＝700,000)溶于蒸馏水中，得到1％(w/v)的溶液，在室温下机械搅拌2小时。然后向溶液中加入0.2％(w/v)1,4-丁二醇二缩水甘油醚(BDDE)，在4℃下机械搅拌96小时。之后将4倍体积的去离子水加入到反应体系中，并将pH调节至11.0。在4℃下搅拌6小时后，在反应体系中滴加4倍量的乙醇，并持续搅拌以沉淀产物。用乙醇洗涤沉淀物4次后，将沉淀物在真空干燥箱中干燥96小时，然后在-20℃下保存备用。
实施例8
在磷酸盐缓冲液中制备2％羧甲基壳聚糖水凝胶用于粘弹补充
取实施例1的交联羧甲基壳聚糖粉末3g加入100mL磷酸盐缓冲液中，机械搅拌至粉末完全溶解。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(磷酸盐缓冲液)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的磷酸盐缓冲液以确保最终的羧甲基壳聚糖浓度为20mg/mL。然后将得到的水凝胶通过50μm过滤膜，装入玻璃注射器中，121℃高压灭菌15分钟。至此获得了可用于粘弹补充的2％羧甲基壳聚糖可注射水凝胶。
实施例9
在生理盐水中制备1％羧甲基壳聚糖水凝胶用于粘弹补充
取实施例2的交联羧甲基壳聚糖粉末1.5g加入到100ml 0.9％NaCl溶液中，机械搅拌至粉末完全溶解。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(生理盐水)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的生理盐水以确保最终的羧甲基壳聚糖浓度为10mg/mL。然后将得到的水凝胶通过50μm过滤膜，装入玻璃注射器中，121℃高压灭菌15分钟。至此获得了可用于粘弹补充的1％羧甲基壳聚糖可注射水凝胶。
实施例10
在磷酸盐缓冲液中制备1.5％羧甲基壳聚糖水凝胶用于粘弹补充
取实施例4的交联羧甲基壳聚糖粉末2g加入100mL磷酸盐缓冲液中，机械搅拌至粉末完全溶解。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(磷酸盐缓冲液)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的磷酸盐缓冲液以确保最终的羧甲基壳聚糖浓度为15mg/mL。然后将得到的水凝胶通过50μm过滤膜，装入玻璃注射器中，121℃高压灭菌20分钟。至此获得了可用于粘弹补充的1.5％羧甲基壳聚糖可注射水凝胶。
实施例11
羧甲基壳聚糖水凝胶质量浓度的检测方法
将0.2g(m 1)羧甲基壳聚糖水凝胶溶于2mL 5M HCl溶液(m 2)中，密封后放入105℃的气氛中16小时。然后，将2mL 5M NaOH(m 3)加入到溶液中并充分混合。取上述溶液2mL与0.6mL二亚硝基水杨酸(DNSA)混合，混合物置于100℃沸水浴中5分钟。用UV-Vis分光光度计在520nm处检测反应液的吸光度(OD)。然后将浓度为1.0，1.25，1.5，1.75，2.0，2.25和2.5μmol/mL的葡萄糖胺盐酸盐的标准溶液与DNSA以相同的方式反应，并记录520nm处的OD。根据标准OD—浓度曲线，得到羧甲基壳聚糖水凝胶的葡糖胺盐酸盐浓度(C)。质量浓度(MS)可以通过以下公式计算：
Figure PCTCN2018085546-appb-000004
um是羧甲基壳聚糖的单体单元的分子量，为219。
实施例12
在磷酸盐缓冲盐水中制备1.2％羧甲基壳聚糖水凝胶用于粘弹补充
将质量比为1:3的1,4-二氨基-2-丁酮二盐酸盐和羧甲基壳聚糖(Mw＝600,000)溶于蒸馏水中，得到羧甲基壳聚糖浓度为4％(w/v)的溶液，室温机械搅拌4小时。然后向溶液中加入1％(w/v)EDC/NHS，并通过加入HCl 将pH调节至4.5。然后在25℃下机械搅拌12小时。之后将4倍体积的去离子水加入到反应体系中，并且通过加入NaOH将体系的pH调节至10.0。在4℃下搅拌24小时后，在反应体系中滴加3倍量的乙醇，并持续搅拌以得到沉淀产物。用乙醇洗涤沉淀物3次后，将沉淀物溶解在磷酸盐缓冲液中，使得羧甲基壳聚糖的浓度为20mg/mL。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(磷酸盐缓冲液)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的磷酸盐缓冲液以确保最终的羧甲基壳聚糖浓度为12mg/mL。最后将得到的水凝胶通过50μm过滤膜，注入玻璃注射器中，121℃高压灭菌15分钟。至此获得了可用于粘弹补充的1.2％浓度的羧甲基壳聚糖可注射水凝胶。
实施例13
取实施例6中的交联羧甲基壳聚糖粉末200mg加入到10ml 0.9％NaCl溶液中，机械搅拌至粉末完全溶解。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(生理盐水)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的生理盐水以确保最终的羧甲基壳聚糖浓度为12mg/mL。然后将1％(w/w)的曲安奈德溶于上述体系中，再通过50μm过滤膜，装入玻璃注射器中，121℃高压灭菌15分钟。至此获得了可用于粘弹补充的含有1％曲安奈德的1.2％羧甲基壳聚糖可注射水凝胶。
实施例14
粘弹剂注射到兔关节内的关节
将新西兰兔后腿的前后十字韧带切断制作骨关节炎动物模型。两周后，将实施例8的粘弹补充剂注射到兔的关节。对于每只兔子，将3mL粘弹补充剂注射到后腿之一中，并将3mL 0.9％NaCl溶液注射到另一个后腿中作为对照。观察期为1个月和2个月，每次使用6只兔子。2个月后关节切片染色见图8，从II型胶原的免疫组化染色中可看出，粘弹剂组关节面光滑、软骨组织完整，而生理盐水对照组关节面毛糙、软骨上层磨损较严重。
实施例15
含有软骨细胞的粘弹补充剂注入大鼠关节内
切断Sprague Dawley大鼠后腿的前后十字韧带，构建骨关节炎动物模型。2周后，将实施例10中的粘弹补充剂与软骨细胞(从Sprague Dawley大鼠提取)混合以1,000,000/mL的密度注射入大鼠关节。对于每只大鼠，将0.5mL含有软骨细胞的粘弹补充剂注入后腿之一，并将0.5mL 0.9％NaCl溶液注射到另一个后腿作为对照。观察期为1个月和2个月，每次使用10只大鼠。为抑制免疫排斥反应，大鼠每天皮下注射甲基泼尼松龙(2mg/kg)，硫唑嘌呤(2mg/kg)和甲基泼尼松林(2mg/kg)。在体内研究之前，在所述水凝胶上培养软骨细胞以检查其生物相容性。
实施例16
制备1.5％透明质酸水凝胶用于面部填充
将透明质酸(Mw＝1,600,000)溶解于蒸馏水中，得到2％(w/v)透明质酸溶液。然后在溶液中加入0.1％(w/v)二乙烯基砜(DVS)，在45℃下机械搅拌2小时。之后将3倍去离子水加入到反应体系中，并将pH调节至13.0。在45℃下搅拌4小时后，在反应体系中加入4倍量的酒精，不断搅拌，使产物沉淀。用乙醇洗涤沉淀物3次后，将沉淀物在真空干燥箱中干燥72小时至恒重。将2g以上的干沉淀物溶于100mL磷酸盐缓冲液中。然后将水凝胶放入透析袋(膜截留分子量MWCO＝10,000Da)中，将含水凝胶的透析袋放入10倍透析液(磷酸盐缓冲液)中透析4天，每天更换透析液。透析后，检出水凝胶中透明质酸的含量。向水凝胶中加入额外的磷酸盐缓冲液以确保最终的透明质酸浓度为15mg/mL。凝胶经过50μm过滤膜，注入玻璃注射器，121℃高压灭菌20分钟，得到1.5％透明质酸水凝胶用于面部填充。
实施例17
制备含有0.3％利多卡因的1.5％透明质酸水凝胶用于面部填充
步骤与实施例16相同，只是在经过50μm过滤膜之前，将0.3％(w/w)利多卡因溶解在相应的透明质酸水凝胶中。利多卡因可以缓解面部注射时的疼痛。
实施例18
制备1％羧甲基壳聚糖水凝胶用于粘弹补充
将羧甲基壳聚糖(Mw＝400,000)溶于蒸馏水中以获得2％(w/v)的溶液。然后在溶液中加入0.1％(w/v)二乙烯基砜(DVS)，在45℃下机械搅拌4小时。之后将3倍体积的去离子水加入到反应体系中，并且通过加入KOH将pH调节至12.0。在45℃下搅拌2小时后，反应体系的温度在10分钟内降至-20℃，然后在-45℃的冷冻干燥机中预冷冻4小时。之后开启真空泵，开始程序升温(-10℃12小时，-5℃12小时，0℃6小时，5℃4小时，10℃4小时，20℃2个小时)。冻干产物用乙醇洗涤3次后，溶于磷酸盐缓冲液中，使其浓度为20mg/mL。然后将水凝胶放入透析袋(膜截留分子量MWCO＝6,000Da)中，将含水凝胶的透析袋放入10倍透析液(磷酸盐缓冲盐水)中透析4天，每天更换透析液。透析后，根据实施例11中的方法检测水凝胶的浓度。向水凝胶中加入额外的磷酸盐缓冲液以确保最终的羧甲基壳聚糖浓度为10mg/mL。最后将得到的水凝胶通过50μm过滤膜，注入玻璃注射器中，121℃高压灭菌15分钟。至此可获得用于粘弹补充的1％浓度的羧甲基壳聚糖可注射水凝胶。
实施例19
羧甲基壳聚糖支架的制备
将质量比为1:1的二氨基聚乙二醇(Mw＝500)和羧甲基壳聚糖(Mw＝800,000)溶于蒸馏水中，得到羧甲基壳聚糖的6％(w/v)溶液，在室温下搅拌4小时。然后将3％(w/v)DMTMM加入到溶液中。反应在4℃下进行，持续搅拌4小时。然后将2倍去离子水加入到反应体系中并搅拌均匀，加入NaOH将反应体系的pH调节至9.0。然后将反应体系置于冰水浴中持续搅拌2小时，再置于4℃下24小时。然后反应体系的温度在10分钟以内降至-20℃，采用实施例18中方法进行冷冻干燥。用乙醇洗涤冷冻干燥的支架3次后，将支架在真空干燥箱干燥24小时，然后置于戊二醛蒸气下48小时以提高交联程度从而增强模量。之后，将支架在真空干燥箱中干燥72小时以除去残留的戊二醛。
尽管已经用附图和一些优选实施例对本发明进行了说明，但是本领域技 术人员在不脱离本发明的精神和范围的情况下可以容易地掌握本发明的特征并做出一些等同替换。本领域技术人员可以根据本发明做出各种变化和修改，以适应不同的用途。例如，不使用NaOH或KOH，而替换为使用任何水溶性碱处理反应体系。又或者可以使用可注射HA水凝胶来填充手部而不是面部。
此处描述的实施例只用于说明(作为例证)，技术人员所做的各种修改或变更也应包括在专利申请的实质和范围内以及附加权利要求范畴之内。

Claims (37)

  1. 一种水凝胶的制备方法，其特征在于，所述方法包括步骤：
    (1)使多糖和交联剂进行预交联，得到预交联体系；
    (2)使预交联体系在碱性溶液中进一步交联，得到交联体系；
    (3)将交联体系与有机溶剂混合得到沉淀物或将交联体系冷冻干燥得到冻干物；
    (4)将沉淀物或冻干物溶解于缓冲溶液中得到水凝胶。
  2. 如权利要求1所述的制备方法，其特征在于，进行预交联时使用催化剂。
  3. 如权利要求1所述的制备方法，其特征在于，所述交联剂为具有两个以上与多糖中的羧基和/或羟基反应的官能团的物质；多糖上羧基与交联剂所述官能团(氨基)的摩尔比为2:1至20:1；多糖上羟基与交联剂所述官能团的摩尔比为1：1至30：1。
  4. 如权利要求3所述的制备方法，其特征在于，1种所述交联剂分子可至少与多糖上的2个羧基或2个羟基或1个羧基和1个羟基反应。
  5. 如权利要求1所述的制备方法，其特征在于，所述预交联的反应时间为2-96小时，反应温度为0-50℃；优选0-37℃。
  6. 如权利要求1所述的制备方法，其特征在于，所述预交联的反应开始时多糖的添加比例为1％(w/v)-6％(w/v)。
  7. 如权利要求1所述的制备方法，其特征在于，所述多糖选自羧甲基壳聚糖、透明质酸或其衍生物、纤维素、淀粉、葡聚糖、天冬酰胺、精氨酸、硫酸软骨素、硫酸皮肤素、硫酸角蛋白、肝素或其衍生物。
  8. 如权利要求7所述的制备方法，其特征在于，所述羧甲基壳聚糖的分子量介于100kDa至800kDa之间，羧甲基的DS不低于1.0，乙酰胺基的DD不超过20％；所述透明质衍生物选自透明质酸钠或透明质酸钾，所述透明质酸钠或透明质酸钾的分子量介于100kDa至3000kDa之间。
  9. 如权利要求1所述的制备方法，其特征在于，所述交联剂选自下述的一种或一种以上：草酰二肼、胱胺二盐酸盐、1,3-二氨基-2-丙醇、1,4-二氨基-2-丁酮二盐酸盐、1,11-二氨基-3,6,9-三氧杂十一烷、二氨基聚乙二醇、赖氨酸、谷氨酰胺、天冬酰胺、精氨酸、二乙烯基砜、1,4-丁二醇二缩水甘油 基醚、二乙烯基砜(DVS)和1,4-丁二醇二缩水甘油醚(BDDE)。
  10. 如权利要求2所述的制备方法，其特征在于，所述多糖和催化剂的质量比在1:1至10:1。
  11. 如权利要求10所述的制备方法，其特征在于，所述催化剂选自1-乙基-3-(3'-二甲基氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)或4-(4,6-二甲氧基-1,3,5-三嗪-2-基)三嗪-2-基)-4-甲基吗啉鎓氯化物(DMTMM)。
  12. 如权利要求1所述的制备方法，其特征在于，所述碱性溶液为pH9.0-13.5的水溶性碱性溶液。
  13. 如权利要求12所述的制备方法，其特征在于，所述碱性溶液为NaOH或KOH的水溶液。
  14. 如权利要求1所述的制备方法，其特征在于，所述交联体系和预交联体系的体积比在2：1和4：1之间。
  15. 如权利要求1所述的制备方法，其特征在于，所述进一步交联的反应时间为2-24小时，反应温度为0-50℃。
  16. 如权利要求1所述的制备方法，其特征在于，所述有机溶剂与交联体系的体积比不小于3:1。
  17. 如权利要求1所述的制备方法，其特征在于，所述有机溶剂为可溶于水但不能溶解多糖的溶剂。
  18. 如权利要求17所述的制备方法，其特征在于，所述有机溶剂选自C1-3的醇、丙酮或氯仿；优选甲醇或乙醇。
  19. 如权利要求1所述的制备方法，其特征在于，所述缓冲溶液选自磷酸盐缓冲溶液或0.9％NaCl溶液。
  20. 如权利要求1所述的制备方法，其特征在于，所述沉淀物或冻干物溶解于缓冲溶液的溶解浓度在1％和4％(w/v)之间。
  21. 如权利要求1-20任一项所述的制备方法，其特征在于，所述方法还包括步骤：
    (5)将得到的水凝胶进行透析。
  22. 如权利要求21所述的制备方法，其特征在于，所述透析的截留分子量(MWCO)不小于6000。
  23. 如权利要求21所述的制备方法，其特征在于，透析使用的透析液 与步骤(4)中的缓冲溶液相同；所述透析液与水凝胶的体积比不小于8:1。
  24. 如权利要求21所述的制备方法，其特征在于，所述方法还包括步骤：
    (6)将经过透析的水凝胶进行过滤或高压灭菌。
  25. 如权利要求24所述的制备方法，其特征在于，所述高压灭菌的温度为120-125℃，时间为10-30分钟。
  26. 一种水凝胶，其特征在于，所述水凝胶含有多糖和交联剂；以水凝胶的总重量计，其中含有1.0-2.0wt％多糖。
  27. 如权利要求26所述的水凝胶，其特征在于，所述交联剂为具有两个以上与多糖中的羧基和/或羟基反应的官能团的物质。
  28. 如权利要求26所述的水凝胶，其特征在于，所述多糖选自羧甲基壳聚糖、透明质酸钠或透明质酸钾；所述羧甲基壳聚糖的分子量介于100kDa至800kDa之间，羧甲基的DS不低于1.0，乙酰胺基的DD不超过20％；所述透明质酸钠或透明质酸钾的分子量介于100kDa至3000kDa之间。
  29. 如权利要求26所述的水凝胶，其特征在于，所述交联剂选自下述的一种或一种以上：草酰二肼、胱胺二盐酸盐、1,3-二氨基-2-丙醇、1,4-二氨基-2-丁酮二盐酸盐、1,11-二氨基-3,6,9-三氧杂十一烷、二氨基聚乙二醇、赖氨酸、谷氨酰胺、天冬酰胺、精氨酸、二乙烯基砜、1,4-丁二醇二缩水甘油基醚、二乙烯基砜(DVS)和1,4-丁二醇二缩水甘油醚(BDDE)。
  30. 如权利要求26所述的水凝胶，其特征在于，所述水凝胶中还含有药物和/或细胞。
  31. 如权利要求30所述的水凝胶，其特征在于，所述药物为抗炎药或麻醉剂；所述细胞选自软骨细胞或间充质干细胞。
  32. 如权利要求31所述的水凝胶，其特征在于，所述抗炎药选自曲安奈德、布洛芬和/或双氯芬酸)；所述麻醉剂为利多卡因。
  33. 如权利要求26-32所述的水凝胶，其特征在于，所述水凝胶具有取向层状结构。
  34. 如权利要求33所述的水凝胶，其特征在于，所述水凝胶具有可注射性和热稳定性。
  35. 如权利要求26-29、33或34所述的水凝胶，其特征在于，所述水凝胶通过如权利要求1-25任一项所述的制备方法获得。
  36. 一种如权利要求26-35任一项所述的水凝胶的用途，其特征在于，所述水凝胶用于制备可用于粘弹补充的试剂或用作粘弹补充剂；或用于制备药物输送系统、可用于组织工程的支架或可填充的软组织。
  37. 如权利要求36所述的用途，其特征在于，所述粘弹补充剂包含如权利要求26-35任一项所述的水凝胶和抗炎药、麻醉剂或细胞；所述抗炎药选自曲安奈德、布洛芬和/或双氯芬酸)；所述麻醉剂为利多卡因；所述细胞选自软骨细胞或间充质干细胞。
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WO2021127807A1 (en) * 2019-12-23 2021-07-01 Evonik Industries Ag Dual-crosslinked hydrogel and preparation method thereof
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