WO2017084300A1 - 透明质酸‐甲基纤维素复合凝胶及其制备和应用 - Google Patents

透明质酸‐甲基纤维素复合凝胶及其制备和应用 Download PDF

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WO2017084300A1
WO2017084300A1 PCT/CN2016/084522 CN2016084522W WO2017084300A1 WO 2017084300 A1 WO2017084300 A1 WO 2017084300A1 CN 2016084522 W CN2016084522 W CN 2016084522W WO 2017084300 A1 WO2017084300 A1 WO 2017084300A1
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hyaluronic acid
methylcellulose
methyl cellulose
added
stirring
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PCT/CN2016/084522
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French (fr)
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蔡强
卓凡露
高琴
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清华大学
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Priority claimed from CN201510810221.XA external-priority patent/CN105330902A/zh
Priority claimed from CN201610065326.1A external-priority patent/CN105504316B/zh
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Publication of WO2017084300A1 publication Critical patent/WO2017084300A1/zh

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    • 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
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/08—Cellulose derivatives
    • C08L1/26—Cellulose ethers
    • C08L1/28—Alkyl ethers

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  • the invention belongs to the technical field of biomedical materials and tissue engineering, and particularly relates to a hyaluronic acid-methyl cellulose composite gel and a preparation and application thereof.
  • Hyaluronic Acid is a linear polysaccharide widely distributed in the extracellular matrix, with excellent non-antigenicity and cytocompatibility, and its high viscoelasticity makes it suitable for clinical applications. It has good separation, protection and filling functions and is a popular natural polymer material. When used for repairing damaged nerves in the body, hyaluronic acid is often used in combination with other natural or synthetic materials because of its high water solubility, inability to maintain normal body morphology, and excessive degradation rate.
  • Hybrid hyaluronic acid-methylcellulose complex coagulation can be formed by complex shearing of dilute hyaluronic acid and Methyl Cellulose (MC). Glue (hereinafter referred to as HAMC).
  • HAMC is a sol prior to injection and the gel strength is increased after injection due to an increase in body temperature relative to room temperature.
  • the HAMC complex has a lower gelation temperature and a smaller thixotropic ring when compared to methylcellulose alone, and these effects are attributed to the anionic carboxylate groups of hyaluronic acid.
  • HAMC is non-cell-adherent and has a degradable Sexual and biocompatible in the intrathecal space.
  • the polyethylene glycol molecule is esterified, and then the hyaluronic acid is acetylated in an alkaline environment to produce an amino group, and the two are covalently reacted at room temperature to produce hyaluronic acid-polyethylene glycol derivative (HA). ⁇ PEG).
  • the rheological properties of the derivative material were tested.
  • the present invention provides a hyaluronic acid-methyl cellulose composite gel and its preparation and application.
  • a hyaluronic acid-methylcellulose composite gel comprising methyl cellulose and hyaluronic acid, or methyl cellulose and polyethylene glycol succinimide succinic anhydride
  • the crosslinked active ester obtained by the reaction of acetylated hyaluronic acid has a crosslinked network structure having a pore diameter of from 30 ⁇ m to 100 ⁇ m.
  • the present invention relates to a method for preparing the above hyaluronic acid-methylcellulose composite gel, and the specific steps are as follows:
  • hyaluronic acid is added to the methyl cellulose solution, stirred and dissolved, and a crosslinking agent is added thereto; after the system is uniformly mixed, the pH is adjusted to 7.4 with a NaOH solution, and stirring is continued for 2 hours to obtain a coagulation. A gelatinous substance; the material was refrigerated overnight and lyophilized to obtain a hyaluronic acid-methylcellulose composite hydrogel.
  • the crosslinking agent is adipic acid dihydrazide or polyethylene glycol.
  • the mass ratio of methyl cellulose, sodium chloride, and hyaluronic acid is (5 to 11): 4:1 in this order.
  • the temperature of the ice salt bath is ⁇ 4° C.
  • the stirring time in the ice salt bath is 40 min
  • the heating temperature of the first artificial cerebrospinal fluid is 80 to 90° C.
  • step (2) hyaluronic acid is slowly added in portions.
  • the rotation speed at the time of stirring was 200 rpm to 300 rpm, and the temperature at the time of refrigerating overnight was 4 °C.
  • the hyaluronic acid-methylcellulose composite hydrogel has a gel temperature of less than 37 ° C, a small thixotropic ring area, and is injectable.
  • the degradation rate of the hyaluronic acid-methylcellulose composite hydrogel is continuously reduced, and the total degradation rate in three weeks is 40 to 70%.
  • a method for preparing the above hyaluronic acid-methylcellulose composite gel wherein polyethylene glycol succinimide succinic anhydride is reacted with deacetylated hyaluronic acid to obtain a crosslinked active ester, The crosslinked active ester is crosslinked with methylcellulose to form a hyaluronic acid-methylcellulose composite gel.
  • the specific operation can be:
  • polyethylene glycol succinimide succinic anhydride is formulated into an aqueous solution, added to the deacetylated hyaluronic acid solution, stirred to uniformly mix the solution, reacted at room temperature for 12 h, and then added to the solution with a mass fraction of 95%. Ethanol to precipitate is completely produced, the precipitate is collected by filtration, and the precipitate is washed with ethanol for 3 times or more, and dried under vacuum to obtain a crosslinked active ester;
  • the mass ratio of sodium chloride, sodium phosphate, methyl cellulose, and deacetylated hyaluronic acid is (2 to 4): (0 to 0.05): 7:1.
  • the concentration of the aqueous solution of polyethylene glycol succinimide succinic anhydride is 1 ⁇ 10 -3 mol / L, and the volume ratio of the solution to the deacetylated hyaluronic acid solution is 1:10.
  • the preparation method of the polyethylene glycol succinimide succinic anhydride may be:
  • the molar ratio of polyethylene glycol, succinic anhydride, pyridine, N-hydroxysuccinimide, and dicyclohexylcarbodiimide is 1:1:0.695:2.453:2.563; polyethylene glycol and each solvent
  • the temperature at the time of reflux stirring is 100 ° C, and the stirring time is 4 h; in the steps (1) and (2), the stirring time of the two ice baths is: the first stirring for 30 min, the second Stir for 15 min.
  • the method for preparing the deacetylated hyaluronic acid is: adding a sodium hydroxide solution to an aqueous solution of hyaluronic acid, stirring slowly, allowing the hyaluronic acid to fully contact with the sodium hydroxide solution, and reacting for 2 hours. Deacetylated hyaluronic acid is obtained.
  • the concentration of the aqueous hyaluronic acid solution is 9.8 ⁇ 10 ⁇ 3 mol/L, and the concentration of the sodium hydroxide solution is 1 mol/L; the addition amount of the sodium hydroxide solution is adjusted to a pH value of 12.65 ⁇ 0.05.
  • polyethylene glycol forms polyethylene glycol succinimide succinic anhydride under the action of succinic anhydride and N-hydroxysuccinimide, and then reacts with a deacetylated hyaluronic acid solution to obtain a crosslinked active ester.
  • the co-active ester is reacted with methylcellulose to give the final product, a composite gel.
  • the composite gel maintains the spatial porous structure, good mechanical properties and biocompatibility of the original hyaluronic acid-methylcellulose gel, and reduces its degradation through structural modification from the viewpoint of hyaluronic acid molecules.
  • the rate increases the retention time of the body, so the proportion of methylcellulose component originally used to delay degradation in the composite gel is reduced, so that the gel has better injectability and rheological properties, which is beneficial to the damaged central nervous system.
  • Minimally invasive repair is a method for a deacetylated hyaluronic acid solution.
  • the invention attempts to change the form of a single sodium chloride additive, and a small amount of sodium phosphate is added to reduce the amount of sodium chloride.
  • the method maintains the gelation ability of the composite gel at body temperature, and has an osmotic pressure environment closer to human body fluid, which reduces cell stimulation and obtains better cell compatibility.
  • the invention provides a hyaluronic acid-methyl cellulose composite by physically crosslinking a methyl cellulose having a temperature-sensitive property, a good biocompatibility and a slow degradation in the body, and a hyaluronic acid molecule through polyethylene glycol. Hydrogels.
  • the composite hydrogel has a spatial network structure, and by controlling the concentration of methyl cellulose and the salt additive, the function of the hyaluronic acid-methyl cellulose composite hydrogel to form a gel at body temperature can be realized, and the mechanics can be improved.
  • the performance can control the release of bioactive molecules, prolong the retention time of the gel in vivo, and is beneficial to the regeneration and repair of soft tissue similar to nerve tissue, and has broad application prospects.
  • Both hyaluronic acid and methylcellulose are polymers derived from the organism itself. Because both have natural biocompatibility and non-antigenicity, the combination of the two is injectable and can be fast at a certain temperature. The gel, and complemented, the biodegradability of the composite hydrogel will be superior.
  • the preparation method of the invention has simple preparation process, good repeatability and is suitable for mass production.
  • Example 1 is a microscopic topographical view of the composite gel of Example 1 under scanning electron microscopy.
  • Fig. 2 is a graph showing G'(?) and G"(?) curves of the composite gel of Example 1 at 37 °C.
  • Figure 3 is a graph showing the degradation of the composite gel in Example 1 within 3 weeks.
  • Figure 4 is a graph showing the degradation of a composite gel with 3.75 wt% NaCl added in different cross-linking modes within 3 weeks.
  • Figure 5 is a graph showing the degradation of a composite gel with 4 wt% NaCl added in 3 weeks under different cross-linking modes.
  • Figure 6 is a topographical view of the hyaluronic acid-methylcellulose composite hydrogel of Example 4.
  • Fig. 7 is a G'(T) and G"(T) curve of the hyaluronic acid-methylcellulose composite hydrogel of Example 4.
  • Fig. 8 is a graph showing G'( ⁇ ), G"( ⁇ ) and ⁇ *( ⁇ ) curves of the hyaluronic acid-methylcellulose composite hydrogel of Example 4 at 37 °C.
  • Figure 9 is a graph showing the thixotropic ring test curve of the hyaluronic acid-methylcellulose composite hydrogel of Example 4.
  • Figure 10 is a graph showing the degradation of hyaluronic acid-methylcellulose composite hydrogel of different methylcellulose concentrations in Example 4 within three weeks.
  • Figure 11 is a scanning electron microscope topography of the hyaluronic acid-methylcellulose composite hydrogel of Example 4 after three weeks of degradation.
  • Figure 12 is a topographical view of the hyaluronic acid-methylcellulose composite hydrogel of Example 5.
  • Figure 13 is a topographical view of the hyaluronic acid-methylcellulose composite hydrogel of Example 6.
  • Figure 14 is a topographical view of the hyaluronic acid-methylcellulose composite hydrogel of Example 7.
  • Figure 15 is a topographical view of the hyaluronic acid-methylcellulose composite hydrogel of Example 8.
  • the polyethylene glycol succinimide succinic anhydride is formulated into an aqueous solution having a concentration of 1 ⁇ 10 ⁇ 3 mol/L, and 2 mL is added to 20 mL of the deacetylated hyaluronic acid solution, and the solution is uniformly mixed by stirring. After reacting for 12 h, add 95% by mass of ethanol to the solution to complete the precipitation, collect the precipitate by filtration, wash the precipitate with ethanol for more than 3 times, and dry in vacuo to obtain a cross-linked active ester;
  • the microscopic morphology of the composite gel is shown in Fig. 1.
  • the composite gel of the present invention exhibits a porous structure at a micron scale, and has connectivity between the pores, and the pore diameter ranges from 40 ⁇ m to 60 ⁇ m.
  • Figure 2 shows the rheological test results of the un-freeze-dried composite gel at 37 °C. It can be seen from the figure that as the frequency increases, the storage modulus G' and the loss modulus G" of the gel gradually increase, and a certain linear relationship is satisfied in the logarithmic coordinates.
  • This rheological characteristic and The brain tissue is similar, indicating that the composite gel belongs to a pseudoplastic fluid and has injectability; G' is always greater than G", indicating that the gel has a solid-like property at 37 ° C, and has a certain mechanical strength after gel formation in the body.
  • the temperature rising process shows that the gelation temperature of the composite gel is 24.3 ° C, which meets the requirements of gel formation at body temperature.
  • the degradation rate of the composite gel within 3 weeks is shown in Figure 3. From the 7th day, the degradation rate of the gel is basically stable at 60%, indicating that the crosslinking activity is controlled by the degradation rate.
  • the ester has a certain reduction in the content of methylcellulose (in this case, the methylcellulose content is 7wt%) (the same level of degradation is maintained when no crosslinking agent is used or only unactivated polyethylene glycol is used). Generally, 9 to 11% by weight of methyl cellulose is required.
  • the amount of sodium chloride added was 1.125 g, and sodium phosphate was not added, and the other operations were the same as in the first embodiment.
  • Figure 4 shows the change of in vitro degradation rate (PEG-SS curve) of the composite gel within 3 weeks in the present example.
  • the gel degrades faster in the first week, and then the degradation rate slows down and remains in the range of 50-60%.
  • the degradation rate is significantly lower in the long run by cross-linking the active ester. The retention is better, which is beneficial to the sustained release of the loaded active molecules.
  • the amount of sodium chloride added was 1.2 g, and the other operations were the same as in the second embodiment.
  • Figure 5 shows the change of in vitro degradation rate (PEG-SS curve) of the composite gel within 3 weeks in the present example.
  • the final degradation rate of the gel in the third week is still less than 60%, and the retention is better than the unused crosslinking agent.
  • Pure curve and a gel crosslinked only with unactivated polyethylene glycol (PEG curve).
  • hyaluronic acid was purchased from Huaxi Furida Biomedical Co., Ltd.; bovine serum albumin was purchased from Spain; methylcellulose M20, polyethylene glycol, sodium hydroxide, hydrochloric acid, chlorine Sodium, anhydrous calcium chloride, potassium chloride, magnesium chloride, sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd.; adipic acid dihydrazide, carbodiimide purchased from Aladdin reagent company .
  • the morphology of the composite hydrogel is shown in Fig. 6. It can be seen that the hyaluronic acid-methylcellulose composite hydrogel exhibits a highly porous structure at a micrometer scale, and the pores are connected to each other with an average pore diameter of about 40 ⁇ m. 60 ⁇ m.
  • the measured thixotropic ring is shown in Fig. 9.
  • ⁇ when the size reaches a certain range, the viscosity of the sample begins to decrease rapidly until it is close to 0; when the shear stress decreases, the viscosity decreases. Gradually pick up and increase sharply when the shear stress is small, returning to the initial viscosity value.
  • the two processes do not coincide, and the data points enclose a shuttle ring having a certain area.
  • the thixotropic ring reflects the ability of the fluid to return to its original state after being deformed by the shear force. The smaller the area enclosed by the ring, the shorter the time required for fluid recovery and the better the injectability.
  • the presence of the thixotropic ring is also a good proof that the methylcellulose imparts "shear-thinning" properties to the composite hydrogel and can therefore be used for injection.
  • the degradation curve obtained by simulating the in vitro degradation test of the composite hydrogel is shown in Fig. 10 as "11% MC".
  • the sample degraded rapidly in the first week, and became slower in the second and third weeks, which was consistent with the experimental expectation.
  • the sample has a final degradation rate of about 50%, which is a certain improvement (about 60%) than the results reported in the previous literature, which is beneficial to the controlled release of biologically active molecules.
  • Figure 11 is a scanning electron microscope topography after three weeks of degradation.
  • the average pore diameter was observed from the scanning electron microscope photograph of Fig. 13 to be 80 ⁇ m to 100 ⁇ m.
  • the in vitro degradation test showed that after three weeks of degradation, the final degradation rate was about 40% (Fig. 10 "5% MC" curve).

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Abstract

一种透明质酸-甲基纤维素复合凝胶，由甲基纤维素和透明质酸，或甲基纤维素与聚乙二醇琥珀酰亚胺琥珀酸酐和去乙酰化透明质酸反应得到的交联活性酯交联而得到，所述复合凝胶具有交联网络结构，孔径为30μm～100μm。

Description

透明质酸‐甲基纤维素复合凝胶及其制备和应用
本申请要求在2015年11月20日提交中国专利局、申请号为201510810221.X、发明名称为“一种透明质酸‐甲基纤维素复合水凝胶及其制备和应用”的中国专利申请的优先权，以及在2016年01月29日提交中国专利局、申请号为201610065326.1、发明名称为“一种透明质酸‐甲基纤维素复合凝胶的活性酯交联方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医用材料及组织工程技术领域，具体涉及一种透明质酸‐甲基纤维素复合凝胶及其制备和应用。
背景技术
透明质酸(Hyaluronic Acid，HA)是一种线性多糖，广泛存在于细胞外基质中，具有优异的非抗原性和细胞相容性等，同时其溶液的高粘弹性使之在临床医学领域应用中具有良好的分离、保护、充填作用，是一种备受青睐的天然高分子材料。当用于体内的受损神经修复时，透明质酸存在水溶性过高、不能维持正常的体内形态以及降解速度过快等不足，因此常与其它天然或人工合成材料结合使用。
通过复合剪切自稀的透明质酸与热逆性的甲基纤维素(Methyl Cellulose，MC)可以形成一种快速凝胶化的可注射凝胶——透明质酸‐甲基纤维素复合凝胶(以下简称HAMC)。HAMC在注射前是一种溶胶，在注射后由于体内温度相对于室温温度的升高，凝胶强度增强。HAMC复合物相比于甲基纤维素单独存在时拥有更低的凝胶化温度和较小的触变环，这些影响归因于透明质酸的阴离子羧酸盐基团。除了可以快速凝胶化以外，HAMC是非细胞粘附的，具有可降解 性，并在鞘内空间具备生物相容性。
研究结果表明，相对于透明质酸自身，经过了化学修饰后得到的透明质酸衍生物，其化学稳定性将会提高，抗降解性能也将被改善，在动物体内的半衰期有所延长，但其生物相容性以及生物活性不会有大的改变。将聚乙二醇分子进行酯化，再使透明质酸在碱性环境中经乙酰化处理产生氨基，二者于室温下共价反应即可产生透明质酸‐聚乙二醇衍生物(HA‐PEG)。对该衍生物材料进行流变性能的测试，结果显示，改性后的透明质酸衍生物材料属于非牛顿流体，具有高聚物的粘弹性，符合医用生物材料要求。
发明内容
为了解决现有技术中的弊端和不足，本发明提供一种透明质酸‐甲基纤维素复合凝胶及其制备和应用。
作为本发明的一个方面，涉及一种透明质酸‐甲基纤维素复合凝胶，由甲基纤维素和透明质酸，或甲基纤维素与聚乙二醇琥珀酰亚胺琥珀酸酐和去乙酰化透明质酸反应得到的交联活性酯交联，所述复合凝胶具有交联网络结构，孔径为30μm～100μm。
作为本发明的另一个方面，涉及制备上述透明质酸‐甲基纤维素复合凝胶的方法，具体步骤如下：
(1)在搅拌条件下，将第一份人工脑脊液加入锥形瓶中，加热，并向其中加入氯化钠；待完全溶解后，加入甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，加入第二份人工脑脊液，置于冰盐浴中，搅拌，得到甲基纤维素溶液，冷藏过夜；
(2)至少15h后，向甲基纤维素溶液中加入透明质酸，搅拌溶解，向其中加入交联剂；待体系混合均匀后，用NaOH溶液将pH调节为7.4，继续搅拌2h，得到凝胶状物质；将该物质冷藏过夜，并对其进行冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶。
具体地，所述交联剂为己二酸二酰肼或者聚乙二醇。
具体地，甲基纤维素、氯化钠、透明质酸的质量之比依次为(5～11)：4：1。
具体地，步骤(1)中，所述冰盐浴的温度为‐4℃，在冰盐浴中的搅拌时间为40min；第一份人工脑脊液的加热温度为80～90℃。
具体地，步骤(2)中，缓慢分次加入透明质酸。
具体地，步骤(1)和步骤(2)中，搅拌时的转速均为200rpm～300rpm，冷藏过夜的温度均为4℃。
作为本发明的再一方面，涉及上述透明质酸‐甲基纤维素复合凝胶在软组织修复方面的应用。
具体地，所述透明质酸‐甲基纤维素复合水凝胶的凝胶温度低于37℃，触变环面积小，具有可注射性。
具体地，在三周内，所述透明质酸‐甲基纤维素复合水凝胶的降解速率不断变小，三周的总降解率为40～70％。
作为本发明的又一方面，涉及制备上述透明质酸‐甲基纤维素复合凝胶的方法，聚乙二醇琥珀酰亚胺琥珀酸酐和去乙酰化透明质酸反应得到交联活性酯，所述交联活性酯与甲基纤维素交联，形成透明质酸‐甲基纤维素复合凝胶。
具体操作可以是：
(1)将预先配制好的第一份人工脑脊液加热至90℃，加入一定量的氯化钠、磷酸钠和甲基纤维素，搅拌均匀后再加入等体积的第二份人工脑脊液，‐4℃冰盐浴中搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜；
(2)将聚乙二醇琥珀酰亚胺琥珀酸酐配制成水溶液，加入去乙酰化透明质酸溶液中，搅拌使溶液混合均匀，室温下反应12h，再向溶液中加入质量分数为95％的乙醇至沉淀产生完全，过滤收集沉淀，用乙醇洗涤沉淀3次以上，真空干燥，得到交联活性酯；
(3)称取一定量的交联活性酯加入甲基纤维素溶液中，调节pH至7.4，搅拌 2h以上使交联活性酯充分分散且溶解均匀，4℃冷藏过夜，并对其冷冻干燥，得到透明质酸‐甲基纤维素复合凝胶。
具体地，氯化钠、磷酸钠、甲基纤维素、去乙酰化透明质酸的质量之比依次为(2～4)：(0～0.05)：7：1。
具体地，步骤(2)中，聚乙二醇琥珀酰亚胺琥珀酸酐水溶液的浓度为1×10‐3mol/L，与去乙酰化透明质酸溶液的体积比为1：10。
具体地，所述聚乙二醇琥珀酰亚胺琥珀酸酐的制备方法可以为：
(1)将分子量为6000的聚乙二醇，溶解于二氧六环中，以吡啶作为催化剂，加入琥珀酸酐，加热状态下回流搅拌，反应完毕后冷却，在快速搅拌下滴加无水乙醚至沉淀不再产生，之后置于冰浴中搅拌，过滤后将沉淀充分溶解于二氯甲烷中，再次滴加无水乙醚至沉淀不再产生，冰浴搅拌，将过滤得到的沉淀真空干燥；
(2)将步骤(1)得到的沉淀与N‐羟基琥珀亚酰胺溶于含有二环己基碳化二亚胺的N,N‐二甲基甲酰胺中，搅拌12h后，在快速搅拌下滴加无水乙醚至沉淀不再产生，之后置于冰浴中搅拌，过滤后将沉淀充分溶解于二氯甲烷中，再次滴加无水乙醚至沉淀不再产生，冰浴搅拌，过滤，用无水乙醚多次洗涤，将沉淀进行真空干燥，得到所述的聚乙二醇琥珀酰亚胺琥珀酸酐。
具体地，聚乙二醇、琥珀酸酐、吡啶、N‐羟基琥珀亚酰胺、二环己基碳化二亚胺的摩尔比依次为1：1：0.695：2.453：2.563；聚乙二醇与各溶剂的质量体积比为聚乙二醇：二氧六环：步骤(1)中所用的二氯甲烷：步骤(2)中所用的二氯甲烷：N,N‐二甲基甲酰胺＝0.48g：20mL：30mL：30mL：1mL。
具体地，步骤(1)中，回流搅拌时的温度为100℃，搅拌时间为4h；步骤(1)和步骤(2)中，两次冰浴搅拌的时间均为：首次搅拌30min，第二次搅拌15min。
具体地，所述去乙酰化透明质酸的制备方法为：向透明质酸的水溶液中滴加氢氧化钠溶液，缓慢搅拌，使透明质酸与氢氧化钠溶液充分接触，反应2h， 得到去乙酰化透明质酸。
具体地，所述透明质酸水溶液的浓度为9.8×10‐3mol/L，氢氧化钠溶液的浓度为1mol/L；氢氧化钠溶液的添加量以调节pH值至12.65±0.05为准。
本发明至少实现了如下有益效果：
本发明中，聚乙二醇在琥珀酸酐和N‐羟基琥珀酰亚胺作用下形成聚乙二醇琥珀酰亚胺琥珀酸酐，然后与去乙酰化透明质酸溶液反应得到交联活性酯，交联活性酯与甲基纤维素反应得到终产物——复合凝胶。所述复合凝胶保持了原透明质酸‐甲基纤维素凝胶的空间多孔结构、良好的力学性能和生物相容性，且从透明质酸分子的角度出发，通过结构改造降低了其降解速率，延长了体内存留时间，因此复合凝胶中原本用于延缓降解的甲基纤维素成分比例得以减少，使凝胶具有更好的注射性和流变学性能，有利于受损中枢神经的微创修复。
本发明在对透明质酸‐甲基纤维素复合凝胶成胶温度的调控中，尝试改变单一的氯化钠添加剂形式，加入少量磷酸钠以减少氯化钠的用量。该方法使复合凝胶保持了体温下成胶能力，并具有更接近人体体液的渗透压环境，减少对细胞的刺激，获得更好的细胞相容性。
本发明通过将具有温敏特性、生物相容性良好、体内降解较缓慢的甲基纤维素与透明质酸分子通过聚乙二醇进行物理交联反应，得到透明质酸‐甲基纤维素复合水凝胶。所述复合水凝胶具有空间网络状结构，通过控制甲基纤维素以及盐类添加剂的浓度，既可以实现透明质酸‐甲基纤维素复合水凝胶在体温下成胶的功能，提高力学性能，又能对生物活性分子进行控制释放，延长凝胶的体内留存时间，有利于类似于神经组织的软组织的再生修复，具有广阔的应用前景。
透明质酸和甲基纤维素均为来源于生物自身的聚合物，因两者均具有天然的生物相容性和非抗原性，两者的复合物具备可注射性，在一定温度下能够快速凝胶，且互补之下复合水凝胶的生物降解性将更加优越。
本发明所述的制备方法制备工艺简单，重复性好，适合批量生产。
附图说明
图1为实施例1中复合凝胶在扫描电镜下的微观形貌图。
图2为实施例1中复合凝胶在37℃下的G’(ω)和G”(ω)曲线图。
图3为实施例1中复合凝胶在3周内的降解情况曲线图。
图4为不同交联方式下添加3.75wt％NaCl的复合凝胶在3周内的降解情况曲线图。
图5为不同交联方式下添加4wt％NaCl的复合凝胶在3周内的降解情况曲线图。
图6为实施例4中透明质酸‐甲基纤维素复合水凝胶的形貌图。
图7为实施例4中透明质酸‐甲基纤维素复合水凝胶的G’(T)和G”(T)曲线。
图8为实施例4中透明质酸‐甲基纤维素复合水凝胶在37℃下的G’(ω)、G”(ω)和η*(ω)曲线。
图9为实施例4中透明质酸‐甲基纤维素复合水凝胶的触变环测试曲线。
图10为实施例4不同甲基纤维素浓度的透明质酸‐甲基纤维素复合水凝胶在三周内的降解情况曲线。
图11为实施例4中透明质酸‐甲基纤维素复合水凝胶经过三周降解后的扫描电镜形貌图。
图12为实施例5中透明质酸‐甲基纤维素复合水凝胶的形貌图。
图13为实施例6中透明质酸‐甲基纤维素复合水凝胶的形貌图。
图14为实施例7中透明质酸‐甲基纤维素复合水凝胶的形貌图。
图15为实施例8中透明质酸‐甲基纤维素复合水凝胶的形貌图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人 员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
(一)制备聚乙二醇琥珀酰亚胺琥珀酸酐(PEG‐SS)：
(1)将2.4g聚乙二醇(分子量为6000)溶解于100mL二氧六环中，加入0.022g吡啶作为催化剂，再加入0.04g琥珀酸酐，在100℃下回流搅拌4h，反应完毕后冷却，在快速搅拌下滴加无水乙醚至沉淀不再产生，之后置于冰浴中搅拌30min，过滤后将沉淀充分溶解于150mL二氯甲烷中，再次滴加无水乙醚至沉淀不再产生，冰浴搅拌15min，将过滤得到的沉淀真空干燥；
(2)将步骤(1)得到的沉淀与0.1129g N‐羟基琥珀亚酰胺溶于含有0.2114g二环己基碳化二亚胺的5mL N,N‐二甲基甲酰胺中，搅拌12h后，在快速搅拌下滴加无水乙醚至沉淀不再产生，之后置于冰浴中搅拌30min，过滤后将沉淀充分溶解于150mL二氯甲烷中，再次滴加无水乙醚至沉淀不再产生，冰浴搅拌15min，过滤，用无水乙醚多次洗涤，将沉淀进行真空干燥，得到聚乙二醇琥珀酰亚胺琥珀酸酐。
(二)制备去乙酰化透明质酸：
向9.8×10‐3mol/L的透明质酸水溶液中滴加1mol/L的NaOH溶液，利用pH计检测使溶液pH值为12.65±0.05，缓慢搅拌，使透明质酸与氢氧化钠溶液充分接触，反应2h，得到去乙酰化透明质酸。
(三)透明质酸‐甲基纤维素复合凝胶的活性酯交联方法：
(1)取预先配制好的第一份人工脑脊液15mL，加热至90℃，加入0.6g氯化钠、0.015g磷酸钠和2.1g甲基纤维素，搅拌均匀后再加入15mL第二份人工脑脊液，‐4℃冰盐浴中搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜；
(2)将聚乙二醇琥珀酰亚胺琥珀酸酐配制成浓度为1×10‐3mol/L的水溶液，取2mL加入20mL去乙酰化透明质酸溶液中，搅拌使溶液混合均匀，室温下反 应12h，再向溶液中加入质量分数为95％的乙醇至沉淀产生完全，过滤收集沉淀，用乙醇洗涤沉淀3次以上，真空干燥，得到交联活性酯；
(3)称取0.3g交联活性酯加入30mL甲基纤维素溶液中，调节pH至7.4，搅拌2h以上使交联活性酯充分分散且溶解均匀，4℃冷藏过夜，并对其冷冻干燥，得到透明质酸‐甲基纤维素复合凝胶。
复合凝胶的微观形貌如图1所示，微米尺度下本发明所述的复合凝胶呈现出多孔结构，且孔隙间有连通性，孔径范围在40μm～60μm。
图2给出了未冷冻干燥的复合凝胶在37℃下的流变测试结果。从图中可以看出，随着频率的增大，凝胶的存储模量G’和损失模量G”均逐渐增大，在对数坐标中满足一定的线性关系，此流变学特征与脑组织相似，说明复合凝胶属于假塑性流体，具备可注射性；G’始终大于G”，说明凝胶在37℃时具有类固体性质，体内成胶后具有一定机械强度。升温过程显示复合凝胶的成胶温度为24.3℃，满足体温下成胶的需求。
体外降解实验中，复合凝胶在3周内的降解率变化如图3所示，从第7天起凝胶的降解率基本稳定在60％，说明在降解速度的控制上，采用交联活性酯为甲基纤维素含量的降低(本例中甲基纤维素含量为7wt％)起到了一定的弥补作用(不加交联剂或仅用未活化聚乙二醇时，维持同等降解率水平一般需要9～11wt％的甲基纤维素)。
实施例2
步骤(三)的第(1)步中，氯化钠加入量为1.125g，未加入磷酸钠，其他操作均与实施例1相同。
图4为本实施例中复合凝胶3周内的体外降解率变化情况(PEG‐SS曲线)，凝胶在第1周降解较快，之后降解速度放缓并维持在50～60％的范围内。对比未使用交联剂(Pure曲线)和仅用未活化聚乙二醇交联(PEG曲线)的凝胶，从长期来看，通过交联活性酯进行交联，其降解率明显更低，存留情况较好，有利于所负载的活性分子的持续释放。
实施例3
步骤(三)的第(1)步中，氯化钠加入量为1.2g，其他操作均与实施例2相同。
图5为本实施例中复合凝胶3周内的体外降解率变化情况(PEG‐SS曲线)，第3周凝胶的最终降解率仍低于60％，存留情况优于未使用交联剂(Pure曲线)和仅用未活化聚乙二醇交联(PEG曲线)的凝胶。
本发明所使用的试剂中，透明质酸购买于华熙福瑞达生物医药有限公司；牛血清白蛋白购买于Spain公司；甲基纤维素M20、聚乙二醇、氢氧化钠、盐酸、氯化钠、无水氯化钙、氯化钾、氯化镁、磷酸二氢钠以及磷酸氢二钠均购买于国药集团化学试剂有限公司；己二酸二酰肼、碳化二亚胺购买于Aladdin试剂公司。
实施例4
(1)在磁力搅拌(转速300rpm)下，将50mL预先配制好的人工脑脊液加入锥形瓶中，在恒温油浴锅中加热至90℃，向其中加入4g氯化钠；待完全溶解后，加入11g甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，再加入50mL人工脑脊液，置于‐4℃冰盐浴中，以200rpm的转速搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜。
(2)24h后向甲基纤维素溶液中缓慢分次加入1g透明质酸粉末，搅拌溶解，向其中逐滴加入0.4g/L的PEG溶液2mL；用1M NaOH溶液将pH调节为约7.4后，继续搅拌2h，得到凝胶状物质；将该物质在4℃下冷藏过夜，并对其进行真空冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶。
复合水凝胶形貌如图6所示，可以看出透明质酸‐甲基纤维素复合水凝胶在微米尺度下表现出高度疏松的多孔结构，这些孔隙相互连通，平均孔径约为40μm～60μm。
复合水凝胶流变性能测试如图2～4所示。图7中，随着温度上升，样品的存储模量G’和损失模量G”均增大，G’从几十Pa增到约1000Pa，而G”从 几十Pa只提高到100Pa上下，增长速度明显不如G’快。各样品的两条曲线均交于30℃以下，满足37℃成胶要求。
由图8可知G’和G”随着频率的增大而增大，损失模量G”约在100～600Pa的范围内，存储模量G’则从约400Pa上升至1000Pa以上，始终比G”大得多，再次说明凝胶在37℃下具有典型的类固体特性。粘度η*随频率的增大而减小，从几百降到几十Pa s，且在对数坐标中有明显的线性关系，是甲基纤维素溶液作为假塑性流体“剪切变稀”性质的体现。透明质酸‐甲基纤维素复合水凝胶的模量特点与脑组织相似，但脑组织的粘度不随频率变化，这一点有所不同。
测得的触变环如图9所示，在剪切应力τ增大的过程里，其大小达到某一范围时，样品粘度开始迅速降低，直至接近于0；当剪切应力减小时粘度又逐渐回升，并在剪切应力很小的时候剧增，恢复到一开始的粘度值。两个过程不重合，数据点围成一个具有一定面积的梭型环。触变环反映的是流体受剪切力发生形变后恢复原状的能力，环围成的面积越小，表明流体恢复所需的时间越短，可注射性越好。同时触变环的存在也是甲基纤维素使复合水凝胶拥有“剪切变稀”性质，因而能被用于注射的很好的证明。
通过模拟复合水凝胶的体外降解试验，试验研究，得到的降解曲线如图10中“11％MC”所示。从图中可看出，样品在第1周内降解迅速，第2、3周则变得较为缓慢，与实验的预期是相符的。样品在经过三周的降解后最终降解率约50％，比以往文献报道中的结果有了一定的改善(约60％)，有利于对生物活性分子的控制释放。图11为经过三周降解后的扫描电镜形貌图。
实施例5
(1)在磁力搅拌(转速300rpm)下，将50mL预先配制好的人工脑脊液加入锥形瓶中，在恒温油浴锅中加热至90℃，向其中加入4g氯化钠；待完全溶解后，加入9g甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，再加入50mL人工脑脊液，置于‐4℃冰盐浴中，以200rpm的转速 搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜。
(2)24h后向甲基纤维素溶液中缓慢分次加入1g透明质酸粉末，搅拌溶解，向其中逐滴加入0.4g/L PEG溶液2mL；用1M NaOH溶液将pH调节为约7.4后，继续搅拌2h，得到凝胶状物质；将该物质在4℃下冷藏过夜，并对其进行真空冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶。
扫描电镜下的微观形貌如图12所示，平均孔径为50μm～70μm。体外降解试验表明，样品经过三周的降解后，最终的降解率约70％(图10“9％MC”曲线)。
实施例6
(1)在磁力搅拌(转速300rpm)下，将50mL预先配制好的人工脑脊液加入锥形瓶中，在恒温油浴锅中加热至90℃，向其中加入4g氯化钠；待完全溶解后，加入5g甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，再加入50mL人工脑脊液，置于‐4℃冰盐浴中，以200rpm的转速搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜。
(2)24h后向甲基纤维素溶液中缓慢分次加入1g透明质酸粉末，搅拌溶解，向其中逐滴加入0.4g/L PEG溶液2mL；用1M NaOH溶液将pH调节为约7.4后，继续搅拌2h，得到凝胶状物质；将该物质在4℃下冷藏过夜，并对其进行真空冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶。
从图13的扫描电镜照片中可观测到其平均孔径为80μm～100μm。体外降解试验表明，样品经过三周的降解后，最终的降解率约40％(图10“5％MC”曲线)。
实施例7
(1)在磁力搅拌(转速300rpm)下，将50mL预先配制好的人工脑脊液加入锥形瓶中，在恒温油浴锅中加热至90℃，向其中加入4g氯化钠；待完全溶解后，加入11g甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，再加入50mL人工脑脊液，置于‐4℃冰盐浴中，以200rpm的 转速搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜。
(2)24h后向甲基纤维素溶液中缓慢分次加入1g透明质酸粉末，搅拌溶解，向其中逐滴加入1M盐酸至pH为约4.5；依次加入0.3g己二酸二酰肼和0.3g碳化二亚胺，搅拌20min；用1M NaOH溶液将pH调节为约7.4后，继续搅拌2h，得到凝胶状物质；将该物质在4℃下冷藏过夜，并对其进行真空冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶，平均孔径为40μm～70μm(图14)。
实施例8
(1)在磁力搅拌(转速300rpm)下，将50mL预先配制好的人工脑脊液加入锥形瓶中，在恒温油浴锅中加热至90℃，向其中加入4g氯化钠；待完全溶解后，加入9g甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，再加入50mL人工脑脊液，置于‐4℃冰盐浴中，以200rpm的转速搅拌40min，得到甲基纤维素溶液，4℃冷藏过夜。
(2)24h后向甲基纤维素溶液中缓慢分次加入1g透明质酸粉末，搅拌溶解，向其中逐滴加入1M盐酸至pH为约4.5；依次加入0.3g己二酸二酰肼和0.3g碳化二亚胺，搅拌20min；用1M NaOH溶液将pH调节为约7.4后，继续搅拌2h，得到凝胶状物质；将该物质在4℃下冷藏过夜，并对其进行真空冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶，平均孔径为60μm～80μm(图15)。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (7)

  1. 一种透明质酸‐甲基纤维素复合凝胶，其特征在于，由甲基纤维素和透明质酸，或甲基纤维素与聚乙二醇琥珀酰亚胺琥珀酸酐和去乙酰化透明质酸反应得到的交联活性酯交联，所述复合凝胶具有交联网络结构，孔径为30μm～100μm。
  2. 制备权利要求1所述的透明质酸‐甲基纤维素复合水凝胶的方法，其特征在于，具体步骤如下：
    (1)在搅拌条件下，将第一份人工脑脊液加入锥形瓶中，加热，并向其中加入氯化钠；待完全溶解后，加入甲基纤维素，继续加热；甲基纤维素粉末分散均匀并完全润湿后，取出锥形瓶，加入第二份人工脑脊液，置于冰盐浴中，搅拌，得到甲基纤维素溶液，冷藏过夜；
    (2)至少15h后，向甲基纤维素溶液中加入透明质酸，搅拌溶解，向其中加入交联剂；待体系混合均匀后，用NaOH溶液将pH调节为7.4，继续搅拌2h，得到凝胶状物质；将该物质冷藏过夜，并对其进行冷冻干燥，得到透明质酸‐甲基纤维素复合水凝胶。
  3. 根据权利要求2所述的制备方法，其特征在于，所述交联剂为己二酸二酰肼或者聚乙二醇。
  4. 根据权利要求2所述的制备方法，其特征在于，甲基纤维素、氯化钠、透明质酸的质量之比依次为(5～11)：4：1。
  5. 根据权利要求2所述的制备方法，其特征在于，步骤(1)中，所述冰盐浴的温度为‐4℃，在冰盐浴中的搅拌时间为40min；第一份人工脑脊液的加热温度为80～90℃。
  6. 根据权利要求2所述的制备方法，其特征在于，步骤(2)中，缓慢分次加入透明质酸。
  7. 根据权利要求2所述的制备方法，其特征在于，步骤(1)和步骤(2)中，搅拌时的转速均为200rpm～300rpm，冷藏过夜的温度均为4℃。
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CN115154409A (zh) * 2022-07-04 2022-10-11 杭州师范大学钱江学院 一种负载nmn的埃洛石/羧甲基纤维素钠/可德胶复合水凝胶及其应用
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