技术领域technical field
本发明属于生物医用材料技术领域，涉及一种pH敏感型复合药物载体材料及其制备方法。The invention belongs to the technical field of biomedical materials, and relates to a pH-sensitive composite drug carrier material and a preparation method thereof.
背景技术Background technique
pH敏感型聚合物载体材料能够感知病灶处酸碱度信号并产生响应，实现对药物的pH敏感型靶向控制释放，因此，pH敏感型聚合物载体材料在医药领域具有诱人的应用前景，吸引了研究人员越来越多的目光。中国专利CN103214635A公开的pH敏感型超疏水三嵌段共聚物对不同pH环境有灵敏的响应性，包覆抗癌药物喜树碱之后，在模拟肿瘤部位载体可产生响应而释放喜树碱。中国专利CN101708335A公开的一种pH敏感树枝状聚合物可用于包裹抗肿瘤药物，在体内具有很强的pH敏感性能，到达肿瘤部位后可以产生快速响应。然而由于病灶处的异常酸碱度信号在短时间内很难通过机体的自我修复回归正常水平，当pH敏感型聚合物载体材料感知病灶处异常酸碱度信号后，会长时间处于响应状态，导致局部药物快速积累，甚至由于药物的蓄积而引发严重的副作用，极大的限制了其临床应用。硼酸盐生物玻璃是一类新近开发的生物活性玻璃，作为骨修复材料具有诸如生物活性高、可完全降解、良好的骨传导和骨诱导等许多性能。有关文献报道硼酸盐玻璃降解过程中释放出的离子可以使得局部微环境形成弱碱性，因而将其制备成药物载体后，可在释药的过程中调节病灶处的异常酸碱度信号。因此，结合pH敏感型聚合物材料以及硼酸盐玻璃的特性，从材料设计的角度出发，可将硼酸盐玻璃与某些pH敏感型聚合物材料复合，使得硼酸盐玻璃成为病灶处酸碱度的调节剂，在释药过程中调节微环境的酸碱度，进而反馈调控pH敏感型聚合物构象变化而实现“打开”、“关闭”效果，即使得载体材料释药一定时间后自行停止或减缓释药，控制药物释放程度，待重新感知到响应信号后再次释药，实现载体的自身反馈调节作用，达到药物缓控释效果。The pH-sensitive polymer carrier material can sense and respond to the pH signal at the lesion, and realize the pH-sensitive targeted and controlled release of the drug. Therefore, the pH-sensitive polymer carrier material has an attractive application prospect in the medical field, attracting Researchers are increasingly looking. The pH-sensitive superhydrophobic tri-block copolymer disclosed in Chinese patent CN103214635A has sensitive responsiveness to different pH environments. After coating the anticancer drug camptothecin, the carrier can respond and release camptothecin at the simulated tumor site. A pH-sensitive dendritic polymer disclosed in Chinese patent CN101708335A can be used to encapsulate anti-tumor drugs, has strong pH-sensitivity in vivo, and can produce a rapid response after reaching the tumor site. However, because the abnormal pH signal at the lesion is difficult to return to the normal level through the body's self-repair in a short period of time, when the pH-sensitive polymer carrier material senses the abnormal pH signal at the lesion, it will be in a response state for a long time, resulting in rapid local drug recovery. Accumulation, and even cause serious side effects due to the accumulation of drugs, which greatly limits its clinical application. Borate bioglass is a kind of newly developed bioactive glass, which has many properties such as high bioactivity, complete degradability, good osteoconduction and osteoinduction as a bone repair material. It has been reported in relevant literature that the ions released during the degradation of borate glass can make the local microenvironment weakly alkaline. Therefore, after it is prepared as a drug carrier, it can regulate the abnormal pH signal at the lesion during the drug release process. Therefore, combining the characteristics of pH-sensitive polymer materials and borate glass, from the perspective of material design, borate glass can be combined with some pH-sensitive polymer materials, so that borate glass becomes Regulators that adjust the pH of the microenvironment during the release process, and then feedback the conformational changes of the pH-sensitive polymer to achieve the "open" and "close" effects, that is, the release of the carrier material will stop or slow down after a certain period of time. Release the drug, control the degree of drug release, release the drug again after the response signal is re-perceived, realize the self-feedback regulation of the carrier, and achieve the effect of slow and controlled release of the drug.
此外，硼酸盐玻璃作为药物载体时，由于其主要依靠与药物机械混合进行载药，因而其药物负载能力较差，存在初期爆发性释放以及缓释周期短等缺点，即使利用生物可降解高分子材料对其包覆，依然存在着药物突释的现象，极大的限制了其临床应用。多孔结构磷酸盐药物载体材料具有良好的药物负载性能和缓释效果，有关文献报道利用硼酸盐玻璃原位转化法可制备出多孔羟基磷灰石微球，作为人重组骨发生蛋白2(rhBMP-2)的载体，其体外缓释周期可达到30天以上且无明显突释现象。因此利用原位转化法，在玻璃表面转化生成具有一定厚度的多孔磷酸盐材料，利用其对药物进行负载，在改善药物负载能力和缓释效果方面具有重要意义。In addition, when borate glass is used as a drug carrier, because it mainly relies on mechanical mixing with drugs for drug loading, its drug loading capacity is poor, and there are shortcomings such as initial burst release and short sustained release period. When it is coated with molecular materials, there is still the phenomenon of sudden drug release, which greatly limits its clinical application. Porous phosphate drug carrier materials have good drug loading performance and sustained release effect. According to relevant literature reports, porous hydroxyapatite microspheres can be prepared by borate glass in situ conversion method, which can be used as human recombinant osteogenetic protein 2 (rhBMP -2) the carrier, its in vitro sustained release period can reach more than 30 days and there is no obvious burst release phenomenon. Therefore, using the in-situ conversion method to generate a porous phosphate material with a certain thickness on the glass surface, and using it to load the drug is of great significance in improving the drug loading capacity and sustained release effect.
因此，将表面为磷酸盐材料的硼酸盐玻璃微球作为复合药物载体无机部分进行药物负载，将某些具有pH敏感特性的聚合物材料作为复合药物载体有机部分包覆在微球表面，综合利用核壳结构硼酸盐玻璃微球和pH敏感型聚合物的特性，是一种改善载体药物负载性能和缓控释效果的理想途径。Therefore, borate glass microspheres with phosphate materials on the surface are used as the inorganic part of the composite drug carrier for drug loading, and some polymer materials with pH-sensitive properties are used as the organic part of the composite drug carrier to coat the surface of the microspheres. Utilizing the properties of borate glass microspheres with core-shell structure and pH-sensitive polymers is an ideal way to improve the drug-loading performance and sustained-release effect of carriers.
发明内容Contents of the invention
针对现有技术的缺陷，本发明的目的是提供一种基于硼酸盐玻璃微球的pH敏感型复合药物载体材料，其中P-bg微球为复合药物载体材料的无机部分，pH敏感型聚合物作为复合药物载体的有机部分，对微环境的酸碱度变化做出及时响应，将盐酸万古霉素等药物装载到P-bg微球中，并利用pH敏感型聚合物对P-bg微球进行包覆，制备复合药物载体，实现药物的缓控释效果，并用于骨缺损治疗。For the defects of the prior art, the purpose of this invention is to provide a kind of pH-sensitive composite drug carrier material based on borate glass microspheres, wherein P-bg microspheres are the inorganic part of the composite drug carrier material, and the pH-sensitive polymer As the organic part of the composite drug carrier, it responds in time to the pH change of the microenvironment, loads vancomycin hydrochloride and other drugs into the P-bg microspheres, and uses the pH-sensitive polymer to carry out the P-bg microspheres. Coating, preparing composite drug carrier, realizing the slow and controlled release effect of the drug, and used for the treatment of bone defects.
本发明的另一个目的是提供一种上述pH敏感型复合药物载体材料的制备方法。Another object of the present invention is to provide a method for preparing the above pH-sensitive composite drug carrier material.
为了实现上述目的，本发明的技术方案如下：In order to achieve the above object, the technical scheme of the present invention is as follows:
一种pH敏感型复合药物载体材料，由以下质量百分含量的组分制成：A pH-sensitive composite drug carrier material, made of the following components in mass percentage:
核壳结构硼酸盐玻璃微球(P-bg) 70～90wt％，Core-shell structure borate glass microspheres (P-bg) 70～90wt%,
pH敏感型聚合物 9～27wt％，pH sensitive polymer 9～27wt%,
药物 1～3wt％。Drug 1～3wt%.
所述核壳结构硼酸盐玻璃微球是由以下质量百分含量的组分制成：The borate glass microspheres with a core-shell structure are made of the following components in mass percentage:
多孔磷酸盐壳层材料(P) 40～70wt％，Porous phosphate shell material (P) 40～70wt%,
硼酸盐玻璃内核材料(bg) 30～60wt％。Borate glass core material (bg) 30-60wt%.
所述核壳结构硼酸盐玻璃微球的尺寸为100～150μm。The size of the core-shell structure borate glass microsphere is 100-150 μm.
所述pH敏感型聚合物为阳离子型聚合物，其通常含有可在弱酸性条件下产生质子化、而在弱碱性条件下产生去质子化的氨基基团，如壳聚糖(CS)及其接枝共聚物、组氨酸(His)接枝共聚物以及聚甲基丙烯酸N,N-二甲氨基乙酯(PDMAMEA)接枝共聚物等。The pH-sensitive polymer is a cationic polymer, which usually contains amino groups that can be protonated under weakly acidic conditions and deprotonated under weakly alkaline conditions, such as chitosan (CS) and Its graft copolymer, histidine (His) graft copolymer and polymethacrylate N,N-dimethylaminoethyl ester (PDMAMEA) graft copolymer, etc.
所述药物为盐酸万古霉素、重组人骨形态发生蛋白2(rhBMP-2)或替考拉丁等骨疾病治疗用药物。The medicine is vancomycin hydrochloride, recombinant human bone morphogenetic protein 2 (rhBMP-2) or teicodin and other medicines for treating bone diseases.
一种上述pH敏感型复合药物载体材料的制备方法，包括以下步骤：首先将质量百分含量为1～3wt％的药物装载入质量百分含量为70～90wt％的核壳结构硼酸盐玻璃微球中；然后将载有药物的核壳结构硼酸盐玻璃微球与质量百分含量为9～27wt％的pH敏感型聚合物溶液混合均匀，最后干燥得到pH敏感型复合药物载体材料。A method for preparing the above-mentioned pH-sensitive composite drug carrier material, comprising the following steps: first loading a drug with a mass percentage of 1 to 3 wt % into a core-shell structure borate with a mass percentage of 70 to 90 wt % in glass microspheres; then mix the drug-loaded core-shell structure borate glass microspheres with a pH-sensitive polymer solution with a mass percentage of 9 to 27 wt%, and finally dry to obtain a pH-sensitive composite drug carrier material .
所述药物在核壳结构硼酸盐玻璃微球中的装载量为10～1000μg/g。The loading amount of the drug in the core-shell structure borate glass microsphere is 10-1000 μg/g.
所述核壳结构硼酸盐玻璃微球与pH敏感型聚合物溶液的固液比为(1～5)/10(g/ml)。The solid-liquid ratio of the core-shell structure borate glass microspheres to the pH-sensitive polymer solution is (1-5)/10 (g/ml).
所述核壳结构硼酸盐玻璃微球的制备方法包括以下步骤：将质量比为(1～5)：(1～5)：(14～21)的Na2CO3、CaCO3和H3BO3充分混合，在温度为1100～1150℃的条件下加热熔融30～40min，将熔化的玻璃液淬冷制得硼酸盐玻璃，破碎后获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球(NCB玻璃微球)，将玻璃微球加入到浓度为0.10～0.25mol/L、pH为9.0～9.5的磷酸盐溶液中，玻璃微球与磷酸盐溶液的比例为(1～3)/100(g/mL)，在温度为25～40℃的条件下浸泡3～12h，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，在温度为60～90℃的条件下干燥12～24h，最后将干燥后的微球置于温度为300～500℃的条件下煅烧2～3h，制得核壳结构硼酸盐玻璃微球。The preparation method of the core-shell structure borate glass microspheres comprises the following steps: Na2 CO3 , CaCO3 and H3 with a mass ratio of (1-5):(1-5):(14-21) Mix BO3 thoroughly, heat and melt at a temperature of1100-1150 °C for 30-40 minutes, quench the molten glass to obtain borate glass, and obtain glass particles with a particle size of 100-150 μm after crushing. The glass particles are made into glass microspheres (NCB glass microspheres) by the spray ball method, and the glass microspheres are added to the phosphate solution with a concentration of 0.10-0.25mol/L and a pH of 9.0-9.5, and the glass microspheres and phosphate The ratio of the solution is (1~3)/100(g/mL), soak for 3~12 hours at a temperature of 25~40°C, then remove the soaking solution, wash it with deionized water three times, and then wash it with absolute ethanol Wash once, dry at a temperature of 60-90°C for 12-24 hours, and finally place the dried microspheres at a temperature of 300-500°C for 2-3 hours to obtain borate glass with a core-shell structure Microspheres.
所述火焰喷球法包括以下步骤：将所获得的粒径为100～150μm的玻璃颗粒放入振荡给料器中，以一定频率将玻璃颗粒送入丁烷火焰并随火焰飘落在收集器皿中；将所收集的颗粒再次送入振荡给料器中，如此重复操作3～5次，直至在光学显微镜视野范围内，90～100％的玻璃颗粒均形成球体即可。The flame spray ball method includes the following steps: put the obtained glass particles with a particle size of 100-150 μm into a vibrating feeder, send the glass particles into a butane flame at a certain frequency and fall into a collecting vessel with the flame Send the collected particles into the vibrating feeder again, and repeat the operation for 3 to 5 times until 90 to 100% of the glass particles form spheres within the field of view of the optical microscope.
所述磷酸盐为K2HPO4或Na2HPO4等。The phosphate is K2 HPO4 or Na2 HPO4 or the like.
所述药物装载入核壳结构硼酸盐玻璃微球中包括以下步骤：将药物溶于磷酸盐缓冲溶液中，制成浓度为(0.01～50)g/L的水相溶液；在0～4℃的环境中，将核壳结构硼酸盐玻璃微球按(1～5)/100(g/mL)的比例浸泡于上述药物的水相溶液中，将其置于真空环境下，保持真空度0.001～0.1Mpa，30～60min后取出，用磷酸盐缓冲溶液冲洗微球，经冷冻干燥后得到载有药物的核壳结构硼酸盐玻璃微球。The loading of the drug into borate glass microspheres with a core-shell structure comprises the following steps: dissolving the drug in a phosphate buffer solution to prepare an aqueous phase solution with a concentration of (0.01-50) g/L; In an environment of 4°C, soak the core-shell structure borate glass microspheres in the aqueous phase solution of the above drug at a ratio of (1-5)/100 (g/mL), place them in a vacuum environment, and keep The vacuum degree is 0.001-0.1Mpa, and it is taken out after 30-60 minutes. The microspheres are rinsed with phosphate buffer solution, and freeze-dried to obtain drug-loaded borate glass microspheres with a core-shell structure.
所述pH敏感型聚合物溶液(以壳聚糖及其接枝共聚物为例)的制备方法包括以下步骤：The preparation method of described pH-sensitive polymer solution (taking chitosan and graft copolymer thereof as example) comprises the following steps:
将壳聚糖溶于浓度为0.5～1mol/L的乙酸溶液中，搅拌1～3h制备20～30g/L壳聚糖/乙酸混合溶液；然后将β-甘油磷酸二钠溶于去离子水中，搅拌制备500～560g/L的β-甘油磷酸二钠溶液；最后将制备的壳聚糖/乙酸混合溶液与β-甘油磷酸二钠溶液按(7～9):1的体积比混合制备pH敏感型聚合物溶液，置于0～4℃的冰箱冷藏备用；Dissolve chitosan in an acetic acid solution with a concentration of 0.5-1 mol/L, stir for 1-3 hours to prepare a 20-30 g/L chitosan/acetic acid mixed solution; then dissolve β-glycerophosphate disodium in deionized water, Stir to prepare 500-560g/L β-glycerol phosphate disodium solution; finally mix the prepared chitosan/acetic acid mixed solution with β-glycerol phosphate disodium solution at a volume ratio of (7-9):1 to prepare pH-sensitive Type polymer solution, put it in the refrigerator at 0～4℃ for later use;
或：将壳聚糖、冰醋酸和去离子水按比例为(1～2)/3/100(g/ml/ml)混合加热搅拌使其完全溶解，通N2时间为15～30min置换出空气，升温至50～60℃后在氮气保护下按(1～5)/4(mL/mL)的比例依次加入浓度为0.010～0.015g/mL硝酸铈铵溶液和甲基丙烯酸N,N-二甲氨基乙酯单体，甲基丙烯酸N,N-二甲氨基乙酯单体与壳聚糖的比例为(1～8)/1(mL/g)，恒温反应5～7h，反应结束后冷却至室温，在丙酮中沉淀得到粗产物，将粗产物在丙酮中回流5～10h以除去聚甲基丙烯酸N,N-二甲氨基乙酯均聚物，最后置于40～50℃真空烘箱中干燥至恒重得到纯接枝共聚物，将接枝共聚物溶于浓度为0.5～1mol/L的乙酸溶液中，浓度为20～30g/L，磁力搅拌12～24h，得到pH敏感型聚合物溶液。Or: mix chitosan, glacial acetic acid and deionized water in a ratio of (1-2)/3/100 (g/ml/ml) and heat and stir to dissolve completely, and passN2 for 15-30 minutes to replace Air, after heating up to 50-60°C, add cerium ammonium nitrate solution with a concentration of 0.010-0.015g/mL and methacrylic acid N,N- Dimethylaminoethyl ester monomer, the ratio of N,N-dimethylaminoethyl methacrylate monomer to chitosan is (1~8)/1(mL/g), constant temperature reaction for 5~7h, the reaction is over After cooling to room temperature, the crude product was obtained by precipitation in acetone, and the crude product was refluxed in acetone for 5-10 hours to remove poly-N,N-dimethylaminoethyl methacrylate homopolymer, and finally placed in a vacuum at 40-50°C Dry in an oven to constant weight to obtain pure graft copolymer, dissolve the graft copolymer in acetic acid solution with a concentration of 0.5-1mol/L at a concentration of 20-30g/L, stir magnetically for 12-24 hours, and obtain a pH-sensitive type polymer solution.
所述干燥是在温度为35～45℃的条件下真空干燥5～10h或在温度为-21～-10℃的条件下冷冻干燥3～24h。The drying is vacuum drying at a temperature of 35-45°C for 5-10 hours or freeze-drying at a temperature of -21-10°C for 3-24 hours.
与现有技术相比，本发明具有如下优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
1、本发明所制备的pH敏感型复合药物载体材料具有良好的药物缓释性能，P-bg微球因其多孔壳层对药物具有较好的负载能力和缓释作用，包覆pH敏感型聚合物后，进一步提高了复合药物载体的缓释性能。1. The pH-sensitive composite drug carrier material prepared by the present invention has good drug slow-release properties. Because of its porous shell, P-bg microspheres have good loading capacity and slow-release effect on drugs, and the coated pH-sensitive After the polymer, the slow-release performance of the composite drug carrier is further improved.
2、本发明所制备的pH敏感型复合药物载体材料可以在释药过程中调节病灶处酸碱度信号，进而调控载体pH敏感型聚合物的构象变化而实现“打开”、“关闭”效果，从而实现药物的缓控释效果。2. The pH-sensitive composite drug carrier material prepared by the present invention can adjust the pH signal at the lesion during the drug release process, and then regulate the conformational changes of the pH-sensitive polymer of the carrier to achieve the "open" and "close" effects, thereby realizing Drug release effect.
3、本发明所制备的pH敏感型复合药物载体材料归属于一种新型药物缓释系统，该药物缓释系统利用载体材料本身在释药过程中的降解作用，改变环境的刺激信号，进而反馈调节载体的释药过程，实现载体自身反馈调节作用。3. The pH-sensitive composite drug carrier material prepared by the present invention belongs to a new type of drug sustained-release system. The drug sustained-release system uses the degradation of the carrier material itself during the drug release process to change the stimulus signal of the environment, and then feedback Regulate the drug release process of the carrier to realize the self-feedback regulation of the carrier.
4、本发明所制备的pH敏感型复合药物载体材料具有良好的生物活性和生物降解性能，可用于制备骨填充物，用于骨组织修复。4. The pH-sensitive composite drug carrier material prepared by the present invention has good bioactivity and biodegradability, and can be used to prepare bone fillers and repair bone tissue.
5、本发明所制备的pH敏感型复合药物载体材料，利用多孔磷酸盐壳层材料部分进行药物负载，而硼酸盐玻璃部分则作为微环境酸碱度的调节剂，在释药过程中调节微环境pH值。5. The pH-sensitive composite drug carrier material prepared by the present invention uses the porous phosphate shell material part to carry out drug loading, while the borate glass part is used as a regulator of microenvironmental pH to adjust the microenvironment during the drug release process pH.
附图说明Description of drawings
图1为本发明实施例的NCB玻璃微球的光学显微图片。Fig. 1 is the optical micrograph of the NCB glass microsphere of the embodiment of the present invention.
图2为本发明实施例的核壳结构硼酸盐玻璃微球的扫描电镜图片。Fig. 2 is a scanning electron microscope picture of borate glass microspheres with a core-shell structure according to an embodiment of the present invention.
图3为本发明实施例的核壳结构硼酸盐玻璃微球中多孔磷酸盐壳层的扫描电镜图片。Fig. 3 is a scanning electron microscope image of the porous phosphate shell layer in borate glass microspheres with a core-shell structure according to an embodiment of the present invention.
图4为本发明实施例的pH敏感型复合药物载体的扫描电镜图片。Fig. 4 is a scanning electron micrograph of the pH-sensitive composite drug carrier of the embodiment of the present invention.
图5为本发明实施例的PBS浸泡液pH值随复合药物载体浸泡时间的变化曲线示意图。Fig. 5 is a schematic diagram of the change curve of the pH value of the PBS soaking solution with the soaking time of the composite drug carrier according to the embodiment of the present invention.
图6为本发明实施例的载盐酸万古霉素pH敏感型复合药物载体缓释曲线示意图。Fig. 6 is a schematic diagram of the sustained-release curve of the pH-sensitive composite drug carrier loaded with vancomycin hydrochloride according to an embodiment of the present invention.
图7为本发明实施例的pH敏感型复合药物载体的缓控释机理示意图。Fig. 7 is a schematic diagram of the sustained and controlled release mechanism of the pH-sensitive composite drug carrier of the embodiment of the present invention.
具体实施方式Detailed ways
下面结合附图所示实施例对本发明作进一步详细的说明。The present invention will be described in further detail below in conjunction with the embodiments shown in the accompanying drawings.
以下所用的火焰喷球法包括以下步骤：将所获得的粒径为100～150μm的玻璃颗粒放入振荡给料器中，将玻璃颗粒送入丁烷火焰并随火焰飘落在收集器皿中；将所收集的颗粒再次送入振荡给料器中，如此重复操作3～5次，直至在光学显微镜视野范围内，90～100％的玻璃颗粒均形成球体即可。The flame spray ball method used below comprises the following steps: putting the obtained glass particles with a particle size of 100-150 μm into an oscillating feeder, sending the glass particles into a butane flame and falling into a collection container with the flame; The collected particles are sent to the vibrating feeder again, and the operation is repeated 3-5 times until 90-100% of the glass particles form spheres within the field of view of the optical microscope.
以下实施例中符号表示利用pH敏感型聚合物材料对核壳结构硼酸盐玻璃微球进行包覆。The symbols in the following examples indicate that the pH-sensitive polymer material is used to coat the core-shell borate glass microspheres.
实施例1Example 1
载盐酸万古霉素pH敏感型CSP-bg复合药物载体材料的制备过程如下：(1)P-bg微球的制备：称取21.20gNa2CO3、20.02gCaCO3和74.20gH3BO3粉料，于研钵中充分混合。将混合的粉料置于铂金坩埚中，在1150℃的硅碳棒高温炉中，加热熔融30min。将熔化的玻璃液淬冷于冷钢板上，制得硼酸盐玻璃。将玻璃块破碎后，获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球，如图1所示，图1为本发明实施例的NCB玻璃微球的光学显微图片。称取1g玻璃微球，加入到100ml浓度为0.20mol/L，pH为9.0的K2HPO4溶液中，静置于37℃恒温箱中，浸泡3h后，微球失重率约为30％左右，微球内部剩余硼酸盐玻璃含量约为60％左右，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，置于60℃烘箱中干燥24h，最后将干燥后的微球置于300℃煅烧2h，制得P-bg微球。The preparation process of pH-sensitive CSP-bg composite drug carrier material loaded with vancomycin hydrochloride is as follows: (1) Preparation of P-bg microspheres: Weigh 21.20g Na2 CO3 , 20.02g CaCO3 and 74.20g H3 BO3 powder , Mix thoroughly in a mortar. The mixed powder was placed in a platinum crucible, and heated and melted for 30 minutes in a silicon carbide rod high-temperature furnace at 1150°C. The molten glass is quenched on a cold steel plate to produce borate glass. After the glass block is broken, the glass particles with a particle size of 100 to 150 μm are obtained, and the glass particles are made into glass microspheres by the flame spray ball method, as shown in Figure 1, which is a diagram of the NCB glass microspheres of the embodiment of the present invention. Optical micrograph. Weigh 1g of glass microspheres, add them to 100ml of K2 HPO4 solution with a concentration of 0.20mol/L and a pH of 9.0, and place it in a constant temperature box at 37°C. After soaking for 3 hours, the weight loss rate of the microspheres is about 30%. , the remaining borate glass content inside the microspheres is about 60%, then remove the soaking solution, wash three times with deionized water, and then wash once with absolute ethanol, dry in an oven at 60°C for 24 hours, and finally dry the The microspheres were calcined at 300°C for 2h to obtain P-bg microspheres.
(2)盐酸万古霉素在P-bg微球中的装载：为了验证复合材料的药物缓释性能，将盐酸万古霉素装载到P-bg微球上。具体方法为：将盐酸万古霉素溶于磷酸盐缓冲溶液中，制成浓度为1g/L的水相溶液；将1g P-bg微球样品浸泡于50mL盐酸万古霉素的水相溶液中，将其置于真空环境下，保持真空度0.05Mpa左右，使盐酸万古霉素溶液在压力作用下扩散进入P-bg微球的多孔表面及其内部，60min后取出，用PBS磷酸盐缓冲溶液冲洗微球，经冷冻干燥后保存备用，盐酸万古霉素在P-bg微球中的装载量约为10mg/g。(2) Loading of vancomycin hydrochloride in P-bg microspheres: In order to verify the drug sustained release performance of the composite material, vancomycin hydrochloride was loaded on P-bg microspheres. The specific method is: dissolving vancomycin hydrochloride in phosphate buffer solution to make a concentration of 1g/L aqueous phase solution; soaking 1g of P-bg microsphere sample in the aqueous phase solution of 50mL vancomycin hydrochloride, Put it in a vacuum environment, keep a vacuum of about 0.05Mpa, make the vancomycin hydrochloride solution diffuse into the porous surface and interior of the P-bg microspheres under pressure, take it out after 60 minutes, and rinse with PBS phosphate buffer solution The microspheres are stored for future use after freeze-drying, and the loading amount of vancomycin hydrochloride in the P-bg microspheres is about 10 mg/g.
(3)壳聚糖包覆溶液的制备：将壳聚糖溶于浓度为1mol/L的乙酸溶液中，磁力搅拌1h至溶液澄清，制备20g/L的壳聚糖/乙酸混合溶液；然后，将β-甘油磷酸二钠溶于去离子水中，磁力搅拌至溶液澄清透明，制备560g/L的β-甘油磷酸二钠盐溶液；最后，将制备的壳聚糖/乙酸溶液与β-甘油磷酸钠溶液按9:1的体积比混合，制备壳聚糖包覆溶液，并置于4℃的冰箱冷藏备用。(3) Preparation of chitosan coating solution: chitosan is dissolved in the acetic acid solution with a concentration of 1mol/L, magnetically stirred for 1h until the solution is clarified, and the chitosan/acetic acid mixed solution of 20g/L is prepared; then, Dissolve β-glycerophosphate disodium in deionized water, stir magnetically until the solution is clear and transparent, and prepare a 560 g/L β-glycerophosphate disodium salt solution; finally, mix the prepared chitosan/acetic acid solution with β-glycerophosphate The sodium solution was mixed at a volume ratio of 9:1 to prepare a chitosan coating solution, which was stored in a refrigerator at 4°C for later use.
(4)载万古霉素pH敏感型CSP-bg复合药物载体材料的制备：将载有盐酸万古霉素的P-bg微球与上述壳聚糖包覆液以固液比1/5(g/mL)混合并摇匀，然后将其置于真空干燥箱中，37℃干燥6h，即可制得载盐酸万古霉素pH敏感型复合药物载体材料。(4) Preparation of vancomycin-loaded pH-sensitive CSP-bg composite drug carrier material: P-bg microspheres loaded with vancomycin hydrochloride and the above-mentioned chitosan coating solution were mixed at a solid-to-liquid ratio of 1/5 (g /mL) were mixed and shaken evenly, and then placed in a vacuum drying oven at 37°C for 6 hours to prepare a pH-sensitive composite drug carrier material loaded with vancomycin hydrochloride.
本发明所制备的NCB玻璃微球光学显微照片如图1所示，图1为本发明实施例的NCB玻璃微球的光学显微图片，由图1可知，NCB玻璃微球呈规则的球形，直径约为100μm。本发明所制备的P-bg微球的扫描电镜结果如图2所示，图2为本发明实施例的核壳结构硼酸盐玻璃微球的扫描电镜图片，由图2可知，微球均由多孔磷酸盐壳层和硼酸盐玻璃内核构成。P-bg微球球壳的扫描电镜结果如图3所示，图3为本发明实施例的核壳结构硼酸盐玻璃微球中多孔磷酸盐壳层的扫描电镜图片。由图3可知，微球球壳由纳米尺寸的球状颗粒组成且呈多孔结构。本发明所制备的CSP-bg复合药物载体的扫描电镜结果如图4所示，图4为本发明实施例的pH敏感型复合药物载体材料的扫描电镜图片。由图4可知，复合药物载体由pH敏感型聚合物膜、多孔磷酸盐壳层以及硼酸盐玻璃内核构成。测试CSP-bg复合药物载体对微环境酸碱度的影响，结果如图5所示，图5为本发明实施例的PBS浸泡液的pH值随pH敏感型复合药物载体材料浸泡时间的变化曲线示意图。由图5可知，本发明所制备的CSP-bg复合药物载体对环境pH具有良好的调控作用，浸泡24h后，溶液pH由6.0升至8.5。测试载万古霉素CSP-bg复合药物载体在弱酸性环境下(pH＝6.0)的药物缓释性能，结果如图6所示，图6为本发明实施例的载盐酸万古霉素pH敏感型复合药物载体材料的体外缓释曲线示意图。由图6可知，复合药物载体的缓释周期可达2周左右，表现出良好的药物缓控释效果。本发明所制备的CSP-bg复合药物载体缓控释原理如图7所示，图7为本发明实施例所制备的pH敏感型复合药物载体材料的药物缓控释原理示意图。由图7可知，本发明所制备的基于硼酸盐玻璃微球的pH敏感型复合药物载体材料可以在释药过程中调节环境pH信号，进而调控pH敏感型聚合物的构象变化而实现载体的“打开”、“关闭”效果，即载体自身反馈调节作用，从而实现药物的缓控释效果，其具体过程如下：当复合药物载体感受到病灶处弱酸性疾病信号后，载体表面聚合物由于质子化作用呈溶胀状态，药物从微球表面多孔壳层处加速释放；与此同时，微球内部硼酸盐玻璃产生降解并逐渐调控病灶处酸碱度，当病灶处酸碱度信号被硼酸盐玻璃调节到一定程度后，载体表面聚合物由于去质子化作用呈收缩状态，阻碍药物释放，同时硼酸盐玻璃降解变缓；由于病变在短时间内难以至治愈，因此将持续产生疾病信号，当环境酸碱度再次恢复到疾病信号状态，复合药物载体表面聚合物感受到疾病信号后会再次产生质子化作用并呈溶胀状态，药物加速释放，与此同时硼酸盐玻璃降解变快，如此循环响应直至药物完全释放，既满足了pH敏感型药物释放的要求，又有效地调控了药物的释放速度。The prepared NCB glass microsphere optical micrograph of the present invention is as shown in Figure 1, and Fig. 1 is the optical micrograph of the NCB glass microsphere of the embodiment of the present invention, as can be seen from Fig. 1, NCB glass microsphere is the spherical shape of rule , about 100 μm in diameter. The scanning electron microscope result of the P-bg microsphere prepared by the present invention is shown in Figure 2, and Figure 2 is the scanning electron microscope picture of the core-shell structure borate glass microsphere of the embodiment of the present invention, as can be seen from Figure 2, the microsphere Consists of a porous phosphate shell and a borate glass core. The scanning electron microscope results of the spherical shell of the P-bg microsphere are shown in FIG. 3 , which is a scanning electron microscope picture of the porous phosphate shell layer in the core-shell structure borate glass microsphere of the embodiment of the present invention. It can be seen from Figure 3 that the microsphere shell is composed of nanometer-sized spherical particles and has a porous structure. The scanning electron microscope results of the CSP-bg composite drug carrier prepared in the present invention are shown in Figure 4, and Figure 4 is the scanning electron microscope picture of the pH-sensitive composite drug carrier material of the embodiment of the present invention. It can be seen from Figure 4 that the composite drug carrier is composed of a pH-sensitive polymer membrane, a porous phosphate shell, and a borate glass core. The influence of the CSP-bg composite drug carrier on the pH of the microenvironment was tested, and the results are shown in Figure 5, which is a schematic diagram of the change curve of the pH value of the PBS soaking solution according to the soaking time of the pH-sensitive composite drug carrier material in the embodiment of the present invention. It can be seen from Figure 5 that the CSP-bg composite drug carrier prepared by the present invention has a good regulating effect on the pH of the environment. After soaking for 24 hours, the pH of the solution rose from 6.0 to 8.5. Test the drug sustained release performance of the vancomycin CSP-bg composite drug carrier in a weakly acidic environment (pH=6.0), the results are shown in Figure 6, and Figure 6 is the vancomycin hydrochloride pH-sensitive type loaded in the embodiment of the present invention Schematic diagram of the in vitro sustained release curve of the composite drug carrier material. It can be seen from Figure 6 that the sustained release period of the composite drug carrier can reach about 2 weeks, showing a good drug sustained and controlled release effect. The principle of sustained and controlled release of the CSP-bg composite drug carrier prepared in the present invention is shown in Figure 7, which is a schematic diagram of the principle of sustained and controlled drug release of the pH-sensitive composite drug carrier material prepared in the embodiment of the present invention. It can be seen from Figure 7 that the pH-sensitive composite drug carrier material based on borate glass microspheres prepared by the present invention can adjust the environmental pH signal during the drug release process, and then regulate the conformational changes of the pH-sensitive polymer to realize the carrier The effect of "opening" and "closing" is the self-feedback regulation of the carrier, so as to realize the slow and controlled release effect of the drug. The specific process is as follows: The chemical reaction is in a swelling state, and the drug is released from the porous shell on the surface of the microsphere; at the same time, the borate glass inside the microsphere is degraded and gradually regulates the pH of the lesion. When the pH signal of the lesion is adjusted by the borate glass to After a certain level, the polymer on the surface of the carrier shrinks due to deprotonation, which hinders the release of the drug, and at the same time, the degradation of the borate glass slows down; because the disease is difficult to heal in a short time, it will continue to produce disease signals. When the pH of the environment Return to the state of disease signal again, the polymer on the surface of the composite drug carrier will again produce protonation and swell after sensing the disease signal, and the drug will be released at an accelerated rate. The release not only meets the requirements of pH-sensitive drug release, but also effectively regulates the release rate of the drug.
实施例2Example 2
载rhBMP-2pH敏感型CSP-bg复合药物载体材料的制备过程如下：The preparation process of rhBMP-2pH-sensitive CSP-bg composite drug carrier material is as follows:
(1)P-bg微球的制备：称取21.20gNa2CO3、20.02gCaCO3和74.20gH3BO3粉料，于研钵中充分混合。将混合的粉料置于铂金坩埚中，在1150℃的硅碳棒高温炉中，加热熔融30min。将熔化的玻璃液淬冷于冷钢板上，制得硼酸盐玻璃。将玻璃块破碎后，获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球。称取1g球化后的玻璃微球，加入到100ml浓度为0.20mol/L，pH为9.0的K2HPO4溶液中，静置于37℃恒温箱中，浸泡6h后，微球失重率为50％，微球内部剩余硼酸盐玻璃含量为20％，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，置于80℃烘箱中干燥24h，最后将干燥后的微球置于400℃煅烧2h，制得P-bg微球。(1) Preparation of P-bg microspheres: Weigh 21.20 g of Na2 CO3 , 20.02 g of CaCO3 and 74.20 g of H3 BO3 powder, and mix them thoroughly in a mortar. The mixed powder was placed in a platinum crucible, and heated and melted for 30 minutes in a silicon carbide rod high-temperature furnace at 1150°C. The molten glass is quenched on a cold steel plate to produce borate glass. After the glass block is crushed, glass particles with a particle size of 100-150 μm are obtained, and the glass particles are made into glass microspheres by a flame spray ball method. Weigh 1 g of spheroidized glass microspheres, add them to 100 ml of K2 HPO4 solution with a concentration of 0.20 mol/L and a pH of 9.0, and place them in a constant temperature box at 37°C. After soaking for 6 hours, the weight loss rate of the microspheres is 50%, and the remaining borate glass content inside the microspheres is 20%, then remove the soaking solution, wash three times with deionized water, then wash once with absolute ethanol, dry in an oven at 80°C for 24 hours, and finally dry the The microspheres were calcined at 400°C for 2 hours to obtain P-bg microspheres.
(2)重组人骨形态发生蛋白2(rhBMP-2)在P-bg微球中的装载：为了验证复合材料的药物缓释性能，将rhBMP-2装载到P-bg微球上。具体方法为：将rhBMP-2溶于PBS磷酸盐缓冲液中，制成浓度为0.0l g/L的水相溶液；在0～4℃的环境中，将1g P-bg微球样品浸泡于50mLrhBMP-2的水相溶液中，将其放入真空干燥箱内，保持真空度0.05MPa，使rhBMP-2溶液在压力作用下扩散进入P-bg微球，30min后取出，用PBS磷酸盐缓冲液冲洗微球，经冷冻干燥后保存备用，rhBMP-2在P-bg微球中的装载量约为10μg/g。(2) Loading of recombinant human bone morphogenetic protein 2 (rhBMP-2) in P-bg microspheres: In order to verify the sustained drug release performance of the composite material, rhBMP-2 was loaded onto P-bg microspheres. The specific method is as follows: dissolve rhBMP-2 in PBS phosphate buffer solution to make an aqueous phase solution with a concentration of 0.01 g/L; soak 1 g of P-bg microsphere samples in an environment of 0-4 °C 50mL rhBMP-2 aqueous phase solution, put it into a vacuum drying oven, keep the vacuum degree 0.05MPa, make the rhBMP-2 solution diffuse into the P-bg microspheres under pressure, take it out after 30min, buffer with PBS phosphate The microspheres were rinsed with liquid, freeze-dried and stored for later use. The loading amount of rhBMP-2 in P-bg microspheres was about 10 μg/g.
(3)壳聚糖包覆溶液的制备：将壳聚糖溶于浓度为1mol/L的乙酸溶液中，磁力搅拌1小时至溶液澄清，制备20g/L的壳聚糖/乙酸混合溶液；然后，将β-甘油磷酸二钠溶于去离子水中，磁力搅拌至溶液澄清透明，制备560g/L的β-甘油磷酸二钠盐溶液；最后，将制备的壳聚糖/乙酸溶液与β-甘油磷酸盐溶液按9:1的体积比混合，制备壳聚糖包覆液，并置于4℃的冰箱冷藏备用。(3) preparation of chitosan coating solution: chitosan is dissolved in the acetic acid solution that concentration is 1mol/L, and magnetic stirring is to solution clarification for 1 hour, prepares the chitosan/acetic acid mixed solution of 20g/L; Then , Dissolve β-glycerophosphate disodium in deionized water, stir magnetically until the solution is clear and transparent, and prepare a 560g/L β-glycerophosphate disodium salt solution; finally, mix the prepared chitosan/acetic acid solution with β-glycerol The phosphate solution was mixed at a volume ratio of 9:1 to prepare a chitosan coating solution, which was stored in a refrigerator at 4°C for later use.
(4)载rhBMP-2pH敏感型CSP-bg复合药物载体材料的制备：将载有rhBMP-2的P-bg微球与上述壳聚糖包覆液以固液比1/5(g/mL)混合并摇匀，然后将其置于-21℃冷冻干燥24h即可制得载盐酸万古霉素pH敏感型复合药物载体材料。(4) Preparation of pH-sensitive CSP-bg composite drug carrier material loaded with rhBMP-2: P-bg microspheres loaded with rhBMP-2 and the above-mentioned chitosan coating solution were mixed at a solid-liquid ratio of 1/5 (g/mL ) mixed and shaken evenly, and then freeze-dried at -21°C for 24 hours to prepare a pH-sensitive composite drug carrier material loaded with vancomycin hydrochloride.
实施例3Example 3
载盐酸万古霉素pH敏感型CS-g-PDMAEMAP-bg复合药物载体材料的制备过程如下：The preparation process of the pH-sensitive CS-g-PDMAEMAP-bg composite drug carrier material loaded with vancomycin hydrochloride is as follows:
(1)P-bg微球的制备：称取21.20gNa2CO3、20.02gCaCO3和74.20gH3BO3粉料，于研钵中充分混合，将混合的粉料置于铂金坩埚中，在1150℃的硅碳棒高温炉中，加热熔融30min。将熔化的玻璃液淬冷于冷钢板上，制得硼酸盐玻璃。将玻璃块破碎后，获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球。称取1g球化后的玻璃微球，加入到100ml浓度为0.20mol/L，pH为9.0的K2HPO4溶液中，静置于37℃恒温箱中，浸泡3h后，微球失重率约为30％左右，微球内部剩余硼酸盐玻璃含量约为60％左右，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，置于70℃烘箱中干燥24h，最后将干燥后的微球置于400℃煅烧2h，制得P-bg微球。(1) Preparation of P-bg microspheres: Weigh 21.20g Na2 CO3 , 20.02g CaCO3 and 74.20g H3 BO3 powders, mix them thoroughly in a mortar, put the mixed powders in a platinum crucible, Heat and melt for 30 minutes in a silicon carbide rod high-temperature furnace at 1150°C. The molten glass is quenched on a cold steel plate to produce borate glass. After the glass block is crushed, glass particles with a particle size of 100-150 μm are obtained, and the glass particles are made into glass microspheres by a flame spray ball method. Weigh 1g of spheroidized glass microspheres, add them to 100ml of K2 HPO4 solution with a concentration of 0.20mol/L and a pH of 9.0, and place them in a constant temperature box at 37°C. After soaking for 3 hours, the weight loss rate of the microspheres is about The remaining borate glass content in the microspheres is about 30%, and the remaining borate glass content in the microspheres is about 60%. Then remove the soaking solution, wash it with deionized water three times, and then wash it with absolute ethanol once, and dry it in an oven at 70°C for 24 hours. Finally, the dried microspheres were calcined at 400° C. for 2 h to obtain P-bg microspheres.
(2)盐酸万古霉素在P-bg微球中的装载：为了验证复合材料的药物缓释性能，将盐酸万古霉素装载到P-bg微球上。具体方法为：将盐酸万古霉素溶于磷酸盐缓冲溶液中，制成浓度为1g/L的水相溶液；将1g P-bg微球样品浸泡于50mL盐酸万古霉素的水相溶液中，将其置于真空环境下，保持真空度0.05Mpa左右，使盐酸万古霉素溶液在压力作用下扩散进入P-bg微球的多孔表面及其内部，60min后取出，用PBS磷酸盐缓冲溶液冲洗微球，经冷冻干燥后保存备用，盐酸万古霉素在P-bg微球中的装载量约为10mg/g。(2) Loading of vancomycin hydrochloride in P-bg microspheres: In order to verify the drug sustained release performance of the composite material, vancomycin hydrochloride was loaded on P-bg microspheres. The specific method is: dissolving vancomycin hydrochloride in phosphate buffer solution to make a concentration of 1g/L aqueous phase solution; soaking 1g of P-bg microsphere sample in the aqueous phase solution of 50mL vancomycin hydrochloride, Put it in a vacuum environment, keep a vacuum of about 0.05Mpa, make the vancomycin hydrochloride solution diffuse into the porous surface and interior of the P-bg microspheres under pressure, take it out after 60 minutes, and rinse with PBS phosphate buffer solution The microspheres are stored for future use after freeze-drying, and the loading amount of vancomycin hydrochloride in the P-bg microspheres is about 10 mg/g.
(3)CS-g-PDMAEMA包覆溶液的制备：将0.5g壳聚糖，1.5mL冰醋酸和50mL去离子水置于圆底烧瓶中，加热搅拌使其完全溶解，通N215min排除反应瓶中的空气，升温至55℃后在氮气保护下依次加入硝酸铈铵溶液和一定量的DMAEMA单体，升温至50～60℃后在氩气保护下加入2mL浓度为0.010g/mL的硝酸铈铵溶液和2mL甲基丙烯酸N,N-二甲氨基乙酯单体，恒温反应5h，反应结束后冷却至室温。在丙酮中沉淀得到粗产物，将粗产物在丙酮中回流5h以除去PDMAEMA均聚物，最后在真空烘箱中50℃干燥至恒量得到纯接枝共聚物。将CS-g-PDMAEMA溶于浓度为1mol/L的乙酸溶液中，磁力搅拌24h，制备20g/L的CS-g-PDMAEMA溶液。(3) Preparation of CS-g-PDMAEMA coating solution: put 0.5g chitosan, 1.5mL glacial acetic acid and 50mL deionized water in a round bottom flask, heat and stir to dissolve completely, and pass N2 for 15min to eliminate the reaction After heating up to 55°C, add cerium ammonium nitrate solution and a certain amount of DMAEMA monomer sequentially under nitrogen protection, and add 2mL of nitric acid with a concentration of 0.010g/mL under argon protection after heating up to 50-60°C Cerium ammonium solution and 2 mL of N,N-dimethylaminoethyl methacrylate monomer were reacted at constant temperature for 5 hours, and cooled to room temperature after the reaction. The crude product was obtained by precipitation in acetone, and the crude product was refluxed in acetone for 5 h to remove the PDMAEMA homopolymer, and finally dried in a vacuum oven at 50°C to a constant weight to obtain a pure graft copolymer. Dissolve CS-g-PDMAEMA in acetic acid solution with a concentration of 1mol/L, and magnetically stir for 24h to prepare 20g/L CS-g-PDMAEMA solution.
(4)载盐酸万古霉素pH敏感型CS-g-PDMAEMAP-bg复合药物载体材料的制备：将200μl CS-g-PDMAEMA溶液加入100mg载药P-bg微球并混合均匀，置于真空干燥箱中，40℃干燥6h即可获得pH敏感型复合药物载体材料。(4) Preparation of vancomycin hydrochloride-loaded pH-sensitive CS-g-PDMAEMAP-bg composite drug carrier material: add 200 μl CS-g-PDMAEMA solution to 100 mg drug-loaded P-bg microspheres and mix well, place in vacuum drying In an oven, dry at 40°C for 6 hours to obtain a pH-sensitive composite drug carrier material.
实施例4Example 4
载盐酸万古霉素pH敏感型CS-g-PDMAEMAP-bg复合药物载体材料的制备过程如下：The preparation process of the pH-sensitive CS-g-PDMAEMAP-bg composite drug carrier material loaded with vancomycin hydrochloride is as follows:
(1)P-bg微球的制备：称取17.10gNa2CO3、17.87gCaCO3和142.14gH3BO3粉料，于研钵中充分混合，将混合的粉料置于铂金坩埚中，在1100℃的硅碳棒高温炉中，加热熔融30min。将熔化的玻璃液淬冷于冷钢板上，制得硼酸盐玻璃。将玻璃块破碎后，获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球。称取1g球化后的玻璃微球，加入到100ml浓度为0.20mol/L，pH为9.0的K2HPO4溶液中，静置于37℃恒温箱中，浸泡3h后，微球失重率约为30％左右，微球内部剩余硼酸盐玻璃含量约为60％左右，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，置于80℃烘箱中干燥24h，最后将干燥后的微球置于500℃煅烧2h，制得P-bg微球。(1) Preparation of P-bg microspheres: Weigh 17.10g Na2 CO3 , 17.87g CaCO3 and 142.14g H3 BO3 powders, mix them thoroughly in a mortar, put the mixed powders in a platinum crucible, Heat and melt for 30 minutes in a silicon carbide rod high-temperature furnace at 1100°C. The molten glass is quenched on a cold steel plate to produce borate glass. After the glass block is crushed, glass particles with a particle size of 100-150 μm are obtained, and the glass particles are made into glass microspheres by a flame spray ball method. Weigh 1g of spheroidized glass microspheres, add them to 100ml of K2 HPO4 solution with a concentration of 0.20mol/L and a pH of 9.0, and place them in a constant temperature box at 37°C. After soaking for 3 hours, the weight loss rate of the microspheres is about The remaining borate glass content in the microspheres is about 30%, and the remaining borate glass content in the microspheres is about 60%. Then remove the soaking solution, wash it with deionized water three times, and then wash it with absolute ethanol once, and dry it in an oven at 80°C for 24 hours. Finally, the dried microspheres were calcined at 500° C. for 2 h to obtain P-bg microspheres.
(2)盐酸万古霉素在P-bg微球中的装载：为了验证复合材料的药物缓释性能，将盐酸万古霉素装载到P-bg微球上。具体方法为：将盐酸万古霉素溶于磷酸盐缓冲溶液中，制成浓度为1g/L的水相溶液；将1g P-bg微球样品浸泡于50mL盐酸万古霉素的水相溶液中，将其置于真空环境下，保持真空度0.05Mpa左右，使盐酸万古霉素溶液在压力作用下扩散进入P-bg微球的多孔表面及其内部，60min后取出，用PBS磷酸盐缓冲溶液冲洗微球，经冷冻干燥后保存备用，盐酸万古霉素在P-bg微球中的装载量约为10mg/g。(2) Loading of vancomycin hydrochloride in P-bg microspheres: In order to verify the drug sustained release performance of the composite material, vancomycin hydrochloride was loaded on P-bg microspheres. The specific method is: dissolving vancomycin hydrochloride in phosphate buffer solution to make a concentration of 1g/L aqueous phase solution; soaking 1g of P-bg microsphere sample in the aqueous phase solution of 50mL vancomycin hydrochloride, Put it in a vacuum environment, keep a vacuum of about 0.05Mpa, make the vancomycin hydrochloride solution diffuse into the porous surface and interior of the P-bg microspheres under pressure, take it out after 60 minutes, and rinse with PBS phosphate buffer solution The microspheres are stored for future use after freeze-drying, and the loading amount of vancomycin hydrochloride in the P-bg microspheres is about 10 mg/g.
(3)CS-g-PDMAEMA包覆溶液的制备：将0.5g壳聚糖，1.5mL冰醋酸和50mL去离子水置于圆底烧瓶中，加热搅拌使其完全溶解，通N225min排除反应瓶中的空气，升温至50～60℃后在氮气保护下加入3mL浓度为0.010g/mL的硝酸铈铵溶液和3mL甲基丙烯酸N,N-二甲氨基乙酯单体，恒温反应5h，反应结束后冷却至室温。在丙酮中沉淀得到粗产物，将粗产物在丙酮中回流5h以除去PDMAEMA均聚物，最后在真空烘箱中50℃干燥至恒量得到纯接枝共聚物。将CS-g-PDMAEMA溶于浓度为0.5mol/L的乙酸溶液中，磁力搅拌24h，制备30g/L的CS-g-PDMAEMA溶液。(3) Preparation of CS-g-PDMAEMA coating solution: put 0.5g chitosan, 1.5mL glacial acetic acid and 50mL deionized water in a round bottom flask, heat and stir to dissolve completely, pass N2 for 25min to eliminate the reaction The air in the bottle, after heating up to 50-60°C, add 3mL cerium ammonium nitrate solution with a concentration of 0.010g/mL and 3mL N,N-dimethylaminoethyl methacrylate monomer under nitrogen protection, and react at constant temperature for 5h. Cool to room temperature after the reaction. The crude product was obtained by precipitation in acetone, and the crude product was refluxed in acetone for 5 h to remove the PDMAEMA homopolymer, and finally dried in a vacuum oven at 50°C to a constant weight to obtain a pure graft copolymer. Dissolve CS-g-PDMAEMA in acetic acid solution with a concentration of 0.5mol/L, and magnetically stir for 24h to prepare 30g/L CS-g-PDMAEMA solution.
(4)载盐酸万古霉素pH敏感型CS-g-PDMAEMAP-bg复合药物载体材料的制备：将200μl CS-g-PDMAEMA溶液加入100mg载药P-bg微球并混合均匀，置于真空干燥箱中，40℃干燥6h即可获得pH敏感型复合药物载体材料。(4) Preparation of vancomycin hydrochloride-loaded pH-sensitive CS-g-PDMAEMAP-bg composite drug carrier material: add 200 μl CS-g-PDMAEMA solution to 100 mg drug-loaded P-bg microspheres and mix well, place in vacuum drying In an oven, dry at 40°C for 6 hours to obtain a pH-sensitive composite drug carrier material.
实施例5Example 5
载rhBMP-2pH敏感型CSP-bg复合药物载体材料的制备过程如下：The preparation process of rhBMP-2pH-sensitive CSP-bg composite drug carrier material is as follows:
(1)P-bg微球的制备：称取17.10gNa2CO3、17.87gCaCO3和142.14gH3BO3粉料，于研钵中充分混合。将混合的粉料置于铂金坩埚中，在1125℃的硅碳棒高温炉中，加热熔融30min。将熔化的玻璃液淬冷于冷钢板上，制得硼酸盐玻璃。将玻璃块破碎后，获得粒径为100～150μm的玻璃颗粒，利用火焰喷球法将玻璃颗粒制成玻璃微球。称取1g球化后的玻璃微球，加入到100mL浓度为0.25mol/L，pH为9.5的K2HPO4溶液中，静置于37℃恒温箱中，浸泡6h后，微球失重率为50％，微球内部剩余硼酸盐玻璃含量为20％，然后移去浸泡液，用去离子水清洗三次，再用无水乙醇清洗一次，置于90℃烘箱中干燥12h，最后将干燥后的微球置于500℃煅烧2h，制得P-bg微球。(1) Preparation of P-bg microspheres: Weigh 17.10g Na2 CO 3 , 17.87g CaCO3 and 142.14g H3 BO3 powders, and mix them thoroughly in a mortar. The mixed powder was placed in a platinum crucible, and heated and melted for 30 minutes in a silicon carbide rod high-temperature furnace at 1125°C. The molten glass is quenched on a cold steel plate to produce borate glass. After the glass block is crushed, glass particles with a particle size of 100-150 μm are obtained, and the glass particles are made into glass microspheres by a flame spray ball method. Weigh 1 g of spheroidized glass microspheres, add them to 100 mL of K2 HPO4 solution with a concentration of 0.25 mol/L and a pH of 9.5, and place them in a constant temperature box at 37°C. After soaking for 6 hours, the weight loss rate of the microspheres is 50%, the remaining borate glass content inside the microspheres is 20%, then remove the soaking solution, wash three times with deionized water, and then wash once with absolute ethanol, dry in an oven at 90°C for 12 hours, and finally dry the The microspheres were calcined at 500°C for 2 hours to obtain P-bg microspheres.
(2)重组人骨形态发生蛋白2(rhBMP-2)在P-bg微球中的装载：为了验证复合材料的药物缓释性能，将rhBMP-2装载到P-bg微球上。具体方法为:将rhBMP-2溶于PBS磷酸盐缓冲液中，制成浓度为0.0l g/L的水相溶液；在0～4℃的环境中，将1g P-bg微球样品浸泡于50mLrhBMP-2的水相溶液中，将其放入真空干燥箱内，保持真空度0.05MPa，使rhBMP-2溶液在压力作用下扩散进入P-bg微球，30min后取出，用PBS磷酸盐缓冲液冲洗微球，经冷冻干燥后保存备用，rhBMP-2在P-bg微球中的装载量约为10μg/g。(2) Loading of recombinant human bone morphogenetic protein 2 (rhBMP-2) in P-bg microspheres: In order to verify the sustained drug release performance of the composite material, rhBMP-2 was loaded onto P-bg microspheres. The specific method is: dissolve rhBMP-2 in PBS phosphate buffer solution to make an aqueous phase solution with a concentration of 0.01 g/L; soak 1 g of P-bg microsphere samples in an environment of 0-4 °C 50mL rhBMP-2 aqueous phase solution, put it into a vacuum drying oven, keep the vacuum degree 0.05MPa, make the rhBMP-2 solution diffuse into the P-bg microspheres under pressure, take it out after 30min, buffer with PBS phosphate The microspheres were rinsed with liquid, freeze-dried and stored for later use. The loading amount of rhBMP-2 in P-bg microspheres was about 10 μg/g.
(3)壳聚糖包覆溶液的制备：将壳聚糖溶于浓度为1mol/L的乙酸溶液中，磁力搅拌1小时至溶液澄清，制备20g/L的壳聚糖/乙酸混合溶液；然后，将β-甘油磷酸二钠溶于去离子水中，磁力搅拌至溶液澄清透明，制备560g/L的β-甘油磷酸二钠盐溶液；最后，将制备的壳聚糖/乙酸溶液与β-甘油磷酸盐溶液按9:1的体积比混合，制备壳聚糖包覆液，并置于4℃的冰箱冷藏备用。(3) preparation of chitosan coating solution: chitosan is dissolved in the acetic acid solution that concentration is 1mol/L, and magnetic stirring is to solution clarification for 1 hour, prepares the chitosan/acetic acid mixed solution of 20g/L; Then , Dissolve β-glycerophosphate disodium in deionized water, stir magnetically until the solution is clear and transparent, and prepare a 560g/L β-glycerophosphate disodium salt solution; finally, mix the prepared chitosan/acetic acid solution with β-glycerol The phosphate solution was mixed at a volume ratio of 9:1 to prepare a chitosan coating solution, which was stored in a refrigerator at 4°C for later use.
(4)载rhBMP-2pH敏感型CSP-bg复合药物载体材料的制备：将载有rhBMP-2的P-bg微球与上述壳聚糖包覆液以固液比1/5(g/mL)混合并摇匀，然后将其置于-21℃冷冻干燥12h即可制得载盐酸万古霉素pH敏感型复合药物载体材料。(4) Preparation of pH-sensitive CSP-bg composite drug carrier material loaded with rhBMP-2: P-bg microspheres loaded with rhBMP-2 and the above-mentioned chitosan coating solution were mixed at a solid-liquid ratio of 1/5 (g/mL ) mixed and shaken evenly, and then freeze-dried at -21°C for 12 hours to prepare a pH-sensitive composite drug carrier material loaded with vancomycin hydrochloride.
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于这里的实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。The above description of the embodiments is for those of ordinary skill in the art to understand and apply the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the embodiments herein. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.
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| CN201510319406.0ACN104940897A (en) | 2015-06-11 | 2015-06-11 | PH sensitive type compound drug carrier material and preparation method thereof |
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| WD01 | Invention patent application deemed withdrawn after publication | Application publication date:20150930 | |
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