RFP-PLGA骨缓释复合体的初步实验研究

发布时间：2018-04-26 03:28

本文选题：利福平(RFP) + 聚乳酸-羟基乙酸共聚物(PLGA)　；参考：《中南大学》2013年博士论文

【摘要】：目的：①拟通过O/w乳化溶剂挥发法制备RFP-PLGA微球,以形态、粒径分布、包封率和载药量为指标考察各处方因素的影响,并筛选出最优工艺；②拟采用前期优化处方制备缓释微球,与骨支架结合,并观察微球的骨填充情况及体外释放行为；③采用新西兰大白兔作为动物模型,对RFP-PLGA骨缓释复合体中RFP在体内的分布及缓释行为进行了相关研究,同时考察了该复合物在体内的相容性。为探讨术后脊柱结核患者抗结核治疗,减少其副作用提供实验依据。方法：①通过乳化-溶剂挥发法制备缓释微球,以粒径、载药量、包封率为考察指标对处方中的不同因素如PLGA浓度,表面活性剂的种类及浓度,油水比,药载比进行了单因素考察。并选取重要的影响因素进行正交实验对处方进一步优化,得出各影响因素对制备过程影响强度,并筛选出最优处方；②采用超声振荡法将RFP-PLGA缓释微球灌入异体骨内,再浇盖一层壳聚糖溶液,真空干燥喷金后,用电子扫描显微镜和骨密度仪分别观察RFP-PLGA缓释复合体的填充状态、外观表面形态及灌注前后骨密度的变化；体外释放实验分成三组：A组RFP晶体、B组RFP缓释微球及C组RFP-PLGA骨缓释复合体,并采用透析袋法进行体外释放研究；③体内药动学研究分成A组RFP口服给药组、B组RFP-PLGA骨缓释复合体组、C组RFP晶体-骨复合体组,后两组植入兔腰椎骨性缺损处,并通过大体观察、影像学观察、骨密度观察、组织分布结果、生化指标检测、组织形态学观察六个方面进行对比。采用HPLC-UV法测定血浆及各组织中的药物浓度。采用SPSS17.0软件包进行分析,结果以均数±标准差即x±S表示,采用多种统计学分析方法,如t检验、方差分析及组间比较的SNK-q检验、LSD-t检验等,检验水准a=0.05,P值0.05为差异有显著性意义。结果：①通过单因素考察得知对考察指标具有较大影响的处方因素,在此基础上对处方进行了优化,结果中各处方因素的影响强度如下：药载比PVA浓度PLGA浓度油水比,并确定较优工艺为：PLGA浓度15mg·ml-1、药载比1：2、PVA浓度2%、油水比1：5；平行制得3批微球粒径为12.23±0.98μ m；载药量、包封产率分别为(19.63±0.62)%、(59.49±2.62)%,微球冷冻干燥后,再分散性及外观良好； ②体外释放显示：A组RFP晶体在24h内释放基本完全,B组RFP缓释微球组及C组RFP.PLGA骨缓释复合体组释药平缓,其中RFP缓释微球组31d累计释药率达到近95%,释放进行动力学方程拟合,显示符合零级动力学方程F=0.168*t;RFP-PLGA骨缓释复合体组约31d累计释药率达到近92.31%,未见无明显的突释,进行动力学方程拟合,显示符合零级动力学方程F=0.15*t；骨密度检测：三组灌注前后及B、C组骨密度无明显统计学差异(P0.05)； ③体内实验表明：A组即口服给药组虽然在局部腰椎体组织中能维持较高的有效浓度,但其在肝、脾、心、肺等脏器中也存在较高的浓度;C组即RFP晶体-骨复合体植入组,尽管在维持靶组织有效浓度的同时减小药物在其他脏器中的分布,但维持时间甚短,15天后已低于最低检测浓度；相对而言,B组RFP-PLGA骨缓释复合体植入新西兰大兔给药后,3天时RFP浓度为11±0.7μg/ml-1,在7天时RFP浓度为13.3±0.4μg/ml-1,在15天时RFP浓度为8.5±1.6μg/ml-1,在30天时RFP浓度为1.6±0.4μg/ml-1,在40天时RFP浓度为0.45±0.21μg/ml-1,均明显高于有效抑菌浓度,在体内组织中缓慢下降,不仅其他脏器中的药物浓度低,而且40天时腰椎椎体RFP浓度仍可维持有效抑菌浓度,相对于C组在局部腰椎椎体组织中RFP的有效浓度持续时间较长；骨密度检测：a.术后第7天：三组统计学比较分析：P0.05,无明显统计学意义；b.术后第30天：A组与B、C组比较：P0.05,有明显统计学意义；B组与C组比较：P0.05,无明显统计学意义；c.术后第7天与第30天不同组别比较：A7组与A30组比较：P0.05,无明显统计学意义,B7组与B30组比较：P0.05,有明显统计学意义,C7组与C30组比较：P0.05,有明显统计学意义；肝毒性检测：三组给药前后比较未见明显统计学差异；RFP-PLGA骨缓释复合体行内植入腰椎骨性组织后,腰椎和腰大肌组织均未见明显炎症反应,亦未见相关正常组织的破坏或形态异常。结论： ①1、乳化-溶剂挥发法中诸多因素对微球成形和质量具有明显影响；2、筛选出最优处方即PLGA浓度15mg·mL-1、PVA浓度2%、药载比1：2及油水比1：5。 ②RFP微球形态良好,粒径分布均匀,超声波振荡的方法可以与多孔隙的骨制备成RFP-PLGA骨缓释复合体,其填充效果好,三组灌注前后骨密度无明显统计学差异,具有良好的体外缓释释放性能,检测各时间点的浓度均在有效抑菌浓度以上。 ③RFP-PLGA骨缓释复合体体内椎旁植入后,局部组织内RFP抗结核有效浓度可维持在持续到约40天以上,无明显心、肝、肺及脾等组织蓄积作用及毒性,骨密度无明显影响,组织相容性良好。
[Abstract]:Objective: (1) to prepare RFP-PLGA microspheres by O/w emulsification solvent evaporation method, the effects of various prescription factors were investigated by morphology, particle size distribution, encapsulation efficiency and drug loading, and the optimal process was screened. (3) using New Zealand white rabbits as animal models, the distribution and release behavior of RFP in the RFP-PLGA bone sustained release complex were studied, and the compatibility of the compound in the body was investigated, and the experimental basis was provided to explore the anti tuberculosis treatment and reduce the side effects of postoperative spinal tuberculosis.
Methods: (1) the sustained release microspheres were prepared by emulsification solvent evaporation method. The particle size, drug loading and encapsulation rate were used to investigate the different factors in the prescription, such as the concentration of PLGA, the species and concentration of surfactant, the ratio of oil to water and the ratio of drug load. The effect of various factors on the strength of the preparation process was obtained and the optimal prescription was screened. Secondly, the RFP-PLGA microspheres were injected into the allogenic bone by ultrasonic oscillation, and then a chitosan solution was poured and covered. After the vacuum drying, the filling state of the RFP-PLGA sustained release complex was observed by the electron scanning microscope and the bone densitometer, and the appearance surface shape was observed. The changes of bone density before and after perfusion were divided into three groups: group A RFP, B group RFP sustained-release microspheres and C group RFP-PLGA bone slow release complex, and the release study was carried out by dialysis bag method, and the pharmacokinetic study in vivo was divided into A group RFP oral administration group, B group RFP-PLGA bone slow release complex group, C group RFP crystal bone compound The body group and the latter two groups were implanted in the bone defect of the lumbar spine, and compared the six aspects of the gross observation, the imaging observation, the bone density observation, the tissue distribution, the biochemical index detection and the histomorphology observation. The concentration of the drugs in the plasma and the tissues was measured by the HPLC-UV method. The results were analyzed with the SPSS17.0 software package. The standard deviation is x + S, using a variety of statistical analysis methods, such as t test, variance analysis and SNK-q test of group comparison, LSD-t test, test level a=0.05, P value 0.05 is significant difference.
Results: (1) on the basis of single factor investigation, the prescription factors which had great influence on the index were optimized. On this basis, the prescription was optimized. The influence intensity of each prescription factor was as follows: the ratio of PVA concentration PLGA concentration to oil and water ratio was as follows: PLGA concentration 15mg. Ml-1, 1:2, PVA concentration 2%, and oil and water ratio 1 5: the diameter of the 3 batch microspheres was 12.23 + 0.98 mu m, the drug loading, the encapsulation yield was (19.63 + 0.62)%, (59.49 + 2.62)% respectively, and the microspheres were redispersible and the appearance was good after the microspheres were frozen and dried.
In vitro release, the release of RFP crystals in group A was basically complete in 24h. The release of drug release in group B RFP sustained-release microsphere group and C group RFP.PLGA bone slow release complex was slow, and the cumulative release rate of 31d in RFP sustained-release microspheres reached nearly 95%, and the release was fitted by kinetic equation, showing F=0.168*t of zero order kinetic equation and RFP-PLGA bone sustained release complex group. The cumulative release rate of about 31d reached nearly 92.31%, and no obvious sudden release was found. The kinetic equation was fitted to fit the zero order kinetic equation F=0.15*t. Bone mineral density was detected in the three groups before and after perfusion and B, and there was no significant difference in bone mineral density in group C (P0.05).
(3) in vivo experiments showed that, although the A group, the oral administration group, can maintain a high effective concentration in the local lumbar vertebra tissue, it also has a high concentration in the liver, spleen, heart, lung and other viscera. Group C, RFP crystal bone complex implantation group, although it can reduce the distribution of drug in other organs while maintaining the effective concentration of the target tissue. The maintenance time was very short, 15 days later, it was lower than the minimum detection concentration. Relative, the concentration of RFP was 11 + 0.7 mu g/ml-1 at 3 days and the concentration of RFP was 13.3 + 0.4 g/ml-1 at 7 days, and at 15 days, the concentration of RFP was 8.5 + 1.6 mu g/ml-1, and the concentration of RFP was 1.6 + 0.4 g/ml-1 and RF on 30 days, RF, at the time of 15 days. The concentration of P was 0.45 + 0.21 mu g/ml-1, which was obviously higher than the effective inhibitory concentration and decreased slowly in the body tissues, not only the drug concentration in other organs was low, but also the effective inhibitory concentration of the lumbar vertebra RFP concentration was still maintained at 40 days. Compared with the group C in the local lumbar vertebra tissue, the effective concentration of RFP was longer and the bone density was detected. Seventh days after A.: three groups of statistical analysis: P0.05, no significant statistical significance; thirtieth days after B.: group A and B, C group: P0.05, there was significant statistical significance; B group and C group: P0.05, no significant statistical significance; C. postoperative seventh days and thirtieth days to compare: A7 group and the group comparison: there is no obvious unification Study significance, group B7 and group B30 compared with group P0.05, there was significant statistical significance, C7 group compared with the C30 group: P0.05, there was significant statistical significance; hepatotoxicity test: three groups before and after the drug delivery was not significantly different; RFP-PLGA bone slow release complex after the implantation of lumbar vertebra bone tissue, lumbar and the lumbar major muscle tissue are not obvious inflammation There was no damage or abnormal morphology of normal tissues.
Conclusion:
(1) 1, many factors in the emulsification solvent evaporation method have obvious influence on the formation and quality of microspheres. 2, the optimum prescription is PLGA concentration 15mg mL-1, PVA concentration 2%, drug load ratio 1:2 and oil and water ratio 1:5.
The morphology of RFP microspheres was good and the particle size distribution was uniform. The method of ultrasonic oscillation could be prepared with the porous bone to prepare the sustained release complex of RFP-PLGA bone, and its filling effect was good. There was no significant difference in bone density between the three groups before and after perfusion. It had good release performance in vitro, and the concentration of each time point was above the effective bacteriostasis concentration.
(3) after the paravertebral implantation in the RFP-PLGA bone sustained release complex, the effective concentration of RFP in the local tissue was maintained for more than 40 days, without obvious heart, liver, lung and spleen and other tissue accumulation and toxicity, bone density had no obvious influence, and the histocompatibility was good.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

【参考文献】