bFGF-PLGA缓释微球促进兔静脉皮瓣成活的实验研究
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摘要
目的应用W/O/W复乳溶剂蒸发法制备碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)-聚乳酸-聚羟基乙酸共聚物(Polylactic-co-glycolic acid,bFGF-PLGA)缓释微球,考察其对新西兰大白兔静脉皮瓣成活的影响。
方法本文首先建立了BCA法测定缓释微球中牛血清白蛋白( BSA )含量,以及ELISA法测定bFGF-PLGA缓释微球模拟体外释放bFGF的含量,研究了以BSA作为模型药物制备注射用PLGA含药微球,并应用正交设计试验进一步优化bFGF微球的制备工艺,采用优化处方制备bFGF-PLGA微球,并考察其外观形态和粒径分布,检测其载药量和包封率,研究其体外释药的性质;以新西兰大白兔为实验模型制作侧腹壁静脉皮瓣,并术前5天分别皮内注入bFGF-PLGA(Ⅰ组),bFGF+空微球悬浊液组(Ⅱ组)和生理盐水(Ⅲ组)。术后,①计算术后7天皮瓣的成活面积;②取兔皮肤标本行CD34+免疫组化染色,检测CD34+表达情况及平均血管数。
结果所制微球表面光滑圆整,球体均匀度好,无黏连现象。微球粒径的98%分布在12.50～43.49μm之间,平均粒径为26.93μm,径距0.611±6.60。载药量为[(23.11±0.44)×10~(-3)]%,包封率为(86.51±0.83) %。突释期内微球的体外释放度为27.78% , 30d后体外累积释放度高达81.56% ,微球的体外释药规律符合Higuichi方程( r =0.997)。缓释微球组、空白微球组和生理盐水组术后7天的皮瓣平均面积分别为72.03±1.81、51.03±1.77、50.49±2.36%( P < 0.05) ,平均血管数目分别为34.01±2.30、23.81±3.03、22.56±2.26个/cm2 ( P < 0.05)。
结论1.所制备的bFGF-PLGA缓释微球表征良好,载药量和包封率高;微球通过较长时间地持续释放活性bFGF。
2.术前5天局部皮下注射bFGF-PLGA缓释微球持续释放bFGF,有效促进新生血管的形成,增加皮瓣的血运,提高缺缺血皮瓣的成活面积,是一种简单有效的细胞因子治疗方法。
Objective To prepare the basic fibroblast growth factor (bFGF)-Polylactic-co- glycolic acid(PLGA)sustained release microspheres (MS) by W/O/W multiple emulsion evaporation method, and investigate their effects on the survival of venous flaps.
Methods In this study, BCA and ELISA were established in the determination of sustained-release microspheres content of bovine serum albumin BSA and bFGF-PLGA microspheres slow-release of bFGF in vitro release of simulated.BSA were used as a model protein drug and fabricated protein loaded PLGA microspheres were prepared for injectable cell delivery used as microspheres.The formulation of bFGF-PLGA [poly( lactic-co- glycolic -acid)]-MS was optimized by orthogonal design. The morphology,size distribution, drug loading volume encapsulation efficiency and in vitro drug release behavior of bFGF-PLGA-MS being measured. In 24 New Zealand white rabbits , lateral abdominal wall skin flap supplied by the epigastric vein was created, and five days before operation, respectively, intradermal injection of bFGF-PLGA microspheres (Ⅰgroup) , bFGF solution or bFGF- impregnated PLGA (Ⅱgroup) and normal saline (Ⅲgroup). After seven days,①calculation of the survival area of skin;②Rabbit skin samples from CD34+ immunohistochemical staining to detect the expression of CD34+ blood vessels, and the average number of vascular.
Results The bFGF MS prepared based on optimized formulation exhibited well-defind properties,with the even and uniform sphere in appearance, regular particles without adhesion, about 98% of particles with a size distribution between 12.50 to 43.49μm,with a mean particle size of 26.93μm and size span of 0.611±6.60. The drug loading volume and encap sulation efficiency of bFGF MS reached [(23.11±0.44 )×10~(-3)]% and (86.51±0.83) % , respectively. In the burst release phase, the rate of in vitro drug release amounted to 27.78% , but rose to 81.56% accumulatively 30 days later.The in vitro drug release of bFGF MS corresponded with Higuichi equation ( r =0.997). after 7 days, The sustained-release microspheres, blank microspheres and normal saline group, the average survival of the flap were 72.03±1.81、51.03±1.77、50.49±2.36% (P <0.05), the average number of blood vessels were 34.01±2.30、23.81±3.03、22.56±2.26 /cm2 (P <0.05).
Conclusion
1.bFGF MS with good morphology, high drug loading volume and encapsulation efficiency can be obtained using optimized formulation.
2. 5 preoperative days,local subcutaneous injection of the bFGF MS can promote the survival of venous flaps through a long period due to sustained release of bFGF.
方法本文首先建立了BCA法测定缓释微球中牛血清白蛋白( BSA )含量,以及ELISA法测定bFGF-PLGA缓释微球模拟体外释放bFGF的含量,研究了以BSA作为模型药物制备注射用PLGA含药微球,并应用正交设计试验进一步优化bFGF微球的制备工艺,采用优化处方制备bFGF-PLGA微球,并考察其外观形态和粒径分布,检测其载药量和包封率,研究其体外释药的性质;以新西兰大白兔为实验模型制作侧腹壁静脉皮瓣,并术前5天分别皮内注入bFGF-PLGA(Ⅰ组),bFGF+空微球悬浊液组(Ⅱ组)和生理盐水(Ⅲ组)。术后,①计算术后7天皮瓣的成活面积;②取兔皮肤标本行CD34+免疫组化染色,检测CD34+表达情况及平均血管数。
结果所制微球表面光滑圆整,球体均匀度好,无黏连现象。微球粒径的98%分布在12.50～43.49μm之间,平均粒径为26.93μm,径距0.611±6.60。载药量为[(23.11±0.44)×10~(-3)]%,包封率为(86.51±0.83) %。突释期内微球的体外释放度为27.78% , 30d后体外累积释放度高达81.56% ,微球的体外释药规律符合Higuichi方程( r =0.997)。缓释微球组、空白微球组和生理盐水组术后7天的皮瓣平均面积分别为72.03±1.81、51.03±1.77、50.49±2.36%( P < 0.05) ,平均血管数目分别为34.01±2.30、23.81±3.03、22.56±2.26个/cm2 ( P < 0.05)。
结论1.所制备的bFGF-PLGA缓释微球表征良好,载药量和包封率高;微球通过较长时间地持续释放活性bFGF。
2.术前5天局部皮下注射bFGF-PLGA缓释微球持续释放bFGF,有效促进新生血管的形成,增加皮瓣的血运,提高缺缺血皮瓣的成活面积,是一种简单有效的细胞因子治疗方法。
Objective To prepare the basic fibroblast growth factor (bFGF)-Polylactic-co- glycolic acid(PLGA)sustained release microspheres (MS) by W/O/W multiple emulsion evaporation method, and investigate their effects on the survival of venous flaps.
Methods In this study, BCA and ELISA were established in the determination of sustained-release microspheres content of bovine serum albumin BSA and bFGF-PLGA microspheres slow-release of bFGF in vitro release of simulated.BSA were used as a model protein drug and fabricated protein loaded PLGA microspheres were prepared for injectable cell delivery used as microspheres.The formulation of bFGF-PLGA [poly( lactic-co- glycolic -acid)]-MS was optimized by orthogonal design. The morphology,size distribution, drug loading volume encapsulation efficiency and in vitro drug release behavior of bFGF-PLGA-MS being measured. In 24 New Zealand white rabbits , lateral abdominal wall skin flap supplied by the epigastric vein was created, and five days before operation, respectively, intradermal injection of bFGF-PLGA microspheres (Ⅰgroup) , bFGF solution or bFGF- impregnated PLGA (Ⅱgroup) and normal saline (Ⅲgroup). After seven days,①calculation of the survival area of skin;②Rabbit skin samples from CD34+ immunohistochemical staining to detect the expression of CD34+ blood vessels, and the average number of vascular.
Results The bFGF MS prepared based on optimized formulation exhibited well-defind properties,with the even and uniform sphere in appearance, regular particles without adhesion, about 98% of particles with a size distribution between 12.50 to 43.49μm,with a mean particle size of 26.93μm and size span of 0.611±6.60. The drug loading volume and encap sulation efficiency of bFGF MS reached [(23.11±0.44 )×10~(-3)]% and (86.51±0.83) % , respectively. In the burst release phase, the rate of in vitro drug release amounted to 27.78% , but rose to 81.56% accumulatively 30 days later.The in vitro drug release of bFGF MS corresponded with Higuichi equation ( r =0.997). after 7 days, The sustained-release microspheres, blank microspheres and normal saline group, the average survival of the flap were 72.03±1.81、51.03±1.77、50.49±2.36% (P <0.05), the average number of blood vessels were 34.01±2.30、23.81±3.03、22.56±2.26 /cm2 (P <0.05).
Conclusion
1.bFGF MS with good morphology, high drug loading volume and encapsulation efficiency can be obtained using optimized formulation.
2. 5 preoperative days,local subcutaneous injection of the bFGF MS can promote the survival of venous flaps through a long period due to sustained release of bFGF.
引文
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[2] Schumacher B, Pecher P,Specht B U ,et al. Induction of neoangiogenisis in ischemic myocardium by human growth factor - first clinical results of a new treatment of coronary heart disease[J ].Circulation ,1998, 97 : 645-50.
[3] Ay I,Sugimori H , Finklestein S P. Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl- 2 expression in the ischemic brain following middle cerebral artery occlusion in rats1 Brain Res[J]. Brain Res Mol Brain Res,2001,7(1):71-80.
[4] Ishiguro N ,Yabe Y, Shimizu T , et al. Basic fibroblast growth factor has a beneficial effect to the viability of random skin flaps in rats[J]. Ann Plast Surg ,1994 ,32:356-360.
[5]陈文元,傅小兵,等.碱性成纤维细胞生长因子对皮瓣存活的影响[J].中华整形烧伤外科杂志,1998,14:115- 117.
[6] Nissen NN,Polverini PJ,Gamelli RL,et al. Basic fibroblast growth factor mediates angiogenic activity in early surgical wounds. Surgery,1996 ,119(4):457-65.
[7]赵焕童,李湘洲,王歧刚,等.碱性成纤维细胞生长因子对大鼠真皮下血管网皮片愈合的影响.中国临床康复,2003;7(23)3168-3169.
[8] Duan H, Shen B, He Q, et al. Preparation of bFGF-PLGA sustained release microspheres and studies on their release characteristics and biologic activity in vitro [J].Chin Pharm, 2004; 139: 196-8.
[9] Akiko Kamijo.In vitro release test system of (D,L-lactic-glycolic) acid copolymer microcapsules for sustained release of LHRH agonist (leuprorelin)[J].Journal of Controlled Release,1996,40(1):269-276.
[10] Singh L, Kumar V, Ratner B D. Generation of porous microcellular 85/15 poly (DL- lactide- co- glycolide) foams for biomedical applications [J] . Biomaterials,2004 ,25(13): 1611.
[11]尹东锋,钟延强.影响PLA、PLGA微球中的蛋白、多肽类药物释放的因素[J].中国药学杂志,2006,41(14):1049.
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