碱性成纤维细胞生长因子-聚乳酸-聚羟基乙酸共聚物微球的体外释药规律:具有促进兔静脉皮瓣成活的作用?

In vitro drug release rule of basic fibroblast growth factor-poly(lactic-co-glycolic-acid) copolymer microspheres:Promotion of the venous flap survival in rabbits?

背景:与正常生理性皮瓣相比,静脉皮瓣的主要优点在于摆脱了动脉血管分布区域对传统轴型皮瓣的供区与受区的限制,但其成活率不稳定。目的:探讨碱性成纤维细胞生长因子-聚乳酸-聚羟基乙酸共聚物(bFGF-polylactic-co-glycolicacid,bFGF-PLGA)缓释微球对兔静脉皮瓣成活的影响。设计、时间及地点:随机对照动物实验,于2008-05/10在南华大学完成。材料:健康新西兰大白兔24只,随机分为3组:bFGF-PLGA缓释微球组、空微球组、空白对照组,8只/组。方法:应用正交设计试验进一步优化碱性成纤维细胞生长因子微球的制备工艺,采用优化处方制备bFGF-PLGA微球。3组兔均制作侧腹壁静脉皮瓣,术前5d,bFGF-PLGA缓释微球组向皮瓣皮内注射28.85g/L优化处方后的bFGF-PLGA微球3mL(含碱性成纤维细胞生长因子20μg),空微球组同法注射同等质量空微球+等量碱性成纤维细胞生长因子混合溶液,空白对照组注射等量生理盐水。主要观察指标:考察bFGF-PLGA微球外观形态和粒径分布,检测其载药量、包封率、体外释药性质。术后7d检测皮瓣成活率,取兔皮肤标本行CD34+免疫组化染色,检测CD34+的表达及皮瓣内微血管密度。结果:所制微球表面光滑圆整,球体均匀度好,无粘连现象;微球粒径98%分布在12.50～43.49μm,平均粒径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)。空微球组、空白对照组的静脉皮瓣成活率及皮瓣内微血管密度基本相似(P=0.597,P=0.336),但均明显低于bFGF-PLGA缓释微球组(P=0.000)。免疫组化染色结果显示,bFGF-PLGA能够促进皮瓣与周围建立血供关系,改善皮瓣的成活,并表达较为丰富的CD34+。结论:应用W/O/W复乳-干燥法可成功制备表征良好、载药量和包封率高的bFGF-PLGA微球,该微球通过较长时间地持续释放活性碱性成纤维细胞生长因子,可明显促进兔静脉皮瓣的存活。

BACKGROUND：Compared with normal physiological flap,main advantage of venous skin flap is that it throws off the limitation of arterial vascular territory on donor site and recipient site of traditional axial skin flap.However,its survival rate is unstable.OBJECTIVE：To explore effects of basic fibroblast growth factor （bFGF）-[poly（lactic-co-glycolic-acid）] （PLGA）-sustained release microspheres on the survival of rabbit venous flaps.DESIGN,TIME AND SETTING：A randomized controlled animal study was performed at the University of South China from May to October 2008.MATERIALS：A total of 24 healthy New Zealand rabbits were equally randomly assigned to bFGF-PLGA sustained release microsphere,blank microsphere and blank control groups.METHODS：The formulation of bFGF microspheres was optimized by orthogonal design.bFGF-PLGA microspheres were prepared by optimized method.Lateral abdominal wall skin flap was created in rabbits from 3 groups.Five days before operation,28.85 g/L bFGF-PLGA microspheres 3 mL （containing bFGF 20 μg） was intradermally injected into rabbits from the bFGF-PLGA sustained release microsphere group.An equal volume of blank microsphere ＋ bFGF was injected in the rabbits of the blank microsphere group.Rabbits from the blank control group were infused with the same volume of saline.MAIN OUTCOME MEASURES：Morphology and particle distribution of bFGF-PLGA microspheres,drug loading volume,encapsulation efficiency,in vitro drug release characteristics were measured.After seven days,the survival area of skin was determined.Rabbit skin samples received CD34＋ immunohistochemical staining to detect the expression of CD34＋ and average number of blood vessels.RESULTS：The bFGF microsphere prepared based on optimized formulation exhibited well-defined 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 43.49 μm,with a mean particle size of 26.93 μm and size span of （0.611 ± 6.60）.The drug loading volume and encapsulation efficiency of bFGF microsphere 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 microsphere corresponded with Higuichi equation （r=0.997）.The sustained-release microspheres,blank microspheres and normal saline group,the average survival of the flap and the average number of blood vessels were similar （P=0.597,P=0.336）,but still significantly lower than the bFGF-PLGA sustained release microsphere group （P=0.000）.Results of immunohistochemical staining revealed that bFGF-PLGA promoted blood supple between flap and surroundings,improved flap survival and abundant CD34＋ expression.CONCLUSION：bFGF microsphere with good morphology,high drug loading volume and encapsulation efficiency can be obtained using W/O/W multiple emulsion evaporation method.The bFGF microsphere can promote the survival of rabbit venous flaps through a long period due to sustained release of bFGF.