塞来昔布-聚乳酸-羟基醋酸共聚物微球的缓释性及其对实验性脉络膜新生血管抑制作用的在体研究

The sustained releasing ability of CEL-PLGA-MS in vitro and its inhibitory effects on experimental choroidal neovascularization after intravitreal injection

背景 脉络膜新生血管（CNV）是多种眼底病变共同的病理基础,虽然以往有多种方法进行治疗,但均有其缺点.研究表明,塞来昔布可以治疗实验性CNV,而寻求塞来昔布新的剂型和给药途径可为临床上研发治疗CNV疾病的缓释药物提供依据. 目的 评价体外释放实验中塞来昔布-聚乳酸-羟基醋酸共聚物微球（CEL-PLGA-MS）的缓释性能及其玻璃体腔注射后对实验性CNV的抑制作用. 方法 采用扫描电子显微镜观察CEL-PLGA-MS的形态,用激光粒度分析仪测量其粒径,应用高效液相色谱法（HPLC）测定其载药量及体外释放情况.选取72只雄性棕色挪威（BN）大鼠,用氪激光光凝法建立大鼠右眼CNV模型,将模型眼按照随机数字表法随机分为CEL-PLGA-MS组、塞来昔布组、空白PLGA组和PBS组,每组9只大鼠.根据分组的不同,大鼠玻璃体腔分别注射8μl含塞来昔布320 μmol/L PLGA微球、80 μmol/L塞来昔布、空白PLGA微球及0.01 moL/L PBS.于玻璃体腔注射后14 d,采用荧光素眼底血管造影（FFA）法观察各组大鼠CNV生成情况,OCT测量各组视网膜脉络膜纤维血管增生（FVP）厚度,制备视网膜色素上皮（RPE）-脉络膜-巩膜复合物标本,光学显微镜下观察各组大鼠FVP的形态结构.分别于玻璃体腔注射后7d、28 d,采用逆转录PCR （RT-PCR）法测定并比较各组大鼠激光斑区域血管内皮生长因子（VEGF）mRNA和环氧合酶2（COX-2）mRNA的相对表达量（RQ）.结果 CEL-PLGA-MS的平均粒径为2 467.9 nm,平均载药量为7.77％,体外释放可持续45 d,累积释放百分率为80.91％.玻璃体腔注射后CEL-PLGA-MS在玻璃体中呈团状.注射后14d,OCT测量显示空白PLGA组、PBS组、塞来昔布组和CEL-PLGA-MS组平均FVP厚度值分别为（94.67±4.64）、（98.56±4.72）、（71.00±4.77）、（50.44±.3.01） μm,其中空白PLGA组和PBS组大鼠平均FVP厚度值均明显高于CEL-PLGA-MS组和塞来昔布组,差异均有统计学意义（均P＜0.01）,CEL-PLGA-MS组平均FVP厚度值明显低于塞来昔布组,差异有统计学意义（P＜0.01）.FFA晚期可见空白PLGA组和PBS组视网膜光凝斑区大量荧光素渗漏,呈强荧光,而塞来昔布组和CEL-PLGA-MS组荧光素渗漏量少.光学显微镜下视网膜光凝区中空白PLGA组和PBS组纤维血管组织较塞来昔布组和CEL-PLGA-MS组厚.玻璃体腔注射后7d及28 d空白PLGA组大鼠RPE-脉络膜-巩膜标本中COX-2 mRNA和VEGF mRNA的RQ值均明显高于塞来昔布组和CEL-PLGA-MS组,差异均有统计学意义（均P＜0.01）;玻璃体腔注射后7d塞来昔布组大鼠RPE-脉络膜-巩膜标本中COX-2 mRNA RQ值明显低于CEL-PLGA-MS组,而注射后28 d塞来昔布组COX-2 mRNA和VEGFmRNA RQ值均明显高于CEL-PLGA-MS组,差异均有统计学意义（均P＜0.01）. 结论 CEL-PLGA-MS形态规则,粒径均匀,体外实验有良好的缓释效果;大鼠玻璃体注射CEL-PLGA-MS后能抑制实验性CNV,其抑制作用较塞来昔布更持久.

Background Choroidal neovascularization （CNV） is a common pathological basis of many ocular fundus diseases.Some treating methods are proved to be effective on CNV but there exist their own shortages.Celecoxib can inhibit experimental neovescularization.Sustained release drug of celecoxib and application approach can offer a basis for the therapy of CNV.Objective This study was to evaluate the sustained release ability of celecoxib-poly lactide-co-glycolide microsphere （CEL-PLGA-MS） in vitro and its inhibitory ability on experimental CNV in vivo.Methods CEL-PLGA-MS was prepared by Hebei Medical University and examined under the scanning electron microscope.The size of CEL-PLGA-MS was measured by Laser Particle Size Analyzer.The drugloading in vitro releasing was monitored by high performance liquid chromatograph （HPLC）.Experimental CNV was induced by laser photocoagulation of retina in the right eyes of 72 male brown Norway （BN） rats and then were randomized into the CEL-PLGA-MS group,celecoxib group,blank PLGA group and PBS group.CEL-PLGA-MS with 320 μmol/L celecoxib,80 μmol/L celecoxib,blank PLGA microspheres solution and 0.01 mol/L PBS was intravitreally injected separately according to the grouping.CNV was assessed by fundus fluorescein angiography （FFA） on the 14th day after injection.The fibrovascular proliferation （FVP） thickness at photocoagulation spots was measured by OCT.The retinal pigment epithelium （RPE）-choroid-sclera sections were prepared for the histopathologieal examination of FVP.On the 7th and 28th day after intravitreal injection,the relative expression levels of VEGF mRNA and COX-2 mRNA in the photocoagulation area were detected by reverse transcription PCR （RTPCR）.The use and feeding of the experimental animals were followed by the ARVO statement.Results CELPLGA-MS showed the spherical shape with the mean size of 2 467.9 nm and the drug-loading of 7.77% and the drugrelease rate of 80.91% in vitro for 45 days.It presented the controllable release characteristics.CEL-PLGA-MS agglomerated in vitreous body after injection.On the 14th day after intravitreal injection,the mean FVP thicknesses were （94.67±4.64）,（98.56±4.72）,（71.00±4.77）,（50.44±3.01） μm in the blank PLGA microspheres group,PBS group,celecoxib group and CEL-PLGA-MS group,respectively,showing significant increases in mean FVP thickness in the blank PLGA microspheres group and PBS group compared with the celecoxib group and CEL-PLGAMS group （all at P〈0.01）,and the CEL-PLGA-MS group appeared a lower mean FVP thickness value than the celecoxib group （P〈0.01）.FFA revealed a large number of strong hyperfluorescences at the photocoagulation area in the rat eyes of the blank PLGA microspheres group and PBS group;while only weak hyperfluorescences were seen in the eelecoxib group and CEL-PLGA-MS group.Histopathological examinations verified the same results in the FVP thickness to OCT image.The relative expression levels of COX-2 mRNA and VEGF mRNA in the RPE-choroid-sclera were all significantly elevated in the blank PLGA microspheres group compared with the celecoxib group and CELPLGA-MS group both on the 7th and 28th day after intravitreal injection （all at P〈0.01）.On the 7th day after injection,the relative expression levels of COX-2 mRNA were lower on the 7th day and the relative expression levels of COX-2 mRNA and VEGF mRNA were higher on the 28th day in the celecoxib group in comparison with the CEL-PLGA-MS group （all at P〈0.01）.Conclusions CEL-PLGA-MSs are even in size with the spherical shape and controllable release characteristics in vitro.CEL-PLGA-MS can inhibit experimental CNV and was more durable effective than celecoxib after intravitrea] injection.