【作者】 徐建锋；

【导师】 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2007， 硕士

【摘要】 研究背景:脉络膜新生血管(choroidal neovascularization, CNV)生成是许多致盲性眼底病晚期共有的病理改变。目前临床上治疗CNV的方法主要有激光光凝术、PDT、TTT、手术切除新生血管膜或黄斑转位及放射治疗等,但上述方法均有不足之处。随着人们对CNV生成机制认识的深入,新生血管抑制剂已成为近年来治疗CNV相关疾病的研究热点。糖皮质激素(如地塞米松和曲安奈德)为外源性新生血管抑制剂,已用于CNV相关疾病的治疗。然而,由于眼球壁的阻挡和血-视网膜屏障等因素的存在,糖皮质激素经大剂量系统给药后方能达到治疗效果,但极易引起全身性毒副作用;局部滴眼效果差,且很难到达眼后节。同时,由于药物在玻璃体内的半衰期较短、代谢速度过快、以及患者个体差异较大等原因,玻璃体内注射需多次给药后方可维持有效治疗浓度,且易引起青光眼、白内障、玻璃体出血、视网膜脱离及眼内炎等并发症,因而限制了它在眼后节疾病方面的应用。因此,研制一种既有利于治疗而毒副作用降至最低限度的糖皮质激素给药体系是最为理想的方法之一。理想的控释制剂应该是以一种持续的方式释药,并超越所需要的时间期限。目前,以聚乳酸-羟基乙酸共聚物[poly(D,L-lactide-co-glycolide), PLGA]为材料制成的控释给药系统如植入体、微球及纳米粒等已获得广泛应用。采用具有生物降解性PLGA合成的纳米给药系统可控制药物的释放速率和机体靶向性给药,从而最佳优化药物的治疗效果、减少毒副反应以及消除重复注射所致的并发症。PLGA具有良好的生物相容性,更重要的是其降解速率和药物的释放可通过聚合体的物理化学性质如分子量、亲水性和丙交酯与乙醇酸的比值来控制等。与较大的聚合物相比,它的优势在于不仅可以免除植入和取出时所需的手术操作,而且其降解作用的时间范围可从数月至数年。目的:探讨玻璃体内注射醋酸地塞米松(dexamethasone acetate, DA)-PLGA纳米粒对实验性脉络膜新生血管(choroidal neovascularization, CNV)的抑制效果,并评价其在不同眼组织内的释药模式。方法:采用改良的乳化/溶剂蒸发法制备载药量为50%的DA-PLGA纳米粒。雄性BN大鼠180只,每只动物选取一只眼作为实验眼,采用激光光凝法建立CNV模型,对侧眼未处理。随机将实验眼分7组,即50μg、100μg、200μg和400μg DA-PLGA纳米粒组(各30只)、100μg DA组(30只)、空白PLGA纳米粒组(15只)和生理盐水对照组(15只)。各组分别给予玻璃体内注射的剂量为10μL。光凝后各个时间点行临床检查及眼底照相以观察药物在玻璃体内的消退时间。于光凝后1、3、7、14、21、28和56d,采用反相高效液相色谱法(reversed-phase high-performance liquid chromatography, RP-HPLC)检测各组大鼠眼角膜、虹膜、玻璃体和脉络膜视网膜组织内DA的药物浓度。各组于激光前和光凝后第7、14、21、28和56d行闪光视网膜电图检查(flash-electroretinography, F-ERG)评估视网膜功能。光凝后14d和56d,通过荧光素眼底血管造影(fluorescein fundus angiography, FFA)观察CNV的生成率;之后处死动物,摘除眼球制作标本,进行光学显微镜和透射电子显微镜观察。结果:光凝后1d,临床检查发现DA-PLGA纳米粒、空白PLGA纳米粒和DA玻璃体内注射后均可引起明显的玻璃体混浊,之后药物逐渐吸收并沉积在玻璃体腔下方。DA组消退较迅速,至光凝后14d时药物基本完全吸收,玻璃体间质变清;PLGA纳米粒组消退非常缓慢,至光凝后56d时在玻璃体腔偏下方仍可见少量残留的药物团块。DA-PLGA纳米粒在实验眼玻璃体和脉络膜视网膜组织中的释药模式呈“3相”,即初始突释、稳态释药和再次突释,持续释药时间至少56d。DA原药的释药模式呈“单相”,释药时间为14d左右。在实验全过程中,角膜组织内始终未检测到DA,而虹膜组织仅在给药后第1d测得较低浓度的DA。F-ERG检查显示各组在激光前和激光后不同时间点a波和b波的振幅值和峰时值无明显差异(P>0.05)。光凝后14d和56d,50μg、100μg、200μg和400μg DA-PLGA纳米粒组、DA组、空白PLGA纳米粒组和生理盐水对照组CNV的生成率分别为47.4%/46.2%、28.2%/25.0%、15.8%/15.0%、7.9%/7.7%、31.6%/35%、65.8%/64.1%和65.8%/65.0%。所有DA-PLGA纳米粒组和DA组CNV的生成率较空白PLGA纳米粒组和生理盐水对照组明显降低(P<0.01,χ~2分割法),但各治疗组内无明显差异(P>0.05)。其中,400μgDA-PLGA纳米粒组CNV的生成率明显低于50μg和100μgDA-PLGA纳米粒组(P<0.05)。光凝后56d,DA组中部分原来无荧光素渗漏的光凝斑又重新出现荧光素渗漏。光凝后56d,光凝区纤维血管组织增生(fibrovascular proliferation, FVP)的平均厚度在50μg、100μg、200μg和400μg DA-PLGA纳米粒组、DA组、空白PLGA纳米粒组和生理盐水对照组中分别为84.77±9.79μm、69.52±10.52μm、53.17±13.83μm、38.39±7.87μm、70.49±12.39μm、103.86±16.36μm和105.11±13.70μm。所有DA-PLGA纳米粒组和DA组FVP的平均厚度较空白PLGA纳米粒组和生理盐水对照组显著变薄(P<0.01,SNK-q检验)。不同剂量DA-PLGA纳米粒组组间两两比较均有显著性差异(P<0.05)。透射电子显微镜检查发现,光凝后14d,400μgDA-PLGA纳米粒组和DA组的RPE层紊乱、外节膜盘结构模糊,视网膜节细胞层和神经纤维层的线粒体明显空泡化,内质网肿胀,高尔基复合体排列紊乱,微管肿胀及微丝排列紊乱。至光凝后56d,400μgDA-PLGA纳米粒组RPE层、外节膜盘、视网膜节细胞层和神经纤维层的超微结构基本恢复正常,而DA组未见明显改变。50μg、100μg、200μgDA-PLGA纳米粒组的RPE层、外节膜盘、内外核层及内外丛状层的超微结构均无明显异常。结论:玻璃体内注射DA-PLGA纳米粒可呈剂量依赖性地抑制实验性CNV的生长。与DA原药相比,DA-PLGA纳米粒具有毒性低、缓释及药效持久等特性,有可能成为治疗CNV相关疾病的理想手段之一。更多还原

【Abstract】 Background: Choroidal neovascularization (CNV) is a common pathological endpoint in a host of blind oculopathies. Several therapies in clinical status are available to treat CNV including laser photocoagulation, photodynamic therapy (PDT), transpupillary thermotherapy (TTT), macular translocation or surgical removal and radiation therapy. However, above-mentioned treatments on CNV have some insufficient aspects. As the pathogenesis of CNV is better understood, the anti-neovasular agents for CNV-related diseases has been developed the researched hot point for the past few years. Glucocorticoid, for example dexamethasone and triamcinolone, as an exogenous neovascular inhibitors, has been applied to treat the CNV-related diseases. However, it is difficult to deliver effectively doses of drugs to the posterior part of the eyes. Systemic administration necessitates large doses of the drug, resulting in general side effects. Topical eye drops penetrate poorly into the posterior part of the eye, because of lacrimation and the length of the diffusion path. Intravitreal injection must be administered multiple to maintain drug concentrations within a therapeutic range for a long period of time and sometimes causes a series of complications, such as glaucoma, cataract, vitreous hemorrhage, retinal detachment or endophthalmitis. The development of drug delivery systems to facilitate therapeutic efficacy and to minimize side effects may be the ideal method.Controlled delivery of drugs via poly(D,L-lactide-co-glycolide) (PLGA) polymers as implants, microspheres, and nanoparticles has gained wide acceptance. An ideal controlled release formulation should release the entrapped drugs in a continuous manner over desired time periods. Biodegradable nanoparticles formulated from PLGA and polyvinyl alcohol (PVA) polymers are being extensively investigated for various drug delivery applications. They are used to control drug release rates and to target drugs to specific sites in the body, thereby optimizing their therapeutic response, decreasing toxic side effects, and eliminating the inconvenience of repeated injections. PLGA is biocompatible, and more importantly, the degradation rates of PLGA and the accompanying release of encapsulated drugs can be controlled by the polymer’s physicochemical properties such as molecular weight, hydrophilicity, and the ratio of lactide to glycolide. Its advantages is not only no surgical procedures for implantation in contrast to large polymer implants and removal but also their degradation rate ranges from months to years.Purpose: To investigate the inhibitory efficacy of intravitreal dexamethasone actate (DA)-loaded by Poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles on choroidal neovascularization (CNV) in a laser-induced rat model,and to assess its release mode.Methods: DA-loaded with PLGA nanoparticles containing 50% DA was prepared using an emulsification/solvent evaporation method with slight modifications. CNV was unilaterally induced by laser photocoagulation in male Brown Norway rats. One hundred and eighty rats were randomly divided into seven groups and the 10μL volume of intravitreal drug suspension was performed the same day after photocoagulation. Different dosages of sterilised DA-loaded PLGA nanoparticles suspension were evaluated: i.e. 50μg (n=30), 100μg (n=30), 200μg (n=30), 400μg (n=30). The others groups as control were 100μg DA (n=30), blank PLGA nanoparticles (n=15) and normal saline (n=15). For each animal studied, the experimental eye received one of aforementioned suspensions. The animals were sacrificed on days 1, 3, 7, 14, 21, 28 and 56 after intravitreal injection, then the eyes were enucleated and frozen in -80℃. The cornea, iris, vitreous and choroid-retina of eyes were retrieved and DA levels were determined using a reversed phase high-performance liquid chromatography (RP-HPLC) method. Flash electroretinography (F-ERG) recordings and transmission electron microscope (TEM) were performed to assess retinal toxicity. Fluorescein fundus angiography (FFA) was performed to evaluate the incidence of CNV on days 14 and 56. The animals were sacrificed at 14 and 56 days, then eyecups were processed for histological analysis.Results: At 1 day after photocoagulation, intravitreal DA-loaded PLGA nanoparticles, blank PLGA nanoparticles and DA suspensions caused immediately the obvious vitreous opacitas in clinical examination. While the time extended, the drug suspension was gradually absorbed and deposited in the beneath of vitreous cavity. DA was fast resolved and almost complete absorbtion at 14 days after photocoagulation. However, PLGA nanoparticles suspension was very slowly absorbed so that small amounts of remanent drug clumping could be observed in the beneath of vitreous at 56 days. By RP-HPLC method, the pharmacokinetics of DA-loaded PLGA nanoparticles in vitreous and choroid-retina of the experimental eyes showed a triphasic pattern: i.e. an initial burst, a lag phase with relatively permanent release rate and another burst, and controlled release at least 56 days. In the cornea, DA was below the detection limit during the observed period. Although DA was slightly detected on day 1 after photocoagulation, it hasn’t been determined after 3 days in the iris. During the examination of F-ERG, the results showed no different significant comparing with the a- and b-wave’s amplitude and implicit time between pro and after laser photocoagulation in various groups.On day 14 and 56 after photocoagulation, the incidence of CNV was 47.4% or 46.2% for 50μg, 28.2% or 25.0% for 100μg, 15.8% or 15.0% for 200μg and 7.9% or 7.7% for 400μg DA-loaded PLGA nanoparticles, 31.6% or 35.0% for DA, 65.8% or 64.1% for blank PLGA nanoparticles and 65.8% or 65.0% for normal saline group, respectively. These results demonstrated that the treated groups (including DA-loaded PLGA nanoparticles and DA groups) compared with the blank PLGA nanoparticles and normal saline groups (as control) showed a statistically significant decrease in the formation of CNV (P<0.05). On day 56, the mean thickness of FVP in the recovered lesions was 84.77±9.79μm (n=10), 69.52±10.52μm (n=9), 53.17±13.83μm (n=8), 38.39±7.87μm (n=7), 70.49±12.39μm (n=9), 103.86±16.36μm (n=10) and 105.11±13.70μm (n=11), respectively, in the 50μg, 100μg, 200μg, 400μg DA-loaded PLGA nanoparticles, DA, blank PLGA nanoparticles and normal saline group. All treated groups inhibited FVP relative to controls in a statistically significant fashion (P<0.01). The inhibition of DA-loaded PLGA nanoparticles on experimental CNV showed a dose-dependent effect (P<0.05).On day 14, TEM revealed the disorder of RPE, indefinite of outer segment membrane disc apparente vacuolization of mitochondria, swelling of endoplasmic reticulum, disorded arrangement of Golg’s complex, derangement of microtubule and microfilament in 400μg-treated PLGA nanoparticles and DA groups. The changes of ultrastructure were almost recovered in RPE, outer segment membrane disc, retinal ganglionic and nerve fiber layer in the 400μg DA-loaded PLGA nanoparticles group on day 56, but there were an invisible changes in the DA group. No signs of ultrastructure destruction in the RPE, outer segment membrane disc, inner and outer nuclear layer, and inner and outer plexiform layer were detected in 50μg, 100μg and 200μg DA-loaded PLGA nanoparticles groups.Conclusions: These results suggest that DA-loaded PLGA nanoparticles can dose-dependently inhibit the development of experimental CNV. Compared with DA, DA-loaded PLGA nanoparticles may be a promising drug delivery system (DDS), which possessed the characteristics of low drug toxicity, controlled release and prolonged action and may have potential as a treatment modality for CNV. .更多还原