聚乙交酯-丙交酯泡沫材料的生物学评价

为了解聚乙交酯-丙交酯(PLGA)多孔聚合物支架材料进一步作为组织工程种子细胞支架材料的安全性,通过细胞毒性试验和肌内植入试验,对PLGA的生物相容性和可降解性进行了检测和评价。结果表明,PLGA泡沫材料的毒级为0～1级,在体外细胞培养试验中显示了良好的细胞相容性;术后各时间点,材料植入处肌组织无明显炎症反应,随着时间推移,泡沫材料占位空间逐渐缩小,证实了植入材料的可降解性。说明PLGA可以作为组织工程研究的细胞外基质材料。     

兽用吡喹酮-聚乳酸-羟基乙酸缓释植入棒含量测定及释放度考察

建立了吡喹酮-聚乳酸-羟基乙酸（吡喹酮-PLGA）缓释植入棒含量测定的HPLC法,并对其释放度进行了考察。以甲醇-水（100：40,V／V）为流动相,采用GraceC18反相色谱柱（4．6mm250mm,5μm）,流速1．0mL／min,紫外检测波长263nm,在此试验条件下,吡喹酮在10—200μg／mL范围内与峰面积线性关系良好,r=0．9999;平均回收率为99．48％,RSD为0．53％（n=9）。三批样品的含量分别为98．96％、98．67％和98．81％,该方法简单、快捷、辅料无干扰、准确度高,适于吡喹酮-PLGA植入棒的含量测定。吡喹酮-PLGA植入棒以骨架溶蚀释放机制缓慢释放,释药期可达三周,释放度高于300μg／d。     

甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物纳米微球作为口蹄疫病毒核酸疫苗递送载体的特性评价

旨在对制备的甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物[poly （D，L-lactide-co-glycolide），PLGA]纳米微球作为口蹄疫病毒（foot-and-mouth disease virus，FMDV）核酸疫苗递送载体进行评价。采用西佛碱反应和元素分析制备具有一定取代度的甘露糖修饰的壳聚糖衍生物（mannose modified chitosan，MCS），然后，经双重乳化挥发法制备得到甘露糖修饰的壳聚糖PLGA纳米微球（MCS-PLGA-NPs）。采用纳米粒径仪检测MCS-PLGA-NPs粒径分布和表面电势（zeta）、扫描电镜考察其形态、琼脂糖凝胶电泳观察其对质粒的吸附和吸附质粒后抵抗核酸酶降解能力、CCK-8法检测MCS-PLGA-NPs的细胞毒性、激光共聚焦观察巨噬细胞对MCS-PLGA-NPs-质粒DNA复合物的摄取、荧光显微镜和Western blot验证MCS-PLGA-NPs加载质粒DNA在细胞中的表达。元素分析结果表明，成功制备了取代度为5%~10%的MCS。纳米粒径测定和扫描电镜结果表明，MCS-PLGA-NPs的zeta为正值、粒径分布均匀且形态规则呈球形。琼脂糖凝胶电泳结果显示，MCS-PLGA-NPs吸附质粒的能力随着其质量的增加而增强并且可以在一定程度上抵抗核酸酶降解质粒DNA。在细胞毒性试验中，不同浓度的MCS-PLGA-NPs与RAW264.7细胞共孵育24 h后，细胞存活率仍在85%以上。在细胞摄取试验中，用激光共聚焦显微镜可以明显观察到质粒DNA结合到纳米微球表面被RAW264.7细胞摄取。荧光显微镜和Western blot试验证明MCS-PLGA-NPs加载质粒DNA可以在细胞中进行表达。综上表明，本研究成功制备了MCS以及具有递送核酸疫苗能力的MCS-PLGA-NPs，为FMDV核酸疫苗的递送研究提供了新的方向和见解，也为该递送载体携带特定抗原靶向抗原递呈细胞表面甘露糖受体以及应用于动物免疫的研究奠定基础。     

乳鼠心肌细胞的体外三维培养

【目的】探讨应用PLGA泡沫支架材料,在体外建造组织工程化心肌组织的可行性。【方法】采用顺序消化及差速贴壁法分离纯化乳鼠心肌细胞,将分离所得的心肌细胞接种于PLGA多孔支架材料上,观测复合体内心肌细胞的生长状况、超微结构、细胞代谢率及细胞组分的变化,并将其与正常心肌和二维培养的心肌细胞进行比较。【结果】复合体内的细胞具有心肌细胞的特殊超微结构,免疫组化显示其中的α-横纹肌肌动蛋白染色呈强阳性,与二维培养的细胞相比,细胞代谢更加旺盛。【结论】采用组织工程技术有可能在体外培育出组织工程化心肌组织。     

无乳链球菌乳酸-羟基乙酸共聚物微球的制备及其体外释放特点分析

以乳酸-羟基乙酸共聚物（PLGA）为材料,采用复乳溶剂挥发法制备无乳链球菌（Streptococcus agalactiae）全菌及超声破碎后的上清微球疫苗。显微镜观察显示随着 PLGA 质量浓度、PVA（聚乙烯醇）质量浓度和外水相体积的增加,上清微球平均粒径均随之增大。最终确定上清微球制备条件为 PLGA 质量浓度25 mg·mL -1、PVA 质量浓度1 mg·mL -1、外水相体积20 mL。全菌微球制备条件与上述的相比,仅 PVA 质量浓度调整为2 mg·mL -1。扫描电镜观察显示全菌和上清微球平均粒径分别为9.4μm 和3.9μm,微球均呈球形。BCA（二喹啉甲酸）法分析显示包封率分别为68.07％和63.49％;载药量分别为5.49＆#215;108个·mg -1和3.58％;28 d 体外累积释放量分别为64.2％和82.8％。     

Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells’ luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2–3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.     

Pyraclostrobin-loaded poly (lactic-co-glycolic acid) nanospheres: Preparation and characteristics

We used poly(lactic-co-glycolic acid)(PLGA) as a carrier polymer for pyraclostrobin-loaded nanospheres. Using the ultrasound emulsification-solvent evaporation method, the physicochemical characteristics and release properties of the pyraclostrobin-loaded nanospheres were studied by dialysis. The optimal nanospheres prepared had a diameter of 0.6 μm, an active ingredient loading of 17.2%, and a loading rate of 89.7%. Infrared spectroscopy data and differential scanning calorimetry revealed that pyraclostrobin was successfully embedded in the carrier PLGA, and photostability tests indicated enhanced ultraviolet resistance of pyraclostrobin-loaded PLGA nanospheres nanospheres. Release property testing indicated that smaller particles had a faster release rate. Nanospheres also had a faster release rate in slightly acidic and slightly basic environments than in a neutral condition. Agitated nanospheres had a faster release rate than immobile nanospheres. The cumulative release kinetics of pyraclostrobin-loaded nanospheres was consistent with the first order kinetic equation and the Weibull equation.     

Ultrastructural and Immunohistochemical Studies on Uptake and Distribution of FITC-Conjugated PLGA Nanoparticles Administered Intratracheally in Rats

Polylactide-glycolide (PLGA) nanoparticles have been developed as pulmonary drug delivery carriers. To investigate their behavior, small- (d50 = 74 nm) and large-sized (d50 = 250 nm) FITC-conjugated PLGA nanoparticles were intratracheally administered to rats and were traced for 5, 30 and 60 minutes and 24 hours after administration (HAT). Immunohistochemically, a, FITC-positive reaction was observed in type-I alveolar epithelial cells (type-I AEC), endothelial cells and alveolar macrophages in the lungs from 5 minutes after treatment (MAT) to 24 HAT in both nanoparticle groups. In the kidneys, a positive reaction was observed in proximal tubular epithelial cells at 30 MAT; the reaction peaked at 60 MAT and was reduced at 24 HAT, while no positive reaction was seen in other sites. Ultrascructurally, the number of membrane-bound vesicles, which were approximately 70 nm in size and hard to distinguish from pinocytic vesicles, apparently increased in type-I AEC and endothelial cells at 5 MAT in the small-sized group, in comparison with the control group receiving physiological saline. The number of vesicles in the large-sized group was almost same as that in the control group. On the other hand, in both nanoparticle groups, lysosomes filled with nanoparticles appeared in alveolar macrophages from 30 MAT to 24 HAT. These results indicate that PLGA nanoparticles might be quickly transferred from the alveolar space to the blood vessel via type-I alveolar epithelial cells and excreted into urine, and that there is a threshold for particle size, less than approximately 70 nm in diameter, with regard to absorption through the alveolar wall.