聚乳酸-羟基乙酸微球内葡激酶突变体(K35R)的稳定性研究

 目的研究微球内葡激酶突变体(K35R,DGR)的存在状态和影响其稳定性的主要因素。方法使用傅里叶变换红外光谱法(FT-IR)研究了微球内DGR的二级结构的变化,并纤溶活性测定法考察了pH和聚乳酸-羟基乙酸(PLGA)表面对DGR稳定性的影响。结果①包封的DGR的二级结构明显发生了改变,但是,这种改变是β-折叠受到微扰的结果,并未影响微球内DGR的稳定性;②体外释放时,微球内PLGA的降解产物产生的酸性微环境会导致DGR变性,在内水相中加入Mg(OH)₂纳米粒可以抑制DGR的变性聚集;③对PLGA吸附DGR的研究表明,PLGA表面对DGR的吸附是离子相互作用和疏水相互作用共同起作用的结果,也是造成DGR失活的重要原因。结论PLGA对DGR的包封会导致蛋白质药物的二级结构发生变化,但是并不影响微球内DGR的稳定性;导致微球内DGR变性失活的主要原因是微球内的酸性微环境,碱性添加物可以抑制这种变性失活。PLGA表面对蛋白质药物的非特异性吸附也会导致部分DGR的失活。     

复乳法制备胰岛素PLGA纳米粒影响包封率因素考察

以Poloxamer188为乳化剂，乙酸乙酯为有机溶剂，采用复乳法制备了胰岛素乳酸／羟基乙酸共聚物（PLGA）纳米粒，考察了乳化剂和PLGA的浓度。内水相中胰岛素的浓度为pH，溶剂挥发方法和内水相中加入聚乙烯醇（PVA）等实验各因素对胰岛素PLGA纳米粒包封率的影响。结果表明，乳化剂的浓度较高，PLGA的浓度较小，内水相的pH接近胰岛素的pI(5．3），胰岛素的浓度较低，缩短有机溶剂挥发时间及内水相中加入PVA有利于提高胰岛素的包封率，经实验条件优化后制备的胰岛素PLGA纳米纳平均粒径为149．6nm，多分散性系数小于0．1，包封率提高到42．8％。     

经等离子体修饰并复合BMSCs的组织工程神经研究

目的探讨经等离子体修饰的PLGA神经导管复合BMSCs后修复大鼠坐骨神经缺损的疗效。方法取30只新生SD大鼠股骨及胫骨骨髓,培养获得原代BMSCs,并传至第3代。将PGA和PLA按90∶10摩尔比例混合,制备PLGA神经导管,对其进行等离子体修饰。取30只4周龄SD大鼠,制备右后肢坐骨神经1cm缺损模型。按修复材料不同随机分成3组(n=10),实验组:第3代BMSCs复合经等离子体修饰PLGA神经导管;对照组:第3代BMSCs复合普通PLGA神经导管;自体组:自体神经。术后6周观察大鼠的行走步态变化,测定坐骨神经功能指数(sciatic function index,SFI)、电生理变化、腓肠肌湿重恢复率,采用再生神经轴突图像分析评价神经修复效果。结果术后大鼠均存活至实验完成。术后6周,实验组和自体组大鼠行走步态恢复情况较对照组好。实验组、对照组及自体组SFI分别为—51.02±6.54、—58.73±7.87及—48.73±3.95,组间比较差异均有统计学意义(P<0.05)。实验组运动神经传导速度及波幅明显高于对照组(P<0.05),但与自体组差别不明显(P>0.05),对照组与自体组差异有统计学意义(P<0.01)。实验组、对照组及自体组腓肠肌湿重恢复率分别为56.13%±4.27%、43.14%±6.52%、59.47%±3.85%,组间差异均有统计学意义(P<0.05)。实验组有髓神经纤维密度、直径及髓鞘厚度均高于对照组(P<0.05),低于自体组(P<0.05);对照组与自体组差异有统计学意义(P<0.01)。结论复合BMSCs的等离子体PLGA神经导管能有效促进周围神经的再生,为研制新型的组织工程神经修复周围神经缺损提供新的思路。     

Local injection of thrombin‐related peptide (TP508) in PPF/PLGA microparticles–enhanced bone formation during distraction osteogenesis

We have previously demonstrated that injections of the thrombin‐related peptide, TP508, into the lengthening gap have significantly enhanced bone consolidation in a rabbit model of distraction osteogenesis. This study was to further test the effect of a single TP508 injection in slow release preparation on bone formation during distraction osteogenesis. Rabbits had left tibiae lengthened unilateral lengthener at rate of 1.4 mm/day for 6 days. TP508 was injected into as the following: Group 1, TP508 in saline; Group 2, in PPF/PLGA [poly(propylene fumarate)/poly(D,L ‐lactic‐co‐glycolic acid)] microparticles; and Group 3, dextran gel only. All the animals were killed 2 weeks after lengthening. On radiographies, more bone was formed in the two TP508‐treated groups at first and secnd week postlengthening than that of the control Group 3. Microcomputed tomography (microCT) at 2 weeks indicated that the most advanced bone formation and remodeling was seen in Group 2. The mean volumetric BMD of the regenerates was significantly higher in the TP508 treated groups compared to the control group (p < 0.05). Histological evaluations supported the radiographic and the microCT results. In conclusion, we have demonstrated that a single injection of small amount of TP508 (300 µg) at the end of lengthening phases has significantly enhanced bone consolidation process in a rabbit model of distraction osteogenesis. The delivery of TP508 in PPF/PLGA microparticles appears to lead to a better quality bone formation over the saline delivery, further examinations are needed to confirm if PPF/PLGA microparticles may be desirable drug delivery form in augmenting bone formation. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:539–546, 2008     

干扰素/聚乳酸-羟基乙酸共聚物及壳聚糖/聚乳酸-羟基乙酸共聚物复合膜对椎板切除后脊髓c-fos表达的影响

背景：应用硬膜外阻隔材料是预防椎板切除术后硬膜外瘢痕粘连的一种方法,而制备既有机械阻隔能力,又有抑制成纤维细胞能力的复合膜是其研究方向。目的：制备壳聚糖/聚乳酸-羟基乙酸共聚物（poly（lactic-co-glycolic acid）,PLGA）与干扰素/PLGA复合膜,观察其减少脊髓伤害性刺激因子c-fos表达的作用。设计、时间及地点：2005-05/2007-04在中科院长春应用化学研究所完成材料制备,随机对照动物实验在解放军第二○八医院实验动物室完成。材料：PLGA膜、壳聚糖/PLGA及干扰素/PLGA复合膜由中科院长春应用化学研究所制备。方法：大耳白兔120只行L2及L4椎板切除,然后随机分为6组,每组20只,于椎板缺损区处理如下：①空白对照组：硬膜外不放任何物质。②自体游离脂肪组：取皮下脂肪贴于硬膜表面。③透明质酸钠组：透明质酸钠1mL涂于硬膜外。④PLGA膜组、壳聚糖/PLGA及干扰素/PLGA复合膜组：分别将各种膜修剪成合适大小植于硬膜外。主要观察指标：术后2,4,6,8周处死动物,取L1～2节段脊髓,对L2脊髓横切片并行免疫组化染色,显微镜下观察并计数Fos样免疫反应阳性细胞。结果：脊髓内有c-fos的表达,随时间的增长,其表达量逐渐增多,主要在脊髓背角浅层;实验各组Fos样免疫反应阳性细胞计数均低于空白对照组（P〈0.01）,晚期以壳聚糖/PLGA复合膜及IFN/PLGA复合膜组的表达量少,与其他组比较差异有显著性意义（P〈0.01）。结论：壳聚糖/PLGA与干扰素/PLGA复合膜可减少椎板切除术后脊髓c-fos的表达。     

几种组织工程支架材料生物力学性能的研究

背景：采用工程学的方法来构建人体组织及器官的替代品以及介入治疗的器械是目前生物组织工程领域的研究热点,而材料的选择是其核心。所制备的移植物,至少要能够赖受住移植过程中的各种外力而不至于破碎。故对被作为原料应用于组织工程领域的常用物质的力学性质进行评价,可以为组织工程研究中的材料选择提供依据。 目的：考察9种组织工程支架材料的力学性能,为组织工程支架的设计、制作的选材提供力学依据。设计：重复测量试验。单位：重庆大学生物工程学院。 材料：以聚乳酸-羟基乙酸共聚物（PLGA）、胶原、海藻酸钠、脱细胞猪皮、脱细胞血管、脱细胞猪皮-PLGA的复合体、脱细胞血管-聚乳酸/聚羟乙酸共聚物的复合体以及经改性的壳聚糖和明胶作为考察材料。 方法：实验于2006-04/2007-03在重庆大学生物工程学院生物流变学实验室完成。制备上述9种材料试件,同时对壳聚糖和明胶进行改性。然后用INSTRON 1011拉伸实验机对其进行拉伸试验。拉伸速度为10mm/min,在应变范围（0.005-0.02）内计算试件的杨氏模量,并获得最大应变和最大应力。主要观察指标：9种材料的最大应变、最大应力及杨氏模量 结果：①最大应变：脆性依次增大的顺序是：脱细胞猪皮〉脱细胞猪皮-PLGA〉脱细胞血管〉海藻酸钠〉脱细胞血管-PLGA〉胶原〉明胶（P〈0.05）;壳聚糖和PLGA的断裂伸长率比其他7种材料高出很多,分别为133%和276%,差异有显著性意义（P〈0.05）。在与PLGA复合后,脱细胞血管和脱细胞猪皮的断裂伸长率均变小,即脆性增加。②最大应力：脱细胞猪皮以及与PLGA共混后的最大应力即断裂强度均明显高于其他材料,且与PLGA共混后的脱细胞猪皮的断裂强度也略高于脱细胞猪皮。明胶、壳聚糖和脱细胞血管的断裂强度大体相当,介于7.67-9.51MPa。胶原和海藻酸钠的断裂强度最低,介于1.16-1.40MPa。同时,与PLGA共混后的脱细胞血管的断裂强度明显低于脱细胞血管,差异有显著性意义（P〈0.05）。③杨氏模量：明胶的杨氏模量及硬度最大,而且远远大于其他各种材料。脱细胞猪皮的杨氏模量及硬度最低。与PLGA共混后的脱细胞血管及脱细胞猪皮的硬度均增大,差异有显著性意义（P〈0.05）,并于PLGA相当。除明胶以外,其他各材料硬度由大到小的排列顺序依次是：脱细胞血管-PLGA、PLGA、脱细胞猪皮-PLGA、脱细胞血管、壳聚糖、海藻酸钠、胶原、脱细胞猪皮。 结论：①脱细胞血管及脱细胞猪皮具有良好的力学性质。②与组织来源的材料即脱细胞血管及脱细胞猪皮相比,PLGA的韧性较好,强度较低,硬度偏高。③海藻酸钠、明胶、壳聚糖的力学性质有望通过与PLGA的复合而得到改善。     

Control of neonatal human dermal fibroblast migration on poly(lactic‐co‐glycolic acid)‐coated surfaces by electrotaxis

Many types of cells respond to applied direct current electric fields (dcEFs) by directional cell migration, a phenomenon called galvanotaxis or electrotaxis. In this study, electrotaxis was used to control cell migration. We designed a new electrotaxis incubator and chamber system to facilitate long‐term (> 12 h) observation and to allow for alterations to the direction of the current. Poly(lactic‐co‐glycolic acid) (PLGA) was coated onto surfaces to mimic a commonly used tissue‐engineering scaffolding environment. Neonatal human dermal fibroblasts (nHDFs) were grown on PLGA‐coated surfaces and exposed to EFs at increasing currents in the range 0–1 V/cm. These cells migrated toward the cathode during 3 h of dcEF stimulation; however, the migration speed decreased with increasing electric fields. Cells exposed to dcEFs in the range 1–2 V/cm showed no changes to migration speed or x forward migration indices (xFMIs) and the cells continued to move toward the cathode. nHDFs showed directional migration towards the cathode in direct current (dc) EFs (1 V/cm) and they moved in the opposite direction when the polarity of the dcEF was reversed. Reorganization of the actin cytoskeleton and polarization of the Golgi apparatus were evaluated by immunostaining, which showed that the actin cytoskeleton elongated towards the cathode and the Golgi apparatus polarized in the direction of the dcEF. This study revealed that cell migration could potentially be controlled on PLGA scaffolds through electrotaxis. Copyright © 2015 John Wiley & Sons, Ltd.     

Collagen type I–PLGA film as an efficient substratum for corneal endothelial cells regeneration

Surface modulations of desired biological construct design for regenerative medical therapy is considered to be highly crucial for cell growth and the subsequent regeneration of biologically competent tissues. In this study, we fabricated stable, transparent, collagen type‐I‐coated PLGA films (Col I–PLGA) as a potential substratum for the regeneration of corneal endothelial cells. Morphological and structural properties were analysed by FE–SEM, AFM, FTIR, contact angle, etc., and in vitro biocompatibility of the Col I–PLGA films was further tested in primary rabbit corneal endothelial cells (rCEnCs) as models. Compared with bare PLGA films, the Col I–PLGA films displayed the requisite surface roughness, with higher Ra (nm) values, transparency, good hydrophilicity, stability and water uptake. Next, cultured rCEnCs on Col I–PLGA films showed the characteristic polygonal shape of rCEnCs with enhanced initial attachment, proliferation and expression of mRNAs. Collectively, these results indicate that Col I–PLGA can be employed as a suitable alternative for high‐quality corneal tissue expansion and transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

三种注射用新辅料对Beagle犬类过敏及过敏反应研究

目的研究3种注射用新辅料乙交酯-丙交酯共聚物（PLGA）、乙二醇单甲醚-聚乳酸嵌段共聚物（mPEG-PDLLA）和羟丙基-β-环糊精（HP-β-CD）能否引起Beagle犬产生类过敏或过敏反应,为临床研究和应用提供依据。方法使用Beagle犬于1、3、5d致敏共3次,末次致敏后第14d激发。观察给药后反应症状,检测常规血液学指标、血浆中组胺、IgE、IgG和IgM含量。结果上述各项指标在首次给药后和激发日激发后均未见明显异常改变。结论 PLGA、mPEG-PDLLA和HP-β-CD在本实验剂量下不能引起Beagle犬产生类过敏或过敏反应。     

pH-responsive nanoparticles releasing tenofovir intended for the prevention of HIV transmission

This study is designed to test the hypothesis that tenofovir (TNF) or tenofovir disoproxil fumarate (TDF) loaded nanoparticles (NPs) prepared with a blend of poly(lactic-co-glycolic acid) (PLGA) and methacrylic acid copolymer (Eudragit® S-100, or S-100) are noncytotoxic and exhibit significant pH-responsive release of anti-HIV microbicides in the presence of human semen fluid simulant (SFS). After NPs preparation by emulsification diffusion, their size, encapsulation efficiency (EE%), drug release profile, morphology, and cytotoxicity are characterized by dynamic light scattering, spectrophotometry, transmission electron microscopy, and cellular viability assay/transepithelial electrical resistance measurement, respectively. Cellular uptake was elucidated by fluorescence spectroscopy and confocal microscopy. The NPs have an average size of 250 nm, maximal EE% of 16.1% and 37.2% for TNF and TDF, respectively. There is a 4-fold increase in the drug release rate from the 75% S-100 blend in the presence of SFS over 72 h. At a concentration up to 10 mg/ml, the PLGA/S-100 NPs are noncytotoxic for 48 h to vaginal endocervical/epithelial cells and Lactobacillus crispatus. The particle uptake (∼50% in 24 h) by these vaginal cell lines mostly occurred through caveolin-mediated pathway. These data suggest the promise of using PLGA/S-100 NPs as an alternative controlled drug delivery system in intravaginal delivery of an anti-HIV/AIDS microbicide.