Silk fibroin layer-by-layer microcapsules for localized gene delivery

Herein, we describe the delivery of plasmid DNA (pDNA) using silk fibroin (SF) layer-by-layer assembled microcapsules. Deposition of fluorescently labeled SF onto polystyrene (PS) template particles resulted in increasing fluorescence intensity and decreasing surface charge in correlation to SF layer number. After removal of the PS core, hollow, monodisperse, and structurally stable SF microcapsules of variable size and shell thickness were obtained. Plasmid DNA encoding for enhanced green fluorescent protein (eGFP) was loaded onto 1 or 4 μm capsules, either by incorporation of pDNA within the innermost layer of the shell or by adsorption to the microcapsules surface, and in vitro pDNA release, cytotoxicty and eGFP expression were studied. Sustained pDNA release over 3 days was observed using both loading techniques, being accelerated in the presence of protease. DNA loaded SF microcapsules resulted in efficient cell transfection along with low cytotoxicity after 3 days incubation compared to treatment with pDNA/branched polyethylenimine complexes. Among the tested conditions highest transfection efficiencies were achieved using 1 μm capsules where pDNA was adsorbed to the capsule surface. Our results suggest that SF microcapsules are suitable for the localized delivery of pDNA, combining low cytotoxicity and high transfection efficiency.     

阿司匹林壳聚糖-海藻酸钠微囊处方优化与释药机制研究

目的:制备阿司匹林壳聚糖-海藻酸钠微囊(ACSPM),并研究其处方优化与释药机制。方法:设计正交试验,以包封率为指标优化ACSPM处方并制备微囊,测定其释放度并通过释放动力学模型方程拟合探讨其释药机制。结果:所得微囊大小及含量均匀,最优处方中海藻酸钠浓度、壳聚糖浓度、海藻酸钠与阿司匹林比例分别为3.0%、1.0%、1∶4,体外释放符合Higuchi方程和Peppas方程。结论:该微囊制备工艺方法简单,释药机制以药物扩散为主兼有骨架溶蚀的non-Fickian过程。     

海藻酸-壳聚糖-聚乙烯乙二醇微囊生物相容性的研究

目的比较海藻酸-壳聚糖-聚乙烯乙二醇微囊(ACP微囊)和海藻酸-聚赖氨酸-海藻酸微囊(APA微囊)的生物相容性。方法(1)两种微囊(50、100和200个)与健康人血清共浴,检测微囊对补体的激活程度。(2)1000个APA和ACP微囊分别植入Wistar大鼠的腹腔,4d和3周时统计取出的微囊数和微囊的纤维化率。(3)Wistar大鼠胰岛用ACP微囊和APA微囊包裹,分别贯续置于含3.3mmol/L和16.7mmol/L葡萄糖的Hank's溶液中培养,测定培养液中胰岛素的浓度。结果(1)ACP微囊组残余补体活性高于APA微囊组。(2)4d时,ACP和APA微囊的取出数分别是845.0±40.4和807.6±45.7(P>0.05),囊周纤维化率分别是16.40%和65.68%(P<0.05);3周时两种微囊的取出数分别为715.0±133.0和367.5±105.6(P<0.05),囊周纤维化率为27.8%和83.9%(P<0.05)。(3)在含3.3mmol/L葡萄糖的Hank's液中,未微囊胰岛组、APA和ACP微囊化胰岛组的胰岛素浓度分别是(123.48±4.70)mIU/L、(110.11±12.18)mIU/L和(110.90±11.95)mIU/L,当葡萄糖浓度为16.7mmol/L时,胰岛素浓度分别是(754.75±13.81)mIU/L、(689.30±27.71)mIU/L和(684.28±70.10)mIU/L。结论海藻酸-壳聚糖-聚乙烯乙二醇微囊的生物相容性要优于海藻酸-聚赖氨酸-海藻酸微囊,前者更适合应用于微囊化胰岛移植。     

包裹骨形态发生蛋白-2基因转染干细胞的微囊化方法和蛋白释放效应

目的观察转基因骨髓间充质干细胞的微囊化包裹方法和骨形态发生蛋白-2(BMP- 2)释放效应。方法利用高压微电场制备由海藻酸纳和多聚赖胺酸组成的微囊(细胞浓度为3×10~6个/ml,海藻酸钠浓度为1.5%)包被转β-gal(报告基因)或BMP-2基因的骨髓间充质干细胞,根据X-gal染色观察细胞在微囊内的存活情况,ELISA方法检测其所释放的BMP-2蛋白量,共培养方法检测其生物学活性。结果7、14、21、28 d时微囊内转基因细胞X-gal染色均为阳性。包裹后30 d,培养液上清中仍可检测到BMP-2蛋白。转BMP-2基因微囊化细胞与未包裹骨髓间充质干细胞共培养10 d后,ALP活性定量检测结果表明,Adv-hBMP-2转染细胞组ALP活性为(52.537±1.774)U/ml,明显高于未转染细胞组的(42.336±0.857)U/ml,差异有统计学意义(P<0.05)。结论微囊化转基因干细胞生长良好,能持续分泌BMP-2蛋白并促进干细胞向成骨细胞方向分化。     

诺氟沙星控释胶囊的制备及体外释放度试验

目的 制备诺氟沙星控释胶囊，减少给药次数，提高疗效。方法 采用二氯甲－正己烷系统制备诺氟沙星微囊，再加微囊重量20％的诺氟沙星原料药制成控释胶囊，并进行体外释放度研究。结果 诺氟沙星控释胶囊释药基本达零级过程，通过零级方程拟合，r=0.9675。加入诺氟沙星原料药作为速释部分可提高控释胶囊的释药速度。结论 用诺氟沙星微囊制成控释胶囊，方法可行，达到了控释目的。     

肝内移植微囊对肝纤维化的影响:经门静脉及肝动脉途径对比

目的通过比较经门静脉及肝动脉两种途径肝内移植海藻酸钠微囊对肝纤维化影响的差别,探求安全、简便的肝内胰岛移植途径。方法实验犬30只随机分为V、A两组,每组15只,分别作为经门静脉移植组及经肝动脉移植组,V、A两组分别随机分为三个小组,每小组5只,分别移植海藻酸钠微囊8000个/kg、16000个/kg、32000个/kg,观察移植前、后血清肝纤维化标志物透明质酸(HA)的变化,并对肝脏进行病理组织学检查。结果血清纤维化指标:V组:血清HA值在移植后逐渐升高,移植前、后有明显差别(P<0.01);且各移植量组间存在显著性差异(P<0.01)。A组:血清HA值在移植后轻度升高,但移植前后无显著差异(P>0.05),且各移植量组间的差异无统计学意义(P>0.05)。相同移植量Vn及An组间血清HA值的比较差异有显著性(P<0.01)。肝脏病理组织学检查:移植术后12周时,V3组肝脏组织学检查发现汇管区少量胶原纤维沉积,肝细胞浊肿,间质见炎性细胞浸润;而A组肝脏组织学检查未见明显异常。结论经肝动脉移植微囊对肝脏损伤小,有望成为一种相对简单、安全的肝内胰岛移植途径。     

微囊化成骨细胞诱导骨髓间充质干细胞体外成骨分化的量效研究

目的探讨微囊化成骨细胞体系细胞添加量对诱导骨髓间充质干细胞体外成骨分化的影响。方法制备含有成骨细胞的海藻酸钠-聚赖氨酸-海藻酸钠微囊,与间充质干细胞的共培养,在1,7,14 d通过细胞增殖量、碱性磷酸酶活性、实时定量PCR等检测,统计结果并分析。结果各时段细胞增量无统计学意义;碱性磷酸酶活性随细胞浓度增加表现出递增趋势,且骨钙蛋白mRNA的表达结果基本一致。结论微囊化成骨细胞在低浓度下已能促进骨髓间充质干细胞成骨分化,浓度升高时促分化能力越强,但达到一定浓度后继续提高时,对成骨分化的作用则不再增强。     

酮康唑微囊对部分真菌的抑菌实验

目的　考察临床分离的几种致病真菌对酮康唑微囊的体外敏感性。方法　采用试管稀释法和琼脂稀释法进行体外药效学研究。结果　 2 1株念珠菌对酮康唑、酮康唑微囊、氟康唑片的MIC分别为 0 .0 2～ 2 .5、6 .2 5～ 10 0、0 .319～ 10 0 μg·ml-1;1株酵母菌对这 3种药物的MIC分别为 0 .0 8～ 0 .16、12 .5～ 2 5、2 5 μg·ml-1结论　酮康唑微囊具有一定的体外抗真菌活性。     

In vivo evaluation of mitomycin C-Polylactic acid microcapsules via canine intrahepatic arterial infusion

The therapeutic advantages of a microcapsule dosage form (poly-d, l-lactic acid; 150 ± 25 μm; 5% drug loading) for the intrahepatic arterial delivery of mitomycin C (MMC) were evaluated in dogs on the basis of measurements of the hepatic and peripheral venous concentrations. Hepatic arterial and venous catheters were placed via the femoral in the left lobe of the liver in mongrel dogs. MMC was administered via hepatic arterial catheter at a dose of 0.25 mg/kg in solution with or without embolization and in the microencapsulated form. Pharmacokinetic parameters were obtained from the hepatic and peripheral venous MMC concentrations and novel parameters were developed to permit comparison of the systemic and hepatic exposures to MMC. Ratios of area under the serum concentration-time profiles (AUC), maximum serum concentration (C_max), and mean residence times (MRT) between the hepatic and peripheral veins indicate that embolization with MMC microcapsules results in an up to 2.4-fold reduction in the systemic drug exposure.