阳离子脂质体载药系统的制备及其体外成像治疗一体化应用研究

目的 构建阳离子脂质体?阿霉素纳米复合物(CLPs?DOX)并将其应用于癌细胞成像及治疗一体化研究.方法 本实验拟采用薄膜分散一锅法合成CLPs?DOX;利用电位分析仪、荧光分析仪、纳米颗粒追踪技术、荧光显微技术分析CLPs?DOX的电位、荧光强度、粒径分布和癌细胞的成像效果;细胞毒性试验验证CLPs?DOX对乳腺癌细...     

整合素αvβ3受体靶向载药脂质体的制备及体外靶向性

背景:含RGD序列的多肽是多种整合素的识别位点,以其相对分子质量小、稳定、易于制备,且无免疫原性等优点被广泛用于纳米靶向药物传递系统的设计.目的:制备以RGD环五肽为配基的整合素αvβ3载药脂质体,通过体外细胞学实验证实其受体靶向性.方法:使用人工合成的RGD环五肽作为靶向分子探针,通过高压均质法制备靶向整合素αvβ3载药脂质体,采用扫描电镜和激光粒度分析仪检测纳米颗粒形态和粒径;以流式细胞分析观察其对血管平滑肌细胞的特异性标记,并考察荷载药物的离体缓释能力以及体外靶向能力.结果与结论:合成的靶向载药脂质体粒径为(175±6) nm,包封率为(96.33±1.02)%,体外溶出时间超过5 d.靶向载药脂质体对整合素αvβ3具有较高的特异性亲和力,可通过受体介导的内吞作用进入细胞内.提示制备的靶向整合素αvβ3载药脂质体,具有较高的药物包封率及缓释性,能与整合素αvβ3受体特异性结合,是一种新型的受体介导靶向制剂.     

超声微泡破裂法联合阳离子脂质体介导基因转染的实验研究

目的 探讨超声微泡破裂法联合阳离子脂质体(cationic liposome,CL)介导绿色荧光蛋白质粒在肝癌细胞(HepG2)基因转染的可行性,并探索最佳转染条件.方法 依次采用培养液中是否含有血清和不同的CL浓度、不同超声辐照时间点、不同纳米级脂质微泡造影剂(nano-liposomal bubble,NB)浓度等处理因素进行细胞基因转染.荧光显微镜和流式细胞仪检测基因转染效率,CCK-8法检测细胞活性,以获得优化的转染参数.结果 血清能降低CL的细胞毒性,但对基因转染效率无明显影响,CL与质粒DNA质量比4∶1时可以达到相对高效低毒的转染效果,转染率(17.71±0.79)％,存活率(91.28±0.76)％.CL联合1h时间点辐照超声可以提高转染率至(24.85±0.78)％(P＜0.01),加入10％的NB可进一步提高转染率至(32.47±4.01)％(P＜0.05).结论 超声微泡破裂法可以有效增强CL介导的基因转染,联合应用为基因治疗提供了新思路.     

包裹ICG的靶向阳离子纳米粒的制备及体外显像

目的 制备一种包裹ICG的靶向相变型阳离子脂质纳米粒(INP),连接CD105抗体,检测其光热效应、体外相变、光声及超声显像规律,并证实纳米粒抗体连接成功。方法 采用双乳化法制备包裹ICG及液态氟碳(PFP)的阳离子脂质纳米粒;链霉亲和素法连接CD105抗体,流式细胞量化及激光共聚焦下观察抗体连接情况;脉冲激光激发观察其光热效应及相变情况;体外Lifu辐照致相变后记录超声显影效果;光声仪检测光声显像能力。结果 制备的INP平均粒径(354.2±93.85nm),平均电位(25.2±3.29mv),与CD105抗体结合率为99.89%,纳米粒具有良好光热效应,一定浓度下2W/cm2激光辐照180s温度可升至63℃,可发生液气相变。体外超声及光声显影效果佳。结论 成功制备了包裹ICG的靶向相变型阳离子脂质纳米粒(INP),其光热效应好、光声显影及增强超声显影效果佳,纳米粒与CD105抗体结合率高。     

Mechanistic studies of sequential injection of cationic liposome and plasmid DNA.

We previously reported that sequential injection of cationic liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al., 2001, Mol. Ther. 3, 673-682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex-serum mixtures (LDS sample). LSD yielded 80% lower TNF-alpha levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-alpha levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, liposomes, and DNA showed that DNA could bind negatively charged liposome-serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.     

携RGDS的靶向超声造影剂的制备及鉴定

目的制备血栓靶向脂膜超声造影剂,并对其理化特性及靶向作用进行鉴定.方法将脂膜超声造影剂通过酰胺键共价键键合的方式与血栓靶向短肽片段(RGDS)进行结合.制备产物通过流式细胞仪进行携带率和稳定性的检测;对内源性凝血途径产生的血栓进行寻靶特性研究.结果流式细胞仪显示携带RGDS的脂膜超声造影剂其波长发生了明显变化,携带率达到82%;激光共聚焦显微镜显示携带RGDS的脂膜超声造影剂对离体血栓具有很强的靶向性和稳定性.结论采用共价键键合的化学修饰方法成功制备了亲血栓靶向脂膜超声造影剂.     

Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome.

Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy.     

胶原在骨组织工程应用中的体外制备及生物相容性评价

目的：归纳、总结近年来胶原在骨组织工程各环节中应用所取得的成果，分析胶原分子结构与功能的关系，阐述胶原作为一种天然的生物材料所具备的巨大优势和存在的不足，并进一步探讨胶原在骨组织工程中应用的新途径。资料来源：应用计算机检索Medline数据库、中文科技期刊数据库（维普资讯网）、CNKI数字图书馆1995—01／2004—12相关胶原的文献。检索词“组织工程，Ⅰ型胶原”．限定文章语言种类为中文。资料选择：对资料进行初审，纳入标准：①与骨组织工程相关文献。②不排除是否为随机、盲法等论证推荐的文章。排除标准：重复研究、综述文献。资料提炼：共收集到43篇与骨组织工程相关的文章。排除其中研究内容相似的文章，以近5年内发表在较权威杂志者优先。对符合标准的25篇文献进行分析。资料综合：通过对入选的文献进行分析、整理，将胶原在骨组织工程中的应用按组织工程三要素相对应的内容进行分类和综合：支架、微载体和接种媒介。另外，Ⅰ型胶原是一种极其重要的细胞信息载体，具有类似生物因子的作用。虽未对其进行归类，但对其作用机制进行了总结。结论：胶原及其产品以其优良的生物相容性、适宜的可降解性、弱的抗原性在骨组织工程中得到广泛应用，但仍存在一些固有缺陷。对胶原进行改性和复合，将有助于克服天然胶原的不足，生产出更多的、符合骨组织工程需要的新产品。     

胶原在骨组织工程应用中的体外制备及生物相容性评价

目的:归纳、总结近年来胶原在骨组织工程各环节中应用所取得的成果,分析胶原分子结构与功能的关系,阐述胶原作为一种天然的生物材料所具备的巨大优势和存在的不足,并进一步探讨胶原在骨组织工程中应用的新途径。资料来源:应用计算机检索Medline数据库、中文科技期刊数据库(维普资讯网)、CNKI数字图书馆1995-01/2004-12相关胶原的文献。检索词“组织工程,I型胶原”,限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①与骨组织工程相关文献。②不排除是否为随机、盲法等论证推荐的文章。排除标准:重复研究、综述文献。资料提炼:共收集到43篇与骨组织工程相关的文章。排除其中研究内容相似的文章,以近5年内发表在较权威杂志者优先。对符合标准的25篇文献进行分析。资料综合:通过对入选的文献进行分析、整理,将胶原在骨组织工程中的应用按组织工程三要素相对应的内容进行分类和综合:支架、微载体和接种媒介。另外,I型胶原是一种极其重要的细胞信息载体,具有类似生物因子的作用。虽未对其进行归类,但对其作用机制进行了总结。结论:胶原及其产品以其优良的生物相容性、适宜的可降解性、弱的抗原性在骨组织工程中得到广泛应用,但仍存在一些固有缺陷。对胶原进行改性和复合,将有助于克服天然胶原的不足,生产出更多的、符合骨组织工程需要的新产品。     

In vitro and in vivo evaluation of novel cationic liposomes utilized for cancer gene therapy.

Advanced peritoneal carcinomatoses is very difficult to treat. We have explored the potential therapeutic application of gene therapy using cationic liposomes in this disease. The lacZ gene was introduced in vitro into ovarian and endometrial cancer cells using cationic liposomes. The transfection efficiency was similar to that of commercially available liposomes in serum-free medium (11.0-20.9% vs. 5.4-26.0%). In serum-containing medium, the efficiency was 1.9-18.1%, which is comparable with the efficiency in serum-free medium. However, the efficiency of commercial liposomes decreased drastically to between 0.1% and 4.7% in the serum-containing medium. When cultured cells were transfected with the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) was added, the anti-tumor effect of GCV was 47-640 times greater than when the same experiment was performed with lacZ gene. Evaluation of anti-tumor effect was performed with the MTT assay. In vivo, the HRA and mEIIL ascitic mice were treated with HSV-tk gene and GCV using the peritoneal route, a significant prolongation of the mean survival time was observed by Kaplan-Meier analysis (16-18 days and 15-30 days, respectively, p < 0.05). These results indicate a potential role for gene therapy in the treatment of advanced intraperitoneal carcinomatoses using the novel cationic liposomes.     

阳离子脂质体介导重组人骨形态发生蛋白-2基因转染的兔骨骼肌干细胞体外成骨分化的实验研究

目的初步探讨阳离子脂质体介导的重组人骨形态发生蛋白-2(rhBMP-2)基因对兔骨骼肌干细胞(SMSCs)体外成骨活性的影响。方法pEGFP-rhBMP-2的扩增、浓度及纯度鉴定。脂质体包裹质粒DNA，转染SMSCs后测定转染率。检测rhBMP-2、碱性磷酸酶(ALP)和Ⅰ型胶原的表达。结果脂质体介导pEGFP-rhBMP-2SMSCs转染后最大转染率为14．18％，rhBMP-2、ALP和Ⅰ型胶原的表达呈阳性，而pEGFP转染的细胞和未染细胞rhBMP-2、ALP和Ⅰ型胶原的表达弱阳性。结论SMSCs是一种较理想的骨组织工程种子细胞。脂质体介导质粒rhBMP-2的基因转移安全性好，但转染率相对较低。     

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.