RGD修饰载紫杉醇脂质体用于抗脑胶质瘤的体外研究

目的制备RGD修饰载紫杉醇脂质体并考察其体外抗脑胶质瘤治疗效果。方法采用薄膜分散法制备RGD修饰载紫杉醇脂质体（RGD—LP-PTX）,考察其理化性质。用鼠源性脑胶质瘤c6细胞考察肿瘤细胞对脂质体的摄取效率。MTT实验考察脂质体对肿瘤细胞的增殖抑制率。结果制备得到的RGD—LP粒径在120nm左右,PD,〈0.3。C6细胞对RGD．LP的摄取能力显著〉LP;RGD-LP-PTX对C6细胞的生长的抑制作用显著强于LP。结论经过RGD修饰后,脂质体具有良好的脑胶质瘤细胞靶向性,且能够显著增强栽药脂质体对脑胶质瘤细胞的增殖抑制作用。     

表面修饰的LPC纳米粒介导的siRNA肿瘤靶向递送(英文)

本研究构建了能够靶向肿瘤的新型纳米粒(脂质体-鱼精蛋白-硫酸软骨素纳米粒,LPC-NP)。该纳米粒粒径约90 nm,zata电位约+35 mV。采用后插法对LPC纳米粒进行DSPE-PEG_(2000)或DSPE-PEG_(2000)-T7修饰。T7是与转铁蛋白功能类似的七肽,能够靶向转铁蛋白受体过度表达的乳腺癌细胞MCF-7。PEG修饰可显著降低血清对LPC纳米粒的聚集作用,T7修饰的纳米粒显著提高siRNA的细胞摄取和基因沉默效率。体外细胞毒实验表明抗EGFR siRNA显著抑制MCF-7细胞生长。实验结果表明经T7肽修饰的LPC纳米粒有望成为RNA干扰肿瘤治疗的递送载体。     

RGD类似物修饰的阿霉素隐形脂质体的制备及体外细胞结合试验

目的研究用精氨酸-甘氨酸-天冬氨酸(RGD)类似物(RGDm)修饰隐形脂质体(SL)，以增加抗癌药物在肿瘤部位积蓄的同时，增加抗癌药物向肿瘤细胞内的传递。方法合成RGDm，将其通过PEG链与二硬脂酰磷脂酰乙醇胺(DSPE)连接形成导向化合物DSPE-PEG-RGDm，在此基础上制备RGDm修饰的隐形脂质体(RGDm-SL)，阿霉素(DOX)作为模型药物通过硫酸铵梯度法装载。体外实验中，用pH探针(BCECF-AM)标记黑色素瘤细胞，通过细胞黏附试验考察导向化合物与肿瘤细胞的黏附情况；通过流式细胞实验和激光共聚焦显微实验考察肿瘤细胞对SL包封的阿霉素(SL-DOX)及RGDm-SL包封的阿霉素(RGDm-SL-DOX)的结合或摄取情况。结果与DSPE-PEG相比，黑色素瘤细胞与导向化合物DSPE-PEG-RGDm的黏附显著增加，过量游离RGDm的加入能抑制其黏附；与SL-DOX相比，RGDm-SL-DOX与黑色素瘤细胞共同孵育后，细胞对阿霉素的结合及摄取均显著增加。结论RGDm修饰的隐形脂质体可作为肿瘤靶向的载体通过受体介导的方式促进抗肿瘤药物向肿瘤细胞内的传递。     

甘草次酸修饰的阿霉素靶向脂质体的制备及体外抑瘤活性考察

目的：制备甘草次酸（GA）靶向阿霉素脂质体（GA-DOX-Lip）,并初步考察其体外抑瘤活性。方法：采用硫酸铵梯度载药法制备GA-DOX-Lip,单因素考察其处方工艺;采用微柱离心法和粒径仪考察脂质体包封率及其理化性质;通过荧光显微镜和流式细胞仪考察BEL-7402肝癌细胞对脂质体的摄取情况并用MTT法评价其体外杀伤效果。结果：GA-DOX-Lip形态圆整,粒径约为120 nm,包封率达到95%以上,72 h释放约20%;体外细胞摄取量和细胞杀伤效果均显著高于阿霉素普通脂质体。结论：甘草次酸修饰的脂质体有望成为肝肿瘤靶向的新型载体,促进抗肿瘤药物向肿瘤细胞内的递送。     

阿霉素脂质体对克服肿瘤多药耐药的细胞学体外评价

目的:评价市售阿霉素脂质体体外逆转肿瘤多药耐药(MDR)的作用。方法:MTT法比较阿霉素和阿霉素脂质体体外对敏感和耐药细胞的细胞毒作用;荧光显微镜观察2组药物对敏感和耐药细胞的影响;流式细胞仪检测2组分别在细胞内的积累和外排情况。结果:细胞毒性试验结果显示,尽管在敏感细胞中,阿霉素脂质体IC50远高于阿霉素溶液组,对于耐药细胞株,阿霉素脂质体的IC50与游离阿霉素无显著性差异(P>0.05);细胞荧光染色显示,阿霉素脂质体较溶液在耐药细胞核中有更强的红染;细胞摄取试验显示,相同浓度下,阿霉素脂质体较溶液组在耐药细胞中积累量差异无显著性(P>0.05),但外排试验显示,在耐药细胞KBv200中,相同浓度下的脂质体较溶液具有更强的细胞滞留能力(P<0.05)。结论:阿霉素脂质体较阿霉素溶液在体外能更多进入耐药细胞核,并表现出更强的药物滞留能力,可部分克服多药耐药。     

两种RGD肽修饰的新型阿霉素脂质体的体外表征

目的作为配体,肽对于多种受体显示出良好的靶向性,例如在肿瘤表面过度表达的整合素家族受体。本文主要研究和表征分别用精氨酸-甘氨酸-天冬氨酸(RGD)三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)五肽修饰的载药脂质体。方法分别采用RGD和GRGDS对包载阿霉素的立体稳定脂质体(SSL-doxorubicin)进行修饰,以制备RGD-SSL-doxorubicin和GRGDS-SSL-doxorubicin。在体外表征试验中,测定了各种脂质体的包封率、粒径、Zeta电位和释放率,采用SRB试验研究了各脂质体对卵巢癌细胞的细胞毒作用,并应用流式细胞仪和共聚焦显微镜考察了肿瘤细胞对各脂质体包封的阿霉素的摄取情况。结果所有脂质体的包封率均在95%以上,采用RGD或GRGDS进行的修饰并未影响长循环脂质体的包封率。各种脂质体的平均粒径在105.7±3.5nm和130.5±3.0nm之间,Zeta电位在–3.3±0.3和–6.1±0.3mV之间,在模仿体内环境的释放介质(含胎牛血清)中,12小时内约有2/5的阿霉素从脂质体中释放。与游离阿霉素相比,修饰后的脂质体对肿瘤细胞的抑制率略有下降;在研究对阿霉素摄取的流式细胞试验和共聚焦试验中,也有类似现象出现。将各种脂质体分别加入肿瘤细胞后,阿霉素主要分布于SKOV-3的细胞核。结论本研究成功制备了两种分别用精氨酸-甘氨酸-天冬氨酸(RGD)三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)五肽修饰的阿霉素脂质体。体外表征结果显示,该修饰不会显著改变立体稳定脂质体的性质。     

RGD肽修饰的异长春花碱-粉防己碱脂质体的制备及体外抑瘤作用研究

目的:制备RGD肽修饰的异长春花碱(VRB)-粉防己碱(TET)脂质体,研究其对脑胶质瘤C6细胞的抑制作用。方法:采用薄膜分散法和硫酸铵梯度法制备RGD肽修饰的VRB-TET脂质体,观察其形态和粒径分布,测定其中VRB的含量;以磺酰罗丹明B法分别测定空白靶向脂质体、VRB普通脂质体和RGD肽修饰的VRB-TET脂质体对C6细胞的抑制作用。结果:所制RGD肽修饰的VRB-TET脂质体呈圆球状或类圆球状,表面光滑,粒径为120 nm左右,其中VRB的平均含量为28.27μg/m L(RSD=0.38%,n=3)。空白靶向脂质体对C6细胞生长无显著影响;RGD肽修饰的VRB-TET脂质体能明显抑制C6细胞生长,其作用后细胞存活率明显低于VRB普通脂质体(P<0.05)。结论:成功制得RGD肽修饰的VRB-TET脂质体,其具有明显的抑制C6细胞生长的作用。     

阿霉素纳米粒对MRP介导的膀胱肿瘤多药耐药细胞株EJ/MRP多药耐药性的逆转作用

目的 研究阿霉素纳米粒对多药耐药相关蛋白(MRP)介导的膀胱肿瘤多药耐药的逆转作用.方法 采用四甲基偶氮唑盐(MTT)法测定药物的体外杀伤作用,应用流式细胞术测定细胞内药物浓度.结果 阿霉素纳米粒对EJ细胞的细胞毒作用与阿霉素相似,EJ/MRP 细胞对阿霉素纳米粒较阿霉素敏感4.00倍.结论 阿霉素纳米粒通过增加耐药细胞内阿霉素浓度而有效逆转多药耐药.     

肝癌靶向的阳离子脂质体共递送多西他赛和siRNA的研究

肝细胞癌是全世界最常见的恶性肿瘤之一,居癌症相关死亡原因第三位。通过存纳米载体系统上修饰靶向配体可将药物主动靶向至肿瘤细胞。随着药物制剂学的发展,采用纳米给药系统共递送化疗药物与基因药物成为了治疗肿瘤的一种手段。本研究通过薄膜超声法制备了共包载多西他赛（DTx）和小干扰RNA（siRNA）的脂质体,并用SP94对其进行修饰。血清稳定性试验表明,脂质体能较好地保护siRNA免受血清中核酸酶的降解。与未修饰的阳离子脂质体相比,SP94修饰的阳离子脂质体显著增强了制剂的在肿瘤细胞内的摄取和对肿瘤细胞的抗增殖作用,表明了SP94的主动靶向作用。本课题成功构建了主动靶向肝癌的共包载DTx和siRNA的阳离子纳米粒给药系统,为肝细胞癌的治疗提供了新的研究思路。     

葡萄糖受体靶向的钆喷酸葡胺长循环脂质体的肿瘤细胞靶向研究

目的：制备葡萄糖受体靶向的钆喷酸葡胺（GdDTPA）长循环脂质体，并研究其对高表达葡萄糖受体肿瘤细胞的靶向性。方法：合成新型含葡萄糖表面活性剂（N-棕榈酰葡萄糖胺），利用逆向蒸发法制备Gd—DTPA脂质体、PEG修饰Gd—DTPA脂质体、葡萄糖修饰Gd-DTPA脂质体以及葡萄糖PEG修饰GdDTPA脂质体。将4种脂质体分别与前列腺癌细胞一起培养，用MRI检测前列腺癌细胞摄取的Gd-DTPA浓度。结果：成功合成新型含葡萄糖表面活性荆（N棕榈酰葡萄糖胺），以及制备4种Gd-DTPA脂质体。前列腺癌细胞对4种脂质体都有摄取，每种脂质体MRI检测的SBTIW信号值分别为Gd-DTPA脂质体362、PEG修饰Gd-DTPA脂质体299、葡萄糖修饰GdDTPA脂质体397、葡萄糖PEG修饰GdDTPA脂质体377。结论：葡萄糖的修饰有利于脂质体对肿瘤细胞的靶向。     

Enhanced anticancer effect of doxorubicin by TPGS-coated liposomes with Bcl-2 siRNA-corona for dual suppression of drug resistance

Multiple drug resistance (MDR) is a tough problem in developing hepatocellular carcinoma (HCC) therapy. Here, we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin (Dox) i.e., Bcl-2 siRNA/Dox-TPGS-LPs, to enhance anticancer effect of Dox in HCC-MDR. TPGS i.e., d-α-tocopheryl polyethylene glycol 1000 succinate, inhibited P-glycoprotein (P-gp) efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein. The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy. The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGS-LPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum. In drug-resistant cells, TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes. In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model. MTT assay confirmed the IC₅₀ value of Dox was 20–50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells. Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo. Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS- (or siRNA-) linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells, and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice. In conclusion, TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.     

Inhibition of VEGF expression in A431 and MDA-MB-231 tumour cells by cationic lipid-mediated siRNA delivery

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.     

Galactose-modified cationic liposomes as a liver-targeting delivery system for small interfering RNA

We have developed a galactose-modified cationic liposome for delivery of small interfering RNA (siRNA) to the liver. The liposomes were designed to be transported into hepatocytes via the asialoglycoprotein receptor, which recognizes galactose residues. The liposomes contained a novel galactose-modified lipid, 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-N-(1-deoxylactito-1-yl)ethanolamine (GDOPE). Delivery of siRNA to hepatocytes by the liposomes was evaluated by measuring the gene-silencing activity of liposome : siRNA complexes in two human hepatoma cell lines. A formulation with a cationic lipid : GDOPE ratio of 3 : 5 by weight, LIC-G5, showed the strongest activity. In mice, intravenous injection of LIC-G5 complexed with (3)H-labeled siRNA led to accumulation of radioactivity in the liver. When the hepatic cellular uptake was determined after intravenous injection into mice followed by collagenase liver perfusion, the distribution of siRNA to parenchymal cells was 1.9 times higher when LIC-G5 rather than nongalactosylated LIC was used as the carrier. The concentration of siRNA accumulated was 45 μg/ml, 30 times the concentration that produced strong gene silencing in vitro and therefore presumably sufficient for a therapeutic effect. Because increasing the cationic-lipid content of a liposome carrier generally enhances the uptake of siRNA by the liver at the expense of increased cell toxicity, we used only a moderate amount of cationic lipid in our galactose-modified carrier. LIC-G5 enhanced the uptake of siRNA by the liver without cytotoxic effects and is a promising candidate delivery system for liver-targeted siRNA therapy.     

Survivin siRNA对卵巢癌细胞侵袭的影响

目的：探讨survivin靶向小分子干扰RNA（siRNA）对卵巢癌细胞侵袭的影响。方法：在survivin基因的编码区内根据干涉位点的设计原则,体外转录合成survivin siRNA,转染人卵巢上皮性癌细胞株SKOV3细胞,用Westernblot方法鉴定干涉位点对survivin基因蛋白的干扰效果;细胞侵袭实验（transwell）检测细胞体外侵袭能力的变化。结果：survivin siRNA能有效抑制survivin蛋白的表达,siRNA组survivin蛋白的相对表达水平分别与空白对照组、非特异性组相比,差异均有统计学意义（P〈0.05）。特异性干涉组细胞的侵袭能力明显低于非特异性干涉组及空白对照组,差异均具有统计学意义（P〈0.001）。结论：特异性的survivin siRNA能有效降低卵巢癌细胞survivin的表达,抑制细胞侵袭能力,这为卵巢癌的基因治疗提供了一种新策略。     

Novel cationic cholesterol derivative-based liposomes for serum-enhanced delivery of siRNA

Most cationic liposomes used for gene delivery suffer from reduced transfection efficiency in the presence of serum. In this study, we report serum-enhanced delivery efficiency of siRNA via the use of newly synthesized liposomes that contain cationic lipids. Two cholesterol derivatives, cholesteryloxypropan-1-amine (COPA) and cholesteryl-2-aminoethylcarbamate (CAEC), were synthesized. A fluorescein label was then used to visualize cellular uptake of small interfering RNA (siRNA) via COPA or CAEC-based liposomes. The presence of serum had different effects on the cellular delivery of siRNA when siRNA was complexed to different cationic liposomes. CAEC-based liposomes showed significantly reduced cellular delivery of siRNA in serum-containing media as compared to serum-free media. Conversely, COPA-based liposomes (COPA-L) provided serum-enhanced delivery of siRNA in Hepa1-6, A549, and Hela cell lines. Following delivery of the oncogene survivin-specific siRNA, COPA-L reduced the mRNA expression levels of the target gene more efficiently than did Lipofectamine 2000. The delivery of green fluorescent protein-specific siRNA with COPA-L reduced the expression of green fluorescent protein in 293T stable cell lines. The apoptosis of Hepa1-6 significantly increased by delivery of survivin-specific siRNA by COPA-L. Additionally, Hepa1-6, A549, and Hela cells were >80% viable after treatment with COPA-L. These results suggest that the newly synthesized cholesterol derivative, COPA-L, could be further developed as a serum-enhanced delivery system of siRNA.     

lncRNA HOST2通过PI3K/Akt途径调控卵巢癌SKOV3 细胞对顺铂的敏感性

摘要：目的 探讨下调长链非编码RNA（lncRNA）上皮性卵巢癌转录因子2（HOST2）表达对人卵巢癌SKOV3细 胞顺铂敏感性的影响及其作用机制。方法 体外培养人卵巢癌SKOV3细胞,取对数生长期细胞分为空白对照组、阴 性对照组和siRNA干扰组,其中阴性对照组和siRNA干扰组SKOV3细胞应用Lipofectamine 2000分别转染阴性对照 siRNA 和 lncRNA HOST2-siRNA,空白对照组未进行转染。采用 qPCR 法检测各组细胞 lncRNA HOST2 的表达; Western blot检测磷脂酰肌醇3-激酶（PI3K）/蛋白质丝氨酸苏氨酸激酶（Akt）信号通路相关蛋白Akt、p-Akt （S473）、pAkt（S380）及Bcl-2的表达;CCK-8法检测经不同浓度（0、20、40、60、80、100 μmol/L）顺铂作用后细胞的存活情况,并计算半抑制浓度（IC50）;流式细胞术检测经20 μmol/L顺铂作用后的细胞凋亡率。结果 与空白对照组和阴性对照组 比较,siRNA 干扰组细胞 lncRNA HOST2 表达及 p-Akt（S473）、p-Akt（S380）和 Bcl-2 蛋白表达水平均降低（均 P＜ 0.05）;3组Akt蛋白表达水平差异无统计学意义（P＞0.05）。空白对照组、阴性对照组和siRNA干扰组细胞存活率均 随顺铂药物浓度的增加而降低,IC50分别为（59.58±5.97）、（51.42±5.22）和（39.75±5.31）μmol/L。siRNA干扰组细胞凋 亡率（12.42%±1.46%）明显高于空白对照组（7.53%±1.25%）和阴性对照组（8.16%±1.31%）。结论 下调 lncRNA HOST2表达可增强人卵巢癌SKOV3细胞对顺铂的敏感性,其机制与抑制PI3K/Akt信号通路相关蛋白的表达有关     

A bioassay for the activity of PSC 833 in human serum for modulation of P-glycoprotein-mediated multidrug resistance

We established a rapid and sensitive ex vivo bioassay to detect the multidrug resistance (MDR)-inhibitory activity of SDZ PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833)) in two RPMI 8226 human myeloma sublines (parent 8226 and doxorubicin-resistant subline Dox6) in 75% human serum. In vitro sensitivity of the tumor to doxorubicin was determined by 3-h drug exposure growth inhibition assay (MTT assay). PSC 833 in serum restored the IC50 of doxorubicin in the P-glycoprotein (P-gp)-positive resistant subline to the same level as in the sensitive cells at 1 microg/ml, which has been shown to be an achievable concentration in clinical trials. In addition, the cytotoxic effect of doxorubicin was enhanced by PSC 833 in the sera of the patient in whom the blood level was 705.7 ng/ml. However, 10 microg/ml PSC 833 in serum does not cause a complete recovery in the IC90 of doxorubicin in the resistant sublines. This MDR-inhibitory activity was supported by the finding that PSC 833 in serum does not increase accumulation of rhodamine 123 in doxorubicin-resistant cells in an in vitro functional assay. The present study provides evidence that PSC 833 in human serum is effective to modulate P-gp-mediated MDR but insufficient for the reversal of MDR from the clinicopharmacological point of view.     

Intratracheal <Emphasis Type="BoldItalic">Versus</Emphasis> Intravenous Liposomal Delivery of siRNA,Antisense Oligonucleotides and Anticancer Drug

Purpose  To compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA). Methods  “Neutral” and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer. Results  Liposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously. Conclusions  The present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.