卡莫氟固脂纳米粒对人大肠癌细胞多药耐药的逆转的机制研究

研究卡莫氟固脂纳米粒对人大肠癌细胞多药耐药的逆转作用,并对其逆转机制进行了初步探讨.采用MTT法比较了卡莫氟不同药物形式的细胞毒作用;采用RT-PCR技术考察卡莫氟固脂纳米粒对敏感和耐药人大肠癌细胞中mdr 1和MRP的基因表达的影响.卡莫氟固脂纳米粒能下调耐药人大肠癌细胞中mdr 1的mRNA表达,对其中的MRP的mRNA的表达没有显著影响.卡莫氟固脂纳米粒能逆转人大肠癌细胞由mdr 1 介导的多药耐药,而对MRP介导的多药耐药没有显著影响.     

卡莫氟固脂纳米粒的制备及其体外逆转人大肠癌细胞多药耐药性研究

目的 研制卡莫氟固脂纳米粒（HCFU-SLN）并检测其相关参数,考察其对大肠癌细胞（CCL-187）多药耐药的逆转作用。方法 采用溶剂乳化法制备HCFU-SLN,透射电镜观察其外部特征,激光粒度仪测定其粒径和粒径分布,MTT法考察其对CCL-187多药耐药的逆转作用。结果HCFU-SLN外观圆整,平均粒经为（172.9±70.5） nm,包封率为92.0%。其对敏感细胞的细胞毒作用与游离卡莫氟的作用差异无显著性,对耐药细胞的杀伤力较游离卡莫氟显著增强（P＜0.05＝,CCL-187对HCFU SLN的敏感性是对游离卡莫氟的5.5倍。结论卡莫氟固脂纳米粒可有效逆转人大肠癌细胞多药耐药。     

具有活性羧基末端的长循环脂质体的制备和分布

目的　研究长循环免疫脂质体(immunoliposomes,IML)的制备方法, 体外靶细胞杀伤活性和在小鼠体内的组织分布。方法　合成和纯化了1个带末端羧基的磷脂酰乙醇胺(PE)的聚乙二醇衍生物(DPPE-PEG3000-COOH),掺入脂质体中制成长循环脂质体;通过羧基活泼酯化,将膀胱癌单克隆抗体BDI-1或小鼠IgG共价连接到该脂质体表面制成免疫脂质体,体外肿瘤细胞杀伤实验检测载阿霉素免疫脂质体(ADM-BDI-1-IML)特异杀伤靶细胞的能力。用同位素氚示踪法测量免疫脂质体在小鼠的组织分布。结果　抗体在脂质体上的结合率可达30％。体外肿瘤细胞杀伤实验证明载阿霉素免疫脂质体有选择性杀伤靶细胞人膀胱癌细胞EJ的能力。和普通脂质体相比,免疫脂质体在血中的滞留时间明显延长,并减少了在肝、脾的聚集。结论　长循环免疫脂质体在血中有较长的滞留时间,在体外有特异寻靶活性,载阿霉素免疫脂质体有选择性杀伤靶细胞的活性,这些性质为其在体内主动寻靶和选择性杀伤靶肿瘤细胞提供了必要条件。     

第三代载药免疫脂质体及体内外寻靶研究

目的　研究载阿霉素第三代免疫脂质体的制备及体内外寻靶、抑瘤效果。方法　设计将人膀胱癌单抗与聚乙二醇羧酸(PEG-COOH)端相联,使构成的脂质体既充分发挥PEG的保护功能,延长药物血循环时间,又使单抗伸展在外部充分发挥其寻靶作用,即第三代免疫脂质体(IML)。进而研究载抗癌药的免疫脂质体的制备方案,制备出阿霉素免疫脂质体(IML-ADM)使达到对药物高包封、高稳定,又不降低单抗活性的目的。以人膀胱癌靶细胞EJ和人直肠癌非靶细胞LOVO进行体外杀伤和体内肿瘤的抑瘤实验。结果　IML-ADM对EJ细胞和LOVO细胞杀伤,及对EJ细胞移植瘤体的抑制与对照组比较均有显著性差异。结论　证实脂质体载药以单抗制导达到主动靶向给药是可行的     

人鼠嵌合抗肿瘤细胞核单抗-空间稳定脂质体的制备及其体外靶向

目的研究人鼠嵌合抗肿瘤细胞核单抗(chTNT-3)-空间稳定脂质体的制备方法、免疫活性及体外靶向性。方法合成聚乙二醇末端带吡啶二硫丙酰基的磷脂衍生物(PDP-PEG-HSPE)，制备含PDP-PEG-HSPE的空间稳定脂质体，经二巯基苏糖醇还原后共价连接马来酰亚胺衍生化抗体。荧光胺法和钼蓝法测定脂质体与抗体的连接效率及抗体密度，激光散射粒度仪测定其粒径分布，ELISA法检测脂质体表面的抗体免疫活性。体外实验考察该免疫脂质体与固定Raji细胞的结合活性。结果chTNT-3-空间稳定脂质体的粒径分布为(115±33) nm。当初始Ab/PDP-PEG-HSPE=1∶10时，脂质体与抗体的连接效率为71%，抗体密度为106 μg Ab/μmol PL。chTNT-3经化学修饰后连接到脂质体表面，其免疫活性基本保留。chTNT-3-空间稳定脂质体能特异性地结合固定Raji细胞。结论通过PDP-PEG-HSPE法共价连接抗体制备的chTNT-3-空间稳定脂质体能基本保留chTNT-3的免疫活性，具有体外靶向细胞核抗原的能力。     

蜂毒多肽空间稳定免疫脂质体的制备及体外对肿瘤细胞的选择性

蜂毒多肽在肿瘤治疗中的应用引起研究者的极大兴趣。本研究使用大豆磷脂、胆固醇、羧酸化PEG-胆固醇制备了蜂毒多肽空间稳定脂质体，并将二硫键稳定抗人肝癌单链抗体联结PEG-胆固醇末端。使用酶联免疫法考察了蜂毒多肽空间稳定免疫脂质体的活性。蜂毒多肽空间稳定免疫脂质体有较高的肿瘤细胞选择性。体外实验证明，其对SMMC-7721细胞的杀伤能力远强于蜂毒多肽空间脂质体，而对Hela细胞的杀伤能力与蜂毒多肽空间脂质体无区别。蜂毒多肽空间稳定免疫脂质体对肿瘤细胞的选择性，可使其成为一种有效的靶向制剂。     

脂质体VEGFR2胞外区基因肿瘤疫苗的制备及其免疫激活体外抗肿瘤活性研究

目的:制备脂质体(LP)携带VEGFR2胞外区(exVEGFR2)肿瘤抗原pCMV质粒复合物基因疫苗,为肿瘤免疫治疗提供新的主动免疫疗法。方法:RT-PCR扩增含2个KpnⅠ和XbaⅠ限制性酶切位点的exVEGFR2序列,将其与pCMV质粒连接,构建pCMV/exVEGFR2质粒。Western blot检测exVEGFR2体外表达水平。用制备好的脂质体pCMV/exVEGFR2复合物(LP-pCMV/exVEGFR2)免疫C57BL/6小鼠,ELISA法检测其免疫激活活性。利用51 Cr释放实验分析CTLs介导的细胞毒活性。结果:成功扩增到exVEGFR2序列,并构建pCMV/exVEGFR2质粒;只有在pCMV/exVEGFR2转染COS-7细胞中,检测到与目的蛋白序列大小一致的相对分子质量为90000的VEGFR2特异性条带。LP-pCMV/exVEGFR2免疫小鼠6周后,ELISA法检测到强烈的特异性抗VEGFR2免疫激活效应;其免疫T细胞能够有效地介导体外VEGFR2阳性的CT-26结肠癌细胞毒性。结论:LP-pCMV/exVEGFR2能够有效地激活小鼠产生特异性抗VEGFR2免疫应答效应,并产生体外抗肿瘤细胞免疫毒性,为后期研究主动免疫治疗VEGFR2阳性肿瘤奠定实验基础。     

脂质体制剂对环胞苷二棕榈酸酯抗癌活性的影响

目的 研究3′,5′-环胞苷二棕榈酸酯(DPC)制成脂质体剂型后其抗癌活性的变化。方法 将DPC制成脂质体剂型,分别与其母体药物阿糖胞苷脂质体和游离的DPC对照进行药效学的研究,比较它们对小鼠腹水型肝癌的抑制率。结果 DPC脂质体的抑制率明显高于阿糖胞苷脂质体(P<0.05);十分明显地高于游离的DPC(P<0.01)。结论 DPC载于脂质体上其抗癌活性明显提高,药物吸收难的问题得以解决。     

3种剂量卡莫氟联合亚叶酸钙和奥沙利铂治疗晚期大肠癌的近期疗效比较

目的:探讨卡莫氟联合亚叶酸钙和奥沙利铂治疗晚期大肠癌的量效关系,以寻求更高效、低毒的优化治疗方案。方法:将我院90例大肠癌患者随机均分为A、B、C组,分别口服卡莫氟150mg,tid;100mg,tid;100mg,bid。并联合亚叶酸钙和奥沙利铂治疗,观察并比较3组近期疗效、毒副反应、临床受益反应,并综合分析评价。结果与结论:口服卡莫氟(100mg,tid)联合亚叶酸钙和奥沙利铂方案疗效高(有效率达53.3%)、不良反应发生率低,是一高效、低毒的治疗晚期大肠癌的优化方案。     

硒化桐酸的抗癌作用

将作者合成的硒化桐酸分别制成乳剂和多相脂质体,进行动物体内外抗癌活性研究。体外实验表明硒化桐油无抑癌活性,而硒化桐酸有抑癌活性。体内实验表明硒化桐酸对ARS腹水瘤和S_(180)实体瘤有明显抑制作用;它的多相脂质体对S_(180)实体瘤有较强的抑制作用。     

Characterization of in vivo immunoliposome targeting to pulmonary endothelium

Two rat monoclonal antibodies, 34A and 201B, which specifically bind to a surface glycoprotein (gp112) of the pulmonary endothelial cell surface, have been coupled to unilamellar liposomes of approximately 0.25 microns in diameter. The 34A- and 201B-liposomes (monoclonal antibodies 273-34A and 411-201B, respectively), but not antibody-free liposomes and liposomes coupled to 14, a nonspecific monoclonal antibody, accumulate efficiently (approximately 30% injected dose) in the lung of mice which have been injected via the tail vein. Immunoliposome targeting to lung is demonstrated both by using a 125I-labeled lipid marker and an entrapped water-soluble marker. Lung accumulation of 34A-liposomes is completely blocked by a preincubation of free antibody 34A, but not antibody 14, indicating that the immunoliposome accumulation at the target site is immunospecific. Time course studies have revealed that 34A-liposomes bind to lung antigens within 1 min after injection, indicating that the target binding takes place during the first few passages of immunoliposomes through the lung capillary bed. Unbound immunoliposomes are taken up by liver and spleen within 3-5 min after injection. The level of lung accumulation increases significantly as the protein:lipid ratio of the immunoliposome increases. Approximately 50% of injected dose is accumulated in lung for 34A-liposomes, with an average of 935 antibody molecules per liposome. Immunoliposomes of larger size accumulate in lung more significantly than those of smaller size. Injection with higher doses also enhances the level of lung accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herceptin-conjugated temperature-sensitive immunoliposomes encapsulating gemcitabine for breast cancer

Gemcitabine (2′,2′-difluorodeoxycytidine, GCT) is an analog of deoxycytidine with cytotoxicity for breast cancer cells. However, because of its hepatotoxicity and other side effects, an efficient drug delivery system is needed for better therapeutic outcomes. A temperature-sensitive PEGylated immunoliposome (TSL) with trastuzumab (or Herceptin) attached encapsulating GCT (Her-PEG-TSL-GCT) was prepared. The mean diameter of the liposome was about 200 nm and the prepared immunoliposome showed the capacity to deliver the payload to the hyperthermic environment. The actual number of antibody molecules attached to one single liposome is about 19, with the GCT encapsulation efficiency of 54.6 ± 3.50 %. This immunoliposome shows a temperature-dependent drug release at around 41–43 °C. Anticancer activity of Her-PEG-TSL-GCT was determined using HER-2 expressing breast cancer cells, SK-BR-3, in vitro and resulted in increased cytotoxicity compared to free GCT (IC₂₀ 11.7 nM) or conventional liposome lacking the targeting antibody. In conclusion, these data show that improved delivery of GCT to breast cancer cells can be achieved by Her-PEG-TSL-GCT in vitro, and this strategy could be used for breast cancer therapy with further studies.     

Targetability of novel immunoliposomes prepared by a new antibody conjugation technique.

In order to develop long-circulating immunoliposomes (IL), which combine sterical stabilization with a superior targetability, we have introduced a new methodology for attaching monoclonal antibodies directly onto the distal ends of liposome-grafted polyethylene glycol (PEG) chains. Therefore, we have synthesized a new PEG-PE derivative, which had been endgroup-functionalized with cyanuric chloride. Antibodies can simply be coupled to this membrane anchor in mild basic conditions (pH 8.8) without the need for previous antibody derivatizations. The coupling results have been determined with consideration to various liposome parameters and have been compared to several established antibody coupling procedures, where antibodies had been linked directly to the liposome surface in the presence of PEG (conventional IL). To investigate the targetability of the resulting new IL, anti E-selectin mAb have been coupled and the degree of binding selectin-containing cells has been analyzed. The terminal coupled antibodies show a 1.8-fold higher degree of in vitro cell binding compared to conventional IL, which has been attributed to the antibody position being more easy accessible at the PEG termini. Furthermore, we have illustrated the liposome surface topology and the coupled antibodies by atomic force microscopy, which for such fluid IL has been used first. These images have finely corresponded to the cell binding results, and have been discussed in terms of antibody position and flexibility at the liposome surface. Copyright     

Pharmacokinetics of Differently Designed Immunoliposome Formulations in Rats with Or Without Hepatic Colon Cancer Metastases

Purpose. Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases). Methods. A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [³H]-cholesteryloleylether or bilayer-anchored 5-fluoro[³H]deoxyuridine-dipalmitate ([³H]FUdR-dP) as a marker. Results. In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages. Conclusions. Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.     

Interaction of Differently Designed Immunoliposomes with Colon Cancer Cells and Kupffer Cells. An in Vitro Comparison

Purpose. Evaluate the effectiveness of distal-end coupling of a tumor-specific antibody to liposomal polyethylene glycol (PEG) chains to improve target binding and reduce interference by macrophage uptake. Methods. Monoclonal antibody CC52, specific for CC531 rat colon carcinoma, was coupled to the bilayer of PEG-liposomes (type I) or to the distal end of bilayer-anchored PEG-chains (type II). Uptake of both (radiolabeled)liposome types by CC531 cells and rat liver macrophages was determined. Results. With increasing antibody density, both immunoliposome types showed increased binding to target cells, but type II liposomes displayed better target recognition than type I. Uptake by macrophages increased with antibody density for both liposome types. Lowest uptake by macrophages was found for type II liposomes at low antibody densities. Unexpectedly, not only for type I but also for type II liposomes, in which the antibody is coupled via its Fc moiety, uptake by macrophages was inhibited by aggregated IgG, indicating involvement of Fc receptors. Also polyinosinic acid, an inhibitor of scavenger receptors, reduced uptake of type II liposomes. Conclusion. Although distal end coupling of antibodies to bilayer-anchored PEG chains in liposomes through the Fc moiety enhances target cell binding, it does not prevent the recognition by Fc receptors on macrophages.     

Possibility of active targeting to tumor tissues with liposomes

In terms of active targeting by immunoliposomes, two anatomical compartments are considerable for targeting sites. One is located a readily accessible site in intravascular, and another is a much less accessible target site located in the extravascular. However, it was made clear that the active targeting with immunoliposomes is determined by two kinetically competing processes, such as binding to the target site and uptake by the RES. To overcome these contradictions, we have designed a new type of long-circulating immunoliposome, which was PEG-immunoliposome attached antibodies at the distal end of PEG chain, so called the pendant type immunoliposome. The pendant type immunoliposome showed much higher targetability than the ordinary immunoliposomes to both targeting sites of lung endothelial cells and solid tumor tissue. This is due to the free PEG chains (not linked to the antibody) effectively avoiding the RES uptake of liposomes, resulting in elevated the blood concentration and enhanced the target binding of immunoliposomes. The presence of free PEG does not interfere with the binding of the terminally linked antibody to the antigen. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to make the pendant type immunoliposomes (34A-PEG-ILP). 34A-PFG-ILP showed significantly higher targeting degree than the ordinary type of immunoliposomes. For targeting to the solid tumor tissue, Fab' fragment of 21B2 antibody which is anti-human CFA and transferrin (TF) were used. Both pendant type immunoliposomes (Fab'-PFG-ILP and TF-PEG-ILP) showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. TF-PEG-ILP was internalized into tumor cells with receptor mediated endocytosis, after extravasation into tumor tissue. The pendant type immunoliposome can escape from the gaps between adjacent endothelial cells and openings at the vessel termini during tumor angiogenesis by passive convective transport much rather than ligand directed targeting. Active targeting to tumor tissue with the pendant type immunoliposome is particularly important for many highly toxic anticancer drugs for cancer chemotherapy. An ultimate goal of pendant type immunoliposome is the incorporation of a fusogenic molecule that would induce fusion of liposome following their binding to the target cells or their internalization by endocytosis. Such liposomal formulations should be useful for endocytotic internalization of plasmid DNA and other bioactive materials.     

A novel and effective approach to generate germline-like monoclonal antibodies by integration of phage and mammalian cell display platforms

Phage display technology allows for rapid selection of antibodies from the large repertoire of human antibody fragments displayed on phages. However, antibody fragments should be converted to IgG for biological characterizations and affinity of antibodies obtained from phage display library is frequently not sufficient for efficient use in clinical settings. Here, we describe a new approach that combines phage and mammalian cell display, enabling simultaneous affinity screening of full-length IgG antibodies. Using this strategy, we successfully obtained a novel germline-like anti-TIM-3 monoclonal antibody named m101, which was revealed to be a potent anti-TIM-3 therapeutic monoclonal antibody via in vitro and in vivo experiments, indicating its effectiveness and power. Thus, this platform can help develop new monoclonal antibody therapeutics with high affinity and low immunogenicity.     

脑靶向空间稳定免疫脂质体研究进展

介绍了脑靶向空间稳定免疫脂质体制剂的研究进展。大部分药物因其水溶性或分子质量超过500u无法透过血脑屏障，而脑靶向空间稳定免疫脂质体能将治疗药物通过相关单抗主动靶向脑部，从而提高脑内药物浓度，降低毒副作用。