Endoglin抗体cytochalasin D超声微泡靶向治疗肿瘤研究

Liposome has been used as carriers to encapsulate cytotoxic agent for cancer therapy, which can also been combined to encapsulate special air for diagnosis and treatment at the same time. Our research group has isolated an acetyl cytochalasin D (aCD) from a tropical plant Cephalotaxus hainanensis Li, which possess better cytotoxic effects than the conventional cytochalasin D. We have encapsulated aCD in the nonaqueous interior of PEG liposomes and proved these encapsulation has tumor-targeting ability in mouse tumor models, which has been published on Eur J Cancer. In addition, study results have demonstrated that endoglin is overexpressed and up-regulated in tumor-associated angiogenic vasculature. In this study, we will made a ultrasonic microbubble that is located in the aqueous interior of PEG liposome, which still keep aCD in nonaqueous interior. Thereafter, we conjugate with a monoclonal antibody against endoglin to made a targeting aCD-carried microbubble (aENDaCDmB) as a model anticancer drug. Because the anti-endolgin mAb on the aENDaCDmB can target the endoglin expressed on the tumor endothelial cells and aCD directly kill tumor cells. In addition, aENDaCDmB still keep as a ultrasound contrast agent for imaging and drug carriers for drug delivery by ultrasound-meidated microbubble disruption technique.Thus, aENDaCDmB will possess "several functions" inducing anti-angiogenesis and targeting cytotoxic to tumor tissues. In this study, we will apply in vitro and in vivo tests to evaluate its ability on tumor tissue-targeting, acknowledge the capabilities on inhibition of tumor cell proliferation and induction of tumor cell apoptosis in comparison with the control microbubbles. High-performance liquid chromatography will be used to observe the biodistribution of the aENDaCDmB in tumor-bearing mice. The antitumor activities of the aENDaCDmB will be investigated in several murine tumor models. Tumor cell apoptosis and angiogenesis will be observed in tumor tissues and mice treated with aENDaCDmB by immunohistological and alginate-encapsulated tumor-cell assays. Our study will potentially provide a novel approach and facilitate the widely acceptation of aCD as cytotoxic agents for cancer therapy.

脂质体包被细胞毒药物已经用于肿瘤治疗，同时包被特殊气体可制成超声微泡用于影像诊断和爆破给药。我们发现从热带真菌提取的乙酰基细胞松弛素D(aCD)包裹于PEG脂质体后位于非水性脂膜内层并有肿瘤靶向治疗活性，明显减少aCD的毒副作用。在此基础上，本项目拟综合载药超声微泡的理念，制备载aCD的脂质体超声微泡，同时将肿瘤血管生成相关的endoglin单抗(aEND)偶联于微泡的外层，获得肿瘤血管靶向并载有aCD的超声微泡（aENDaCDmB）。这样，aENDaCDmB就具有肿瘤靶向、肿瘤组织成像、在影像指导下爆破释放aCD于目标肿瘤组织的多种功能活性。本项目拟设计体内实验了解aENDaCDmB的靶向、B超成像、肿瘤组织爆破释药和细胞毒等活性，在数个小鼠肿瘤模型中研究aENDaCDmB治疗肿瘤的效果，了解是否有效诱导肿瘤细胞凋（死）亡，是否有效避免aCD的毒副作用，为肿瘤诊断和治疗提供新的有效手段。

本项目首先制备一个载药（Dox）超声微泡脂质体复合物（MbDox）和相应的对照复合物。对他们的理化特征进行分析后，检测发现MbDox加超声处理（MbDox+US）能更加高效诱导多种肿瘤细胞凋亡；MbDox+US处理诱导分泌或表达更多的内质网应激的标志分子ERp57和磷酸化(p-)elF-2-α，明显高表达或分泌免疫原性死亡的标志分子CRT、HMGB1和ATP。MbDox+US处理的肿瘤细胞上清能更加高效地诱导DC细胞成熟和激活。细胞及小鼠体内实验发现，MbDox+US处理细胞及肿瘤组织中Dox药物浓度更高。将MbDox+US处理的肿瘤细胞（诱导免疫原性死亡）作为疫苗注射小鼠，发现MbDox+US处理的细胞疫苗更明显地抑制肿瘤生长，产生更强的细胞免疫（TCL）和体液免疫反应。分别在免疫正常和免疫缺陷小鼠中建立肿瘤模型并进行超声控制靶向释放药物试验，结果同样发现MbDox+US治疗的肿瘤在两种小鼠中均能明显抑制肿瘤生长，但在免疫正常小鼠中效果更好。对肿瘤组织侵润的淋巴细胞（TIL）进行分析也发现MbDox+US治疗能提高肿瘤组织中分泌IFN-γ的CD8细胞的数量，但调节T细胞（Treg）的数量则明显减少。这些结果表明，超声靶向控释Dox能更好诱导肿瘤免疫原性死亡，改变肿瘤免疫抑制的微环境，是一种有效治疗肿瘤的新模式。.在此基础上，我们还通过抗体和iRGD 多肽制备靶向肿瘤新生血管和超声微泡（MBiRGD/CCR2）进行靶向DNA治疗，同样分析了它们的理化和靶向特性后，进行超声控释DNA的实验。结果表明，双靶向MBiRGD/CCR2具有更好的内皮细胞和肿瘤新生血管的靶向性，超声控释DNA的浓度更高，在细胞和组织中表达更好，说明MBiRGD/CCR2双靶向超声控释是一种更高效的投递基因治疗药物的方法。.本项目还制备了靶向 iRGD 并载有光热治疗剂的脂质体纳米颗粒，在体外细胞试验中发具有αvβ3 整合素的靶向性，并能高效诱导光热反应，在细胞和小鼠肿瘤模型中都发现具有更好的肿瘤治疗作用，并且副作用不明显。此外，我们还制备一种αvβ3靶向的超声微泡，在体内外均可以靶向结合靶分子，在小鼠肿瘤组织中可以进行靶向超声显像。