靶向溶瘤、化疗和体内DC疫苗多策略综合脂质体抗肿瘤药效机理研究

Many approaches have been well studied and accepted as potential strategies for treatment of cancers, including tumor target therapy, oncolytic therapy, chemotherapy, anti-angeogenesis and in vivo DC vaccine. 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 this 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. Herpes simplex virus type 1 (HSV1) is one of the most effective oncolytic virus, which can especially kill tumor cells without damaging normal cells. MIP-3α is a special chemokine of DC and we have used it to prepare in vivo DC vaccine in previous studies. In this study, we conceive a new combinatorial strategy that will reach the purpose of combination of targeting therapy, oncolytic therapy, chemotherapy, anti-angeogenesis and in vivo DC vaccine via a PEG liposome structure. In this study, we will construct a recombinant MIP-3α HSV1 (rHSV1-MIP3α) and encapsulate it in the aqueous interior of PEG liposomes which still keep aCD in nonaqueous interior (HSV1-MIP3α/aCDPL). Thereafter, we conjugate with a monoclonal antibody (mAb) against endoglin (aEND/HSV1-MIP3α/aCDPL) as a model anticancer drug. Because the anti-endolgin mAb on the aEND/HSV1-MIP3α/aCDPL can target the endoglin expressed on the tumor endothelial cells, recombinant rHSV1-MIP3α virus cause oncolysis and express MIP-3α, and aCD directly damage tumor cells. Thus, aEND/HSV1-MIP3α/aCDPL will possess muti-functions, inducing tumor-cell lysis, immune killing of tumror cells, anti-angiogenesis and cytotoxic to tumor cells. In this study, we will apply in vitro tests such as MTT and TUNEL based immunological assays to acknowledge the capabilities of aEND/HSV1-MIP3α/aCDPL in inhibition of tumor cell proliferation and induction of tumor cell apoptosis in comparison with the control liposomes. High-performance liquid chromatography will be used to observe the biodistribution of the aEND/HSV1-MIP3α/aCDPL in tumor-bearing mice. The antitumor activities of the aEND/HSV1-MIP3α/aCDPL will be investigated in several murine tumor models. Tumor cell apoptosis and angiogenesis will be observed in tumor tissues and mice treated with aEND/HSV1-MIP3α/aCDPL by immunohistological and alginate-encapsulated tumor-cell assays. In addition, Western blto and ELISA will be used to prove whether aEND/HSV1-MIP3α/aCDPL cause cellular and humoral immunity in the treated mice. Our study will potentially provide a new combinatorial approach and facilitate the widely acceptation of aCD as cytotoxic agents for tumor therapy.

靶向溶瘤、化疗和DC疫苗是有应用前景的肿瘤治疗策略。我们从热带内生真菌提取了乙酰基细胞松弛素D(aCD)并将其包裹于PEG脂质体，发现aCD位于非水性脂膜内层并有肿瘤靶向治疗活性，可以有效避免aCD的毒副作用。在此基础上，本研究利用病毒拯救技术将DC趋化因子MIP-3α构建于溶瘤作用的单纯疱疹病毒1(HSV1)中，获得重组HSV1-MIP3α病毒，使该重组病毒保留HSV1溶瘤特性的同时还具有分泌MIP-3α的作用。此后将重组病毒HSV1-MIP3α(位于水溶部位)连同aCD（位于内脂膜层）同时包裹于空间结构稳定的PEG脂质体中并与肿瘤血管生成相关的endoglin单抗(aEND)共轭偶合。通过PEG脂质体同时将aEND/aCD/HSV1/MIP3α结合于一体，这种脂质体作为药物就可能同时具有靶向、溶瘤、化疗和DC体内疫苗的多种功能活性。进行体内外实验研究其功能特性以及治疗肿瘤的作用和机理。

在本研究中，我们组合了包括靶向、溶瘤、化疗和体内DC疫苗等多种治疗肿瘤的策略。取得了如下成果：（1）我们发现，重组NDV-MIP3α溶瘤病毒除了保持其溶瘤特性外，还能诱导肿瘤细胞免疫原性死亡，同时能有效表达活性 MIP-3α。在B16和CT26小鼠肿瘤模型中，感染溶瘤NDV-MIP3α病毒能促使DC成熟和活化，注射NDV-MIP3α溶瘤病毒后能有效抑制肿瘤生长，同时诱导产生B16和CT26肿瘤细胞特异的细胞和体液免疫反应。与此同时，我们发现注射NDV-MIP3α溶瘤病毒后还能有效调节免疫反应，使肿瘤组织中免疫抑制的微环境逆转为免疫促进的微环境，其中CD8和CD4细胞均参与了这种调节作用。这些结果说明，重组NDV-MIP3α溶瘤病毒通过增强全身系统的的免疫反应达到抑制治疗肿瘤的目的；（2）我们制备了Dox的脂质体超声微泡复合物 (MbDox)。体内外进行试验发现，超声控释MbDox能增加Dox的靶向吸收能力，超声控释MbDox能增加肿瘤细胞的免疫原性死亡。此外，超声控释MbDox治疗的肿瘤细胞具有更好的促进DC成熟和活化的能力，能明显抑制模型小鼠的肿瘤生长，能诱导更强的以CD8细胞活性，使其分泌TNF-α和INF-γ，具有更强的肿瘤细胞杀伤活性，同样也有改善免疫抑制内环境的作用；（3）我们制备了CytD聚乙二醇（PEG）脂质体（CytD-PEGL），分析其理化特性后试验发现，CytD-PEGL可以有效抑制肿瘤细胞的增殖，抑制增殖，并能有效诱导肿瘤细胞凋亡。在小鼠B16肿瘤模型中发现，CytD-PEGL能够完全溶解于水，具有明显的肿瘤组织靶向特异性，血浆CytD的半衰期明显长于对照小鼠，CytD-PEGL在肿瘤组织能长时间保持药物高浓度。在B16、CT26和H22小鼠肿瘤模型中观察发现，CytD-PEGL可以有效抑制肿瘤生长，明显延长荷瘤小鼠的生存时间，抗肿瘤作用效果和传统的抗肿瘤药物顺铂（DDP）相近，但CytD-PEGL治疗没有明显的副作用。机理研究发现，CytD-PEGL治疗后可以有效诱导肿瘤组织细胞凋亡，效果接近于化疗药物DDP。此外，CytD-PEGL还具有明显的抑制肿瘤血管生成的作用；（4）此外，我们还开展了微泡靶向重组MIP-3α溶瘤病毒、微泡靶向药物脂质体和MIP-3α溶瘤病毒联合等多种组合的研究，发现均有协同增加疗效的作用，结果待在成文报道中。