基于阳离子聚合物的仿生药物载体肿瘤免疫治疗中的研究
发布时间：2018-07-13 12:46
【摘要】：如何将肿瘤新方法安全有效的应用到肿瘤治疗中,是肿瘤治疗面临的重大挑战。纳米技术的发展使高分子纳米药物载体在此领域担任了至关重要的角色。基于环糊精-PEI的阳离子聚合物作为载药体系生物相容性好,且携载基因转染效率较高,但基于环糊精-PEI阳离子聚合物在肿瘤多药耐药和肿瘤免疫治疗方面应用研究较少。因此,本论文对阳离子聚合物及其仿生材料在肿瘤免疫治疗、化疗中的应用作了以下研究,主要分以下三部分:第一部分,以γ-羟丙基-环糊精偶联低分子量PEI600(HP)为主体,金刚烷甲酸修饰的阿霉素(AD)为客体经主客自组装为HPAD超分子纳米材料,通过静电作用携载ETS1siRNA。形成内核为疏水药物,外壳携载siRNA具有核壳结构的协同给药体系(HPAD/siRNA)。HPAD/siETS1经细胞内吞后进入到溶酶体,通过质子海绵效应进行溶酶体逃逸,由于溶酶体酸性环境使药物和siRNA的释放,发挥不同功能。ETS1 siRNA通过干扰ETS1蛋白表达降低MDR1表达水平,以加强乳腺癌耐药细胞药物敏感性,降低阿霉素的泵出。第二部分,设计将β-环糊精偶联低分子量PEI600(CP)与DNA静电结合后形成阳离子聚合物纳米材料(CP/DNA),用CP/DNA包被VEGFR2重组的减毒沙门氏菌(NP/SAL)构建一种新型的肿瘤口服疫苗,并对NP/SAL疫苗进行了活力检测、形态学观察、体外细胞转染水平评估及体内免疫原性、抗肿瘤能力等研究。实验证明NP/SAL通过口服后在NP层的保护下,减毒沙门氏菌免受胃酸环境消化,且聚阳离子材料可以通过"质子海绵效应"导致溶酶体肿胀及膜的破裂,从而有助于细菌逃离吞噬体而到达肠粘膜部位。NP/SAL携带VEGFR2作为口服DNA疫苗能抑制肿瘤血管的形成,有利于抑制肿瘤的生长与转移。体内实验结果证明NP/SAL疫苗能刺激免疫系统产生T细胞和细胞因子,进而能影响VEGF通路,降低血管的形成,最终抑制肿瘤生长。第三部分,将癌细胞膜(CM)和细菌S层蛋白(SLP)与阳离子聚合物HPAD结合,制备了一种新型仿生纳米材料S-CM-HPAD用于黑色素瘤的免疫治疗和化疗的联合治疗。该仿生材料以合成材料HPAD为核,以黑色素瘤细胞膜为外壳形成核壳结构,表面自组装乳酸杆菌的S层蛋白(SLP)。在体内外实验证明了S-CM-HPAD具有癌细胞膜的特征蛋白,能靶向同源肿瘤,增加药物DOX疗效。同时,具有癌细胞膜上抗原,在免疫佐剂SLP的作用下能刺激机体产生抗肿瘤的CD4CD8T细胞和白介素-12、肿瘤坏死因子α、干扰素γ等细胞因子,发挥抗肿瘤免疫效应。通过对小鼠体重和生存率的观察,证明了 S-CM-HPAD生物相容性好,安全性高。
[Abstract]:How to safely and effectively apply the new tumor method to tumor treatment is a major challenge for tumor treatment. With the development of nanotechnology, polymer nano-drug carriers play an important role in this field. Cationic polymer based on cyclodextrin-PEI has good biocompatibility and high transfection efficiency as a drug carrier system. However, the application of cyclodextrin-PEI cationic polymer in tumor multidrug resistance and tumor immunotherapy is less. Therefore, the application of cationic polymer and its biomimetic materials in tumor immunotherapy and chemotherapy was studied as follows: the first part was composed of 纬 -hydroxypropyl-cyclodextrin coupled low molecular weight PEI600 (HP). Adriamycin (AD) modified by adriamycin (AD) was self-assembled by host and guest to form Hpad supramolecular nanomaterials. ETS1siRNA was carried by electrostatic interaction. The core was formed as a hydrophobic drug, and the shell carried core-shell siRNA with core-shell structure (HPAD / siRNA). HPAD / siETS1 entered the lysosome after endocytosis and escaped from lysosome by proton sponge effect. The release of drugs and siRNA was due to the acidic environment of lysosome. By interfering with the expression of ETS1 protein, the expression of MDR1 was reduced by different functions of ETS1 siRNA, in order to enhance the drug sensitivity of breast cancer cells and reduce the pump of doxorubicin. In the second part, 尾 -cyclodextrin coupled low molecular weight PEI600 (CP) was designed to form cationic polymer nanomaterials (CP-DNA) after electrostatic binding with DNA. A novel oral tumor vaccine was constructed by encapsulating the recombinant attenuated Salmonella mutans VEGFR2 (NPP / SAL) with CPR / DNA. The NPP / Sal vaccine was tested for its viability, morphological observation, in vitro cell transfection level evaluation, in vivo immunogenicity and anti-tumor ability. The results show that NPP / Sal can protect attenuated Salmonella from gastric acid environment after oral administration, and that polycationic materials can cause lysosome swelling and membrane rupture through "proton sponge effect". It is helpful for bacteria to escape phagocytosis and reach intestinal mucosa. NPP / Sal carrying VEGFR2 as oral DNA vaccine can inhibit tumor angiogenesis and inhibit tumor growth and metastasis. The results show that NPP Sal vaccine can stimulate the immune system to produce T cells and cytokines, which can affect the VEGF pathway, reduce the formation of blood vessels, and ultimately inhibit tumor growth. In the third part, a novel biomimetic nano-material S-CM-Hpad was prepared by combining cancer cell membrane (CM) and bacterial S layer protein (SLP) with cationic polymer Hpad for immunotherapy and chemotherapy of melanoma. The biomimetic material consisted of synthetic material Hpad and melanoma cell membrane to form core-shell structure and self-assemble S-layer protein (SLP) of Lactobacillus. In vitro and in vivo experiments proved that S-CM-Hpad has the characteristic protein of cancer cell membrane, which can target homologous tumor and increase the therapeutic effect of DOX. At the same time, with the antigen on the membrane of cancer cells, SLP can stimulate the production of anti-tumor CD4CD8T cells, interleukin-12, tumor necrosis factor 伪, interferon 纬 and other cytokines, play an anti-tumor immune effect. It was proved that S-CM-Hpad had good biocompatibility and high safety by observing the body weight and survival rate of mice.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O631;TQ460.1
本文编号：2119414
[Abstract]:How to safely and effectively apply the new tumor method to tumor treatment is a major challenge for tumor treatment. With the development of nanotechnology, polymer nano-drug carriers play an important role in this field. Cationic polymer based on cyclodextrin-PEI has good biocompatibility and high transfection efficiency as a drug carrier system. However, the application of cyclodextrin-PEI cationic polymer in tumor multidrug resistance and tumor immunotherapy is less. Therefore, the application of cationic polymer and its biomimetic materials in tumor immunotherapy and chemotherapy was studied as follows: the first part was composed of 纬 -hydroxypropyl-cyclodextrin coupled low molecular weight PEI600 (HP). Adriamycin (AD) modified by adriamycin (AD) was self-assembled by host and guest to form Hpad supramolecular nanomaterials. ETS1siRNA was carried by electrostatic interaction. The core was formed as a hydrophobic drug, and the shell carried core-shell siRNA with core-shell structure (HPAD / siRNA). HPAD / siETS1 entered the lysosome after endocytosis and escaped from lysosome by proton sponge effect. The release of drugs and siRNA was due to the acidic environment of lysosome. By interfering with the expression of ETS1 protein, the expression of MDR1 was reduced by different functions of ETS1 siRNA, in order to enhance the drug sensitivity of breast cancer cells and reduce the pump of doxorubicin. In the second part, 尾 -cyclodextrin coupled low molecular weight PEI600 (CP) was designed to form cationic polymer nanomaterials (CP-DNA) after electrostatic binding with DNA. A novel oral tumor vaccine was constructed by encapsulating the recombinant attenuated Salmonella mutans VEGFR2 (NPP / SAL) with CPR / DNA. The NPP / Sal vaccine was tested for its viability, morphological observation, in vitro cell transfection level evaluation, in vivo immunogenicity and anti-tumor ability. The results show that NPP / Sal can protect attenuated Salmonella from gastric acid environment after oral administration, and that polycationic materials can cause lysosome swelling and membrane rupture through "proton sponge effect". It is helpful for bacteria to escape phagocytosis and reach intestinal mucosa. NPP / Sal carrying VEGFR2 as oral DNA vaccine can inhibit tumor angiogenesis and inhibit tumor growth and metastasis. The results show that NPP Sal vaccine can stimulate the immune system to produce T cells and cytokines, which can affect the VEGF pathway, reduce the formation of blood vessels, and ultimately inhibit tumor growth. In the third part, a novel biomimetic nano-material S-CM-Hpad was prepared by combining cancer cell membrane (CM) and bacterial S layer protein (SLP) with cationic polymer Hpad for immunotherapy and chemotherapy of melanoma. The biomimetic material consisted of synthetic material Hpad and melanoma cell membrane to form core-shell structure and self-assemble S-layer protein (SLP) of Lactobacillus. In vitro and in vivo experiments proved that S-CM-Hpad has the characteristic protein of cancer cell membrane, which can target homologous tumor and increase the therapeutic effect of DOX. At the same time, with the antigen on the membrane of cancer cells, SLP can stimulate the production of anti-tumor CD4CD8T cells, interleukin-12, tumor necrosis factor 伪, interferon 纬 and other cytokines, play an anti-tumor immune effect. It was proved that S-CM-Hpad had good biocompatibility and high safety by observing the body weight and survival rate of mice.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O631;TQ460.1
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相关期刊论文 前2条
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2 ;Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier[J];World Journal of Gastroenterology;2003年06期
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