聚苹果酸纳米接枝复合物高效肿瘤靶向释药体系的构建

The aim of this project is to construct a kind of multifunctional drug delivery system with ligand(LHRH)-mediated drug targeting, cell penetrating peptides (TAT) and nuclear localizing signals(NLS)-mediated high efficiency endocytosis and active nuclear transport characteristics. By means of the pH difference between normal tissue and tumor tissue, LHRH and TAT/NLS were designed in pH-sensitive charge-reversal nanoconjugate platforms so as to realize carrier cumulated specifically in target site and tumor cell nuclear delivery of drug with high efficiency. Polymalic acid as initating material, 2 groups of PEG-modified comblike nanoconjugates were prepared. One is conjugated with DNA-toxins drug(doxorubicin, DOX or camptothecin, CPT) via a disulfide bond、β-carboxylic acid amide charge-reversal chemical group and ligand; the other is connected with drug, TAT and NLS. By means of electrostatic interaction between the positively charged and negatively charged vehicles, nanocomplexes was self-assembled. The chemical construction was determined by nuclear magnetic resonance and mass spectrum. Micelle formation is monitored by fluorometry in the presence of pyrene as a fluorescent probe. The pH-response characteristics connected with the ratios of polymers and surface charges are studied by photo-physical methods. The cellular uptake and intracellular trafficking of nanocarriers was observed with confocal laser light fluorescent microscopy, distribution in vivo of nanocomplexes and antitumous effect are detected by in vivo imaging system. Pharmacodynamics studies in vitro and in vivo are performed to investigate the pH-response characteristics of nanocomplexes and regularity of drug release, testified the effects of cascade cancer cell nuclear targeting.

本课题旨在构建一种兼有配体(LHRH)主动靶向、穿膜肽(TAT)、核定位信号肽(NLS)介导高效入胞、入核特性的药物运载体系。将LHRH、TAT、NLS依次设计在pH响应型电荷翻转纳米复合物体系中，利用正常组织与肿瘤组织pH值差异，实现载体在靶位特异性累积和将药物高效转运至肿瘤细胞核的多重功能。以聚苹果酸为原料，设计2组PEG化梳状接枝物：一组连接LHRH、β-羧基酰胺化阳离子基团及药物DOX(或CPT)；另一组连接TAT、NLS和药物。利用正负电荷静电作用，自组装成纳米复合物。核磁、质谱等表征接枝物结构，芘荧光探针法研究聚合物在水溶液中的自组装行为；光物理法研究聚合物配比、表面电荷与pH值响应之间的关系；激光共聚焦荧光显微法、在体成像系统检测纳米复合物与（耐药）肿瘤细胞的相互作用、体内分布及抗肿瘤效果。体内外药效学研究纳米粒对肿瘤pH值的特异响应性及释药规律，验证其逐级核靶向释药特性。

本课题构建了一种兼有配体主动靶向、穿膜肽（TAT）介导高效入胞特性的药物运载体系。将配体和TAT设计在pH响应型电荷翻转纳米复合物体系中，利用正常组织与肿瘤组织 pH值差异，实现载体在靶位特异性累积和将药物高效转运至肿瘤细胞的多重功能。核磁、红外等表征接枝物结构，芘荧光探针法研究聚合物在水溶液中的自组装行为；光物理法研究聚合物配比、表面电荷与pH值响应之间的关系；激光共聚焦荧光显微法、在体成像系统检测纳米复合物与（耐药）肿瘤细胞的相互作用、体内分布及抗肿瘤效果。体内外药效学研究纳米粒对肿瘤pH值的特异响应性及释药规律，验证其逐级核靶向释药特性。在完成项目标书设计内容的基础上，申请者构建了多种聚苹果酸纳米药物载体，填补了国内聚苹果酸作为药物载体及相关生物学应用的空白。.1. 将多种功能基团同时接枝于PMLA骨架上，制备抗体介导和具有pH敏感电荷翻转效应的多重功能纳米接枝物，载体表面正电性促进入胞的作用优于抗体介导，两种方式同时修饰具有更好的靶向递送和高效入胞作用。2. 以聚苹果酸为载体，通过自组装和静电吸附，制备了具有双重pH敏感特性的电荷翻转型纳米复合物。该纳米复合物同时对肿瘤胞外及胞内溶酶体/内涵体酸性环境敏感，实现两种作用：①纳米载体感应肿瘤细胞外酸性环境产生电荷翻转，释放带正电荷和TAT修饰的聚合物胶束，高效入胞；②胞内溶酶体酸性环境促使药物在胞内快速释放。3. 制备了具有pH响应特性的TAT保护/去保护的PMLA纳米接枝物。考察了穿膜肽发挥穿膜作用的最小用量（0.3%，mol/mol）及PEG发挥保护作用的最佳用量3.6%（mol/mol），并以聚苹果酸苄基酯（PMLABz）为载体，研究嵌段聚合物胶束（PMLABz-PEG2000-TAT和PMLABz-Hz-PEG3400）对TAT的保护/去保护作用。4. 采用接枝、嵌段两种方式得到不同结构的两亲性共聚物，研究PEG连接在链端或接枝在骨架自组装得到两种不同类型胶束的稳定性，证实平头胶束稳定性略优于星型胶束。控制化疗药物CPT的接枝率可调节聚合物的亲水/疏水性能，得到较好稳定性的聚合物胶束。

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