具有血液滞留时间长和易进入癌细胞的纳米载体

The residence time of nanoparticles in the blood is ofen in conflict with their uptake by cancer cells. The neutral and hydrophilic surfaces can increase nanoparticles' blood circulation time, but can prevent their cellular uptake. Conjugation of tumor-targeting ligands on the surfaces increases the uptake by cancer cells. Unfortunately, the conjugation compromises their blood circulation time. In this proposal, to circumvent this conflict, nanocarriers would be modified in their surfaces using hydrophilic polymers with amine groups at the terminals. The amine groups would be unprotonated at blood's and normal tissues' pH (7.4) and the unprotonated amine groups should remain hydrophilic, ensuring that the nanocarriers have a long blood circulation time and a less tendency to enter normal cells. Upon entering tumor tissues, the amine groups would be protonated due to the slightly acidic environment in tumor tissues, enabling the nanocarriers to have positive charges on their surfaces and subsequently to enter the cancer cells. Furthermore, polymeric micelles with suface amine groups that are sensitive to tumor slight acidic environment, disulfide-crosslinkage that is sensitive to redox potential in cells, and loaded-drugs that can be released responsive to the more acidic environments in endosomes and lysosomes would be prepared. The disulfide-crosslinkage can prevent disassembly of the micelles and thus decreses the release of the loaded drugs in bloodsream. After the micelles enter cancer cells, the loaded drugs would released rapidly responsive redox potential and acidic enviornments in the cells.

纳米粒子载体在血液中的滞留时间和被癌细胞摄取是一对矛盾。中性亲水性的表面延长载体在血液中的滞留时间，但削弱载体被癌细胞摄取；表面偶合识别癌细胞的配体可促进载体进入癌细胞，但又缩短其血液滞留时间。为了解决这一矛盾，本项目用末端含有胺基的亲水性聚合物修饰纳米载体的表面，所选择的胺基在血液和正常组织的pH（7.4）下是非质子化的，而且非质子化的胺基是亲水性的，保证载体在血液中不易被清除，也不易进入正常细胞内。当载体进入肿瘤组织后，肿瘤组织的微酸性环境使胺基质子化使载体表面带正电荷，促使载体被癌细胞摄取。进而研制表面带感应肿瘤微酸性的胺基、含二硫键交联结构和负载的药物感应胞内较强酸性而释放的聚合物胶束，二硫键交联结构保证胶束在血液中不解聚，减小药物在血液中的释放。进入癌细胞后感应胞内还原性和酸性使药物快速释放。

纳米粒子的表面性质对粒子在血液中的滞留时间和被癌细胞摄取的影响往往是相互矛盾的，具有非离子电中性亲水性表面的纳米粒子和带有电中性或微负电性两性离子表面的纳米粒子具有长的血液循环时间，但这样的表面使粒子不易被癌细胞摄取。为了解决这一矛盾，本项目在纳米粒子表面引入吗啉基、或制备壳聚糖的纳米粒子，吗啉基和壳聚糖的氨基的pKa值都处于肿瘤胞外的微酸性pH值到血液的中性的范围内，纳米粒子在血液的中性介质中其表面为电中性或带微负电荷，因此纳米粒子具有长的血液循环时间，血液循环半衰期增加1倍以上。当纳米粒子进入肿瘤组织后，肿瘤胞外微酸性的环境使吗啉基或壳聚糖氨基发生质子化而带正电荷，激光共聚焦和细胞流式实验证明纳米粒子在微酸性环境下易被癌细胞摄取；为了进一步增加血液循环时间，在纳米粒子表面同时引入吗啉基和羧基，通过调节吗啉基和羧基的比例，使纳米粒子在中性环境中带有两性离子且为电中性或带微负电性，因此纳米粒子具有长的血液循环时间。而在肿瘤的微酸性环境中由于吗啉基的质子化使纳米粒子带正电荷，被癌细胞摄取的能力大大增加；为了增加自组装聚合物胶束的稳定性，避免胶束进入血液后因稀释而解聚，同时为了实现所载药物在肿瘤细胞内的快速和完全释放，并增大胶束中药物的负载量，合成核含有疏水基团和羧基、且核含有二硫键交联结构的聚合物胶束，阿霉素通过离子键和疏水作用负载于胶束的核中，因此具有高负载量，负载量高达560mg/g。在血液的中性条件下24h的释放率为27%，当胶束进入癌细胞后，胞内高浓度的谷胱甘肽还原二硫键使胶束解聚，同时内涵体和溶酶体的酸性环境使聚合物上的羧基负离子质子化，使阿霉素与聚合物之间失去离子键，因此负载的阿霉素快速释放，在pH 5和15mmol/L谷胱甘肽的介质中24h的释放率为75%。