黏惰化及酸敏感修饰对纳米粒子黏膜穿透的影响

使用纳米粒子进行疾病的诊断和治疗是当前研究的一个热点. 由于受到黏液层的阻碍, 纳米粒子对于黏膜上皮细胞的进入效果不佳, 从而限制了其对黏膜相关疾病的诊断和治疗. 本文设计合成了一种具有黏惰性的酸敏感纳米粒子(MSNs-pCBMA-DMMA), 可有效穿透黏液层进入黏膜上皮细胞. 首先采用溶胶-凝胶法合成了表面氨基化的介孔二氧化硅纳米粒子(MSNs-NH₂), 然后通过原子转移自由基聚合法(ATRP)使两性离子羧基甜菜碱甲基丙烯酸酯(CBMA)在MSNs-NH₂表面上聚合形成聚羧基甜菜碱甲基丙烯酸酯(pCBMA), 获得惰性化的粒子(MSNs-pCBMA), 最后将酸响应性分子2,3-二甲基马来酸酐(DMMA)修饰于MSNs-pCBMA表面, 制备了MSNs-pCBMA-DMMA. 场发射透射电子显微镜(TEM)、 傅里叶变换红外光谱(FTIR)、 氢核磁共振波谱(¹H NMR)和纳米粒度Zeta电位测定仪等分析结果表明, 本文合成了MSNs-pCBMA-DMMA, 且粒子表面电位随pH值降低显著增加, 在pH=7.4~5.7范围内具有酸敏感能力. Transwell^?小室实验表明, pCBMA的接枝提高了粒子在模拟黏液中的渗透速率, 而DMMA的修饰则进一步增强了粒子的扩散能力, 4 h内MSNs-pCBMA-DMMA的模拟黏液渗透率达到16.3%, 为MSNs-pCBMA的1.9倍, MSNs-NH₂的3倍, 而以MSNs-NH₂的表观渗透系数（P_app）为标准计算得到的MSNs-pCBMA-DMMA的相对表观渗透系数达到了2.96. 细胞毒性试验验证MSNs-pCBMA-DMMA粒子的生物安全性良好. 细胞摄取试验表明, 相比于其它粒子MSNs-pCBMA-DMMA能够更快的被黏膜上皮细胞摄取. 本文所构建的纳米粒子能够快速渗透黏液且易于被黏膜上皮细胞摄取, 为其应用于黏膜相关疾病的活体诊断和治疗提供了基础.

Effects of Muco-inert and Acid-sensitive Modification on Mucosal Penetration of Nanoparticles

Gastric cancer， cervical carcinoma， lung cancer are occurred in mucosa， and the nanoparticles provided a competitive way for the diagnosis and therapy of these diseases. However， the mucus layer which contains mucus and other viscous substances could adsorb and remove the foreign substance， resulting the hindered penetration of nanoparticles. In this paper， a pH-responsive mucus-inert nanoparticle was designed for penetrating mucus and entering mucosal epithelial cells. In brief， mesoporous silica nanoparticles with surface amination（MSNs-NH₂） was prepared by sol-gel method. Then， the zwitterionic polymer was modified to the surface of MSNs-NH₂ by atom transfer radical polymerization to form mucus-inert particles（MSNs-pCBMA）. Acid-sensitive molecule DMMA was modified onto MSNs-pCBMA through amino groups on the particle surface to obtain pH-responsive mucus-inert nanoparticles（MSNs-pCBMA-DMMA）. The results of transmission electron microscopy（TEM）， proton nuclear magnetic resonance（¹H NMR），Fourier transform infrared spectrophotometer（FTIR） and Zeta potential measurements showed that four nanoparticles were synthesized successfully， and MSNs-pCBMA-DMMA exhibited sensitive pH response at pH= 7.4—5.7. A mucus-containing Transwell^? model was used to study the permeability of nanoparticles in mucus. The penetration results showed that within 4 h， the mucus permeability of MSNs-pCBMA-DMMA reached 16.3%， 1.9 times that of MSNs-pCBMA and 3 times that of MSNs-NH₂， and its relative apparent permeability coefficient reached 2.96 calculated with the apparent penetration coefficient（P_app） of MSNs-NH₂ as the reference. In addition， the grafting pCBMA improved the penetration rate of nanoparticles in mucus and the modification of DMMA further improved the penetration rate. Cytotoxic test showed that the viability of cells cultured with 100 μg/mL MSNs- pCBMA-DMMA was 78.3% in 24 h， indicating its good biosafety. The cellular uptake experiment showed that MSNs-pCBMA-DMMA could be endocytosed effectively at 4 h， and exhibited better endocytosis performance compared with other particles. The work proved that the combination of mucus inertia and acid sensitivity can effectively promote the mucosal permeability of nanoparticles， and MSNs-pCBMA-DMMA has excellent mucus permeability. The experimental results would benefit the design of nanoparticles for the diagnosis and therapy of mucosal diseases.