基于表面性质智能转变、高效穿透粘液和上皮细胞屏障的口服纳米粒构建及研究

The rapid capture and clearance of nanocarriers by the mucus layer has become one of the major limitations of oral drug absorption. Although nanocarriers with hydrophobic surface have high mucus-penetrating ability, but their retention time in the epithelial cell surface is short, and the ability to enter the cell is significantly reduced, resulting in unefficient drug absorption improvement. The surface properties of the nanocarriers required to penetrate the mucus diffusion barrier and the epithelial cell absorption barrier are distinctly different. Conventional oral nanocarriers can not solve this contradiction. In this project, multifunctional nanoparticles with smart transformation of surface hydrophilicity/ hydrophobicity, electrification and adhesion would be constructed based on the acidic microenvironment between the surface of jejunum epithelial cells and the mucus. The nanoparticles can penetrate the mucus layer quickly through masking of an acid-sensitive PEG shell. When reach the acidic microenvironment in the surface of the epithelial cells, the PEG shell would shed from the nanoparticles. The positively charged chitooligosaccharides nanoparticles would adhere to the epithelial cells and improve drug absorption through endocytosis or opening the cell bypass. The ability and mechanism of multi-functional nanoparticles to penetrate the mucus layer, acid-sensitive PEG-shell shedding and epithelial cell uptake would be studied. The pharmacokinetics of the nanoparticles as well as its therapeutic effect in the diabetic rats would be also studied. This strategy will provide a new idea to overcome the bottleneck problem of conventional nanocarriers with constant surface properties.

粘液层对纳米载体的快速捕捉和清除，已成为其促药物口服吸收的主要限制之一。虽然表面亲水性的纳米载体粘液穿透能力强，但在上皮细胞表面滞留时间短，且进入细胞能力显著降低，难以充分发挥递药作用。穿透粘液扩散屏障和上皮细胞吸收屏障所需纳米载体表面性质截然不同。常规口服纳米载体无法解决这一矛盾。本项目利用空肠上皮细胞表面与粘液间酸性微环境特性，构建了表面亲/疏水性、带电性和粘附性智能转变的多功能纳米粒。该纳米粒可在酸敏感的PEG外壳掩饰下快速穿透粘液层，到达上皮细胞后在其表面酸性微环境下，智能响应脱落PEG，暴露正电性壳寡糖纳米粒，粘附在上皮细胞表面并通过内吞或打开细胞旁路促药物高效吸收入血。以胰岛素为模型药物，研究多功能纳米粒穿透粘液层、继而酸敏感脱去PEG外壳和跨上皮细胞吸收的能力及机制；大鼠体内药动学及糖尿病模型大鼠的药效学。该策略为克服常规纳米载体固定表面性质存在的递药瓶颈问题提供了新的思路。

纳米载体给药系统的开发与应用，为解决蛋白类药物口服递送的问题提供了可能。然而克服粘液扩散屏障和上皮细胞吸收屏障对纳米载体表面性质的需求截然不同，大大限制其口服递药效率。针对以上矛盾，本项目利用空肠上皮细胞表面的酸性微环境，构建了一种能够智能生物响应，精准调控表面性质，逐级穿越两种屏障达到高效药物吸收的口服纳米给药系统。本项目通过pH敏感的腙键将具有优越粘液穿透性能的PEG2000与高细胞亲和力的聚乳酸-羟基乙酸共聚物（PLGA）相连，合成了具有上皮细胞表面酸性微环境响应能力的智能粘液穿透材料PEG-Hyd-PLGA，并以胰岛素为模型药物，采用复乳/溶剂挥发法制备了包载胰岛素的PEG-Hyd-PLGA纳米粒，以实现逐级依次跨越粘液屏障和肠细胞吸收屏障。所设计的载智能纳米粒粒径为139.6 nm，呈较规则的圆球形，包封率高，且可有效抵抗肠胰酶降解。通过测定不同pH下其粒径及电位变化以及PEG链的脱落效率，证明了智能纳米粒可在酸性环境响应脱落PEG链转变表面性质。用DiD标记的纳米粒进行口服吸收机制研究，与常规粘液穿透纳米粒相比，pH 6.8和7.4时，智能纳米粒同样降低了纳米粒与粘蛋白的相互作用。然而随着pH降低，其酸敏感键断裂，显示出PLGA表面对粘蛋白的吸附性。体外及在体一系列实验表明该纳米粒无明显细胞毒性，且在微酸性环境下逐渐暴露出疏水性内核，增加细胞摄取量，并且该纳米粒能够到达深层粘液处，又在到达空场上皮细胞附近逐渐断裂PEG链，较好地被上皮细胞吸收。药效学研究表明，在体空肠给药后，与对照组相比，智能纳米粒组的降血糖作用最为显著，且作用持久。这些结果表明本项目成功构建了智能纳米递药系统，可通过生物响应智能转变表面性质，同时提高了其跨粘液和上皮细胞吸收的能力。在蛋白多肽类药物的口服给药中具有一定的潜在应用价值，为克服粘液扩散屏障和上皮细胞吸收屏障提供了新的实验依据。