超声介导脂质纳米粒克服粘液层和小肠上皮细胞双重屏障及其胃肠道传递过程的研究

The mucus and intestinal epithelial cell barriers can block the oral absorption of poorly water-soluble drugs and macromolecular drugs, which serves as the key problem of limiting the oral application of drugs. Therefore, based on the strategies of mucus penetrating nanoparticle, cell-penetrating peptides modification, charge reversal as well as ultrasound mediation, this project is planning to prepare the novel lipid-based nanoparticles to overcome the mucus and intestinal epithelial cell barriers, and demonstrate their fate in the gastrointestinal tract. Firstly, lipid-based nanoparticles with various physicochemical properties will be prepared using perfluoropentane as oil phase. The ultrasound responsive and mucus penetrating nanoparticles will be selected for further study. Then, cell-penetrating peptide (DSPE-PEG-TAT) and phosphate group (DSPE-PEG-Pho) will be introduced into the lipid-based nanoparticles, to make their surface charge reversible after hydrolyzed by alkaline phosphatase (IAP). Secondly, we will establish the in vitro barrier models of lipase, mucus and intestinal epithelial cells. The models are used to study the stability of nanoparticles under the hydrolysis of lipase, the mucus penetrating property under ultrasound mediation, the surface charge reversal under IAP hydrolysis as well as the absorption by intestinal epithelial cell. Moreover, the rules will be summarized and the key physicochemical properties will be identified to optimize the prescription. Thirdly, the lipid-based nanoparticles will be labeled by tracers to study the distribution of nanoparticles in the gastrointestinal tract and other organs. At last, the lipid-based nanoparticles will be used to encapsulate model drugs such as paclitaxel, cyclosporin A and insulin. The pharmacokinetics and toxicology of drug loaded nanoparticles will be studied. This project can not only provide the theoretical basis and experimental evidence for lipid-based nanoparticles overcoming barriers of both the mucus layer and the intestinal epithelial cells, but also bring new inspiration to other oral formulations.

粘液层和小肠上皮细胞屏障阻碍水难溶性药物和大分子药物的口服吸收，是限制其口服应用的关键问题。为此，本项目基于粘液层渗透型纳米粒，电荷反转、细胞穿膜肽修饰和超声介导设计策略，拟构建新型脂质纳米粒克服该双重屏障，并阐明其胃肠道传递过程。首先，以全氟戊烷为油相构建不同性质脂质纳米粒，筛选出超声响应的粘液层渗透型纳米粒。引入细胞穿膜肽和磷酸基，使其在IAP作用下电荷反转。其次，建立体外脂肪酶、粘液层和小肠上皮细胞模型，考察脂肪酶水解稳定性、超声作用下粘液层渗透性、IAP作用下电荷反转及小肠上皮细胞吸收情况，总结规律并确定关键指标，优化处方。再次，示踪剂标记脂质纳米粒，体内考察其在超声作用下胃肠道和组织器官中的分布。最后，将脂质纳米粒包载模型药物紫杉醇、环孢菌素A和胰岛素，进行药代动力学与安全性评价。本项目不仅为脂质纳米制剂克服上述双重屏障提供理论基础和实验依据，而且为其他口服制剂的研究提供新思路。

粘液层和小肠上皮细胞屏障阻碍水难溶性药物和大分子药物的口服吸收，是限制其口服应用的关键问题。基于此，本项目提出结合粘液层渗透型，电荷反转、细胞穿膜肽修饰和超声介导四重策略，构建新型脂质纳米粒递送系统，以克服双重屏障提高药物口服吸收。在国家自然科学基金青年项目（批准号81903560）资助下，本团队开展了以下研究并取得重要成果。首先，制备了几十种脂质纳米粒（LNP）类型，并从中筛选出Solutol HS15/MCT（1:2，w/w）作为基本纳米粒，进一步引入TAT、磷酸基和全氟己烷，获得超声响应脂质纳米粒TAT-PHO-PFH-LNP，用于后续评价。其次，建立体外粘液层模型，粘液层粘附试验发现纳米粒受益于表面“类病毒”特性，被粘液层吸附的程度最低。粘液层渗透性试验发现，超声响应脂质纳米粒在超声刺激下渗透性提高了几十倍，体现出克服粘液层屏障方面的巨大优势。再次，建立了Caco-2细胞模型和Caco-2/HT29-MTX-E12共培养细胞模型。发现当纳米粒浓度低于0.75 mg/mL，且超声强度低于1.5 W/cm2时，超声刺激对细胞活率没有显著影响；但细胞摄取效率和细胞转运效率在超声刺激后均大幅提高，体现出克服小肠上皮细胞屏障方面的巨大优势。从次，利用小动物活体成像系统观察脂质纳米粒在胃肠道分布情况，发现超声刺激能够加快纳米粒的吸收。最后，抗癌药紫杉醇装入超声响应纳米粒中并考察其口服生物利用度，发现超声响应纳米粒联合超声刺激后，其口服生物利用度显著高于其它组，证明超声刺激能促进药物吸收。此外，大鼠肠组织形态在超声刺激前后并无差异，说明超声刺激不会伤害肠组织。因此，本项目构建了一种新型脂质纳米粒，其联合超声刺激能显著克服小肠粘液层和上皮细胞屏障，促进药物吸收，且安全性较好。本项目的完成不仅为脂质纳米制剂克服上述双重屏障提供理论基础和实验依据，而且为其他口服制剂的研究提供新思路。