细胞穿透肽与磷脂膜之间的相互作用规律与机理研究

The selective permeability of cellular membranes protects the cells from an influx of exogenous molecules, including bioactive molecules such as peptides, proteins, and oligonucleotides. However, the transportation of bioactive molecules, especially the therapeutic drugs, into the cell is very important for the disease treatment. This has been a challenge in the biomedical field. Some strategies have been developed to deliver the therapeutic agents across cellular membranes. These include microinjection, electroporation, liposome and viral-based vectors. However, these methods have various drawbacks, including low efficiency, high toxicity, poor bioavailability, and difficult operation. Since it was discovered in 1988, cell penetrating peptides (CPPs) have received the substantial attention due to they meet the requirements of the ideal drug-carriers. CPPs have been used to facilitate the cellular entry of many bioactive molecules, including DNA, protein, liposome, and drug etc. However, due to the unclear transmembrane mechanism of CPPs, it is impossible to establish very highly efficient CPPs, which largely limits the further development of CPPs. The mechanisms by which CPPs enter cells dependent on not only the structures of CPP, but also the characteristics the cell membrane. In this project, we will firstly establish the comprehensive thermodynamic method and obtain the overall interaction trend of peptide with membrane, and then explore the structural transformation using the microscopic method. By combination of macroscopic and microscopic methods, we will systematically study the effects of peptide structure (amino acid sequence and number, hydrophobic chain length and number, the introduction of cyclic or branched structures) and membrane composition (phospholipid types, cholesterol or glycolipids additions) on the interactions between peptide and membrane, and establish a deeper understanding to the interaction mechanism, thus providing a theoretical basis for the design of more efficient cell-penetrating peptides.

细胞膜的选择通透性会阻止具有治疗作用的药物分子进入细胞内，如何实现药物分子的安全、高效和简易的跨膜转运是生物医学界的研究热点和难点。细胞穿透肽的出现极大地满足了人们对理想跨膜载体的要求，自1988年被发现以来立刻引起了研究者的广泛关注，并已作为载体成功应用到各种活性分子的跨膜转运，但是它的透膜机制尚不明确。深入认识穿透肽的透膜机制是建立高效穿透肽的前提和基础，迫切需要进行深入系统的研究。研究穿透肽与细胞膜相互作用的方式和规律，深入认识各种分子间作用力的贡献对理解作用机制至关重要。因此，本项目将通过建立全面研究穿透肽透膜过程的宏观热力学方法，结合微观手段，系统地研究穿透肽结构变化（氨基酸序列和个数、疏水链长度和个数、引入环状或支状结构）和细胞膜组成变化（磷脂头基电荷、磷脂疏水链特征、胆固醇或糖脂的加入）对多肽与膜相互作用的影响，获得对作用机理的深入认识，为设计更高效的细胞穿透肽提供理论依据。

本项目主要围绕着穿透肽两亲分子与磷脂膜之间的相互作用开展，充分发挥了等温滴定量热在研究复杂分子间相互作用方面的优势，并结合其它多种手段，系统研究了肽两亲分子结构，聚集体结构，磷脂组成等对相互作用的影响，并研究了肽两亲分子对磷脂膜通透性的调节规律。在此基础上，拓展研究了穿透肽两亲分子对真实细菌膜的影响和抗菌活性，氨基酸和两亲分子的水合性质也被关注。主要取得了四个方面的研究进展：（1）建立了穿透肽两亲分子结构/聚集体结构与磷脂膜相互作用方式和强度的关系；（2）利用穿透肽两亲分子调控了磷脂膜通透性，实现了跨膜反应和细菌内吞；（3）获得了氨基酸和两亲分子水合性质的探索性结果，为设计高效穿透肽两亲分子奠定了基础；（4）发现了具有良好破膜能力与抗菌活性的肽两亲分子，有望实现实际应用。因此，本项目的研究目标已经基本实现。本项目既有对基本科学问题的研究，也有对实际功能和应用的探索，取得的研究成果具有重要的科学意义和应用前景。在项目执行过程中也发现了一些新的课题有待深入探究，例如磷脂膜中脂阀的结构和功能；发展具有长效性和选择性的穿透肽两亲分子抗菌剂；以及穿透肽水合性质和功能之间的关系等。项目执行期间在Angew. Chem. Int. Ed.，J. Phys. Chem. Letters，ACS Appl. Mater. Interfaces，ACS Appl. Bio Mater，Langmuir，Soft Matter杂志上共计发表学术论文8篇；待发表论文3篇。申请专利2项。