巨单层磷脂囊泡冷冻干燥关键热物理问题研究

The giant unilamellar phospholipid vesicles (GUVs) play important roles in the scientific research including artificial cells, bioreactors and analytical science. However, their stability at room temperature are very poor. In this project, the freeze-drying method will be used to make GUVs stable and suitable for long-term storage. First, the phase diagram of ternary phospholipid in the presence of disaccharide will be obtained by exploring the interactions between them. Second, the microfluidic chip will be designed to prepare the GUVs. Third, the freeze-drying of GUVs will be carry out to examine the factors that may destroy the GUVs in the freezing and dying procedures. Finally, based on the exploring of long-term stability of freeze-dried GUVs, the mechanism of bilayer injury occurred during the storage will be discovered and an optimized protocol will be obtained. A practical method to predict the storage stability will also be given for the freeze-dried GUVs. The realization of stabilization of GUVs in dry state accommodates greater flexibility in their use after preparation, and thus greatly extends their breadth of downstream applications. In addition, this study is also helpful for revealing the mechanism of the injury of mammalian cell membranes during the freeze drying, which are difficult to obtain on the real cells. It is expected from the present study that some thermophysical criterion will be given to promote the freeze-drying technology of mammalian cells.

巨单层磷脂囊泡（GUVs）在人工细胞、生物反应器和分析科学中具有重要应用价值，但其稳定性问题至今没有解决。本项目提出用冷冻干燥的方法稳定GUVs。首先通过研究复合磷脂与糖的相互作用，获得双分子层糖基质复合系统的相图，然后采用微流体方法制备GUVs，在此基础上开展GUVs的冻干技术研究。探索冷冻干燥各环节对GUVs造成损伤的机理并提出优化的冻干方案。通过对冻干分散系开展长期稳定性测试，考察存储过程中的失稳行为及其影响因素，从分子活动性角度揭示引起GUVs失稳的若干机理，给出稳定性控制方案及预测方法。实现GUVs的冻干存储不仅对提高下游科学研究的效率和灵活性具有重要意义，还能揭示在哺乳动物细胞干燥中无法揭示的细胞膜干燥破坏机理，为目前进展缓慢的细胞冻干技术提供基础的物理学依据。

巨单层磷脂囊泡（GUVs）是优秀的细胞膜模型，在合成生物学和药剂学领域具有重要应用价值，但存在稳定性不佳的问题。本项目针对GUVs冷冻干燥保存中的几个关键热物理问题开展研究，尝试突破冻干方法稳定GUVs。主要研究内容有：磷脂双分子层与冻干保护剂相互作用热力学、微流体方法制备GUVs及其工艺流程的优化、冻干GUVs的冷冻、干燥、存储各环节关键失稳机理揭示等。重要结果有：（1）发现现有的关于干燥保护效果的假说并不相互排斥，而是在不同的干燥阶段具有不同的干燥保护机制，这一发现为细胞及磷脂囊泡的干燥保存提供了更为准确的分子动力学依据；（2）实现了PDMS芯片在低于生理温度范围内的双向精确控温，从而为GUVs的粒径调控提供了新的途径；（3）通过改变冻干分散系的位形基态实现其分子活动性的精确预测，从而为GUVs的长期稳定保存奠定了热物理学基础。