Lyophilization of Lipid Nanoparticles and Lipid-mRNA Complex for Extending the Shelf Life
Rui, Xuehui.
2020
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Thesis (M.S.)--Tufts
University, 2020.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Qiaobing Xu.
Committee: Bing Xu, and Srivalleesha Mallidi.
Keyword: Biomedical engineering.
Lipid nanoparticle, also known as liposome, is a class of novel vector that has been developed for decades because of its inspiring ability in delivering ... read morebiomolecules and drug molecules in vivo and in vitro. Via chemical structural modification, liposomes could obtain other outstanding properties like positive electricity, long circulation timespan in vivo and biodegradability. In those ways, the liposomes could increase the cargo encapsulation efficiency, realize a controlled release of cargo molecules and significantly reduce biotoxicity. However, during storage in aqueous solution, the biodegradable cationic liposomes are exposed to physical as well as chemical property changes and the shelf time is short. Specifically, the liposomes could be ruptured, and unfortunately induce cargo leakage and disfunction of delivery ability. Besides, the liposomes could aggregate in aqueous solution, thus increase the particle size and affect the delivery functions. For the biomolecular cargos, they could be degraded and lose their bio-function during storage in aqueous solution. These instabilities are induced by the aqueous environment. In order to solve the problems above, this thesis developed a lyophilization strategy for a type of biodegradable cationic liposome and investigated the protective ability of lyophilization on liposomes along with encapsulated mRNAs by monitoring the property changes of freeze-dried sample on storage. The first part of this thesis is optimizing the lyophilization recipe and process. The optimization includes buffer selection, excipient selection and lyophilization process control. The physical properties of empty liposomes are viewed as the most significant parameters during lyophilization. The reason why we chose empty liposomes as optimization object is that, the protective effect on empty liposome is viewed as the precondition for the following studies. The particle size, zeta potential and morphology property changes of empty liposomes are investigated and compared before and after lyophilization to get the best recipe. The final determined best recipe would then be applied on the lipid-mRNA complex so as to validate its comprehensive protective effect. The second part of this thesis is for studying the long-term stability of lyophilized samples. In this study, the firefly luciferase mRNA encapsulated liposomes are prepared and lyophilized with the previously optimized recipe and then the lyophilized lipid-mRNA complexes are investigated to validate the protective effect of lyophilization. The lyophilized lipid-mRNA complexes are dissolved and delivered in vitro at different time points to monitor the functionality changes of mRNA-liposomes. Also, the cargo encapsulation efficacy, liposome particle size and mRNA integrity are investigated at different time points to monitor the quality changes on storage. The long-term performance of lyophilized lipid-mRNA complexes is studied in this part. This thesis developed the lipid-mRNA complex into a freeze-dried agent with a longer shelf-time. The function of liposome-mRNA complex could be retained for weeks, which is much longer than the non-lyophilized one. The protective effect of lyophilization on liposome-mRNA complex is prominent. According to the study, the main restriction to the lyophilization outcome is the freezing step. The formation of water crystals in the freezing step is potentially to damage the vesicular structure of liposomes and result in the cargo leakage and reduce its functions, so the freezing process calls for a deeper study and optimization. Hopefully, with further improvements, the lyophilization strategy could be widely applied in liposome agents to make them into a solid anhydrous dosage which is more stable on storage and easier for clinical or laboratory usage.read less - ID:
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