Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes

Abstract

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56 to 150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.

Keywords::

• DNA delivery
• vaccine delivery
• DNA vaccination
• dehydration-rehydration method

Acknowledgments

The authors acknowledge Kees Franken from the Department of Immunohematology and Blood Transfusion and the Department of Infectious Diseases, Leiden University Medical Center (Leiden, The Netherlands), for making the plasmid DNA. Frits Flesch, from the Department of Biomedical Analysis, and Electron Microscopy Utrecht, Faculty of Betasciences, Utrecht University (Utrecht, The Netherlands), is acknowledged for the CryoTEM imaging of the liposomes.

Declaration of interest

The authors declare no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper.