Degradation of plasmid DNA (pDNA) in the endosome compartment and its release to the cytosol are the major hurdles for efficient gene transfection. This is generally addressed by developing transfection reagents that can overcome these limitations. In this article, we present the first report, which suggests that controlling the release of pDNA from endosome is the key for achieving efficient transfection. In this study, we developed chondroitin sulphate (CS) coated DNA-nanoplexes using a modular approach where CS was coated post pDNA/PEI nanoplex formation. To ensure good stability of the nanoplexes, we exploited imine/enamine chemistry by reacting aldehyde-modified chondroitin sulphate (CS-CHO) with free amines of pDNA/PEI complex. With our rational design of the supramolecular gene carrier system we observed efficient cellular uptake, and controlled endosomal pDNA release without any cytotoxicity. On the contrary, burst release of pDNA from endosome (using chloroqine) resulted in significant reduction in gene expression. Unlike pDNA/PEI based transfection, our design resulted in exceptionally high gene transfection efficiency, which is proven in human colorectal cancer cells (HCT116), human embryonic kidney cells (HEK293) and mouse skin-derived mesenchymal stem cells (MSCs) using luciferase protein as reporter gene. This new insight will be valuable in designing next generation of transfection reagents.
Preserving the integrity of plasmid DNA (pDNA) after endocytosis by promoting quick release of pDNA from endosome compartment is believed to be vital for gene transfection. In this article, we present the first evidence that suggests that slow and sustained release of the pDNA from endosome is the key for achieving efficient transfection, which can be achieved by covalent coating of nanoplexes with chondroitin sulphate.
Particles size and ζ-potential analysis of nanoplexes by DLS. (A) Size distribution of pDNA/PEI, pDNA/PEI/CS and pDNA/PEI/CS-CHO nanoplexes at the ratio of N/P = 10. (B) Size distribution of pDNA/PEI/CS and pDNA/PEI/CS-CHO nanoplexes after 24 h incubation at room temperature. (C) Kinetic studies of ζ-potential of pDNA/PEI/CS and pDNA/PEI/CS-CHO nanoplexes.
Endosomes disruption capacity of pDNA/PEI, pDNA/PEI/CS and pDNA/PEI/CS-CHO nanoplexes in HCT116 cells, as indicated by LysoTracker® Red. (A) Representative fluorescence microscopy images after 1 h. (B) Fluorescence intensity of LysoTracker® Red after incubation of nanoplexes for different time points. (C) Luciferase activity in the presence and absence of chloroquine. Scale bar 100 μm.
The present work was supported by Swedish Strategic Research grant ‘StemTherapy’ and EU Framework Program-7 project ‘Biodesign’. We also acknowledge China Scholarship Council (CSC) for supporting HY.
References:
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[2] E. Alton, S. Ferrari, U. Griesenbach, Gene Ther. 2007, 14, 1555.