Abstract
The introduction of the DNA into mammalian cells remains a challenge in gene delivery, particularly in vivo. Viral vectors are unmatched in their efficiency for gene delivery, but may trigger immune responses and cause severe side-reactions. Non-viral vectors are much less efficient. Recently, our group has suggested that a star-shaped structure improves and even transforms the gene delivery capability of synthetic polycations. In this contribution, this effect was systematically studied using a library of highly homogeneous, paramagnetic nano-star polycations with varied arm lengths and grafting densities. Gene delivery was conducted in CHO-K1 cells, using a plasmid encoding a green fluorescent reporter protein. Transfection efficiencies and cytotoxicities varied systematically with the nano-star architecture. The arm density was particularly important, with values of approximately 0.06 arms/nm2 yielding the best results. In addition, a certain fraction of the cells became magnetic during transfection. The gene delivery potential of a nano-star and its ability to render the cells magnetic did not have any correlations. End-capping the polycation arms with di(ethylene glycol) methyl ether methacrylate (PDEGMA) significantly improved serum compatibility under transfection conditions; such nano-stars are potential candidates for future in vivo testing.
Highlights
The introduction of DNA remains a challenge in genetic modification (‘transfection’) of mammalian cells, in particular for in vivo applications
Viral vectors are still unmatched in their efficiency, but trigger immune responses and may show toxic or carcinogenic effects [2]
Plasmid pEGFP-N1 (4.7 kb, Clontech Laboratories, Inc., Mountain View, CA, USA) encoding for the enhanced green fluorescent protein (EGFP) driven by the cytomegalovirus (CMV) immediate early promoter was used as a reporter. pEGFP-N1 was amplified in E. coli DH5α using standard laboratory techniques and purified by the EndoFree Plasmid Kit (Giga Prep) from QIAGEN (Hilden, Germany)
Summary
The introduction of DNA remains a challenge in genetic modification (‘transfection’) of mammalian cells, in particular for in vivo applications. Increasing molar mass is typically associated with improved gene delivery, and with increasing toxicity, while for a given molar mass, non-linear polymers such as branched or dendritic polymers show less cytotoxicity with a comparable transfection efficiency [13,14] In this context, our group has introduced star-shaped, PDMAEMA-based transfection agents (‘nano-stars’) [15], as vectors for gene delivery. A library of highly homogeneous magnetic nano-stars with varied arm length and grafting density was synthesized, including one structure with arms that were end-capped with blocks of poly(diethylene glycol) methyl ether methacrylate) (PDEGMA) for improved blood compatibility Statistical tools, such as the nonparametric Spearman correlation, were used to correlate physicochemical parameters of both the polycations and the corresponding polyplexes with their eventual ability to deliver plasmid DNA to CHO-K1 cells, and with their cytotoxicities and magnetic effects
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