Engineered Polyallylamine Nanoparticles for Efficient In Vitro Transfection
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Cationic polymers (i.e. polyallylamine, poly-L-lysine) having primary amino groups are poor transfection agents and possess high cytotoxicity index when used without any chemical modification and usually entail specific receptor mediated endocytosis or lysosomotropic agents to execute efficient gene delivery. In this report, primary amino groups of polyallylamine (PAA, 17 kDa) were substituted with imidazolyl functions, which are presumed to enhance endosomal release, and thus enhance its gene delivery efficiency and eliminate the requirement of external lysosomotropic agents. Further, systems were cross-linked with polyethylene glycol (PEG) to prepare PAA-IAA-PEG (PIP) nanoparticles and evaluated them in various model cell lines.
Materials and Methods
The efficacy of PIP nanoparticles in delivering a plasmid encoding enhanced green fluorescent protein (EGFP) gene was assessed in COS-1, N2a and HEK293 cell lines, while their cytotoxicity was investigated in COS-1 and HEK293 cell lines. The PAA was chemically modified using imidazolyl moieties and ionically cross-linked with PEG to engineer nanoparticles. The extent of substitution was determined by ninhydrin method. The PIP nanoparticles were further characterized by measuring the particle size (dynamic light scattering and transmission electron microscopy), surface charge (zeta potential), DNA accessibility and buffering capacity. The cytotoxicity was examined using the MTT method.
In vitro transfection efficiency of synthesized nanoparticles is increased up to several folds compared to native polymer even in the presence of serum, while maintaining the cell viability over 100% in COS-1 cells. Nanoparticles possess positive zeta potential between 5.6–13 mV and size range of 185–230 nm in water. The accessibility experiment demonstrated that nanoparticles with higher degree of imidazolyl substitution formed relatively loose complexes with DNA. An acid-base titration showed enhanced buffering capacity of modified PAA.
The PIP nanoparticles reveal tremendous potential as novel delivery system for achieving improved transfection efficiency, while keeping the cells at ease.
Key wordsbuffering capacity DNA accessibility GFP imidazole acetic acid nanoparticles polyallylamine transfection
The authors are thankful to Sophisticated Analytical Instrument Facility, Central Drug Research Institute, Lucknow, India and NMR Laboratory, Indian Institute of Technology, Delhi for NMR analysis. Authors (AP, RKK, SP and AS) gratefully acknowledge the Indian Council for Medical Research (ICMR), the Council of Scientific and Industrial Research (CSIR) and the University Grant Commission (UGC), respectively, for providing financial support.
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