Abstract
Cationic polymers such as polyallylamine (PAA) having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P) ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa) were suitable vectors for gene delivery with low cytotoxicity.
Highlights
Research in recent years has witnessed major concern about gene therapy providing novel approaches to the treatment of many diseases caused by genetic disorders (Guo et al, 2008)
Many deficiencies in several areas including the induction of host inflammatory and immune response are reported for viral vectors, though they provide high gene delivery efficiency (Tros-de-Ilarduya, Sun, Duzgunes, 2010)
The present study aims at enhancing PAA transfection efficiency through replacing primary amino groups with cholesterol, reducing the polymer cytotoxicity, developing polymer hydrophobic interaction with the cell membrane, and maintaining the polymer buffering capacity
Summary
Research in recent years has witnessed major concern about gene therapy providing novel approaches to the treatment of many diseases caused by genetic disorders (Guo et al, 2008). Numerous modifications have been carried out on this polymer to make it an effective and non-toxic carrier, including the preparation of nanocomplexes composed of PAA-dextran-DNA with an average size of about 150 nm, by Nimesh, Kumar, and Chandra (2006). In order to improve the endosomal release and enhance the gene transfer efficiency of the polymer, Pathak, Patnaik, and Gupta (2009) replaced primary amino groups of PAA (17 kDa) with imidazolyl moieties. The present study aims at enhancing PAA transfection efficiency through replacing primary amino groups with cholesterol, reducing the polymer cytotoxicity, developing polymer hydrophobic interaction with the cell membrane, and maintaining the polymer buffering capacity. It is expected that as nanoparticles cross the cell membrane, vector release from the endosome becomes easier (through endosome membrane instability) and polymer toxicity decreases
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