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  • Title: Hemocompatible pullulan-polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency.
    Author: Rekha MR, Sharma CP.
    Journal: Acta Biomater; 2011 Jan; 7(1):370-9. PubMed ID: 20659595.
    Abstract:
    Polyethyleneimine (PEI; 25 kDa)-conjugated pullulans (PPE1, PPE2 and PPE3) were developed and investigated for possible use in gene delivery applications. The cytotoxicity, blood component interactions such as red blood cell/white blood cell aggregation, platelet and complement activation, and protein interaction of the pullulan-conjugated PEI was drastically reduced in comparison to PEI-based nanocomplexes. Based on the blood compatibility studies, PPE1 was selected for further study. The buffering capacity of this derivative was similar to that of PEI, which plays an important role in efficient gene transfection. The particle size, zeta potential, stability in the presence of plasma and resistance to nuclease degradation were evaluated. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of HepG2 cells. Endocytosis inhibitors, confocal laser scanning microscopy and fluorescent labeling techniques were used to visualize the nanoplex uptake mechanism, cellular distribution and nuclear localization. The results from inhibitor experiments in the presence of asialofetuin indicated that the asialoglycoprotein receptor is involved in transfection of hepatocytes with pullulan-PEI complexes. The conjugation of pullulan with PEI did not hinder the plasmid nuclear localization ability of PEI. The transfection efficiency of pullulan conjugate was similar to PEI, with the added advantage of hemocompatibility and non-cytotoxicity. The transfection efficiency of PEI and PPE1 was 1.6- and 2-fold more, respectively, in the presence of serum than in the absence of serum. Therefore, the pullulan-PEI conjugate seems to be a promising gene delivery vector with good hemocompatibility and low toxicity but without compromising the transfection efficacy of PEI.
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