Hemocompatible pullulan–polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency

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 prote...

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Bibliographic Details
Published in:Acta biomaterialia 2011, Vol.7 (1), p.370-379
Main Authors: Rekha, M.R., Sharma, Chandra P.
Format: Article
Language:English
Subjects:
Biocompatible Materials - pharmacology
blood serum
buffering capacity
Buffers
Cell Death - drug effects
complement
Complement Activation - drug effects
Complement Activation - immunology
confocal laser scanning microscopy
cytotoxicity
DNA - metabolism
endocytosis
erythrocytes
fluorescence microscopy
fluorescent labeling
Gene delivery
gene expression
genes
Glucans - chemical synthesis
Glucans - chemistry
Glucans - pharmacology
Hemocompatibility
Hep G2 Cells
hepatocytes
Hepatocytes - drug effects
Hepatocytes - metabolism
human cell lines
Humans
in vitro studies
Intracellular Space - drug effects
Intracellular Space - metabolism
Intracellular trafficking
leukocytes
Liver - cytology
Liver - metabolism
Magnetic Resonance Spectroscopy
Nanoparticles - ultrastructure
Particle Size
plasmids
Plasmids - metabolism
Polyethyleneimine
Polyethyleneimine - chemical synthesis
Polyethyleneimine - chemistry
Polyethyleneimine - pharmacology
Pullulan
Static Electricity
transfection
Transfection - methods
zeta potential
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Summary: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.
ISSN:1742-7061
1878-7568
DOI:10.1016/j.actbio.2010.07.027