Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 627

Modification of bPEI (1,800 Da) with cholesteryl chloroformate produced a
water-soluble lipopolymer (bPEI-Chol) as the percentage of conjugated cholesterol
was about 47% (Han et al. 2001 ). In contrast to PEI25/pDNA complexes, bPEI-
Chol/pDNA polyplexes and bPEI-Chol itself were less toxic to CT-26 colon adeno-
carcinoma and 293 T human embryonic kidney transformed cells. Compared to
25 kDa or 1,800- Da based formulations, bPEI-Chol showed higher transfection
efficiency in both CT-26 and 293 T cells. High levels of GFP expression in Jurkat
cells was also observed for this type of PEI derivatives (Wang et al. 2002a ). Kim
et al. observed the enhanced transfection when cholesterol or myristate was conju-
gated onto 2 kDa PEI (Kim et al. 2001a ). Increased cytotoxicity, however, was
found in this case. N-Dodecylation of primary amino groups of 2,000 Da PEI yields
a low toxic polycation whose transfection efficiency in the presence of serum is 400
times higher than its precursor polymer (Thomas and Klibanov 2002 ). This
derivative was even more effective than PEI25. The same group also found that
N-acylation of PEI25 with alanine nearly doubled the transfection efficiency in the
presence of serum and also lowered its cytotoxicity. Acylation of PEI25 with palm-
itic acid (PA) created amphiphilic PEI derivatives (bPEI-PA) that could form nano-
particles or vesicles (Brownlie et al. 2004 ). The PEGylated bPEI-PA was 10-fold
less toxic while retaining 30% of the transfection efficiency in vitro . After intrave-
nous administration in a mouse model, polyplexes based on the PEGylated bPEI-
PA could mediate the transgene expression of GFP in the liver. However, a recent
report by Incani et al. showed negative results when they using PA modified PEI to
transfer GFP-encoding pDNA to bone marrow stromal cells (Incani et al. 2007 ).
Difference in substitution and cell line may be responsible for this discrepancy. On
the other hand, modification of bPEI using oleic and stearic acid gave promising
results for siRNA delivery (Alshamsan et al. 2009 ). The modified bPEIs showed
3-fold increased siRNA transfection in B16 cells than the parent PEI, which were
superior or comparable to some of the commercially available carriers like jetPEI,
Metafectene, and INTERFERin.
Small hydrophobic alkyl moieties, such as acetyl, butyryl and hexanoyl, are
also able to increase the transfection capability of modified polycations. Study by
Pack's group showed the increased gene delivery efficiency of acetylated PEI25 in
MDA-MB-231 and C2C12 cell lines, upon acetylation of up to 43% of the primary
amines with acetic anhydride (Forrest et al. 2004 ). They also found that transfec-
tion efficiency continued to increase (up to 58-fold in HEK293) with up to 57%
acetylation of primary amines, albeit decreased as the acetylation degree further
increased (Gabrielson and Pack 2006 ). Putnam and coworkers hydrophobically
decorated PEI25 with acetyl, butyryl and hexanoyl by 1-ethyl-3-(3-dimethylamin-
opropyl)carbodiimide (EDAC)/N-hydroxysuccinimide (NHS) mediated conden-
sation reactions between protonated PEI25 and acetate, butanoate, and hexanoate,
respectively (Doody et al. 2006 ). In vitro transfections using HeLa, NIH/3T3 and
Clone 9 cell lines showed that substitution of the primary amines generally
increased transfection efficiency relative to unmodified PEI25. Increasing both the
substitution degree beyond 25 mol% and hydrocarbon length, however, decreased
the delivery efficacy.