Biomedical Engineering Reference
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less branched polymers(Fischer et al.
2002
). However, increasing the branching
degree is found to increase the
in vitro
cytotoxicity as well as the
in vitro
haemolysis
effects on erythrocytes. In addition to the molecular architecture, the molecular
weight of PEI severely influences the polyplex characteristics. Generally, increasing
the molecular weight (MW) of PEI leads to polyplexes with smaller size, while a
decrease in molecular weight may dramatically impair its capability for condensa-
tion. Compared with the high molecular weight PEI (HMW-PEI), a thorough DNA
condensation of low molecular weight PEI (LMW-PEI) occurred at higher N/P
ratios (the molar ratio of PEI nitrogen-to-DNA phosphate) (Kunath et al.
2003b
).
Whereas a considerable results have shown the higher transfection efficiency of PEI
as its MW increased, HMW-PEI encounter a pronounced toxicity both in cell cul-
ture and
in vivo
and a variety of undesired unspecific interactions with the biological
components. Great efforts have been therefore directed towards creating PEI deriva-
tives integrated with higher transfection efficacy and excellent biocompatibility.
3.2.2
Cross-Linking of Low Molecular Weight PEI
Low molecular weight PEI (LMW-PEI) is generally regarded as poor gene vectors,
despite its low toxicity. Cross-linking of LMW-PEI has been considered to be a
simple but effective approach to discover pseudo-HMW-PEIs with low toxicity and
higher transgene efficacy. Theoretically, any molecule with multiple functional
groups (³ 2) that are able to react with amine groups can function as a cross-linker
to synthesize high molecular weight polymers. A larger number of cross-linkers,
including dithiobis(succinimidylpropionate) (DSP) and dimethyl-3,3'-
dithiobispropionimidate·2HCl (DTBP), methylthiirane, diacrylate, glutadialde-
hyde, cyclodextrins (CDs) and their derivatives among them, have been utilized to
produce HMW-PEIs (Table
3
).
Gosselin et al. cross-linked 800 Da PEI using DSP and DTBP, and the disul-
fide bonds in the resulting high molecular weight products could be reduced in
intracellular reducing microenvironments to release their low molecular weight
counterparts that can be cleared more easily from the body (Gosselin et al.
2001
).
In vitro
evaluation using the luciferase reporter gene showed significantly higher
transfection efficiency to Chinese hamster ovary (CHO) cells, compared with that
of 800 Da PEI. The efficacy was dependent on the cross-linking reagent, the
extent of conjugation, and the N/P ratio. At higher N/P ratios, the transgene effi-
ciency of cross-linked PEIs was even comparable to that of PEI25. This may be
the first report regarding the
in vitro
transfection study on cross-linked LMW-
PEI. DSP has also been used by Kissel's group to cross-link LMW-PEI (Neu
et al.
2006
). It was found that polyplexes prepared by the cross-linked PEI dis-
played significantly reduce interactions with major blood components like albu-
min and erythrocytes. No transfection results, however, were reported by these
researchers. Through the ring-opening reaction of LMW-PEI (800 Da) with meth-
ylthiirane, disulfide cross-linked PEI (7,100~8,400 Da based on viscosity mea-
surements) can be synthesized (Peng et al.
2008
). In comparison to HMW-PEI
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