Biomedical Engineering Reference
In-Depth Information
escape of PEI-DNA complexes into the cytosol. The proton sponge hypothesis was
confirmed by finding that bafilomycin A1, a potent inhibitor of endosomal acidifica-
tion, can selectively inhibit transgene expression with PEI polyplexes and not with
cationic lipids
[47]
.
The condensation process and the diameter of polyplexes formed depend on the
N/P ratio (PEI nitrogen
/
DNA phosphate), PEI molecular weights, and the salt con-
centration. Generally, the N/P charge ratio of 6 or higher generates polyplexes of size
50 nm that possess considerable buffering capacity at a lower endosomal pH
[43]
.
Complex formed at lower N/P ratios and higher salt concentration results in aggrega-
tion. As molecular weight PEI increased from 600 to 70,000 Da, transfection effi-
ciency also increased due to easy entry into cells and better protection of the DNA
[48]
. However, high-molecular-weight polymers also exhibit higher cytotoxicity
[49]
.
The optimal molecular weight for PEI polyplex formation is between 5 and 25 kDa
[50]
. PEI revealed superb transfection efficiency
in vitro
; however,
in vivo
transfec-
tion efficiency was modest. Also the toxicity is a major constraint for its clinical use.
Linear PEI is reported to be less toxic than branched PEI. Besides,
in vitro
studies
have shown superior and rapid gene expression with linear PEI-DNA compared to
its branched counterpart. These studies support the fact that linear PEI-DNA com-
plexes are less condensed and so are able to dissociate more efficiently than branched
PEI-DNA complexes, when inside a cell. Studies have demonstrated easier accessi-
bility of the nucleus by linear PEI-DNA complexes than by branched ones
[51]
.
Toxicity is reduced by the presence of serum and by removal of the lower molecu-
lar weight molecules present in the polymer mixture. Purification of PEI conjugates
is mostly carried out by chromatography, which removes lower molecular weight
molecules from higher molecular weight PEI complexes. However, it is demon-
strated that although purified PEI reduced the toxicity both
in vitro
and
in vivo
, it sig-
nificantly reduced transfection activity as free PEI apparently facilitates endosomal
release
[52]
.
PEI exhibits nonspecific interactions via positive-charged complexes interacting
with negatively charged components of the blood, thereby removing particles out of
circulation. The common mechanisms of particle removal are nonspecific binding
to erythrocytes, RES uptake of the particles, and complex segregation by albumin
[53]
. PEG not only reduces the toxicity of PEI but also allows gene transport to sites,
although
in vitro
uptake and transfer is diminished by PEG.
4.2.1.2.1 PEI Conjugates
4.2.1.2.1.1 PEI-PEG
The major concerns in delivery of PEI are cytotoxicity and
aggregation. In an attempt to reduce cytotoxicity and aggregation, PEI-grafted PEGs
(PEI-
g
-PEG) with different PEG grafting ratios were synthesized
[54]
. PEI-
g
-PEG
was more effective than PEI in reducing cell cytotoxicity; however, transfection effi-
ciency remained unchanged. Molecular weight of PEG does not have any significant
effect on cytotoxicity. But the degree of substitution affected cytotoxicity to a greater
extent. The shielding effect provided by PEG has been shown to reduce aggregation
and nonspecific interactions with serum components. Low-molecular-weight PEG
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