Biomedical Engineering Reference
In-Depth Information
ratio. However, the diameters of the M1-complexes (48 ¡ 6 nm) and those of
the M2-complexes (70 ¡ 10 nm) essentially did not change with N/P ratio. The
higher particle density of the M-complexes led to easier internalization via
endocytosis, higher siRNA delivery efficiency, and higher gene-silencing
efficiency. During siRNA transfection experiments in Huh-7 Luc cells, the two
M-complexes knocked the gene down by nearly 50% at both 48 h and 72 h
when the N/P ratio was 60 or 75. This efficiency was comparable to that of
Lipofectamine 2000 and much better than PEI-25 kDa. However, the P-
complex exhibited little gene knockdown at any N/P ratio.
71
Because polyelectrolyte complex nanoparticles suffer from poor stability
under physiological conditions, an effective stabilization method is essential
for the success of polycationic nanoparticle-mediated siRNA delivery.
Nakanishi et al. used sodium triphosphate (TPP) as an ionic crosslinking
agent to stabilize siRNA-containing nanoparticles by co-condensation. siRNA
and TPP were co-encapsulated into a block copolymer, poly(ethylene glycol)-
b-polyphosphoramidate (PEG-b-PPA), to form ternary nanoparticles. The
PEG-b-PPA/siRNA/TPP ternary nanoparticles exhibited high uniformity with
smaller size (80-100 nm) compared with PEG-b-PPA/siRNA nanoparticles
and showed increased stability in physiological ionic strength and serum-
containing medium, due to the stabilization effect from ionic crosslinks
between the negatively charged TPP and cationic PPA segments. The
transfection and gene-silencing efficiency of the TPP-crosslinked nanoparticles
were markedly improved over PEG-b-PPA/siRNA complexes in serum-
containing medium. No significant difference in cell viability was observed
between the nanoparticles prepared with and without TPP co-condensation.
72
High charge densities could cause the cytotoxicity of cationic polymers,
peptides, and liposomes,
73,74
and low molecular weight carriers with fewer
positive charges may be safer than high molecular weight ones with more
positive charges. Thus, low molecular weight polycations have been developed
for siRNA delivery. Ryu et al. evaluated amphiphilic peptides with arginine
and valine residues as siRNA carriers. The peptides were composed of one to
four arginine (R) blocks and six valine (V) blocks with a low charge density. In
aqueous solution, the RV peptides formed micelles with hydrophobic cores
composed of a valine block and a cationic surface composed of an arginine
block. In their previous work, the RV peptides had much lower plasmid DNA
delivery efficiency than PEI, suggesting that stable complexes could not form
because the size of the plasmid DNA was much larger than that of the RV
peptides.
75
However, the RV peptides have similar molecular weights to the
siRNAs, and they may locally increase charge density in aqueous solution
through the formation of micelles. The RV peptides formed more stable
complexes with siRNA than they did with PEI25k; the results also showed that
the R3V6 peptide was more efficient than the R1V6, R2V6, and R4V6 peptides
in silencing reporter genes. R3V6 had the highest siRNA delivery efficiency at
a 1:20 weight ratio and the siVEGF/R3V6 complex suppressed the VEGF
expression by 35% compared with the untreated control. In addition, the
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