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protein as compared
to the PEI-siRNA, at a weight ratio of 3:1 the ND-PEI800:siRNA complex show an
almost twofold higher knockdown in cellular green fluorescent protein
expression. 

        In presence of serum in the treatment
medium, the ND-PEI800:siRNA complex showed better knockdown in green
fluorescent protein expression as well as lower cytotoxicity than LipofectamineTM,
which is a gold standard for in vitro delivery of nucleic
acids. Therefore, NDs have the capacity to improve the transfection ability
of polymers while remaining biocompatible with the cell lines. Gene
delivery is the introduction of genetic material or gene therapeutics into
cells, aiming to exchange the ‘impaired’
gene to regain biological function or add a new gene to trigger additional
functions207. Long ago
viruses have been discovered as primitive
and smart enough to interchange their genetic material into a genome of cells.
Since the delivery of genetic material via viruses (viral
vectors) has been broadly followed in clinics to irreversibly change
cell functions—a permanent transfection.
Although viral vectors have high gene transfection effectiveness, they give
rise to serious safety concerns. That’s why non-viral delivery is also actively
pursued. Non-viral strategies are good to deliver genetic material exclusively
to cytoplasm-transient transfection.
The transiently transfected genetic material occupy
incytoplasm, does not replicate, and is gradually lost when the cells divide.
Transposing DNA in chromosome by non-viral vectors is much less well organized
compared to viral vectors. Genetic material can be transport to the nucleus by
means of passive diffusion of non-viral vectors (nanoparticles)
through the nuclear pore complex (NPC).
The passive diffusion through NPC strongly depends on the net size of the
vector and genetic cargo (preferentially less than 5 nm in
diameter)208. Many different
nanocarriers are studied as gene delivery agents, comprises gold and magnetic
iron oxide nanoparticles (magnetofection). ND-based
gene delivery platforms are attractive
because NDs are biocompatible,and have a rich surface chemistry,
amenable to different modifications to help cell entry and ferry
a gene. ND particle size (2 to 5 nm in diameter)
meets the criteria for a passive diffusion into the nucleus. Unprecedented
examples of ND particle nuclear entry have been express a few years ago with
Fenton treated ND. The Fenton oxidation
leads to ND free of amorphous carbon and of much smaller size (on
average 4.4 nm after the oxidation of 7 nm NDs contained in the soot),
small enough that it can passively penetrate into HeLa cells nuclei. The
reported capability of ND to easily escape from the endosomes is also essential
for delivery of genetic material into nuclei209. Fast
escape from endosome helps to protect genetic material from digesting enzymes. Perhaps,
the most studied application of ND for gene delivery is based on the
non-covalent integration of poly-cationic molecules onto the ND surface
followed by interconnection with
negatively charged nucleic acids. For example, pEGFPLuc plasmids encoding
Luciferase and green fluorescent protein (GFP)
have been successfully delivered in cytoplasm by means of aa ND-PEI vector.
Positively charged PEI-ND have significantly improved transfection as
compared to PEI or ND alone, probably, due to a faster endosomal liberation.
Since high molecular weight cationic vectors show high cellular toxicity, the
authors have optimized the ratio PEI:ND:DNA to attain high transcription rates, while minimizing toxicity.
It is important to sustain the right balance between the quantity of DNA on the
surface of the vector and the DNA-induced reduction of the positive charge of
the structure, which is needed for the efficient endosomal libration and
transcription of genetic material210.
It was measured that 4.1 nm ND particle binds on average 70 branched 800 Da PEI
molecules. Studies have explained that siRNA and ND-PEI ratios (1
to 75 w/ w siRNA to ND-PEI, respectively)
can be tuned to knock down GFP and EWS-Fli1 genes more efficiently
than the well-known liposomal vector Lipofectamine.

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        The potential of ND-PEI to liberate siRNA in
the cytoplasm much faster than other vectors (including
other ND-polycationic complexes) is due to the fast endosomal
release of the vector verified by TEM . The large number of primary and
tertiary amino groups (at least 216 ?mol
g?1)
on the ND-PEI surface results in osmotic influx
of counter-ions through the endosome membrane to protonated ND-PEI complex
leading to endosome swelling and disruption. In addition to ND-PEI complexes, hydrogenated
detonation NDs with zeta potential +55
mV have been studied to electrostatically bind the negatively charged siRNA.
The approximte number of siRNA molecules is 37 per one 7 nm ND particle. The
air oxidized ND-COOH (zeta potential –50
mV) can not exhibit any non-specific
binding of siRNA as expected. Carboxylated derivatives of larger, 20 nm HPHTNDs
have been exploited in a covalent reaction with amino-modified nucleic acid through EDC/NHS
chemistry211. Another
covalent DNA binding technique has been recently demonstrated via a copper-free
coupling of dibenzocy-clooctin-modified
nucleic acid to azido-functionalized 100 nm HPHTND212,213.

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