Publication: The cellular uptake and endosomal escape mechanisms of chitosan-protamine-siRNA nanoplexes for efficient gene transfection and silencing
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Date
2021-09-11
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Abstract
Aim: The use of antisense-based molecules in gene expression inhibition has allowed the
design of a new pathway for therapeutics. In order to ensure that oligonucleotides with gene
silencing potential can be used effectively in therapy, a suitable carrier system is required that
can be transported to the target site. In this study, gene silencing activities of siRNA targeted
to the LacZ gene were compared and the gene delivery capabilities, transfection efficiency,
cellular uptake and endosomal escape mechanisms of nanoplexes prepared with siRNA and
chitosan/protamine polymers were investigated.
Material and methods: Nanoplex formulations were prepared by simple complexation
method of chitosan/protamine polymers and oligonucleotides. The particle size and zeta
potential of the prepared nanoplexes were measured, and their serum and enzyme stability
were investigated. In order to determine the transfection and gene silencing activities of the
selected formulations, HEK293 cells stably expressing beta-gal were prepared, inhibition of
beta-gal protein was measured by enzymatic assay and suppression by X-gal method was
evaluated microscopically. Cellular uptake and endosomal escape mechanisms of nanoplexes
were studied.
Results: Chitosan/Protamine/siLacZ nanoplexes have been observed to protect siRNA against
enzymatic and serum degradation for up to 48 hours. It was observed that the transfection
efficiency was the highest in the formulations prepared together with chitosan/protamine at a
rate of 10/10/1. It was observed that the transfection increased significantly with the increase
in the ratio of chitosan and protamine. Transfection efficiency was found to be 88.60% at a
rate of 10/10/1. In the cellular uptake study, it was observed that the inhibitor that reduced
cellular uptake the most was phenylarsine oxide, and the uptake of siRNA carried by
nanoplexes was 56%. There was no decrease in cellular uptake when chlorpromazine
hydrochloride inhibitor was administered. This indicated that the nanoplexes were not uptake
by clathrin-mediated endocytosis. It was observed that cellular uptake was 75% with
colchicine, this inhibitor decreases cellular uptake by inhibiting microtubules in cells.
Conclusion: It has been shown that cellular uptake and transfection studies with
chitosan/protamine nanoplexes can be used as an effective carrier system for siRNA transport.
Description
Keywords
siRNA, LacZ, cellular uptake, chitosan, protamine, nanoplexes
Citation
ŞALVA E., EKENTOK C., CÖMEZ B., ÖZBAŞ S., AKBUĞA F. J., \"The cellular uptake and endosomal escape mechanisms of chitosan-protamine-siRNA nanoplexes for efficient gene transfection and silencing\", BioTürkiye International Biotechnology Congress, İstanbul, Türkiye, 9 - 11 Eylül 2021