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  • Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

Pharmaceutical research (2014-12-24)
Hao Zheng, Cui Tang, Chunhua Yin
ABSTRACT

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
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Fluorescein isothiocyanate isomer I, ≥97.5% (HPLC)
Sigma-Aldrich
Boc-His-OH, 99%
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, ≥97.5% (HPLC)
Sigma-Aldrich
1,2-Dichloroethane, SAJ first grade, ≥99.0%
Sigma-Aldrich
Deuterium oxide, 70 atom % D
Sigma-Aldrich
Deuterium oxide, 60 atom % D
Sigma-Aldrich
Deuterium oxide, filtered, 99.8 atom % D
Sigma-Aldrich
Deuterium oxide, 99.9 atom % D, contains 1 % (w/w) 3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt (DSS)
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N-Hydroxysuccinimide, 98%
Sigma-Aldrich
Deuterium oxide, 99.9 atom % D, contains 0.75 wt. % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt
Sigma-Aldrich
DL-Cysteine, technical grade
Sigma-Aldrich
Deuterium oxide, 99.9 atom % D, contains 0.05 wt. % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt
Sigma-Aldrich
L-Arginine monohydrochloride, BioUltra, ≥99.5% (AT)
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, BioReagent, suitable for fluorescence, mixture of 2 components, ≥90% (HPLC)
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1,2-Dichloroethane, anhydrous, 99.8%
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L-Glutathione reduced, ≥98.0%
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L-Glutathione reduced, suitable for cell culture, BioReagent, ≥98.0%, powder
Sigma-Aldrich
L-Arginine monohydrochloride, not synthetic, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Arginine monohydrochloride, reagent grade, ≥98% (HPLC), powder
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, ≥90% (HPLC)
Sigma-Aldrich
L-Glutathione reduced, BioXtra, ≥98.0%
Sigma-Aldrich
1,2-Dichloroethane, JIS special grade, ≥99.5%
SAFC
L-Arginine monohydrochloride
Sigma-Aldrich
Deuterium oxide, 99.9 atom % D