Splenic Delivery System of pDNA through Complexes Electrostatically Constructed with Protamine and Chondroitin Sulfate

Abstract

We developed and optimized a novel gene delivery vector constructed electrostatically with an anionic biological component and a cationic biological component. Cationic binary complexes of plasmid DNA (pDNA) with novo-protamine sulfate as a medical product (PRT complexes) demonstrated high gene expression with minimal cytotoxicity, likely related with its total cationic charge. Subsequently, anionic compounds were added to the PRT complexes to form ternary complexes with neutral or anionic charges. Among the anionic compounds examined, chondroitin sulfate sodium (CS) as a medical product encapsulated the PRT complexes to produce stable ternary complexes (CS complexes) at charge ratios of ≥4 with pDNA. CS complexes exhibited high gene expression without cytotoxicity in mouse melanoma cell line, B16-F10 cells, in vitro. An inhibition study with endocytosis inhibitors suggested that PRT complexes were mainly taken up by caveolae-mediated endocytosis, and CS complexes were mainly taken up by clathrin-mediated endocytosis in B16-F10 cells. We found that CS complexes including pDNA encoding Oplophorus gracilirostris luciferase induced selective gene expression in the spleen after intravenous administration into ddY male mice. Thus, we successfully constructed useful gene vectors with biological components as medical products.

Delivery of pDNA to the Lung by Lipopolyplexes Using N-Lauroylsarcosine and Effect on the Pulmonary Fibrosis

“…PEI destabilizes endosomal membranes with its pH buffering capacity, thus improving the cytoplasmic transfer of pDNA [18,19]. DGL also has a high pH buffering capacity because the spatial density of cations is increased by the sterically crowded structure [20]. Contrarily, PLL and PLA demonstrate low endosome escape because of their low pH buffering capacities [21].…”

Biopolymer‐based Carriers for DNA Vaccine Design

“…This phenomenon can be observed in nature,w here electrostatic interaction has been adopted for condensation of DNA, for example around the histone or protamine proteins,w hich are rich in basic amino acid residues. [120,121] Numerous biopolymers,a mong them chitosan, [101,108,109,[122][123][124][125][126] ePLL, [84,127] poly-l-lysine (PLL), [67,105,[128][129][130][131][132][133] protamine, [76,79,80,[134][135][136][137] polyarginine, [67,90,91,138] and polyspermine [89,[139][140][141] (Table 2), bear intrinsic positive charge,making them excellent candidates as DNAc ondensation agents.…”

Biopolymer‐based Carriers for DNA Vaccine Design

“…This phenomenon can be observed in nature,w here electrostatic interaction has been adopted for condensation of DNA, for example around the histone or protamine proteins,w hich are rich in basic amino acid residues. [120,121] Numerous biopolymers,a mong them chitosan, [101,108,109,[122][123][124][125][126] ePLL, [84,127] poly-l-lysine (PLL), [67,105,[128][129][130][131][132][133] protamine, [76,79,80,[134][135][136][137] polyarginine, [67,90,91,138] and polyspermine [89,[139][140][141] (Table 2), bear intrinsic positive charge,making them excellent candidates as DNAc ondensation agents.…”

“…[248] Guan et al introduced NLS derived from simian virus 40 (SV40) large Tantigen to ac ationic HSA-DNAs ystem, significantly increasing its gene expression efficacy in vitro and in vivo. [190] Similarly,p rotamine,a na rginine-rich protein commonly used for DNAcondensation and membrane translocation, has been shown to aid the nucleus uptake when attached to biodegradable anionic polymers such as chondroitin sulfate [135] or hydrophobic moieties such as cholesterol. [83] This increase in nuclear transport is attributed to the presence of NLS-like regions consisting of four to six arginine repeats.…”