A novel gene carrier prepared from triple helical β-glucan and polydeoxyadenylic acid

Abstract

A naturally occurring β-glucan from Lentinus edodes (t-LNT) has been proved to adopt a unique triple helix structure due to strong hydrogen bonding. The single chain of t-LNT (s-LNT) can interact with polydeoxyadenylic acid (poly(dA)) through hydrogen bonding between s-LNT and poly(dA). Based on this, a novel strategy for an efficient gene transfection was developed as follows. The target DNA was attached to poly(dA)₅₀ through a disulfide bond to form DNA–SS–poly(dA)₅₀, and s-LNT was then combined with the poly(dA)₅₀ segment to form a DNA–SS–poly(dA)₅₀/s-LNT complex through hydrogen bonding, which could be taken up by cells followed by the release of target DNA in the cytoplasm via the breaking of the disulfide bond under the treatment of reducing agents such as GSH. The experimental results from the agarose gel retardation assay and circular dichroism demonstrated the formation of the complex DNA–SS–poly(dA)₅₀/s-LNT. Confocal microscopy and flow cytometry indicated that the target DNA was delivered into the cells with high transfection efficiency, which was strongly dependent on molecular weight and the concentration of s-LNT. The uptake mechanism of the complex was ascribed to endocytosis. The significant enhancement of IL-12p40 production in macrophage RAW 264.7 cells stimulated by the transfected CpG DNA with immunomodulating activity further confirmed the feasibility of this system. Additionally, gene transfection with high efficiency could be performed in tumor cells (HeLa). The cytotoxicity of s-LNT against RAW 264.7 and HeLa cells by the MTT assay showed good safety. Overall, s-LNT could be used as an excellent gene carrier, indicating its promising application in gene delivery.