Transfection efficiency and internalization of the gene carrier prepared from a triple-helical β-glucan and polydeoxyadenylic acid in macrophage RAW264.7 cells.

Abstract

A novel gene carrier was prepared from a triple-helical β-glucan LNT (t-LNT) on the basis of the special interaction between the single chain of t-LNT (s-LNT) and polydeoxyadenylic acid (poly(dA)). The effects of the chain length of poly(dA) in the target DNA sequence on the stability and gene transfection efficiency of the gene carrier of the poly(dA)-s-LNT complex were evaluated by agarose gel retardation assay, circular dichroism, flow cytometry, and ELISA kits. All the results demonstrated that the chain length of poly(dA) that produced the strongest interaction with s-LNT was favorable for the stability and gene transfection efficiency. Moreover, the gene transfection efficiency was strikingly dependent on the transfection time and temperature, suggesting the internalization of the gene delivery system via endocytosis. The high expressions of receptors including TLR2 and CR3 (CD11b/CD18) on the surfaces of RAW264.7 cells were confirmed by confocal microscopy and flow cytometry. By blocking TLR2, CD11b, and CD18 with the corresponding specific antibodies, the transfection efficiency was hardly changed at 4 °C or 37 °C. This suggested that the internalization pattern of such a delivery system was not via the usual β-glucan receptor-mediated endocytosis (such as dectin-1, TLR2, and CR3), which was different from the reported uptake mechanism. Overall, this work facilitates the understanding of the pathways of delivery systems involving β-glucans, which has significant implications for potential applications in disease treatment.