Novel gelatin–siloxane nanoparticles decorated by Tat peptide as vectors for gene therapy

Abstract

In principle, the technique of gene delivery involves taking complete or parts of genes that can code specific messages and delivering them to selected cells in the body. Such a transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. A series of gelatin-siloxane nanoparticles (GS NPs) with controlled size and surface charge were synthesized through a two-step sol-gel process. In order to increase the efficiency of cellular uptake, HIV-derived Tat peptide was further grafted to GS NPs. In vitro co-location and endocytosis inhibition experiments suggested that the as-synthesized TG NPs may enter HeLa cells via a combined pathway of lipid-raft- and receptor-dependent endocytosis, and only cause little cell damage. Moreover, this study shows the encapsulation of a plasmid DNA in TG NPs to be obtained as a non-viral gene vector. This kind of encapsulation provides complete protection to the plasmid DNA from the external DNase and serum environment, and generates the hope that the resulting formulation can be developed into a potential vector for effective gene delivery. In order to check this potential, the reporter gene pSVβ-gal was encapsulated, and in vitro transfection efficiency of this system was found to be nearly 130% compared to the commercially available transfection reagent Lipofectamine™.

Uptake pathways and subsequent nucleus targeting of tat-conjugated gelatin-siloxane nanoparticles as nonviral gene vector

“…In a previous paper, we synthesised Tat peptide modified gelatin-siloxane hybrid nanoparticles (Tat-GS NPs) through a two-step sol-gel process, and confirmed the assynthesised Tat-GS NPs, with controlled size and surface charge, were biocompatible [23]. In the current study, we report an optimisation of Tat-GS NPs in culture process, and discuss their uptake pathways and subsequent nucleus targeting for gene expression.…”

“…Hence, the cellular uptake of Tat-GS NPs has the characteristics of both concentration-and incubation time-dependence. In addition, our previous study indicated that cellular uptake of Tat-GS NPs was severely impaired by incubation of cells at 4 C [23]. It is thus suggested that Tat-GS NPs could be uptaken by cells via an energy-dependent internalisation mechanism.…”

“…Tat-GS NPs were synthesised according to our previously reported procedure [23]. Briefly, appropriate amount of APTMS was added to an acidic aqueous solution (pH 3.0), containing 1% gelatin and 1% GPSM at 25 C. Once pH became 9.0, the above mixture was continuous stirred for 24 h. The hybrid gelatin-siloxane nanoparticles (GS NPs) were collected by centrifugation (19,000 rpm, 25 C, 15 min), and purified with deionised water thrice.…”

“…In our previous studies [23], spherical GS NPs were yield through the hydrolysis and condensation of the metal alkoxides of APTMS and GPSM, following the ring-opening reaction of the epoxy groups between GPSM and gelatin. It has been demonstrated that the current GS NPs are in the range of 155-170 nm with a mean diameter of 165 nm and the surface charge is þ24.5 mV [23].…”

“…It has been demonstrated that the current GS NPs are in the range of 155-170 nm with a mean diameter of 165 nm and the surface charge is þ24.5 mV [23]. Conjugation of Tat peptide did not alter the particle shape and size as shown in Figure 1a, but increase surface charge to þ36.4 mV [23]. Moreover, Tat-GS NPs could condense pEGFP-N1 plasmid into compact nanosized particles via the electrostatic interaction (Figure 1b).…”