Preparation of CaP/pDNA nanoparticles by reverse micro-emulsion method: Optimization of formulation variables using experimental design

Abstract

In this study, the CaP/pDNA nanoparticles were prepared using Triton X-100/Butanol/Cyclohexane/Water reverse microemulsion system. Optimization of preparation conditions was based on evaluation of particle size by Box-Behnken design method. The particle sizes of the optimized CaP/pDNA nanoparticles were found to be 60.23 ± 4.72 nm, polydispersity index was 0.252 and pDNA encapsulate efficiency was more than 90%. The optimized CaP/pDNA nanoparticles have pH sensitivity and biocompatibility. Further, optimized CaP/pDNA nanoparticles showed higher transfection efficiency.

Keywords: Box–Behnken design; CaP nanoparticles; Reverse microemulsion method; Transfection and expression; pDNA.

Graphical abstract

Fig. 1

Schematic diagram of nanoparticle preparation by microemulsion.

Fig. 2

3-D surface response diagrams. (A) Response surface plot for the effect of the ratio of the Ca²⁺ to the PO₄³⁻ and the pH on the particle size. (B) Response surface plot for the effect of the pH and the stirring speed on the particle size. (C) Response surface plot for the effect of the ratio of the Ca²⁺ to the PO₄³⁻ and the stirring speed on the particle size.

Fig. 3

Characterization of nanoparticles. (A) The particle size measured by DLS (n = 3). (B) Transmission electron micrographs of nanoparticles.

Fig. 4

The pH-sensitive release of pDNA from nanoparticles at different time points in pH 7.4 and pH 5.5 environment (n = 3).

Fig. 5

In vitro cytotoxicity studies of pDNA, CaP and CaP/pDNA nanoparticles (pDNA at 500 ng/well) with different times (n = 3).

Fig. 6

(A) In vitro transfection and expression of CaP/pDNA nanoparticles at different times (scale bar in all pictures indicates 20 µm). (B) In vitro transfection and expression of pDNA, CaP/pDNA, Lipofectamine after 24 h. (C) Imaging flow cytometry results of pDNA, CaP/pDNA, Lipofectamine transfect and express in HepG-2 cells after 24 h (n = 3).