Reconstitutable charged polymeric (PLGA)(2)-b-PEI micelles for gene therapeutics delivery

Abstract

This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA(36kDa))(2)-b-bPEI(25kDa) (PLGA MW 36 kDa, bPEI M(w) 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100-150 nm) and surface charge (30-40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50-100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for "reconstituted" micelle/pDNA/bPEI(1.8kDa) (WR 1) complexes was 16-fold higher than its "fresh" counterpart. Although transfection levels achieved using "reconstituted" micelle/pDNA/bPEI(1.8kDa) complexes were 3.6-fold lower than control "fresh" bPEI(25kDa)/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than "reconstituted" bPEI(25kDa)/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI(1.8kDa) system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.

Fig. 1

(a) Reaction schematic for (PLGA_36kDa)₂-b-bPEI_25kDa copolymer and (b) schematic representation of the structures of (PLGA_36kDa)₂-b-bPEI_25kDa micelles, micelle/pDNA complexes, and micelle/pDNA/bPEI_LMW complexes.

Fig. 2

Particle size and surface charge of (PLGA_36kDa)₂-b-bPEI_25kDa micelles before and after reconstitution. (n=5; mean ± SD)

Fig. 3

Proton buffering capacity of (PLGA_36kDa)₂-b-bPEI_25kDa micelles and bPEI_25kDa. (n=3; mean ± SD)

Fig. 4

Cytotoxicity of (PLGA_36kDa)₂-b-bPEI_25kDa micelles, bPEI_25kDa, and bPEI_1.8kDa in MCF7 cells. (n=6; mean ± SD)

Fig. 5

Surface shielding effect of bPEI_1.8kDa on gene condensation of micelle/pDNA complexes: (a) agarose gel retardation and (b) dye quenching assay (n=3; mean ± SD).

Fig. 6

Particle size and surface charge of micelle/pDNA complexes and micelle/pDNA/bPEI_1.8kDa complexes before and after reconstitution. (n=5; mean ± SD)

Fig. 7

Transfection efficiency of fresh and reconstituted micelle/pDNA/bPEI_1.8kDa complexes (1 μg of pDNA) in MCF7 cells (5×10⁵ cells at seeding). (n=8; mean ± SD)

Fig. 8

Cytotoxicity of fresh and reconstituted micelle/pDNA/bPEI_1.8kDa complexes (0.5 μg of pDNA) in MCF7 cells (2.5×10⁵ cells at seeding). (n=4; mean ± SD)

Fig. 9

Cellular uptake of “fresh” and “reconstituted” micelle/pDNA/bPEI_1.8kDa (WR 1) and “fresh” bPEI_25kDa/pDNA (N/P 5) complexes in MCF7 cells.

Fig. 10

pDNA-dose dependent transfection characteristics of “reconstituted” micelle/pDNA/bPEI_1.8kDa (WR 1) complexes: (a) transfection efficiency in MCF7 cells (5×10⁵ cells at seeding) and (b) cytotoxicity in MCF7 cells (2.5×10⁵ cells at seeding). (n=4; mean ± SD)