Gene-Directed Enzyme Prodrug Therapy by Dendrimer-Like Mesoporous Silica Nanoparticles against Tumor Cells

Abstract

We report herein a gene-directed enzyme prodrug therapy (GDEPT) system using gated mesoporous silica nanoparticles (MSNs) in an attempt to combine the reduction of side effects characteristic of GDEPT with improved pharmacokinetics promoted by gated MSNs. The system consists of the transfection of cancer cells with a plasmid controlled by the cytomegalovirus promoter, which promotes β-galactosidase (β-gal) expression from the bacterial gene lacZ (CMV-lacZ). Moreover, dendrimer-like mesoporous silica nanoparticles (DMSNs) are loaded with the prodrug doxorubicin modified with a galactose unit through a self-immolative group (DOXO-Gal) and modified with a disulfide-containing polyethyleneglycol gatekeeper. Once in tumor cells, the reducing environment induces disulfide bond rupture in the gatekeeper with the subsequent DOXO-Gal delivery, which is enzymatically converted by β-gal into the cytotoxic doxorubicin drug, causing cell death. The combined treatment of the pair enzyme/DMSNs-prodrug are more effective in killing cells than the free prodrug DOXO-Gal alone in cells transfected with β-gal.

Keywords: DMSNs; GDEPT; drug delivery; tumor treatment.

Scheme 1

Scheme of the mechanism of action of the GDEPT system. Cancer cells are transfected with the plasmid CMV-lacZ to promote β-gal expression. Then, β-gal-expressing cells are treated with DOXO-Gal@DMSN-PEG, which specifically release the prodrug in the cytoplasm of cells. DOXO-Gal is activated after the enzymatic activity of β-gal, yielding the cytotoxic drug doxorubicin and causing cell death.

Scheme 2

Synthesis steps to obtaining the prodrug DOXO-Gal.

Scheme 3

Mechanism of DOXO release after the enzymatic action of β-gal.

Scheme 4

Synthetic route followed to prepare the final solid DOXO-Gal@DMSN-PEG.

Figure 1

(A) Representative TEM images of DMSN (a,b) and DOXO-Gal@DMSN-PEG (d,e). Histogram with particle size distribution measured by TEM of DMSN (c) and DOXO-Gal@DMSN-PEG (f). Size distributions were established upon analysis of 100 nanoparticles. (B) Intensity PSD DLS curves of DMSN, DOXO-Gal@DMSN-PEG, and DMSN-PEG. Samples measured were suspended in distilled water. Brief spin pulses were applied in order to remove large aggregates or sediments. (C) N₂ adsorption–desorption isotherms of DMSN and Rubpy@DMSN; Inset: pore-size distribution of DMSN and Rubpy@DMSN.

Figure 2

Release profile of [Ru(bpy)₃]²⁺ from Rubpy@DMSN-PEG in the absence (a) and presence (b) of GSH 10 µM.

Figure 3

Cytotoxicity profile of the LN18 cell line treated with free DOXO, DOXO-Gal, and DOXO-Gal + β-gal at different concentrations. DOXO and DOXO-Gal were dissolved in DSMO and diluted with DMEM to a final DMSO volume percentage of 0.3%. β-gal was used at 1 mg/mL. Cell viability was studied using WST-1 at 72 h. In this study, the statistical significance was determined by one-way ANOVA and Dunnet post-test (*** p < 0.0002, **** p < 0.0001).

Figure 4

(A) Optical microscope images of X-Gal assay in untransfected (a,b) and transfected (c,d) LN18 cells. (B) Cytotoxicity profile when transfected (grey columns) and untransfected (black columns) cells were treated with free DOXO-Gal, DMSN-PEG, or DOXO-Gal@DMSN-PEG at different concentrations. DOXO-Gal was dissolved in DMSO and diluted with DMEM to a final DMSO volume percentage of 0.3%. Cell viability was studied by WST-1 at 72 h. Statistical significance was determined by one-way ANOVA and Dunnet post-test (**** p < 0.0001; ns = no significance).