The use of nanoparticle-mediated targeted gene silencing and drug delivery to overcome tumor drug resistance

Abstract

Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) enables cancer cells to develop resistance to multiple anticancer drugs. Functional inhibitors of P-gp have shown promising efficacy in early clinical trials, but their long-term safety is yet to be established. A novel approach to overcome drug resistance is to use siRNA-mediated RNA interference to silence the expression of the efflux transporter. Because P-gp plays an important role in the physiological regulation of endogenous and xenobiotic compounds in the body, it is important to deliver P-gp targeted siRNA and anticancer drug specifically to tumor cells. Further, for optimal synergy, both the drug and siRNA may need to be temporally colocalized in the tumor cells. In the current study, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel, along with P-gp targeted siRNA, using poly(D,L-lactide-co-glycolide) nanoparticles to overcome tumor drug resistance. Nanoparticles were surface functionalized with biotin for active tumor targeting. Dual agent nanoparticles encapsulating the combination of paclitaxel and P-gp targeted siRNA showed significantly higher cytotoxicity in vitro than nanoparticles loaded with paclitaxel alone. Enhanced therapeutic efficacy of dual agent nanoparticles could be correlated with effective silencing of the MDR1 gene that encodes for P-gp and with increased accumulation of paclitaxel in drug-resistant tumor cells. In vivo studies in a mouse model of drug-resistant tumor demonstrated significantly greater inhibition of tumor growth following treatment with biotin-functionalized nanoparticles encapsulating both paclitaxel and P-gp targeted siRNA at a paclitaxel dose that was ineffective in the absence of gene silencing. These results suggest that that the combination of P-gp gene silencing and cytotoxic drug delivery using targeted nanoparticles can overcome tumor drug resistance.

Figure 1

SEM image of dual agent nanoparticles without surface functionalization (A) and with biotin functionalization (B). Nanoparticles were platinum-coated for visualization. Bar is 670 nm.

Figure 2

In vitro release of (A) paclitaxel and (B) siRNA targeted to P-gp from dual agent nanoparticles. Legend: Pac-siRNA NP: nanoparticles containing paclitaxel and siRNA; BI Pac-siRNA NP: nanoparticles surface functionalized with biotin and containing paclitaxel and siRNA. Data as mean ± S.D.; n = 3.

Figure 3

Enhanced cytotoxicity of dual agent nanoparticles in JC cells. Cells were incubated with treatments for 24 hrs and the cell viability was determined by MTS assay at the end of 1 day (A) or 5 days (B) after the addition of treatments. Legend: siRNA Soln - siRNA in solution complexed with DharmaFECT; Pac-Soln - paclitaxel in solution; Pac NP - nanoparticles containing paclitaxel; Pac-siRNA Soln - Paclitaxel and siRNA in solution along with DharmaFECT; and Pac-siRNA NP - Nanoparticles containing paclitaxel and P-gp targeted siRNA. Data as mean ± S.D.; n = 10.

Figure 4

Quantitative RT-PCR analysis of MDR1 gene expression in JC cells. Cells were incubated with different treatments, either free in solution (Soln) or encapsulated in nanoparticles (NP). The siRNA solution treatments also contained DharmaFECT. P-gp mRNA levels were normalized to β-actin mRNA levels, and the results are presented as fold-change in the normalized values relative to untreated controls. The experiment was performed in duplicate and the values presented here are mean ± S.D. of the two replicates.

Figure 5

Enhanced paclitaxel accumulation in the presence of P-gp targeted siRNA. JC cells were incubated with nanoparticles containing paclitaxel and siRNA (Pac-siRNA NP), paclitaxel and siRNA in solution with DharmaFECT (Pac-siRNA Soln), nanoparticles containing paclitaxel (Pac NP) or paclitaxel in solution (Pac Soln) at a paclitaxel concentration of 100 nM. Paclitaxel concentration was determined by LC-MS/MS and normalized to total cell protein. Data as mean ± S.D.; n = 3. # P<0.05 Vs Pac NP; * P<0.05 Vs Pac Soln.

Figure 6

Incorporation of biotin on dual agent nanoparticles results in enhanced anticancer effectiveness in vivo. BALB/c mice bearing JC tumors of ~100 mm³ were injected with a single dose of the different treatments through the tail vein. Growth in tumor volume was monitored on a regular basis. Legend: Pac Soln - paclitaxel in solution; Pac NP - Nanoparticles containing paclitaxel; BI Pac NP - Nanoparticles containing paclitaxel and functionalized with biotin; Pac-siRNA Soln - paclitaxel and siRNA in solution with DharmaFECT; Pac-siRNA NP - Nanoparticles containing paclitaxel and siRNA; Pac-SsiRNA NP - Nanoparticles containing paclitaxel and scrambled siRNA and BI Pac-siRNA NP - Nanoparticles containing paclitaxel and siRNA and functionalized with biotin. Data as mean ± S.D.; n = 5