Inhibition of ABCB1 (MDR1) expression by an siRNA nanoparticulate delivery system to overcome drug resistance in osteosarcoma

Abstract

Background: The use of neo-adjuvant chemotherapy in treating osteosarcoma has improved patients' average 5 year survival rate from 20% to 70% in the past 30 years. However, for patients who progress after chemotherapy, its effectiveness diminishes due to the emergence of multi-drug resistance (MDR) after prolonged therapy.

Methodology/principal findings: In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure resulting from MDR, we designed and evaluated a novel drug delivery system for MDR1 siRNA delivery. Novel biocompatible, lipid-modified dextran-based polymeric nanoparticles were used as the platform for MDR1 siRNA delivery; and the efficacy of combination therapy with this system was evaluated. In this study, multi-drug resistant osteosarcoma cell lines (KHOS(R2) and U-2OS(R2)) were treated with the MDR1 siRNA nanocarriers and MDR1 protein (P-gp) expression, drug retention, and immunofluoresence were analyzed. Combination therapy of the MDR1 siRNA loaded nanocarriers with increasing concentrations of doxorubicin was also analyzed. We observed that MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. The results also demonstrated that this approach may be capable of reversing drug resistance by increasing the amount of drug accumulation in MDR cell lines.

Conclusions/significance: Lipid-modified dextran-based polymeric nanoparticles are a promising platform for siRNA delivery. Nanocarriers loaded with MDR1 siRNA are a potential treatment strategy for reversing MDR in osteosarcoma.

Figure 1. Characterization of stearylamine-dextran modified nanoparticles.

500 MHz ¹H NMR spectra of dextran and dextran conjugated stearyl amine after purification using D₂O as the solvent. The spectra clearly show the additional peaks of the long chain fatty amine at ∼1ppm indicating the successful lipid modification of dextran. The % fat modification was estimated to be 7 mole % of dextran.

Figure 2. Effect of EGFP siRNA loaded nanoparticles on BHK-21-EGFP cells was assessed.

EGFP siRNA was efficiently incorporated into cells and effectively inhibited the expression of EGFP in a dose dependent manner (NP: nanoparticle). siPORT™ NeoFX™ Transfection Agent (Ambion) was used as a positive control. Nanoparticles loaded with 100 nM non-specific siRNA (c-siRNA) and unloaded nanoparticles were used as negative controls.

Figure 3. Western blot assay was performed to assess the effect of MDR1 siRNA loaded nanoparticles on expression of P-gp.

The expression of P-gp was significantly decreased after application of 30 nM MDR1 siRNA loaded nanoparticles for KHOS_R2 (A) and 100 nM MDR1 siRNA loaded nanoparticles for U-2OS_R2 (B). P values are shown as follows: *P<0.05.

Figure 4. Western blot assay was performed to assess the duration of MDR1 siRNA loaded nanoparticle.

siRNA transfected with siPORT™ NeoFX™ Transfection Agent was able to suppress P-gp expression for 48 hours (A). Inhibition of P-gp started slower when MDR1 siRNA was loaded in to nanoparticles, but exhibited longer inhibition compared to MDR1 siRNA transfected with commercially available agent (B). P values are shown as follows: *P<0.05.

Figure 5. Effect of MDR1 siRNA loaded nanoparticle on P-gp – mediated uptake and efflux was analyzed using calcein AM.

Cells treated with MDR1 siRNA were shown to decrease calcein AM efflux in a dose-dependent manner as determined by image analysis, and this was confirmed by microplate spectrofluorometer analysis. P values are shown as follows: *P<0.05.

Figure 6. Subcellular distribution of doxorubicin in drug sensitive and resistant osteosarcoma cell lines was analyzed under fluorescence microscope (A-C).

A prominent increase in fluorescence was observed in the nucleus when multidrug resistant cells were treated with doxorubicin after treatment with MDR1 siRNA loaded nanoparticles.

Figure 7. The effect of doxorubicin alone or nanoparticle loaded with MDR1 siRNA on KHOS_R2 and U-2OS_R2 was analyzed.

Varying concentrations of doxorubicin was added and cells were cultured for 5 days. Treatment with nanoparticles loaded with MDR1 siRNA showed increased anti-proliferative activity in both drug resistant osteosarcoma cell lines, KHOS_R2 (A) and U-2OS_R2 (B), in a dose dependent manner. Growth inhibition was assessed by MTT as described under Materials and Methods. The experiment was repeated four times in triplicate.