Targeted dual agent nanoparticles to overcome tumor drug resistance

Tumor drug resistance is a major challenge for the success of chemotherapy in cancer. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp) confers resistance to a broad range of chemically diverse anticancer drugs. The objective of this research was to develop a delivery system that will overcome drug resistance in tumor cells. It was hypothesized that ligand-functionalized, dual agent nanoparticles will effectively overcome P-gp-mediated drug efflux and increase intracellular paclitaxel accumulation, resulting in a more effective inhibition of drug-resistant tumor. To test this hypothesis, poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing an anticancer drug (paclitaxel) along with a P-gp modulator (tariquidar/siRNA) were developed and tested for anticancer activity in vitro and in vivo. Nanoparticles were surface functionalized with folic acid and/or biotin for tumor cell targeting. The specific aims of this research were to (1) develop surface functionalized PLGA nanoparticles with folic acid and biotin as tumor targeting ligands and investigate the tumor targeting effectiveness of ligand functionalized nanoparticles in vitro as well as in vivo; (2) investigate the effectiveness of tumor-targeted dual agent nanoparticles encapsulating paclitaxel and tariquidar in overcoming drug resistance in vitro and in vivo ; and (3) Determine the therapeutic effectiveness of tumor-targeted dual agent nanoparticles encapsulating paclitaxel and siRNA targeted to P-gp in overcoming drug resistance in vitro and in vivo .;Cancer cells overexpress different receptors on cell membrane, which can be utilized for targeting the cancer cell. We initially investigated a facile, single-step surface functionalization technique driven by interfacial activity of block copolymers called Interfacial Activity Assisted Surface Functionalization (IAASF). Using this technique, we formulated nanoparticles containing folic acid and/or biotin on the surface. Scanning electron microscopy and dynamic light scattering studies indicated a particle size in the range of 200-250 nm. Presence of ligands on the surface was confirmed qualitatively by transmission electron microscopy and surface plasmon resonance. Number of biotin molecules on the surface was determined quantitatively using a commercially available biotin quantitation kit. Surface functionalized nanoparticles demonstrated significantly higher uptake (5- to 12-fold) than non-functionalized nanoparticles in vitro in different cancer cell lines. In vivo studies in NCR-NU mice bearing MCF-7 xenografts showed that ligand-functionalized nanoparticles were significantly better in delivering paclitaxel to tumors and in inhibiting tumor growth than non-functionalized nanoparticles.;Dual agent nanoparticles encapsulating tariquidar/cyclosporine-A, both P-gp modulators, along with paclitaxel resulted in significantly greater cytotoxicity and higher paclitaxel accumulation in drug resistant tumor cells than paclitaxel treatment alone. In vivo studies in Balb/c mice bearing drug-resistant JC tumors showed that biotin-functionalized dual agent (paclitaxel-tariquidar) nanoparticles were significantly more effective in inhibiting the growth drug-resistant tumor and improving the survival of tumor-bearing animals.;Based on the success of the dual agent delivery concept, we investigated an alternative approach of delivering paclitaxel along with siRNA targeted to P-gp using targeted nanoparticles to overcome tumor drug resistance. Initial studies suggested that introduction of PEI in PLGA nanoparticle matrix was necessary to enhance encapsulation and improve the release of siRNA from nanoparticles. Incorporation of PEI also enhanced the cellular uptake of nanoparticles and appeared to improve the cytosolic delivery of siRNA. Dual agent nanoparticles containing paclitaxel and P-gp-targeted siRNA enhanced paclitaxel accumulation and cytotoxicity significantly in drug resistant JC cells, indicating effective down-regulation of P-gp. Further, in vivo studies in Balb/c mice bearing drug-resistant JC tumors showed that ligand-functionalized dual agent nanoparticles were significantly more effective in inhibiting the growth drug-resistant tumor and improving the survival of tumor-bearing animals.;In conclusion, ligand-functionalized nanoparticles have shown significant tumor targeting in both drug-sensitive and drug-resistant tumors. Simultaneous delivery of an anticancer drug along with P-gp modulator using tumor targeted nanoparticles has shown great promise in overcoming drug resistance in refractory tumor cells.