Multifunctional nanoemulsion system for combination paclitaxel and curcumin delivery in human glioblastoma cells

Abstract

Glioblastoma multiforme (GBM) is an aggressive form of primary brain tumor that afflicts as many as 12,000 patients per year in the United States. GBM is a very aggressive disease and the average patient life-span is reduced to 6 months after diagnosis. Some of the clinical challenges in GBM include difficulty in initial diagnosis, presence of tumor cell "islets" in the critical areas of the brain, and the role of blood-brain barrier (BBB) in effective transport and distribution of drugs in the brain. Due to the presence of tumor cell islets, complete surgical resection is not always possible. Additionally, poor penetration of many chemotherapeutic agents across the BBB limits availability and distribution for effective treatment of GBM. In addition to the presence of a physical barrier in the form of tight endothelial cell junctions, BBB is also comprised of chemical barriers in the form of expression of the efflux pump, P-glycoprotein (P-gp), and the presence of drug metabolizing enzymes and the vascular endothelial cell surface. GBM also has high incidence of resistance development due to the expression of multidrug resistant protein (MRP). In this study, our main objective was to evaluate co-administration of paclitaxel (PTX) and curcumin (CUR), a modulator of MRP and an inhibitor of nuclear factor kappa B (NF and B) signaling, in oil-in-water nanoemulsion formulations in brain tumor cells. CUR is known to down-regulate both P-gp and MRP expression and also enhances apoptotic activity by inhibition of NF and B mediated intracellular signaling. The nanoemulsion formulations in this study are specifically composed of oils rich in polyunsaturated fatty acids (PUFA) and the surface was modified to enhance BBB penetration. As part of the study, PTX and CUR containing nanoemulsions have been formulated with pine-nut oil, which is rich in linoleic and linolenic acids, an example of omega-6 and omega-3 PUFA, respectively. The optimized formulations were characterized for oil droplet size, surface charge, and observed with transmission electron microscopy (TEM). Human wild-type glioblastoma cells U87 and the MRP-1 expressing resistant glioblastoma cells, T98G, were grown in culture. Baseline expression of MRP-1 in T98G cells was confirmed by Western blot and immunocytochemistry analysis. Intracellular delivery of fluorescently-labeled PTX and CUR using nanoemulsion formulation was examined by fluorescence microscopy. The cell viability upon treatment with PTX and CUR, either alone or in combination, was examined in both of these cell lines using the MTT (conversion of tetrazolium salt to formazan) assay. MRP-1 down-regulation and the inhibition of NF and B pathway was confirmed by Western blot analysis. Qualitative apoptotic activity in U87 and T98G cells was evaluated using TUNEL staining. Pine-nut oil nanoemulsions were successfully formulated with an approximate hydrodynamic diameter of 120 nm and a -40 mV surface charge. Both PTX and CUR were efficiently encapsulated in the oil phase of the nanoemulsion. TEM analysis showed that the oil droplets of the nanoemulsion had a spherical shape and smooth surface morphology. Due to rapid cellular uptake and internalization of PTX and CUR, as assessed by fluorescence microscopy, the nanoemulsion formulations were able to significantly enhance cytotoxicity in both cell lines. Combination of PTX and CUR were found especially to enhance cytotoxicity in MRP-1 gene expressing T98G cells. CUR was found to down-regulate MRP-1 and was found to inhibit NF and B pathway. TUNEL staining images confirmed the apoptosis in the treated U87 and T98G cells. The overall results allowed us to understand the synergistic therapeutic effect upon administration of PTX and CUR using nanoemulsions made with PUFA-rich oil.