(W1130-04-24) Mebendazole Nanosuspension for the Treatment of Glioblastoma

Purpose: Glioblastoma (GBM) is a rapidly growing type of tumor of the brain or spinal cord. The World Health Organization (WHO) assigned GBM as a grade IV brain tumor which represents one of the most lethal human cancers. The major challenge for delivery of drug in any brain related disorder is the selective permeability of blood brain barrier (BBB). Thus, there are very few chemotherapeutic agents with the ability to cross BBB that are used to inhibit the progression of GBM. Overall, there is a dire need of anti-cancer molecules for the treatment of GBM. Repurposing an old molecule while developing a proficient novel formulation could be a promising approach in cancer drug discovery. Mebendazole (MBZ), an antiparasitic agent showed anticancer activity against various tumors including brain cancer. Poor aqueous solubility/bioavailability, multiple polymorphs, and insufficient blood–brain barrier permeability restrict translational efficiency. Nanosuspension (NS) is a unique system consists of drugs and stabilizers having nano-range particle size, generally ranging from 10 to 1000 nm. The aim of our study was to formulate and characterize albumin and polysorbate 80 coated MBZ nanoparticles (NPs) using different glioma cell lines. In this study, we have explored a novel dual centrifuge nano milling technology to formulate MBZ nanocrystals for systemic delivery. The Quality by Design approach was used to optimize drug loading and stabilizer concentration. Polysorbate 80 has been reported to enhance brain uptake of nanoparticles while albumin facilitates stabilization and long circulation.

Methods: Organic solvent free, top-down nano milling process was used to prepare MBZ NPs. 2³ factorial designs optimizing MBZ load vs Human Serum Albumin (HSA)/Polysorbate 80 concentration was employed. The nano-milling was carried out using 0.3 mm Zirconium beads in NETZSCH DeltaVita^® (Figure 1a). The particle size, Poly dispersibility index (PDI) and zeta potential were analyzed by Malvern zeta sizer. Solid state characterization was carried out using DSC and pXRD. Further, the microscopic morphology of MBZ NS was analyzed using transmission electron microscopy (TEM). In vitro cytotoxicity study of MBZ and M­­BZ NPs was evaluated in U-87 and LN-229 using MTT assay. In vitro migration assay was performed to assess the contribution of molecular and cellular mechanisms to cell migration. Clonogenic assay was performed to determine the survival ability of the single cell to grow into colony. To better mimic the in vivo tumor microenvironment, we evaluated the growth, apoptosis, and invasion of 3D multicellular U-87 spheroids following MBZ and MBZ NPs treatment. Ongoing studies are Apoptosis assay, western blot, and in vitro hemolysis assay.

Results: Nanosuspension batches with single stabilizers show rapid phase separation while combination of albumin and polysorbate resulted in stable batch. We have also performed QbD 2³ factorial design which showed optimized batch with drug load of 0.5%, 2.5% of HSA and 0.2% of Polysorbate-80. The optimized MBZ NPs depicted particle size of 177.2 ± 0.2 nm with PDI of 0.2 and zeta potential of -0.76 ± 0.86 mV with maximum stability among all batches. (Figure 1b-c). Processing temperature and drug concentration played a significant role in the %yield. The %yield of optimized MBZ NPs was found to be 70 ± 0.21% (Figure 1d). Furthermore, DSC and TEM confirmed the crystallinity of MBZ in MBZ NPs (Figure 1e-f). In vitro cytotoxicity study showed the IC50 of MBZ NPs were found to be to be lower than that of pure MBZ as shown in (Figure 2a). MBZ NPs shows substantial inhibition in the in vitro migration of the cells and colony formation at lower concentration of MBZ NPs of 0.3 µM in LN-229 cells (Figure 2b-c). MBZ NPs led to substantial reduction in area of 3D spheroids of U-87 over 6 days with marked increase in apoptotic surface. 3D spheroids study clearly depicted that very high concentration of MBZ required for the activity which is achievable using such nano formulation (Figure 3a-3c).

Conclusion: Albumin-polysorbate-coated nanosuspension could be useful parenteral delivery of mebendazole. In vivo studies are sought to investigate the brain uptake and anticancer efficacy in brain tumor xenograft model.

Figure 1. (a) Schematic representation of preparation of mebendazole nanoparticles (MBZ NPs), (b) Particle size of Optimized MBZ NPs, (c) Zeta Potential of MBZ NPs, (d) HPLC chromatogram of MBZ NPs, (e) DSC of Pure MBZ & MBZ NPs, (f) TEM imaging of MBZ NPs.

Figure 2. (a) IC-50 of MBZ and MBZ NPs in LN229 and U-87 cell lines (b) In vitro migration and (c) Clonogenic assay results in LN229 cells.

Figure 3. (a) Bright field microscopic images of U-87 cells 3D multicellular Spheroid treated with MBZ and MBZ NPs for 6 days (b) Growth curve for 3D U-87 spheroids following MBZ and MBZ NPs treatment. (c) Results of cell viability within MBZ and MBZ NPs treated 3D multicellular U-87 spheroids.