(M1030-04-28) Comparative Cell Viability Study for Paclitaxel Loaded PLGA-PEG Nanoparticles in Established U-87 and Patient Derived Primary GBM10, GBM39, GBM43 and GBM75 Glioblastoma Cells

Purpose: The survival benefit observed with the addition of temozolomide to standard radiotherapy was much higher in methylated MGMT(O-6-methylguanine-DNA methyltransferase) gene promoter in glioblastoma, a deadly brain tumor, as compared to patients with MGMT-unmethylated tumors[1]. Standard preclinical research evaluates drug delivery systems in ATCC-established glioblastoma cells; however, immortalized “clean” cell systems do not represent the vast heterogeneity of glioblastoma tumors. Our objective was to evaluate our drug delivery systems in patient derived MGMT methylated and unmethylated cells to obtain a more comprehensive picture of their efficacy. We used MGMT promoter unmethylated GBM10, GBM43, and GBM75 and methylated GBM39 cells, derived from patients, in addition to the ATCC-established U-87 cells, to compare and evaluate cell viability when treated with paclitaxel loaded PLGA-PEG nanoparticles. Paclitaxel, a microtubule stabilizing agent, is highly potent against GBM but unable to penetrate the blood-brain barrier (BBB). Therefore, we used PLGA-PEG nanoparticles which have been shown to enhance drug delivery across BBB[2].

Methods: A modified simple emulsion technique was utilized to produce nanoparticles. A solution of paclitaxel in ethyl acetate was added to a solution of PLGA-PEG in dichloromethane, and the mixture was slowly added to polyvinyl alcohol -Tween 80, in water, at a temperature of 4°C while being sonicated. The colloidal dispersion was placed in a rotary evaporator for one hour to completely remove all organic solvents. The resulting aqueous dispersion was filtered through a mesh filter to eliminate any large polymer precipitates, followed by ultracentrifugation and washing to collect the nanoparticles. The size, polydispersity index (PDI), and zeta potential of PTX-PLGA-PEG nanoparticles were analyzed using a Malvern Zetasizer. Additionally, the morphology of the nanoparticles was characterized using Transmission Electron Microscopy. The loading and encapsulation efficiency were determined by HPLC. In vitro drug release was assessed using a sample and separate approach[3]. We evaluated the cytotoxicity of PTX-PLGA-PEG nanoparticles in five different cell lines, including the established GBM cell line U87 and four primary glioma xenografts derived from patients (GBM10, GBM39, GBM43, and GBM75, obtained from Mayo Clinic Brain Tumor PDX National Resource). All cells were cultured in DMEM supplemented with 10% fetal bovine serum (v/v) and incubated in a 5% CO₂ air humidified atmosphere at 37°C. For each plate, an untreated control column (cells treated with only media) and a treated control with 0.03% DMSO were compared to the positive control (either PTX or encapsulated PTX in nanoparticles). Drug-free nanoparticles were also assessed and showed no effect on any of the tested cell lines. After five days of treatment, cell viability was assessed using a methylene blue assay and absorbance was measured with a microplate reader. All experiments were performed at least three times (n=3).

Results: The PTX-PLGA-PEG nanoparticles exhibited reproducible average encapsulation (70%) and drug loading (65%) efficiencies with low standard deviation. The average particle size ranged from 100-120 nm with a low polydispersity index. The release rate of PTX from the nanoparticles was fast within the first 24h (45.1 ± 6.2% of the initial drug loaded in NPs) but slowed down within the 7 days during the course of study. Total PTX released from the particles after 7 days was 72.5 ± 3.1%. The change of rate of the drug release study may be due to the distribution of the drug localized in the PLGA core vs surface of the nanoparticles. The PTX-PLGA-PEG nanoparticles showed a steady decrease in cell viability with higher potency (compared to free PTX) in the U87, GBM10, GBM43, and GBM75 cell lines. However, this trend was reversed in GBM39 where PTX showed higher potency (IC50 = 6 nM) compared to the PTX-PLGA-PEG nanoparticles (IC50 = 301 nM) (Fig 1). At 10 mg/ml polymers, no cytotoxic activity was observed for the drug-free nanoparticles. The higher cell killing effect of PTX-PLGA-PEG nanoparticles are likely due to the prolonged release of paclitaxel. GBM39 cells present a methylated MGMT, whereas GBM 10, 43 and 75 have a nonmethylated MGMT promoter [4]. Since it has been shown that methylation could affect the transporter ions, it is possible that GBM39 resisted uptake of nanoparticles but allowed PTX molecules. We are currently studying this issue, in detail.

Conclusion: This study showed that encapsulated PTX in PLGA-PEG polymeric nanoparticles exhibited higher potency than the free PTX in four out of five human glioblastoma cell lines, supporting the hypothesis that encapsulation of PTX in nanoparticles improves intracellular penetration and delivery. It is interesting to note that there is a variation of cell death effects in different methylated and unmethylated MGMT GBM cell lines

References: [1] Hegi, Monika E., et al. "MGMT gene silencing and benefit from temozolomide in glioblastoma." New England Journal of Medicine 352.10 (2005): 997-1003.
[2] Chen, Yung-Chu, et al. "Effects of surface modification of PLGA-PEG-PLGA nanoparticles on loperamide delivery efficiency across the blood–brain barrier." Journal of biomaterials applications 27.7 (2013): 909-922.
[3] D'Souza, Susan S., and Patrick P. DeLuca. "Methods to assess in vitro drug release from injectable polymeric particulate systems." Pharmaceutical research 23 (2006): 460-474.
[4] Vaubel, Rachael A., et al. "Genomic and Phenotypic Characterization of a Broad Panel of Patient-Derived Xenografts Reflects the Diversity of Glioblastoma Characterization of Glioblastoma Patient-Derived Xenografts." Clinical Cancer Research 26.5 (2020): 1094-1104.

Figure 1. IC50 (nM) presentation of cellular potency of PTX and PTX-NPs in U87, and PDX GBM10, GBM43, GBM75, and GBM39 cell lines. No reduction in cell viability was observed when the cells were exposed to the control drug-free PLGA-PEG NPs