(W0930-05-29) Enhanced U-87 Glioblastoma Cell death by PLGA-PEG Encapsulated Paclitaxel Nanoparticles

Purpose: Malignant gliomas are the most common primary brain tumors, among which glioblastoma (GBM) is the most aggressive grade IV glioma. The median life expectancy for GBM patients is only 12–15 months even with a treatment combining resection, radiation therapy, and chemotherapy with oncolytic drug temozolomide (TMZ). However, over the course of the treatment, GBM tumor cells acquire resistance to TMZ via DNA repair enzymes. Therefore, there is an unmet need to come up with a more effective treatment for GBM. Paclitaxel (PTX) is among the most active agents against glioblastoma in preclinical models. However, its clinical use has been challenged by the blood-brain barrier (BBB) due to its large hydrophobic structure. Nanoparticle encapsulation of PTX enhances its cellular endocytosis and the ability to cross the BBB, provides stability to PTX, and allows slow release of the drug, helping reduce toxicity to healthy cells. The objective of this study is to develop PLGA-PEG nanoparticles loaded with PTX and determine their cell death effect in U-87 GBM cells. We hypothesize that PTX-PLGA-PEG nanoparticles will be efficient in reducing the viability of U-87 cells compared to PTX alone.

Methods: We used PLGA-PEG biodegradable polymer for encapsulation of the PTX using modified emulsion solvent evaporation method. Briefly, PLGA-PEG was dissolved in methylene chloride while paclitaxel was dissolved in ethyl acetate. Two organic phases were thoroughly mixed and poured slowly with sonication in aqueous solution of polyvinyl alcohol (PVA). The organic solvents were forced evaporated using a rotavapor. Resultant PTX-PLGA-PEG nanoparticles were harvested using two techniques, 1) ultrafiltration and 2) ultracentrifugation. Fig 1 shows a flowchart of the process. PTX-PLGA-PEG nanoparticles were characterized for size, transmission electron microscopy, Zeta potential, and content of PTX. U-87-Luc (ATCC, isolated from malignant glioma, provided by Dr. Pollock, IU) was cultured in Iscove's Modified Dulbecco's Medium containing high glucose, L-Glutamine, HEPES, and supplemented with 10% fetal bovine serum. Cells were grown at 37°C in a 5% CO2 incubator under humidified air. Once cultured, the cells were plated on 96 well plates at 4,000 cells/100 µL. The cells were treated with PTX alone in DMSO/media or PTX-PLGA-PEG dispersed in media and incubated for 72 hours at 37^oC, 5% CO₂. Methylene blue assay was used to determine the viability. All experiments were replicated at least three times and average results reported with + standard deviation. t-Test was used to determine significant effects.

Results: The ratio of polymer (PLGA-PEG) to drug (PTX) had a significant effect on the particle size of the nanoparticles and a 100:5 ratio of polymer: drug produced an average particle size of 89 nm. PVA solution volume also influenced the particle size. Average particle size of 100 nm was obtained with 100 mL of PVA solution. Ultrafiltration process using 0.45 µm filter membrane produced particles about 10 nm smaller with slightly better polydispersity index than the ultracentrifugation process. TEM pictures revealed spherical structures of the nanoparticles (Fig. 2). Average Zeta potential for nanoparticles in DMEM was negative 5.64 mV. Content of PTX was determined using reversed phase HPLC and was used in calculation of cell treatments. Cell viability results with PTX vs. PTX-PLGA-PEG showed significant difference in IC₅₀ value. The average IC₅₀ value for PTX in PLGA-PEG was 21 nM while the average IC₅₀ value for PTX alone (in DMSO/media) was 80 nM (Fig. 3), after normalization for vehicle and blank nanoparticle controls.

Conclusion: The modified emulsion solvent evaporation method was reproducible and produced spherical particles. Polymer: drug ratio of 100:5 and aqueous medium volume of 100 mL were most appropriate for producing small particles with low polydispersity index. Small negative charge on the particles is favorable to keep the particles non-aggregated. Cell viability studies indicated superior efficacy of encapsulated PTX over PTX alone.The PTX-PLGA-PEG nanoparticles were 4-fold effective in inducing cell death in U-87 GBM cells compared to PTX alone.Therefore, PTX-PLGA-PEG nanoparticles could reduce PTX dose in GBM without compromising efficacy and likely reduce toxicity in our forthcoming preclinical studies.

References: Smiley SB, Yun Y, Ayyagari P, Shannon HE, Pollok KE, Vannier MW, Das SK, Veronesi MC. Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma-In Vitro Evaluation in Glioblastoma Stem Cells. Pharmaceutical Research. 2021 Jun;38(6):1067-79.

Acknowledgements: Indiana University School of Medicine (Indiana University School of Medicine Biomedical Research Grant).


Nanoparticle preparation flowchart


Transmission electron micrograph of typical nanoparticles


Comparative IC50 values for PTX and PTX-PLGA-PEG