Category: Formulation and Quality
Purpose: Glioblastoma multiforme (GBM) is the most common and malignant form of astrocytoma. Poor availability of anticancer drugs such as Temozolomide (TMZ) at tumor site is one of the major hurdles in GBM treatment. Although TMZ can cross the BBB, alternative treatment approaches are necessary owing to its side effects. Epidermal growth factor receptor (EGFR) amplification in GBM is often associated with treatment resistance which can be decreased by using erlotinib (selective inhibitor of the EGFR). Thus, a polymeric nanocarrier system encapsulated with TMZ- erlotinib (with subsequent inhibition of EGFR) combination therapy targeted by the magnetic field, might offer the viable alternative to the current invasive therapeutic measures for the GBM treatment.
Methods: Oleic acid magnetic nanoparticles (OMNP) were synthesized using thermal decomposition method. Nano carrier of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid)− methoxy poly(ethylene oxide) (PLA−PEG) was prepared by single emulsion solvent evaporation method. Iron content in the prepared formulation was quantified by colorimetric ferrozine-based assay. TMZ-erlotinib loaded nanoparticles were prepared and characterized for morphology, particle size, zeta potential, encapsulation efficiency and physical stability. Effect of different PVA % was evaluated and the optimum percentage was taken forward to optimize the drug loading and encapsulation efficiency. NMR and FT IR analysis were carried out to confirm the co polymer linkage. Cytotoxicity was evaluated using P-gp expressing SW 620 cell line and SW 620 AD300 resistant cell line. Currently, the in vitro permeability of TMZ-loaded polymeric nanoparticles is being evaluated by BBB tissue model. The effect TMZ-erlotinib loaded nanoparticles on the cellular proliferation of EGFR expressing cell line will also be evaluated using MTT assay. The effect TMZ-erlotinib loaded nanoparticles on the cellular proliferation of U-118 will also be evaluated using MTT assay. Cellular uptake studies will be conducted to validate the cellular toxicity. The inhibition of EGFR and P-gp expression by the dual loaded nanoparticles will be evaluated by western blot analysis.
Results: A uniform red brick dispersion because of conjugation of ferrous and oleic acid was obtained at 100℃ for 20 min followed by 200℃ for 10 min and 260℃ for 10 min. The synthesized OMNP (10- 30 nm) were characterized for magnetic activity. Coating of PEG PLA - PLGA showed marginal change in particle size but substantial change in zeta potential (negative to neutral). Presence of co polymer block linkage on the surface of OMNP was confirmed by proton NMR and FT IR. TEM analysis showed a spherical particle in the range of 10-30 nm. Drug loading was optimized such that the particle size and stability were not affected. An optimum encapsulation efficiency for TMZ was achieved. The plot for TMZ solution and TMZ-erlotininb solution shows that the combination has higher cell killing rate (IC 50 of TMZ is 200 μM whereas combination is 35 μM) and hence the IC 50 values are lower as compared to TMZ alone.
Conclusion: Characterized and optimized TMZ- erlotinib nanoparticles were evaluated for P-gp ,EGFR inhibition in the in vitro model using various cell lines.The cell toxicity and uptake was confirmed for lowered IC 50 values in GBM cell lines as well. Reversal fold will be evaluated further to confirm the in vitro TMZ resistance decreased. The above information will be used to evaluate the quantity of drug loaded nanoparticles in order to check the anticancer property of TMZ-erlotininb nanoparticles in mice model.