Domain Specific Oncoprotective Targeted Nano-Therapy against Receptor tyrosine kinases in Glioblastoma

Abstract

Glioblastoma is ranked as the 4th most aggressive, highly malignant and infiltrative, solid astrocytic brain tumor with distinct histological features. The inevitable recurrence and reactivation of oncogenic tyrosine kinase molecular pathways including EGFR mutations, PDGFR amplification, and VEGFR upregulation proved to be a limitation of presently practiced therapeutic interventions. The current treatment regimens include neurological surgeries, Stupp protocol, chemotherapy, and administration of FDA-approved TMZ-aided tumor progression-free survival for a few months. The dynamic crosstalk between neurovascular unit, tight junctions, and efflux transporters restricts the passive diffusion of chemotherapeutic medications thus limiting the availability and distribution of drug molecules across the selectively permeable blood-brain barrier. The highlighted breakthrough in the development of nano-carrier mediated transport has solved bottlenecks of drug delivery across BBB. Thus, the current study has investigated the role of chitosan nanoparticles as a nano carrier with non-immunogenic nature, increased surface-to-volume ratios, favorable chemical modifications, and site-directed control drug release across BBB.Ionic gelatin method was used to design targeted mucoadhesive chitosan –TPP nanoparticles to open up tight junctions of BBB and to enhance the drug residual period at the target site.Physio-chemical characterizations of chitosan-TPP nanoparticles were performed to assess their size, stability, entrapment efficiency, and release rate.Database of 691 drug formulations having anti-cancerous, anti-inflammatory, and anti-proliferative activities was created through Molecular Operating Environment (MOE) that aided in docking of all compounds with tyrosine kinase receptors (PDGRF α, EGFR,VEGFR).Top hits with lower binding engeries were further scrutinized by applying Lipinski rule 5, Admetsar and DFT analysis which predicted characteristics like drug-likeness, non-carcinogenicity, non-AMES mutagenesis, no rat acute toxicity ,permeability across BBB along with geometrical and electrical properties .After screening ,Kaempferol was marked as the top hit targeting PDGFR α.Dose and time dependent MTT assays were performed on HEK- 293, U87-MG and U251-MG for the validation of insilico results.At highest concentrations,Kaempferol showed only 36% cell viability on U87-MG at 72 hours with an IC50 value (31.41uM ) while no significant difference in cytotoxicity effects were observed between Kampferol and Kampferol loaded nanoparticles. No appreciable significant cytotoxic trend was shown by TMZ and TMZ loaded nanoparticles.Only 31 % cell viability was observed for highest concentration of Kampferol having IC50 value (37.78uM) on U251 –MG at 72 xii hours.Kaempferol loaded nanoparticles showed significant decrease in U-251 MG cell progression as compare to TMZ loaded chitosan nanoparticle. Kaempferol and Kaempferol loaded nanoparticles showed negligible cytotoxic effect whereas TMZ and TMZ loade nanoparticles had some cytotoxicity at higher concentrations on HEK-293 after 72hrs.Cell migration assay performed on U87- MG and U251-MG cell lines showed significant suppression of glioma cells and reduction in the speed of wound healing in a concentration-dependent manner by Kaempferol and Kaempferol loaded chitosan nanoparticles. . Moreover, TMZ and TMZ loaded nanoparticles didn’t show appreciable hindrance in cell migratory activity of U87-MG and U251-MG cell line even at higher concentrations.Conclusively, the therapeutic efficacy spectrum of Kaempferol and Kaempferol encapsulated chitosan nanoparticles decreased the in vitro glioma cell proliferation and growth. With promising computational and experimental outcomes, this study provides a gateway for revealing masked potent domains of natural compounds.