Uptake and Inhibition of P-Glycoprotein-Mediated Efflux Evaluation of Encapsulated Methotrexate Chitosan and Hypromellose Phthalate Nanoparticles for Potential Glioblastoma Treatment

<b>Background:</b> Methotrexate (MTX), a folic acid antagonist used in chemotherapy, faces limitations due to cancer cell resistance, high toxicity, and low bioavailability. <b>Objective:</b> This study developed nanoparticles (NPs) of chitosan (QS) and hydroxypropylmethylcellulose phthalate (HPMCP) to encapsulate MTX for potential effect investigation on glioblastoma cell targeting and P-gp efflux inhibition. <b>Method:</b> NPs were produced by the polyelectrolyte complexation method and were characterized by DLS, PDI, DSC, FTIR, PXRD, MEV, drug release profile, and an in vitro mucoadhesion test. Cell viability, flow cytometry, and LSCM using U251MG (glioblastoma) and CCD 1059Sk (fibroblasts) cells were used to evaluate glioblastoma and the P-gp efflux effect. <b>Results:</b> NPPM29 (QS3:1) showed 91.72% encapsulation efficiency, a mean diameter of 452.6 nm, and a zeta potential of +22.5 mV. DSC, FTIR, and PXRD confirmed the QS-HPMCP supramolecular interaction. Liquid falling mucoadhesion tests demonstrated strong retention of NPPM29 (84%) compared to free MTX (10.5%). In vitro release studies indicated controlled drug release at pH 7.4. Cytotoxicity assays in U251MG revealed enhanced efficacy of NPPM29 <i>(IC</i>₅₀ = 68.79 µg/mL) compared to free MTX (<i>IC</i>₅₀ = 80.54 µg/mL), with minimal impact on fibroblasts, confirming tumor specificity. Flow cytometry and LSCM confirmed improved cellular internalization and P-gp inhibition. <b>Conclusions:</b> These findings highlight the potential of MTX-QS-HPMCP-NPs for glioblastoma therapy.