A c-MET Receptor Targeting Nanoparticle Platform for Combination Therapy Against Hepatocellular Carcinoma

Introduction: Hepatocellular carcinoma (HCC) poses a significant global health challenge, being the third leading cause of cancer death, with over 906,000 new cases and 830,000 fatalities globally in 2020. Conventional HCC treatments are limited by high recurrence rates, inadequate efficacy, limited patient eligibility—particularly applicable only to a subset with early-stage disease and optimal liver function—and chemoresistance, complicating effective disease management. This situation is further exacerbated by the complex interplay of molecular pathways involved in HCC development and progression, such as the MAPK and PI3K/Akt pathways, which not only drive tumor growth but also contribute to the resistance mechanisms against standard treatments. To address these limitations, this study employs a poly(lactic-co-glycolic acid) (PLGA) polymer nanoparticle (NP) platform coated with a pH-responsive carboxymethyl chitosan (CMC) coating, for liver accumulation and anti-cancer therapy. Lenvatinib, a multi-tyrosine kinase inhibitor used in first-line HCC treatment was encapsulated in the PLGA core. Crizotinib, a c-MET receptor inhibitor, was encapsulated in the CMC shell. This drug combination was chosen for their potential to synergistically inhibit the MAPK and PI3K/Akt pathways. c-MET receptor upregulation is associated with tumor onset promotion, proliferation and invasion, and has also emerged as a key player in Lenvatinib resistance in cancer cells. Thus, this study seeks to overcome the intrinsic challenges of current therapies by harnessing pH-responsive NP-based drug delivery for liver accumulation and controlled release, promising improved efficacy against HCC.

Materials and

Methods: PLGA NPs encapsulating Lenvatinib were synthesized using a standard single emulsion solvent-evaporation technique. CMC at different concentrations, i.e., 0.25% (w/v), 0.5% (w/v) and 1% (w/v), was passively coated on the surface of the NPs. Dynamic Light Scattering (DLS) was used to confirm the hydrodynamic diameters, zeta potential and polydispersity index (PDI) of the NPs. The particles dispersed in de-ionized water were incubated for up to 7 days at 37oC, and stability of the formulation was assessed by measuring diameter, zeta potential and PDI using DLS. Fourier Transform Infrared Spectroscopy (FTIR) was used to verify the presence of CMC coating on the NPs. In vitro drug release studies were performed at pH 7.4 and 5.5 to assess the pH responsiveness of CMC-coated NPs in the acidic tumor environment. Cytocompatibility of the formulations was assessed using MTT assays following treatment of c-MET positive HepG2 liver cancer cells with different concentrations of the NPs for 24 hours. Cellular uptake kinetics was determined by incubating HepG2 cells with different concentrations of coumarin-6-dye-loaded NPs for 2 hours following by cell lysis and evaluation of NP fluorescence normalized to cell protein content. The combinatorial effects of the dual drug-loaded NPs were then evaluated by incubating HepG2 cells with single- and dual-drug loaded NPs for 24h, 72h and 5 days and carrying out MTT assays to determine cell viability.

Results, Conclusions, and Discussions: The study presents a comprehensive stability evaluation of PLGA NPs at varying CMC concentrations, which revealed that while NPs at lower concentrations maintain stability over seven days, those at 2.5%CMC concentration exhibit decreasing size and higher PDI values, indicating potential instability and aggregation. FTIR spectroscopy confirmed the successful CMC coating on these NPs, evidenced by the characteristic -OH peak at 3300 cm-1. Release profiles of Lenvatinib from these NPs highlighted a pronounced pH-sensitive release from the 1% CMC-coated NPs, particularly under acidic conditions that simulate tumor environments, contrasting with the immediate release from uncoated NPs, underscoring the importance of CMC concentration. Therefore, PLGA-1%CMC NPs were chosen for further characterization and in vitro assessment. The empty NPs demonstrated high cytocompatibility with >90% of the treated cells viable at all concentrations. Additionally, crizotinib-conjugated PLGA-1% CMC NPs achieved dose-dependent cellular uptake by HepG2 cells. The combination therapy’s effectiveness is demonstrated in Figure 1(a) –(c) where dual drug-loaded NPs (PLGA-CMC 1.0% LT-CZT) demonstrated a significant reduction in cell viability, particularly evident by day 5, establishing a synergistic and enhanced therapeutic potential against HCC.
In conclusion, we report the development of a stable and biocompatible PLGA-CMC NP formulation for dual-drug delivery to treat advanced HCC. The encapsulated Lenvatinib was released in a pH-dependent manner from the formulation. Preliminary studies looking at the combinatorial effects of NP-delivered Lenvatinib and Crizotinib demonstrated greater anti-cancer effects over time than NPs containing a single drug. Ongoing studies are focusing on improving the specificity of the NPs and further assessment of anti-cancer effects of the formulation.

Acknowledgements (Optional): We acknowledge funding support from NIH grant R37CA283937 to JUM.