(M1230-03-17) Chemosensitization of Liver Cancer Cells Using Synergistic Inhibitor Combination Co-loaded in Docosahexaenoic Acid Based Self-Nanoemulsifying System

Purpose: Hepatocellular carcinoma (HCC) is the fifth most common neoplasm in the world. It is also a leading cause of cancer deaths worldwide, accounting for more than 700,000 deaths each year. Sorafenib (SORA) is the first molecular targeted agent approved for the treatment of metastatic or unresectable HCC and remains standard first-line therapy. However, due to the high dose (400–800 mg/day), poor solubility (solubility in water 1.7 µg/mL), and lipophilicity (log P = 3.8) of SORA, its therapeutic applications remain restricted. Moreover, commercial tablet product of sorafenib (Nexavar^®) has reported low oral bioavailability, which might be due to its poor aqueous solubility. With the over-escalating need to improvise the therapeutic efficacy of SORA, different combination therapies including combination with EGFR-TKIs have been explored in various malignancies. In this context, our current study focused on combining two approaches which would effectively aid in overcoming the challenges related to the existing SORA therapy. 1. To enhance the therapeutic efficacy of SORA by combing it with a third generation EGFR-TKI inhibitor Abivertinib (ABV) and evaluate its cytotoxicity using 2D and 3D cell-based models. 2. To combat the issues related to solubility of these drugs by encapsulating them within docosahexaenoic acid (DHA) based self-nanoemulsifying system that would aid in enhanced stability and additional therapeutic advantages (Figure 1).

Methods: Initially, antiproliferative activity of SORA(S), ABV(A) and the combination (SA) was evaluated using MTT assay in HepG2, Hep3B and Huh-6 cells. Effect of DHA addition on anticancer potential was studied using the same protocol. Based on the potency, self-nanoemulsifying preconcentrate comprising of Dimethylacetamide (DMA) as co-solvent, Captex^® 300 EP/NF (MCT) as oily phase and Kolliphor^® ELP as surfactant were used to co-load SORA and ABV each at 30 mg/mL. Physicochemical evaluation was performed using dynamic light scattering (DLS) particle size analyzer. Anticancer activity and stability of nanoemulsion was further improvised by using DHA at its effective concentration. Optimized batch i.e., SA-DHA SNEDDS was characterized using a dynamic light scattering (DLS) particle size analyzer. In HepG2 cells, anti-metastasizing activity of SA-DHA SNEDDS was assessed using clonogenic assay. Apoptosis potentials were computed using flow cytometry. SOR, ABV, SA and SA-DHA SNEDDS were also tested at 5 μM in 3D spheroid model to mimic in vivo tumor conditions. Toxicity of the developed SNEDDS was evaluated on CaCO2 monolayer. Permeability coefficient of SNEDDS was ascertained by performing transwell permeability assay on CaCO2 cells. Development of a solid-SNEDDS tablet and in vitro dissolution study are currently under process.

Results: Successful encapsulation of 3 mg/mL SOR and ABV in aqueous medium was achieved using MCT: Kolliphor ELP ratio of 5:7 with DMA as the co-solvent. SA-DHA SNEDDS revealed a hydrodynamic diameter and polydispersity index of 48.75 nm and 0.156 respectively (Fig. 1). In comparison to the individual IC50s of SORA and ABV, synergistic inhibition was observed at 1:1 combination ratio. Nearly 2 to 7 folds reduction in individual IC50 values of the two drugs was observed in all the cell lines. In the presence of 25μM DHA, substantial decrease in cell viability was noted. Cell-staining using crystal violet depicted ‘anoikis’ in presence of the SNEDDS formulation (Fig. 2). SA-DHA SNEDDS treated cells exhibited 5-6 folds reduction in colony formation compared to alone drug groups. Nearly 32.35% and 49.10% cell apoptosis were observed after SA and SA-DHA SNEDDS treatment, respectively that was quantified by flow cytometry. Most importantly, substantial reduction was observed in spheroid diameter, post treatment. Higher amount of red fluorescence in treatment groups confirmed large number of dead cells compared to control. More than 90% cell viability on CaCO2 monolayer with 2-4 folds increase in drug permeability coefficient was detected in case of SA-DHA SNEDDS (Fig. 3).

Conclusion: This study entails a great promise of SA-DHA SNEDDS serving as an effective intervention therapy for patients with liver cancer. The potential anticancer efficacy of SA together in 2D and 3D in vitro culture models has never been explored so far. With the major physical issues of SA being surmounted by encapsulating it within a DHA based SNEDDS, it could serve as a promising strategy to overcome the limitations of current first-line therapy.


Fig. 1. Development of SA-loaded DHA-SNEDDS. (A) Graphical representation depicting the significance of SA- DHA SNEDDS. (B) Particle size analysis of SA-DHA SNEDDS.


Fig. 2. In vitro characterization of SA-DHA SNEDDS. (A) In vitro cytotoxicity assessment using MTT. SA showed significant reduction in IC50 compared to individual drugs. (B) Cell viability in presence and absence of DHA. DHA aided in significant reduction in HepG2 cell viability (***p≤ 0.001). (C) Cell staining using crystal violet dye. Visual assessment confirmed anoikis behavior of SA based SNEDDS formulation.


Fig. 3. (A) Flow cytometric analysis in HepG2 cells. SA-DHA SNEDDS showed significantly higher number of apoptotic cells. (B) 3D spheroid assay. SA-DHA SNEDDS treated spheroids revealed higher red fluorescence indicating more apoptotic cells. (C) Cytotoxicity assessment on CaCO2 monolayer. No significant toxicity was observed even at 40μM concentration of SOR and ABV in SA-DHA SNEDDS.