(M1230-01-06) Screening of Synergistic Combination of Ferroptosis-Inducing Drugs and Development of Targeted PLGA Nanoparticles for Improved Anti-Cancer Therapy

Purpose: Most anticancer therapies induce cell death through apoptosis, but due to its complex nature, negative regulation of apoptosis is common [1]. To potentially achieve better anti-cancer effects, we propose targeting a newly identified pathway called ferroptosis, which has a narrow axis dependency, less complex regulation, direct control over cell death, and is pro-immunogenic in nature [2]. We screened ferroptosis-inducing drugs in combination to identify a synergistic combination, which we loaded into a tumor-targeted nano-drug delivery system to enhance their anticancer effect. Our screening process revealed that the combination of sorafenib (SOR) and simvastatin (SIM) exhibited a synergistic effect, which we then loaded into carboxy phenylboronic acid (CPBA)-modified PLGA nanoparticles (CPBA-PLGA(SOR+SIM)-NPs). The boronic acid group in CPBA provides tumor targetability to the PLGA-NPs through its interaction with the exocyclic polyol group of sialic acid residues, that are often overexpressed in cancer cells due to hyperglycosylation (Figure 1A) [3].

Methods: The study aimed to evaluate the synergistic effect of SOR, SIM, phenethyl isothiocynate, and trigonelline in MDA-MB-231, A549, and HeLa cancer cells. The identified combination of SOR and SIM was loaded into CPBA-modified PLGA-NPs using the nanoprecipitation method with CPBA-F127 conjugate as an aqueous stabilizer. Biophysical characteristics (size, PDI, % entrapment, morphology etc.) were evaluated. Formulation stability (2-4 °C and room temperature), and drug release (at pH 5.5 and 7.4, using dialysis bag method) was assessed over a pre-determined time period. Cell culture studies were conducted on MDA-MB-231, A549, and HeLa cells, including cytotoxicity (MTT assay), IC₅₀ reversal (in presence of ferrostatin-1), cell uptake, ROS (DCFH-DA dye), GSH assay, MDA assay, and mitochondrial membrane potential determination (JC-1 dye). The pharmacokinetic profile and anticancer activity were evaluated using SD rats and 4T1 cells induced BALB/c mice model. Toxicity study (liver and kidney biomarkers, histology and hemocompatibility) was performed on Swiss albino mice.

Results: During screening, the combination of SOR+SIM (1:1 w/w) showed synergism in MDA-MB-231 (CI value: 0.66), A549 (CI value: 0.54), and HeLa cells (CI value: 0.58) with favourable dose reductions (Figure 1B), indicating its suitability. To prepare CPBA-PLGA(SOR+SIM)-NPs, a CPBA-F127 conjugate was synthesized and confirmed through ¹H-NMR and FTIR spectra. The developed spherical shaped CPBA-PLGA(SOR+SIM)-NPs had a size of 213.1±10.9 nm with a PDI of 0.22±0.03 and showed % entrapment efficiency for SOR and SIM of 77.8±6.1 % and 81.9±7.2 %, respectively. The particles were spherical in shape with a rough periphery due to CPBA modification. The particles demonstrated sustained release behaviour at pH 7.4, with higher drug release at pH 5.5 due to the vulnerability of the ester bond between glycolic acid and lactic acid at lower pH, resulting in rapid PLGA degradation. Particles at pH 7.4 and 5.5 followed Korsmeyer-Peppas kinetics, with anomalous transport (0.45< n < 0.89) and hindered Fickian diffusion (n < 0.45) as drug release mechanisms at pH 7.4 and 5.5, respectively. In cytotoxicity evaluation, CPBA-PLGA(SOR+SIM)-NPs showed 2.4-, 2.2-, and 2.1-fold reduced IC₅₀ when compared to SOR+SIM in MDA-MB-231, A549, and HeLa cells, respectively. Cytotoxicity study was supported by qualitative and quantitative uptake study where, CPBA-PLGA(SOR+SIM)-NPs, showed higher internalization than all other groups. In the presence of ferrostatin-1 (15 µM), a significant increase (P < 0.05) in IC₅₀ confirmed that the combination-loaded particles induced ferroptosis-mediated cell death. Further, a significant increase in ROS and MDA levels (P < 0.01) and a decrease in GSH levels (P < 0.01) in all tested cells, corroborated the induction of ferroptosis-mediated cell death [4]. The JC-1 assay showed that CPBA-PLGA(SOR+SIM)-NPs-treated cells exhibited mitochondrial membrane depolarization, which further assists in inducing ferroptosis (Figure 2) [5]. Pharmacokinetic evaluation of CPBA-PLGA(SOR+SIM)-NPs revealed ~2.5- and 2.6-fold higher AUC_0-∞ and ~1.7- and 2.7-fold higher MRT for SOR and SIM, respectively, when compared to free groups. The tumor volume in the case of CPBA-PLGA(SOR+SIM)-NPs showed ~9.2, 2.5, and 1.6-fold lower tumor volume than the control, SOR+SIM, and PLGA(SOR+SIM)-NPs, respectively. Furthermore, tumor burden was observed to be least, and % tumor inhibition rate was observed to be the highest in the case of CPBA-PLGA(SOR+SIM)-NPs. The toxicity profile of CPBA-PLGA(SOR+SIM)-NPs showed no significant changes in ALT, AST, BUN, and creatinine levels and no histological changes in the isolated organs, with superior hemocompatibility than free drug groups (Figure 3).

Conclusion: The results indicate that CPBA-PLGA(SOR+SIM)-NPs containing the synergistic combination can induce ferroptosis-mediated cell death. Additionally, at the in-vivo level, the boronic acid-mediated tumor-targeting combination showed superior anticancer activity against the 4T1-induced cancer model. In future, the in-vivo anticancer activity in lung and cervical cancer models will also be explored before moving ahead with scale-up for clinical evaluation.

References: [1] Mohammad et al., Seminars in cancer biology, Academic Press, 35 (2015): S78-S103.
[2] Dixon et al., Immunological reviews, 277, no. 1 (2017): 150-157.
[3] Wu et al., Advanced Materials 32, no. 14 (2020): 1903759.
[4] Song et al., Frontiers in oncology 10 (2020): 597434.
[5] Wang et al., European journal of cell biology 99, no. 1 (2020): 151058.

Acknowledgements: The authors would like to acknowledge and express their gratitude to the Director of NIPER, S.A.S. Nagar, India for providing research facilities. The investigation received no funding, and the authors declare no conflicts of interest.

Panel (A) is the graphical abstract of the study conducted. Panel (B) shows the observed combination index (CI) values and dose reduction index (DRI) values for tested combinations in mentioned cells.

Illustrating the data for cell culture studies

Illustrating the data for mentioned pharmacokinetic study, anticancer study, and toxicity studies