(T1230-06-38) Kolliphor® P407-CPBA Functionalized PLGA Nanoparticles for Co-Delivery of Sorafenib and Venetoclax: A Synergistic Strategy Targeting Ferroptosis and Apoptosis in Breast Cancer

Purpose: The global burden of cancer necessitates the development of non-toxic and well-tolerated therapeutic interventions [1]. Recently, ferroptosis, a newly discovered programmed cell death regulate by protein such as glutathione peroxidase-has been implicated in cancer progression [2]. In this context, Sorafenib (SOR), known to induce ferroptosis, offers promising potential. However, both intrinsic and extrinsic factors contributes to the drug resistance, often associated with the monotherapy [3]. SOR resistances has been linked to downstream pathways in cell growth and apoptosis inhibition via BCL2 pathway [4]. Venetoclax (VTX), BCL-2 inhibitor that induces apoptosis has shown potential in overcoming SOR resistance [4]. Hence, combination of SOR and VTX may provide synergistic therapeutic platform by targeting two distinct cell death pathways, potentially overcoming resistance mechanism. Despite this potential, the therapeutic efficacy of drug combination remains suboptimal. Co-loading these agents into nanocarrier could enhance drug solubility, protect them from degradation, improve tissue penetration, reduce off target effects and ensure synchronized pharmacokinetics. Poly(lactic-co-glycolic acid) (PLGA), an FDA approved polymer was employed to formulate nanoparticles (NPs) for controlled release and functionalization potential [5]. Herein, we utilized Kolliphor^® P 407-carboxyl phenyl boronic acid (CPBA) conjugate as a targeting ligand for PLGA-NPs [6]. The CPBA ligand specifically bind to sialic acid receptors, thereby promoting enhanced cellular uptake and improving therapeutic efficacy.

Methods: The Kolliphor^® P 407-CPBA conjugate was synthesized via Steglich esterification reaction and characterized by ¹H NMR and FTIR spectroscopy to confirm conjugation. Nanoprecipitation method was employed to prepare PLGA NPs using P407-CPBA as  both stabilizer and targeting ligand. A two-level fractional design was initially used for screening, followed by Box-Behnken design (BBD) for optimization. The developed NPs were characterized for particle size, size distribution (PDI), morphology, entrapment efficiency.  Optimized NPs were lyophilized using selected cryoprotectant at optimal concentration to ensure stability. In-vitro drug release study was performed in PBS (pH 7.4) and acetate buffer (pH 5.5) to simulate physiological and tumor microenvironments, respectively. Cell-based assays on different cell lines evaluated cellular uptake, cytotoxicity, apoptosis  induction and ROS generation. Hemocompatibility studies and in-vivo acute toxicity studies were performed to evaluate the safety of NPs. A pharmacokinetic study was conducted in Sprague-Dawley rat for evaluating key pharmacokinetic parameters of co-loaded formulation. In-vivo anticancer efficacy study of SOR and VTX co-loaded PLGA-NPs was investigated in 4T1 cell line-induced breast cancer model using female BALB/c mice.

Results: ¹H NMR confirmed CPBA conjugation by absence of peak at 13 ppm. Presence of ester peak at 1730 cm^-1 in FTIR spectra corroborates the synthesis of P407-CPBA conjugate. Factor screening identified polymer concentration and theoretical drug loading, aqueous volume and as significant factor affecting particle size, PDI, Practical drug loading (PDL) of SOR and VTX (Figure 1). These were optimized using BBD, predicting particle size, PDI, % PDL (VTX and SOR) to be183.7 nm , 0.086, 8.59 %,  and 9.37 % respectively (Figure 1). Morphology studies showed smother surface for PLGA (SOR+VTX)-NPs compared to CPBA-PLGA (SOR+VTX)-NPs (Figure 1). Among cryoprotectants (sucrose, dextrose, trehalose and mannitol), 5 % w/v  sucrose yielded lowest reconstituted index (Figure 1). In-vitro drug demonstrated  pH responsive behaviour, with enhanced release at pH 5.5, consistent with tumor microenvironment (Figure 1). Cytotoxicity assay revealed a 4.47-fold, 1.54-fold, 2.28-fold reduction in IC₅₀ compared to free SOR+VTX in MDA-MB231, A549, HeLa cell, respectively. (Figure 2). In the presence of ferrostatin-1 (Ferroptosis inhibitor), a significant increase in IC₅₀ confirmed ferroptosis mediated cell death (Figure 2). Coumarin C6- labelled qualitative uptake study demonstrated higher internalization in CPBA-PLGA-NPs in MDA-MB-231 and A549 cells (Figure 2). Quantitative uptake study demonstrated time dependent uptake of SOR and VTX. (SOR+ VTX) CPBA-PLGA NPs demonstrated 1.163-fold higher apoptotic index compared to free SOR+VTX (Figure 2). Hemocompatibility test showed negligible hemolysis. Serum toxicity marker (AST, ALT, BUN and creatinine) showed no significant difference between CPBA-PLGA NPs and free drugs. Pharmacokinetic analysis revealed higher C_max, AUC and MRT 1.22-fold, 6.05-fold, 3.38-fold for SOR and 1.05-fold,4.93-fold,2.06-fold for VTX compared to free SOR and VTX respectively (Figure 3). In-vivo anticancer study showed reduced tumor volume , tumor weight and tumor burden in CPBA-PLGA(SOR + VTX)-NPs, signifying enhanced therapeutic outcome of  co-loaded CPBA-PLGA NPs (Figure 3).

Conclusion: The co-delivery of Sorafenib and Venetoclax via CPBA-functionalized PLGA nanoparticles demonstrated a synergistic anticancer effect by simultaneously inducing ferroptosis and apoptosis. The formulation enhanced cellular uptake, improved pharmacokinetics, and significantly reduced tumor growth in-vivo, while maintaining safety. This strategy offers a promising platform for effective, targeted treatment of breast and other solid tumors.

References: [1] F. Bray, M. Laversanne, H. Sung, J. Ferlay, R.L. Siegel, I. Soerjomataram, A. Jemal, Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J Clin 74 (2024) 229–263. https://doi.org/10.3322/caac.21834.
[2] G. Rishi, G. Huang, V.N. Subramaniam, Cancer: The role of iron and ferroptosis, The International Journal of Biochemistry & Cell Biology 141 (2021) 106094. https://doi.org/10.1016/j.biocel.2021.106094.
[3] Q. Li, K. Chen, T. Zhang, D. Jiang, L. Chen, J. Jiang, C. Zhang, S. Li, Understanding sorafenib-induced ferroptosis and resistance mechanisms: Implications for cancer therapy, European Journal of Pharmacology 955 (2023) 175913. https://doi.org/10.1016/j.ejphar.2023.175913.
[4] X. Xu, W. Ma, G. Qiu, L. Xuan, C. He, T. Zhang, J. Wang, Q. Liu, Venetoclax Overcomes Sorafenib Resistance in Acute Myeloid Leukemia by Targeting BCL2, Biology (Basel) 12 (2023) 1337. https://doi.org/10.3390/biology12101337.
[5] R. Sun, Y. Chen, Y. Pei, W. Wang, Z. Zhu, Z. Zheng, L. Yang, L. Sun, The drug release of PLGA-based nanoparticles and their application in treatment of gastrointestinal cancers, Heliyon 10 (2024) e38165. https://doi.org/10.1016/j.heliyon.2024.e38165.
[6] R. Feng, W. Wang, L. Zhu, H. Xu, S. Chen, Z. Song, Phenylboronic acid-functionalized F127-oligochitosan conjugate micelles for doxorubicin encapsulation, J Biomed Mater Res B Appl Biomater 108 (2020) 3345–3355. https://doi.org/10.1002/jbm.b.34670.

Acknowledgements: The authors are thankful to the Director NIPER, S.A.S. Nagar, India for providing necessary research facilities. The authors would also like to thank Mr. Rahul Mahajan, NIPER S.A.S. Nagar for his technical assistance throughout the research. The authors did not receive any funding for conducting the present research. The authors declare no conflict of interest.


Figure 1: Pictorial presentation of the purpose and outcomes of the present investigation


Figure 2: Illustration of data mentioned in screening, optimization characterization and evaluation of CPBA-PLGA NPs


Figure 3: Illustration of data mentioned for in-vitro cell culture studies, in-vivo pharmacokinetic study, anti-tumor efficacy study and toxicity study