(M0930-02-11) Exploring Phenylboronic Acid Conjugated pH-Sensitive Liposomes for Targeted Codelivery of Cisplatin-Chlorambucil Prodrug and Venetoclax for Triple-Negative Breast Cancer Management

Purpose: Cisplatin is a potent anti-cancer agent and is highly effective in difficult-to-treat cancers like triple-negative breast cancer (TNBC). Breast cancer gene dysfunction in TNBC leads to defective DNA damage repair system which makes them sensitive to DNA-damaging drugs like cisplatin. However, the clinical utilization of cisplatin is restricted because of its dose-dependent side effects, development of resistance, etc. [1]. Cisplatin prodrugs have proven to overcome such drawbacks and therefore we have synthesized cisplatin-chlorambucil (CP-CBL) prodrug. Further, venetoclax has demonstrated cytotoxic activity in tumor cells that overexpress Bcl-2 protein [2]. In this investigation we have designed phenylboronic acid (PBA) conjugated pH-sensitive liposomes co-loaded with CP-CBL and VTX (CP-CBL + VTX TLPs) to achieve tumor-specific drug delivery as well as synergistic drug effect of CP-CBL and venetoclax (Figure 1). PBA will assist in achieving active tumor targeting through binding of phenylboronate groups to sialic acid residues overexpressed on tumor cells [3].

Methods: Initially, CP-CBL was synthesized by reacting oxoplatin (reaction of cisplatin with hydrogen peroxide) and CBL anhydride [reaction of CBL with N,N′-Dicyclohexylcarbodiimide (DCC) in DCM] in dry DMF. Next, D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) was conjugated with 4-carboxyphenyl boronic acid (PBA) to form TPGS-phenylboronate (TPGS-PBA) by esterification reaction between 4-carboxyphenylboronic acid and TPGS in presence of DCC and 4-Dimethylaminopyridine (DMAP). Both CP-CBL and TPGS-PBA were characterized using NMR and mass spectroscopy. Next, CP-CBL + VTX TLPs were obtained by solvent injection method. Different formulation parameters were optimized. The TLPs were characterized for vesicle size, polydispersity index, % entrapment efficiency and morphology, and further lyophilized by screening suitable cryoprotectants followed by stability analysis (as per ICH guidelines). In vitro studies such as % cumulative drug release (at pH 5.5 and 7.4 for 72h) with its kinetic data analysis (DDsolver), and hemotoxicity analysis were performed. Cell culture studies including cell viability, apoptosis (Annexin V-Cy3 Kit), cellular drug uptake (qualitative and quantitative with inhibitors), ROS analysis (DCFH-DA dye), Mitochondrial membrane potential (JC1 assay), Nuclear staining (Hoechst 33342), DNA damage (8-OHdG levels) and Caspase 3 assays were performed in MDA-MB-231 (human) TNBC cell line [4]. Further pharmacokinetic analysis (using SD rats), anticancer efficacy (using 4T1 cell line-induced tumor model in BALB/c mice) and toxicity studies were carried out at in vivo level (liver and kidney biomarker levels and organ histology imaging) [5].

Results: ¹H and ¹³C NMR spectra showed characteristic peaks of CP-CBL and TPGS-PBA. Molecular ion peaks at m/z 906.81 [M–H]⁺ of CP-CBL, and at 1581, 1625 and 1669 of TPGS-PBA confirmed their successful conjugation. The optimized TLPs composed of DOPE:HSPC:CHEMS:Cholesterol:TPGS-PBA in mole ratio 4:4:2:2:0.5, organic:aqueous ratio of 1:5 where THF+methanol+acetone composed the organic phase and deionised water as dispersion media, and 1:1 w/w VTX:CP-CBL. The TLPs exhibited as spherical vesicles in TEM image with particle size 158.9±12.7 nm, PDI 0.246±0.051 and entrapment efficiency 81.4%(VTX) and 78.2%(CP-CBL). 5% w/v sucrose provided lyophilisation optimum outcomes in terms of redispersibility index, reconstitution score and were stable over 6 months. The TLPs followed Higuchi drug release kinetics with < 70% release (pH 7.4) and >85% release (pH 5.5) for both drugs in 72 hrs. The TLPs were least hemotoxic (~10%) than cisplatin (~38%). The TLPs showed ~2-12 folds higher cell cytotoxicity than other groups. Much higher apoptotic index (1.19) was observed or TLPs than CP-CBL + VTX (0.843) and free cisplatin (0.293). Further, highest cellular drug uptake was found for TLPs than cisplatin and CP-CBL, both in presence and absence of uptake inhibitors indicating role of phenylboronate-sialic acid interaction in TLP internalisation. In ROS analysis study, CP-CBL + VTX resulted in generation of higher levels of ROS compared to free drug combination, while TLPs showed highest ROS level as compared to other groups. The TLPs showcased higher mitochondrial membrane depolarization potential, higher intensity of nuclear condensation, increased 8-OHdG levels and highest level of caspase-3 expression than free drugs indicating higher oxidative stress, DNA strand breaks and apoptosis (Figure 2). The TLPs showed improved pharmacokinetic parameters where the t1/2 increased by ~23.1-folds (CP-CBL) and ~7.7-folds (VTX). The TLPs treated animals showed ~ 25.2-folds higher tumor volume reduction than cisplatin. Finally, the toxicity study revealed significantly lower levels of nephrotoxicity biomarkers (BUN and creatinine), kidney homogenate biomarkers (MDA, IL-6 and TNF) and no major morphological changes in TLP treated group, proving better tolerability of TLPs. This could be attributed to reduced accumulation of TLPs in vital organs due to PBA-mediated active tumor targeting of drugs (Figure 3)

Conclusion: The developed TLPs showcased sustained drug release, improved cytotoxicity, enhanced cellular and nuclear uptake, and were hemocompatible. PBA as a targeting ligand improved the targetability of the TLPs. Also, the TLPs enhanced the pharmacokinetic properties as well as the anticancer efficacy of the drugs. Therefore, the developed formulation proved to be a safe and efficacious system for the management of TNBC.

References: [1] Jin, Juan, et al. Cancer Biology & Therapy 18.6 (2017): 369-378.
[2] Alhoshani, Ali, et al. OncoTargets and therapy 13 (2020): 13357.
[3] SS Pindiprolu, Sai Kiran, et al. Nanomedicine 15.16 (2020): 1551-1565.
[4] Date, Tushar, et al. ACS Biomaterials Science & Engineering 8.6 (2022): 2349-2362.
[5] Chaudhari, Dasharath, et al. ACS Biomaterials Science & Engineering 8.8 (2022): 3473-3484.

Acknowledgements:The authors are thankful to Neon Laboratories Ltd., India, for providing cisplatin as a gift sample for research purposes and also grateful 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 representation of purpose and outcome of the present investigation

Figure 2. Illustration of data for mentioned in vitro and cell culture-based studies

Figure 3. Illustration of data for mentioned in vivo pharmacokinetic, anticancer efficacy and toxicity studies