(M1330-06-36) Exploiting Redox and Lipid Metabolic Vulnerabilities via Albumin-Encapsulated Oxaliplatin–Oleic Acid Prodrug for the Management of Triple-Negative Breast Cancer

Purpose: Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of breast cancers and is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This phenotype renders TNBC unresponsive to endocrine and HER2-targeted therapies, leaving chemotherapy as the systemic treatment backbone. Despite initial chemosensitivity, TNBC remains prone to early relapse, high metastatic rates, and poor survival outcomes. Platinum-based chemotherapies, such as cisplatin and carboplatin, are increasingly utilized due to their DNA crosslinking ability, particularly in tumors harboring BRCA1/2 mutations or homologous recombination deficiencies. Among them, oxaliplatin (OXA) offers unique advantages by forming distinct DNA adducts and inducing immunogenic cell death. However, its clinical application in TNBC remains constrained by its hydrophilic nature (Log P −1.81), limited tumor penetration, systemic toxicity (particularly peripheral neuropathy), and rapid deactivation by elevated intracellular glutathione (GSH) levels—a hallmark characteristic of TNBC cells. Meanwhile, TNBC tumors exhibit heightened lipid metabolism and enhanced fatty acid transport, resulting in a lipid-rich microenvironment that can be exploited for selective drug delivery. To capitalize on this distinctive tumor biology and address the pharmacokinetic and biological limitations, we synthesized a redox-responsive prodrug by conjugating oxaliplatin (Pt(IV) form) to oleic acid (OA), a monounsaturated fatty acid with tumor-targeting and pro-apoptotic properties. For enhanced tumor selectivity, systemic stability, and controlled delivery, the OXA–OA conjugate was encapsulated into genipin-crosslinked albumin nanoparticles (OXA–OA Alb NPs), leveraging albumin's affinity for gp60 and SPARC receptors overexpressed in TNBC. This dual-targeting strategy—combining redox-sensitive drug release with metabolic and receptor-mediated targeting—was designed to maximize therapeutic efficacy while minimizing systemic toxicity.

Methods: The OXA–OA conjugate was synthesized by oxidizing OXA to a Pt(IV) intermediate using hydrogen peroxide, followed by esterification with OA under anhydrous conditions. Structural confirmation was performed via proton nuclear magnetic resonance (^1H NMR), Fourier-transform infrared (FTIR) spectroscopy, and high-resolution mass spectrometry (HRMS). Lipophilicity was determined by octanol–water partitioning (Log P measurement), and redox sensitivity was assessed by incubating the conjugate in 10 mM GSH at 37 °C, monitoring OXA release via high-performance liquid chromatography (HPLC). For NPs fabrication, bovine serum albumin was desolvated with ethanol and crosslinked with genipin to encapsulate the OXA–OA prodrug. OXA–OA Alb NPs were characterized for particle size, polydispersity index (PDI), zeta potential, morphology (transmission electron microscopy), and encapsulation efficiency. Lyophilization with various cryoprotectants (mannitol, sucrose, trehalose, dextrose) was performed to ensure stability, and redispersibility was evaluated by Redispersibility Index (RDI). Hemocompatibility was assessed by hemolysis assays using rat red blood cells (RBCs), and RBC morphology was analyzed via scanning electron microscopy (SEM). In vitro cytotoxicity was evaluated using MTT assays in 4T1 and MDA-MB-231 TNBC cell lines, with IC₅₀ values calculated via CompuSyn software. Cellular uptake was analyzed using Coumarin-6-loaded Alb NPs by confocal microscopy, and apoptosis induction was quantified by Annexin V–FITC/propidium iodide (PI) staining. In vivo antitumor efficacy and systemic safety were tested in orthotopic 4T1-bearing BALB/c mice treated intravenously with free OXA, OXA–OA, or OXA–OA Alb NPs, monitoring tumor growth, serum biochemistry, and histopathology of major organs.

Results: Successful synthesis of the OXA–OA conjugate was confirmed by ^1H NMR, FTIR, and HRMS analyses. The conjugate exhibited increased lipophilicity (Log P: 3.5 ± 0.56) compared to free OXA (−1.81 ± 0.34) and showed rapid GSH-mediated reduction (~90% conversion within 3 hours), supporting selective intracellular activation. Encapsulation into Alb NPs yielded uniform particles (140.5 ± 14.3 nm), low PDI (0.256 ± 0.054), and encapsulation efficiency of 80.5 ± 5.5%, with maintained stability after lyophilization using 5% trehalose (RDI ≈ 1.2). Hemolysis assays showed minimal RBC lysis, and SEM confirmed preserved RBC morphology, indicating excellent blood compatibility. In vitro, OXA–OA Alb NPs significantly enhanced cytotoxicity, reducing IC₅₀ to 0.19 ± 0.36 µg/mL (4T1) and 0.20 ± 0.16 µg/mL (MDA-MB-231), reflecting 25- and 30-fold reductions versus free OXA, and 36- and 43-fold reductions versus free OA. This potentiation was attributed to synergistic interactions between OXA-mediated DNA damage and OA’s lipid-mediated apoptosis, highly effective within TNBC’s lipid-enriched, redox-active environment. Apoptosis indices (1.099 in 4T1 and 1.147 in MDA-MB-231) were 3.13- and 1.81-fold higher than OA, and 1.97- and 1.77-fold higher than OXA, respectively. In vivo, OXA–OA Alb NPs achieved ~90% tumor inhibition and outperformed free OXA and unconjugated OXA–OA. Systemic safety was supported by normal serum levels of ALT, AST, BUN, and creatinine, and histopathological analysis demonstrated preserved organ architecture with markedly reduced tissue disruption compared to other groups.

Conclusion: By integrating a GSH-responsive OXA–OA conjugate into Alb NPs, this study presents a rationally designed, dual-targeting nanotherapeutic that exploits TNBC’s redox imbalance and lipid-dependency. The formulation synergistically enhanced cytotoxicity, improved tumor selectivity, and minimized systemic toxicity, offering a significant therapeutic advantage in preclinical models. These findings support the potential of lipid–metal–protein biocomposites as modular platforms for overcoming pharmacological barriers associated with conventional chemotherapeutics in hard-to-treat cancers and providing a foundation for further exploration in advanced metastatic settings.

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Acknowledgements: The authors gratefully acknowledge the Director, NIPER S.A.S. Nagar, India, for providing the necessary research infrastructure and support. Technical assistance provided by Mr. Rahul Mahajan (NIPER S.A.S. Nagar) is sincerely appreciated. The authors declare that no external funding was received for the conduct of this research. All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC/23/79), NIPER S.A.S. Nagar, and performed in accordance with CCSEA guidelines. The authors declare no conflicts of interest.


Workflow illustrating the synthesis and functional characterization of the OXA-OA conjugate—including lipophilicity, redox sensitivity, and activation kinetics—followed by formulation of OXA-OA Alb NPs and their evaluation through CD spectroscopy, physicochemical characterization, lyophilization stability, in vitro drug release, and hemolysis analysis.


Experimental evaluation of OXA-OA Alb NPs through in vitro studies including cellular uptake, cytotoxicity, and apoptosis assays in 4T1 and MDA-MB-231 breast cancer cells, followed by in vivo assessment of antitumor efficacy and acute toxicity in a murine model.