Abstract 4469: Multimodal liposomal organic nanoparticle for immunomodulation and photodynamic combinatorial therapy for triple negative breast cancer

Background: IL-12 is a potent cytokine with promising pre-clinical efficacy in solid tumors, including triple-negative breast cancer (TNBC), by enhancing anti-tumor immunity through Treg reprogramming and boosting CD8+ T cell function. However, its clinical use is limited by systemic toxicity and challenges in targeted tumor delivery. Combining IL-12 with photodynamic therapy (PDT) represents a potential solution to these issues, optimizing therapeutic outcomes. Concept & Novelty: We developed a cationic tetra-lipid organic nanoparticle to co-encapsulate IL-12 and HPPH (a photosensitizer; λmax = 658 nm). Efficient IL-12 encapsulation has been challenging due to steric hindrance and hydrophilic nature. To overcome this, we employed a bipolar solvent system (CHCl3:CH3OH, 2:1), achieving 98% IL-12 loading. The self-assembly of IL-12 in methanol enables its complexation with chloroform, forming hydrogen-bonding through synergistic solvation that enhances encapsulation efficiency. Methodology: (a) Liposomal nanoparticle (LNP) Synthesis: The cationic core lumen for IL-12 encapsulation in LNP was achieved by dissolving DPPC, cholesterol, and DOTAP in a 13:5:1 molar ratio in CHCl3. IL-12 (2 mg/mL in CH3OH) was added and stirred for 1 hour. Subsequently, DSPE-PEG-2000-NH2 conjugated with HPPH and DiR in CHCl3 added dropwise and stirred under inert conditions. Solvent removed to form lipid cake (15 mg/L total lipid concentration), and hydrated with PBS. LNP achieved with sonication and multiple freeze/thaw cycles. Purified with centrifugation-dialysis. (b) In-Vivo: 4T1 murine model of breast cancer was developed with 20 Balb/c mice. Double-blind segregation in 5 groups (1 test + 4 control) was done. The test group received intravenous (i.v.) injections of IL-12 and HPPH-loaded LNP (IL-12@LNP-DiR@HPPH) and treated with laser. The control groups received either/or free IL-12/IL-12@LNP-DiR/LNP/No treatment. The biodistribution of LNP was monitored through time-dependent IVIS imaging. Results & Discussion: We synthesized 92 nm IL-12@LNP-DiR@HPPH nanoparticles with surface charge of 0-0.4 mV, indicating stability for biological use. After intravenous injection, maximum DiR fluorescence was observed at tumor site at 6 hours. At this time point, photodynamic therapy with IL-12@LNP-DiR@HPPH resulted in 80-94% tumor volume regression and increased cytolytic T cell presence in the tumor, compared to controls. No toxicity, ulceration, or metastasis was observed in the treatment group during the therapeutic window. Conclusion: We present a novel platform that addresses the challenge of multiple drug loading including efficient loading of IL-12 and successful delivery to the tumor site. A crosstalk between IL-12-mediated immune response and photo-triggered ROS generation promises a potential therapeutic strategy for TNBC with interventions. Citation Format: Sayantan Sinha, Dhruv Bhatnagar, Anne Frei, Heather Himburg, Amit Joshi. Multimodal liposomal organic nanoparticle for immunomodulation and photodynamic combinatorial therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4469.