(T1230-01-05) Development of Inhalable Polymeric Nanoemulsions for Delivery of Gene Silencing Therapeutics in Lung Tumors

Purpose: Lung cancer is one of the most common and deadliest cancers globally, accounting for the highest number of cancer-related deaths, with a 5-year survival rate of around 20%. While anti-PD-L1 monoclonal antibody immunotherapy holds promise in combating various cancers [1], studies suggest its efficacy may be compromised by factors such as poor lymphocyte infiltration and the emergence of an immunosuppressive microenvironment [2]. RNA interference via siRNA/miRNA presents novel possibilities in gene silencing therapeutics, particularly in lung cancer, with inhalation delivery offering a targeted, non-invasive approach to directly address specific genetic factors in the lung [3]. We hypothesized that immune check point inhibition using siPD-L1/miR-34a cocktail delivered via inhalation route can enhance anticancer effects through miRNA’s global regulatory role and minimizing systemic exposure. We have developed an emulsion polyplex (EP) as inhalable delivery system for lung cancer treatment, which can deliver si/miRNA encapsulated in PAMD-C@PFOB nanoemulsions (PAMD-C: polymeric AMD3100-Cholesterol, a copolymer for CXCR4 inhibition, PFOB: perfluorooctylbromide).

Methods: PAMD-C was synthesized by Michael addition followed by preparation of PAMD-C@PFOB oil-in-water nano emulsions (1% v/v) using ultrasonication. Physiochemical characterization for size, charge, and stability over time was carried out using dynamic light scattering, followed by assessment of si/miRNA binding ability of PAMD-C@PFOB using agarose gel retardation assay. To understand the extent of CXCR4 antagonism by the emulsion, CXCR4 redistribution assay was performed in U2OS cells expressing GFP-tagged CXCR4 and using SDF1 as the CXCR4 agonist. Both the polymer and RNA were labelled; confocal microscopy and flow cytometry were performed to understand the in vitro cellular uptake of the si/miRNA in double positive populations in H1975 lung cancer cells. Lung metastatic breast cancer model was generated by IV injection of luciferase-tagged 4T1 breast cancer cells to establish lung tumors in Balb/c mice. Bioluminescent imaging was performed to quantify metastases to the lung followed by administration of the nebulized EPs using 20 µg si/miRNA cocktail per mouse.  The biodistribution was determined at 6 h and 24 h post inhalation using whole body and ex vivo IVIS imaging of lungs and other major organs. In the therapeutic study,  i) H&E staining was performed to visualize tumor necrosis, ii) immunofluorescence staining was done on resected tumors to analyze immune cell infiltration and activation, iii) flow cytometry was performed to quantify immune subsets like Tregs and MDSCs, iv) ELISA was performed to measure cytokines reflective of the immune response, and v) RT-PCR was done to evaluate knockdown of PD-L1 and other genes in the pathway.

Results: The developed PAMD-C@PFOB had a size of 170 nm and zeta potential of +20 mV which enabled successful binding of anionic si/miRNA to the nanoemulsion via electrostatic interactions. Using the gel retardation assay we found that at w/w ratio ≥ 2, the RNA was completely bound by the nanoemulsions. The size of the nanoemulsion increased by ~5 nm over 72 h, indicating their kinetic stability. Treatment with PAMD-C@PFOB showed effective CXCR4 targeting in U2OS cells (Figure 1). Flow cytometry results showed 60% of double positive populations indicating successful uptake of siRNA and the polymer in the formulation. Following nebulized delivery of the synthesized EPs to the mice, a five-fold higher lung distribution was achieved compared to other major organs thus showing strong lung delivery and retention of the nanocomplex (Figure 2, 3). The EPs carrying anti-PD-L1 siRNA and miR-34a significantly reduced tumor burden based on histological analysis. When compared with placebo, the EP treatment also prolonged median survival from 20 to 50 days. Analysis of resected tumors showed the EPs increased CD8+ T cell infiltration, calreticulin exposure, and cytotoxic cytokine production, while also reducing immunosuppressive cell populations like Tregs and MDSCs. Tumor levels of fibrosis and immunosuppressive cytokines like IL-10 and TGF-β were also decreased by the EPs.

Conclusion: The inhalation platform developed here enables effective pulmonary delivery of si/miRNA to treat lung cancer by improving siRNA internalization, endosomal escape, lung penetration, and biodistribution compared to conventional polyplexes. CXCR4 antagonism and PD-L1 gene silencing was achieved, thus inhibiting important pathways involved in lung cancer pathology. EPs developed in this study remodeled the tumor microenvironment to enhance anti-tumor immune response, achieving significantly longer survival. In conclusion, this proof-of-concept study demonstrates the potential of using PAMD-C@PFOB to improve the efficacy of pulmonary siRNA therapeutics via inhalation delivery.

References: 1. Zou, Weiping, Jedd D. Wolchok, and Lieping Chen. "PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations." Science translational medicine 8.328 (2016): 328rv4-328rv4.
2. Hegde, Priti S., Vaios Karanikas, and Stefan Evers. "The where, the when, and the how of immune monitoring for cancer immunotherapies in the era of checkpoint inhibition." Clinical Cancer Research 22.8 (2016): 1865-1874.
3. Zhao G, Ho W, Chu J, Xiong X, Hu B, Boakye-Yiadom KO, Xu X, Zhang XQ. Inhalable siRNA Nanoparticles for Enhanced Tumor-Targeting Treatment of KRAS-Mutant Non-Small-Cell Lung Cancer. ACS Appl Mater Interfaces. 2023 Jul 5;15(26):31273-31284. doi: 10.1021/acsami.3c05007. Epub 2023 Jun 24. PMID: 37354089.

Acknowledgements: This work was supported by start-up funds from the University of Nebraska Medical Center(D.O.).

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