(M1230-01-06) Development of Inhalable CXCR4 Targeted Polymeric Nanoemulsions for Delivery of RNAi Therapeutics to Enhance Anti-PD-L1 Immunotherapy in Lung Tumors

Purpose: Lung cancer is one of the most prevalent and lethal malignancies worldwide with a dismal 5-year survival rate of about 20%. While immune checkpoint inhibitors, such as anti-PD-L1 therapy, have demonstrated promising results [1], a significant proportion of patients exhibit suboptimal responses due to an immunosuppressive tumor microenvironment (TME) that impedes adequate lymphocytic infiltration and T-cell activation [2]. Combining various therapeutic modalities such as chemotherapeutic agents, targeted drug delivery to the cancerous cells, and RNA interference via siRNA/miRNA, can potentially supplement anti-PD-L1 immunotherapy, enhancing its efficacy against lung cancer. We hypothesized that inhaled delivery of a siPD-L1/miR-34a cocktail would facilitate lung-specific immune checkpoint inhibition, simultaneously target multiple oncogenic pathways within the TME, and minimize systemic exposure. We utilized 4T1 lung metastasis mouse model to investigate the combined effect of intravenous nab-paclitaxel and the siPD-L1/miR-34a cocktail delivered via inhalation. Low doses of nab-paclitaxel can induce immunogenic cell death by recruiting innate and adaptive immune effectors into the TME [3]. We developed a perfluorocarbon nanoemulsion, PAMD-C@PFOB (PAMD-C: polymeric AMD3100-Cholesterol, PFOB: perfluorooctylbromide) to formulate the RNA cocktail. The nanoemulsion demonstrated stable aerosolization and achieved pulmonary delivery via inhalation. PAMD is a polymeric CXCR4 antagonist hypothesized to target and inhibit the overexpressed CXCR4 receptors in tumors, potentially reducing tumor fibrosis and facilitating increased T-cell infiltration into the tumor site. This proof-of-concept study demonstrates the potential of using inhalation-based RNA delivery systems in tandem with traditional therapeutic approaches to synergistically improve the efficacy of lung cancer treatment.

Methods: PAMD-C was synthesized via Michael addition, followed by the preparation of PAMD-C@PFOB oil-in-water nanoemulsion (1% v/v) using ultrasonication. Physicochemical characterization, including pre- and post-aerosolization size and charge was conducted using dynamic light scattering. The si/miRNA binding ability of PAMD-C@PFOB was assessed through agarose gel retardation assay. To investigate the extent of CXCR4 antagonism by the emulsion, a CXCR4 redistribution assay was performed in U2OS cells expressing GFP-tagged CXCR4, with SDF1 as the CXCR4 agonist. In vitro cellular uptake and intrapulmonary distribution of Cy3 PAMD-C@PFOB/Cy5.5 siRNA-labeled emulsion polyplexes (EPs) were evaluated in H1975 cell lines and frozen lung sections, respectively, using confocal microscopy and flow cytometry. Biodistribution of labeled EPs was estimated through ex vivo fluorescence imaging and quantification at different time points post-aerosolized administration in healthy mice. A lung metastatic breast cancer model was generated by intravenous injection of 2x10⁵ luciferase-tagged 4T1 breast cancer cells in 9-week-old female Balb/c mice. Nab-Paclitaxel was administered intravenously at a safe dose of 15 mg/kg on days 5 and 9 post-tumor implantation, while nebulized EPs (20 µg si/miRNA cocktail per mouse) were administered on days 6, 10, and 14, followed by autopsy on day 16. Bioluminescent imaging was performed on days 9 and 16 to estimate differences in tumor burden across treatment groups, and lung metastatic lesions were quantified after staining with Bouin's solution. H&E and Trichrome staining were performed to visualize tumor necrosis and fibrosis, respectively. Flow cytometry was employed to quantify immune subsets, such as cytotoxic T cells, Tregs and MDSCs.

Results: The nanoemulsions had a mean size of 150 nm and zeta potential of +20 mV, enabling successful electrostatic binding of anionic si/miRNA and demonstrated complete binding of RNA at weight ratios ≥ 2. They exhibited effective CXCR4 targeting and antagonism (Fig. 1a). In vitro studies revealed efficient cellular uptake of EPs in 60% of lung cancer cells (Fig.1b). After aerosolization, the nanoemulsions had a mean diameter of 220 nm which is an order of magnitude smaller than the aerodynamic diameter of aerosolized droplets (2-5 μm), promising for deep-lung delivery of the EPs. Biodistribution studies demonstrated five-fold higher lung accumulation compared to other organs, with peak fluorescence achieved 12 hours post-administration, and confocal microscopy confirming deep lung delivery and retention (Fig. 2a, b). Therapeutic studies confirmed inhalation of EPs carrying siPD-L1/miR-34a, combined with nab-paclitaxel, drastically reduced tumor burden compared to monotherapies (Fig. 3a). H&E and Trichrome staining clearly showed the impact of the combination treatment on reducing tumor burden and fibrosis (Fig. 3b). Flow cytometry analysis of resected tumors revealed increased CD8+ T cell infiltration, elevated cytotoxic cytokine production and reduced populations of Tregs and MDSCs following the combination treatment approach.

Conclusion: We developed a targeted nanoemulsion delivery platform (PAMD-C@PFOB) for pulmonary administration of RNAi therapeutics against lung tumors. The cationic nanoemulsions (~150 nm) efficiently complexed anionic siRNA/miRNA, enabled CXCR4 targeting, and demonstrated effective cellular uptake in vitro. Upon aerosolized administration in healthy mice, the emulsion polyplexes displayed preferential deep lung accumulation upto 24 hours, with minimal off-target biodistribution. In lung tumor model, inhalation of EPs carrying siPD-L1 and miR-34a, combined with nab-paclitaxel chemotherapy, significantly reduced tumor burden compared to monotherapies. This correlated with enhanced cytotoxic T-cell infiltration, elevated anti-tumor cytokines, and reduced immunosuppressive cells within tumors. Overall, this targeted inhalable nanoemulsion strategy presents a promising platform for pulmonary delivery of RNAi-based therapeutics against lung cancer.

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. Zhong H, Han B, Tourkova IL, Lokshin A, Rosenbloom A, Shurin MR, Shurin GV. Low-dose paclitaxel prior to intratumoral dendritic cell vaccine modulates intratumoral cytokine network and lung cancer growth. Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5455-62.

Acknowledgements: Flow cytometry core facility (UNMC), Tissue sciences facility (UNMC), Confocal Microscopy facility (UNMC)

Funding: P30 GM127200, R01 CA235863, R01 DK120533, R01 DK124095


Fig. 1 (a) Assessment of CXCR4 Antagonism of PAMD-C@PFOB by CXCR4 Redistribution Assay (b)In vitro cellular uptake of EPs at different weight ratios by Flow Cytometry



Fig. 2 (a) Biodistribution- Ex vivo fluorescence imaging and quantification of Emulsion Polyplexes in healthy mice at different time points (b) Intra lung distribution of PAMD-C@PFOB/siRNA emulsion polyplexes


Fig. 3 (a) In vivo bioluminescence imaging of mice bearing 4T1.luc lung metastasized tumors after different treatments (b) H&E and Trichrome staining of harvested lungs on day 16