Inhalable Perfluorocarbon RNA Nanocapsules Bypass Immune Clearance While Targeting Lung Epithelial and Lung Tumor Cells

Abstract

Inhalation RNA therapy offers to transform treatment of pulmonary diseases, yet mucus trapping, immune clearance, and navigation of heterogeneous lung tissue architecture still prevents RNA from reaching its target cells. Here, we develop perfluorocarbon (PFC) RNA nanocapsules that show negligible immune clearance, minimal inflammatory response, and efficient mucus transport, while passively homing to lung epithelial and tumor cells. After a single aerosolized dose in orthotopic lung metastasis model, more than 60% of tumor cells and most type II alveolar and bronchial epithelial cells internalized the nanocapsules, with observed pulmonary retention exceeding 48 h. The nanocapsule provoke negligible cytokine release, enabling repeated dosing. Treatment with therapeutic miR34-a suppresses metastatic outgrowth, potentiates anti-tumor immunity, and almost doubles median survival relative to control paclitaxel chemotherapy. By combining unique PFC disposition with RNA versatility, the delivery platform overcomes the main biological barriers for inhalable RNA medicines and opens a translatable path for treating diverse pulmonary diseases.

Keywords: RNA delivery; cancer therapy; inhalable genetic medicine; perfluorocarbon.

Figure 1.. Schematic illustration of the mechanism of action of PFC-based RNA nanocapsules in the treatment of lung metastases.

RNA nanocapsules encapsulating miR34a are formulated by mixing fixed ratios of nanoemulsion to miRNA. RNA nanocapsules are administered by microsprayer aerosolizer, intratracheally. CXCR4 is overexpressed on malignant lung metastatic cells which provides passive homing of the RNA nanocapsules to the sites of metastasis, aiding in the uptake and retention of RNA nanocapsules. Following endocytosis, the miR34a released from RNA nanocapsules suppresses PD-L1 expression and enhances cleavage of Caspase 3, thereby inducing apoptosis and alleviating metastatic tumors. miR34a nanocapsules exhibit multiple therapeutically beneficial mechanisms: we show that they induce apoptosis through increased levels of cleaved Caspase 3 and reduced Ki-67 abundance. Additionally, miR34a nanocapsules reduce the immunosuppressive milieu by reducing PD-L1 expression, allowing for increased tumor lymphocyte infiltration and reduction in granulocytic myeloid derived suppressor cells. Created with Biorender.com.

Figure 2.. Formulation and characterization of PAMD-C@PFOB/miRNA nanocapsules.

a) miRNA condensing ability of PAMD-C@PFOB nanoemulsion by agarose gel retardation assay. b) Mean hydrodynamic particle size and c) mean zeta potential of the RNA nanocapsules at different w/w ratios by DLS. d) In vitro luciferase gene silencing evaluation of PAMD-C@PFOB/siLuc nanocapsules in 4T1.Luc cells pre- and post-aerosolization. e) Representative TEM image of the RNA nanocapsule before aerosolization and representative CryoTEM images of the aerosolized RNA nanocapsules (w/w 4), scale bar = 100 nm in all three images. f) Aerodynamic particle size distribution from APS and (g) Fluorescence intensity from Cy3-labelled aerosolized RNA nanocapsules across different stage cut-off diameters of the NGI at 15 L/min. h) Colloidal stability of the nanoemulsions and the RNA nanocapsules at room temperature for three days. i) Snapshots from AAMD simumlations for various systems (PAMD-C + 1-fluorobutane + excipient). The first snapshot titled ‘Bare’ denotes the reference system with PAMD-C and 1-fluorobutane. The other snapshots are titled using the name of the excipient included in the corresponding simulation. (j) and (k) show the evolution of the % of excipient molecules bound to the core 1-fluorobutane + PAMD-C particle. In (j-k), the solid lines are obtained after median filtering and the shaded regions denote the raw output data. The mean values and SD bars in (b-d) are obtained using n = 3 replicates each. **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Figure 3.. Biodistribution of PAMD-Ch@PFOB/miRNA nanocapsules in healthy and LMBC mice.

(a) Representative ex-vivo fluorescence images of major organs at different time points post aerosolized administration of nanocapsules in healthy and LMBC mice. b) Mean fluorescence intensity of the miRNA retained in the lungs of healthy and LMBC mice at different time points post-aerosolized administration of the nanocapsules (c) Fluorescence intensity from Cy5.5 miRNA with and without complexation to Cy3-PAMD-C@PFOB nanoemulsion. (d) Representative confocal images of lung sections 24 h post administration of labeled RNA nanocapsules in healthy mice. Scale bar = 1000 μm . (e) Representative confocal images of lung sections 24 h post administration of labeled RNA nanocapsules in LMBC mice injected with 4T1-Luc-mCherry cells. Scale bar = 500 μm. n = 3 per group . * p < 0.05, ns-not significant.

Figure 4.. Cellular distribution and safety assessment of miRNA nanocapsules in healthy and LMBC-mCherry mouse lungs.

a) Pulmonary immune response profile following RNA nanocapsule administration. Analysis of lung tissue protein lysates from healthy controls, placebo-treated (DI H₂O 60 μl), and RNA nanocapsule-treated (20 μg miRNA) mice at 12h post-administration (n = 3). A panel of 40 immune mediators was evaluated, including cytokines (TNF, IL-6, IL-12p70, IL-1β, IFNγ, IL-2, IL-17, IL-5, IL-16, TIMP1, TREM1, IL-23, IL-10, IL-1ra, IL-13 and IL-4), chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL12, CCL17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL12 and CXCL13), growth factors (G-CSF, GM-CSF, M-CSF, IL-3 and IL-7) and cell adhesion molecules (CD54 and complement component 5a) (Data are presented as mean fold change relative to healthy controls. Raw images are shown in Supplementary Figure 8. (b) Flow cytometric analysis of immune cell populations showing uptake of Cy5.5-labeled nanocapsule miRNA in healthy mouse lungs 4 h post-administration. Distribution of Cy5.5 labeled nanocapsule miRNA in healthy mouse lung CD45− populations, showing (c) percentage uptake per cell type and (d) percentage of total administered dose. (e,f) Similar analysis in LMBC mouse lungs showing (e) cellular uptake and (f) total dose distribution in CD45− subtypes. (g) Uptake of Cy3- labeled nanocapsule polymer and Cy5.5- labeled nanocapsule miRNA by 4T1-mCherry tumor cells isolated from the lungs of LMBC mice.

Figure 5.. In vivo antitumor efficacy of the miR-34a nanocapsules in LMBC mice.

(a) Experimental timeline showing the dosing regimen for LMBC mice (b) In vivo bioluminescence imaging and quantification of total bioluminescent flux from the lungs of LMBC mice post different treatments. (c) H&E staining of lung tissues collected on day 16 (scale bar, 5mm). Representative images of immunohistochemical staining across different treatment groups and Percentage of positively stained area for (d) Epcam (e) Cleaved Caspase 3 (CC3) (f) Ki67. n = 3 mice per group. Areas in the sections having metastatic tumors only were quantified. ImageJ was used for quantification of the images. Three fields for each section was quantified. Scale bar = 300 μm. (g) Survival curves after different treatments (n=5) analyzed using log-rank test. Median survival: placebo (24 days), nab-ptx (35 days), miR34a nanocapsule (47 days) (h) Plots representing percentage body weight change in the different treatment groups (n = 5). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05

Figure 6.. Effect of miR34a nanocapsule treatment on the levels of select immunological effectors in LMBC mice.

Representative IHC staining and Percentage of positively stained area for (a) CD45 and (b) F4/80. Levels of (c) Cytotoxic T-cells, (d) Helper T-cells, (e) G-MDSCs, and (f) NK cells across different treatment groups. Pulmonary mRNA levels of (g) PDL1 and (h) CXCR4. GAPDH was used as an internal control. n = 3, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05