(M1430-03-17) Design and Development of Folate Decorated Nanoemulsions as the New Pain Nanomedicines

Purpose: There is an urgent need for an alternate pain-relieving strategy to solve the opioid crisis, which resulted in a staggering 28.5% increase in opioid overdose-related mortality in 2021. In a recent study on the chronic nerve constriction injury (CCI) rat neuropathic pain model, we demonstrated that administration of NSAID-loaded nanomedicine formulated as a theranostic nanoemulsion (NE) significantly reduces pain for up to 4 days following a single low dose (0.24 mg/kg of drug, 100-fold lower than a typical systemic dose in animals) (1, 2). Here we present a novel theranostic NE that is designed to directly engage with activated macrophages, key immune drivers for inflammatory pain. Active macrophage targeting has the potential to improve pain management and imaging agents in the theranostic nanoemulsions support monitoring of therapeutic efficacy. Folate receptor beta (FR-β) is specifically expressed by proinflammatory macrophages (M1 phenotype), and folic acid (FA) has a nanomolar affinity for the FR- β (Kd 0.1–1.0 nM). The goal was to take leverage of this affinity and design, develop, and conduct a preliminary in vitro assessment of the theranostic NE anchored with a folic acid derivative for the delivery of celecoxib (CXB) that specifically inhibits Prostaglandin-endoperoxide synthase 2 (COX-2) in activated macrophages. The NE manufacturing process was designed to be scalable to provide reproducible results.

Methods: Nanoemulsions were prepared by dissolving CXB and folic acid derivative in an optimized solubilizer overnight. The lipophilic near-infrared fluorescent dye, perfluorocarbon (PCE), and non-ionic surfactant solution were combined, and the mixture was processed on a microfluidizer (Microfluidics, M110S) under controlled pressure and at reduced temperature. The developed nanoemulsion was evaluated for long-term colloidal and photo stability. Male macrophages (RAW 264.7) were cultured and nanoemulsions were screened for cell viability using CellTiter-Glo® ATP assay. Drug entrapment and release were assessed by validated HPLC methods. The effects of nanoemulsions on pro-inflammatory markers expressed by macrophages were evaluated using in vitro ELISAs. The impact of surface conjugation with folic acid derivative on nanoemulsion uptake in macrophages was evaluated by fluorescence microscopy and FACS analysis.

Results: The developed triphasic nanoemulsions were constituted of 3 immiscible liquids: perfluorocarbons (PFCs), hydrocarbon oil, and water resulting in small-sized droplets (120 to 180 nm) stabilized by non-ionic surfactants. The average size distribution, surface charge, and drug loading were unaffected by surface conjugation, incorporation of drug, PFCs and dyes (Figure 1A and B). The triphasic nanoemulsions maintained exceptional colloidal stability under elevated temperatures and when mixed with biological media. We observed that, as opposed to free CXB which was toxic even at a low concentration (64 μM), the highest concentration of the CXB loaded nanoemulsion (256 μM) did not alter macrophage cell viability (Figure 2A). The formulated theranostic NE demonstrated a receptor mediated uptake mechanism parallel with phagocytosis (Figure 2B). Tested by ELISA, the anti-inflammatory action of nanoemulsions on macrophages demonstrated a dose dependent decrease in PEG2, the main COX-2 enzyme product. Further, ELISA also showed TNF-alpha released from LPS activated macrophages was reduced in response to nanoemulsions (Figure 3). Similarly, a decrease in IL-6 proinflammatory cytokines was observed in the presence of LPS.

Conclusion: Based on the in-vitro findings, CXB-Folic acid conjugated-NE holds the potential to be tested in vivo owing to the improved safety profile as compared to the free drug. The folate decorated theranostic nanoemulsion shows increased receptor mediated uptake in macrophages and exerts an anti-inflammatory action by suppressing PgE2 release from LPS activated macrophages in a dose-dependent fashion. The presented invitro data shows promise for future M1 targeted anti-inflammatory theranostic nanomedicine developments.

References: 1. Janjic, J. M., Vasudeva, K., Saleem, M., Stevens, A., Liu, L., Patel, S., and Pollock, J. A. (2018) Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion delivery to neuroinflammation of the sciatic nerve in rat. Journal of neuroimmunology 318, 72-79
2. Muzamil Saleem, Brooke Deal, Emily Nehl, Jelena M. Janjic, and Pollock, J. A. (2019) Nanomedicine-driven neuropathic pain elief in a rat model is associated with macrophage polarity and mast cell activation. Acta Neuropathologica Communications in press

Acknowledgements: This research was funded by the CDMRP awards: W81XWH-19-1-0828 (PI: Gorantla/Janjic/Vemula), W81XWH-20-1-0854 (PI: Janjic) and W81XWH-20-1-0730 (PI: Janjic/Gorantla/Hitchens). We want to thank Prof. Lauren O’ Odonnel’s laboratory at Duquesne University for providing training and access to the flowcytometer in support of this project.


Figure 1: A) Average size distribution by intensity and B) average zeta potential distribution for folate decorated theranostic nanoemulsions compared to nanoemulsions without folate on the droplet surface. The figure demonstrates surface conjugation of folic acid derivative has no effect on size and zeta potential of nanoemulsion. Data collected by Dynamic Light Scattering on Zeta Sizer Nano (Malvern, UK). Data represents the average ± SD (n=3). Graphs constructed using Graph Pad Prism.


Figure 2: Effect of nanoemulsions on macrophage viability and uptake. A) Cell viability measured by Cell Titer Glo luminescence assay in male macrophages (RAW 264.7) exposed to (a) Celecoxib-free unconjugated nanoemulsion; (b) Celecoxib-loaded folic acid conjugated nanoemulsion; (c) free drug (celecoxib) in DMSO; or (d) DMSO for 24h. Data represents the average ± SD (n=6). Graphs constructed using Graph Pad Prism. B) Macrophage uptake evaluated by FACS indicating receptor-mediated uptake due to surface folic acid conjugation.


Figure 3: Effect of nanoemulsions on pro-inflammatory markers. A) Inhibition of PgE2 release from male macrophages exposed to CXB-loaded folic acid conjugated nanoemulsion, CXB-NE (0.4 –40 μM/mL of culture media) and drug free nanoemulsion (DF-NE) volume matched to highest dose in presence of lipopolysaccharide (LPS) (B) Inhibition of TNF-alpha in dose-dependent manner in response to CXB loaded nanoemulsion with drug free nanoemulsion (DF-NE) used as a negative control. ***p < 0.0005. Data represents the average ± SD (n=3). Graphs constructed using Graph Pad Prism.