Targeted modulation of macrophage-driven inflammation using etomoxir loaded liposomes

Abstract

Abstract Background and Aim: Etomoxir is an inhibitor of carnitine palmitoyltransferase-1 (CPT1) located on the outer face of the inner mitochondrial membrane. Recently, etomoxir demonstrated its ability to inhibit production of mitochondrial reactive oxygen species (mROS) during bacterial infection in a zebrafish larvae model by preventing the uptake of fatty acids as fuel for β-oxidation. The aim of this study was to develop a liposomal formulation as a drug delivery system for specific targeting to macrophages. Methods: A rapid and reliable reversed phase HPLC method utilising UV detection at 230 nm was developed and validated for the determination of etomoxir concentrations. Three liposome formulations were labelled with Marina Blue and developed, via thin film hydration, along with post insertion of Poloxamer 188 (forming P188-L) or poly(ethylene glycol) 2000 (forming PEG-L) or conjugation with 4-aminophenyl-α-D-mannopyranoside (forming MAN-L). Blank surface modified liposomes were evaluated for cellular uptake within a murine macrophage cell line, RAW 264.7 and within transfected zebrafish macrophages expressing mpeg1:EGFP. Etomoxir was passively loaded into the lipid bilayer of P188-L and observed in storage for long term stability. The expression of genes (irg1 and il-1b) following injection of surface modified liposomes into zebrafish was determined by whole mount insitu hybridisation. The efficacy of etomoxir loaded P188-L (ETO-P188-L) was determined following co-injection with monosodium urate crystals; the latter was used to induce production of mROS in macrophages. Quantification of mROS production was sequentially determined by direct correlation of MitoSOX fluorescence intensity. Results and discussion: The HPLC method developed for determination of etomoxir comprised of acetonitrile, methanol and KH2PO4 (10 mM, pH 2.5) (40:30:30, v/v) as mobile phase and achieved a retention time of 8.1 minutes. The low pH of 2.5 was essential in order to maintain the etomoxir, a weak acid with a pKa of 4.4, in its unionized state for a satisfactory retention time. Optimal particle size (1.2 μm) was achieved by extruding liposomal formulation through 2 cycles of 1 μm membrane filters. MAN-L produced the highest cellular uptake within murine macrophages in vitro, while within zebrafish macrophages P188L was observed to achieve the highest cellular uptake thus was the only formulation to be selected for drug loading for anti-inflammatory study. Loading 107 μg/mL of etomoxir into P188-L achieved entrapment efficiency (EE) and drug loading (DL) of 44% and 0.19% respectively. ETO-P188-L stored at 4 °C, was stable for 2 weeks and showed slight increasing in size by day 30. The in-situ hybridisation indicated P188-L and PEG-L produced no activation of either gene, while MAN-L produced slight activation of il-1b. MitoSOX fluorescence intensity was significantly reduced in zebrafish’s co-injected with ETO-P188-L compared to blank P188-L formulations, suppressing the mROS driven production of pro-inflammatory cytokines. Conclusion: As the first kind of its study, ETO-P188-L demonstrated specific uptake into zebrafish macrophages and significantly reduced the production of mROS. Further investigation is required to improve entrapment efficiency and specific cell targeting, possibly improving delivery of therapeutic drugs into macrophage.