Research On The Intervention Of ALI Cytokine Storm Based On Curcumin Liposome Targeting M1 Macrophages

Objective:Acute lung injury/acute respiratory distress syndrome(ALI/ARDs)is a common complication of COVID-19,and the respiratory failure caused by it is a major cause of death in infected patients.The cytokine storm triggered by the massive release of a variety of inflammatory factors is an important cause of ALI/ARDs,which can cause excessive inflammation and violent attacks by the immune system on the body.In two fatal patients subject to necropsy,the lungs had inflammatory infiltration dominated by Ml alveolar macrophages.Therefore,M1 alveolar macrophages play an important role in the formation of cytokine storm and have potential target value in the treatment of ALI/ARDs.In this study,a safe and efficient pulmonary drug delivery system,L-arginine modified curcumin liposomes(Arg-Cur-Lip),was developed using L-arginine as the ligand and curcumin as the model drug.Arg-Cur-Lip can accurately deliver drugs to inflammatory lesions,and has great potential to interfere the formation of cytokine storm and enhance the therapeutic effect of curcumin by targeting M1 alveolar macrophages.It provided new ideas and theoretical support for the development of the drug delivery system to interfere with the cytokine storm of acute lung infectious diseases such as COVID-19.Methods:(1)Curcumin liposomes(Cur-Lip)were prepared by the thin-film hydration method.L-arginine was conjugated to Cur-Lip by EDC/NHS reaction to obtain Arg-Cur-Lip.The appearance,microscopic morphology,particle size,Zeta potential,entrapment efficiency,and modification efficiency of Arg-Cur-Lip were evaluated.(2)The targeting effect of ArgCur-Lip on M1 macrophages,the difference of M0,M1 and M2 macrophages in the uptake of Arg-Cur-Lip,and cellular internalization mechanism were evaluated by flow cytometry and laser confocal microscopy.(3)L-arginine modified liposomes containing DiR(Arg-DiR-Lip)were prepared,and Arg-DiR-Lip was delivered to the lungs of the rat model of ALI through a quantitative pulmonary liquid nebulizer.In vivo fluorescence imaging was applied for determining the fluorescence in rat lungs and tissues,and evaluating its lung targeting.(4)The anti-inflammatory effect of Arg-Cur-Lip on LPS-induced M1 macrophage was evaluated by Total Nitric Oxide Assay Kit,Enzyme-linked Immunoassay Kit(ELISA)and real-time fluorescent quantitative PCR(qRT-PCR).(5)Arg-Cur-Lip was delivered to the lungs of the rat model of ALI through a quantitative pulmonary liquid nebulizer,and the therapeutic effect of Arg-Cur-Lip was preliminarily evaluated.Results:(1)Arg-Cur-Lip was stable yellow homogeneous liquids.Arg-Cur-Lip has ideal particle size(142.73± 13.47 nm),narrow size distribution(PDI,0.17 ± 0.03),favorable stability(Zeta potential,-24.67±0.56 mV)and high entrapment efficiency(103.27± 3.86%).The modification efficiency of L-arginine to the amino group on the liposome is 19.15 ± 5.51%.(2)Arg-Cur-Lip group has stronger fluorescence intensity in M1 macrophages compared with CurLip group and has the ability to target M1 macrophages.Meanwhile,M1 macrophages had higher uptake for Arg-Cur-Lip compared with M0 and M2 macrophages.M1 macrophages were pre-treated with chlorpromazine(CPZ),5-(N-ethyl-N-isopropyl)amiloride(EIPA),or methyl-?-cyclodextrin(M-?-CD),the uptake ratio was 34.77%,95.92%,and 91.07%,respectively,indicating that cellular internalization mechanism of Arg-Cur-Lip primarily relied on clathrin mediated endocytosis.(3)The results of in vivo fluorescence imaging showed that the fluorescence intensity of the lungs in Arg-DiR-Lip-treated rats was apparently higher than that in DiR-Lip-treated rats at all observed time points after administration.(4)Cur-Lip and Arg-Cur-Lip significantly inhibited LPS-induced inflammation in vitro,including downregulated the expression levels of NO,the protein expression levels of TNF-? and IL-6,and the mRNA expression levels of TNFA,IL-6 and IL1B.However,compared with the Cur-Lip group,Arg-Cur-Lip only showed a stronger inhibitory effect on the expression of NO,both the protein and mRNA level of inflammatory factors were no significant difference.(5)Cur-Lip and ArgCur-Lip have potential therapeutic effects in the rat model of ALI through alleviated LPSinduced lung tissue damage and inhibited the expression of TNF-? and IL-6.The efficacy of Arg-Cur-Lip was slightly stronger than Cur-Lip,but there is no statistical difference in the expression levels of TNF-? and IL-6 between Arg-Cur-Lip and Cur-Lip.Conclusions:In this study,a new pulmonary drug delivery system,L-arginine modified liposomes,was developed for targeted delivery of curcumin to M1 macrophages.The results proved that L-arginine modified liposomes exhibit an ideal particle size,favorable stability,and preferential accumulation in M1 macrophages and the lung.Further,Arg-Cur-Lip alleviated LPS-induced the inflammatory response in RAW 264.7 cells and LPS-induced ALI in rats.In summary,Arg-Cur-Lip,designed to interfere cytokine storm of acute lung injury,can achieve the drug's targeting for inflammatory cells/environment and has a strong anti-inflammatory effect.It provided new ideas and theoretical support for the development of the drug delivery system to interfere with the cytokine storm of acute lung infectious diseases such as COVID-19.