Study On The Antimalarial Effect Of Erythrocyte Membrane Biomimetic Targeting Artemether Liposome

Research purposesMalaria is a vector-borne infectious disease that poses a serious health hazard to human,of as now the morbidity and mortality of malaria in the world have not been effectively controlled.In the long-term battle against malaria,Chinese scientists extracted artemisinin,a sesquiterpene lactone compound with an internal peroxy bridge structure,from Artemisia annual.Compared with traditional antimalarial drugs,it plays an important role in multiple stages of Plasmodium in erythrocyticcycle.But there still remain some problems in artemisinin,such as low bioavailability,poor solubility,and short half-life time.Artemether is one of the main derivatives of artemisinin with relatively stable chemical properties and more powerful antimalarial effect than artemisinin,it is the main component of Novartis’ antimalarial drug-Coartem,which is used for the research of biomimetic nano-targeted preparations to improve its bioavailability,increase targeting and extend the half-life time so as to enhance the antimalarial effect in this project.Biomimetic nano-drug delivery technology has very broad prospects in struggling in complex infectious diseases,studies have found that Plasmodium merozoites invade normal erythrocytes as a result of a series of ligand-receptor interactions.For example,the GPI-anchored merozoite surface protein 1(MSP1)on the surface of merozoites can bind to heparan sulfate(HS)on the membrane of normal erythrocytes to mediate the invasion of red blood cells by Plasmodium.CD47,a transmembrane glycoprotein,is expressed on the surface of erythrocyte membranes,which can form a signal complex with SIRPα on the surface of immune cells and then send out a "Don’t eat me" signal to suppress the immune phagocytosis of red blood cells.In this study,normal erythrocyte membranes were stripped,encapsulated in artemether liposomes,of which PS targeting peptides were modified on the surface to prepare artemether-containing erythrocyte membrane biomimetic targeting liposomes.On the one hand,the free merozoites in the blood are captured by receptors on the surface of erythrocyte membrane(such as HS),prohibiting the merozoites from repeatedly infecting normal erythrocytes and holding back the periodic attacks of malaria;on the other hand,PS targeting peptides are used to target the everted PS of the erythrocytes infected by Plasmodium,thereby artemether-containing biomimetic targeting liposomes are delivered into infected erythrocytes to take antimalarial effect of artemether.This project is expected to prepare biomimetic liposomes for multiple growth stages Plasmodium with more safe and effective therapeutic effect of malaria,which provide experimental basis for the improvement of artemisinin-based drugs in anti-malarial infection.MethodsWe prepared artemether liposomes(Lip-ARM)by ethanol injection method,then extracted the erythrocyte membrane by hypotonic method,and encapsulated the erythrocyte membrane on the surface of artemether liposome by physical extrusion method to prepare erythrocyte membrane-incorporated liposomes(EM-ARM).Three PS targeting peptides(PS Targeting peptide,PTP)were solid-phase synthesized,the sequence was CLIKKPF(P1),CPGDLSR(P2)and CSVSVGMKPSPRP(P3),respectively.They were coupled with functionalized phospholipids to compound DSPE-PEG2000-PTP,by which EM-ARM was modified,we constructed PS targeting peptide modified biomimetic liposomes(PEM-ARM),and then characterized the prepared liposomes,such as distribution of particle size,zeta potential,stability,encapsulation efficiency and drug loading rate,etc.By constructing an ANKA-infected malaria model of mouse,prescription screening of PEM-ARM was carried out.Firstly,the morphology of erythrocytes infected by malaria parasites was observed through the Giemsa staining method,then infected erythrocytes were detached by percoll cell separation solution,and merozoites were extracted by hypotonic hemolysis.The uptaking of biomimetic liposomes with different erythrocyte membrane concentrations by merozoites was compared and PEM-ARM by infected red blood cells in mice with different infection rates(5%.10%,and 10-20%)was analyzed by flow cytometry,including normal red blood cells,rings,trophozoites and merozoites,so as to screen out the best PS targeting peptide for subsequent experiments.Confirming the endocytic pathway of PEM-ARM:different methods for suppressing uptaking(such as endocytosis inhibitors,low temperature,and Ca+-containing solutions)were used to deal with infected erythr-ocytes,and then internalisation of PEM-ARM was determined by flow cytometry.Investigating the effect of PEM-ARM on Plasmodium at different growth stages:PEM-ARM was incubated with merozoites and infected erythrocytes respectively,and the localization of liposomes in the cells was observed with a laser confocal microscope.Taking ANKA-infected malaria model of mice as the research objects,the targeting in vivo,distribution in tissues and organs,antimalarial efficacy of biomimetic liposomes were studied.Firstly,the metabolic distribution and targeting of liposomes in the body was investigated,and then figuring out the in vivo efficacy of biomimetic liposomes through survival and weight curves,mitochondrial activity,oxidative stress ability,organ coefficients of significant organs in mice(heart,liver,spleen,lung,kidney,brain,etc.),lung appearances,immunohistochemical analysis of TNF and IL-6,tunel staining for apoptosis,cytoadherence assay between infected erythrocytes and normal lung tissues,cytoadherence assay between merozoites and normal erythrocytes,etc.Finally preliminary investigation of the biological safety of liposomes had been done through hemolysis test,uptaking and cytotoxicity test of MLE-12 cell,hematoxylin-eosin staining.ResultsThe erythrocyte membrane biomimetic liposomes were successfully prepared with uniform distribution of particle size,perfect stability,drug loading rate and encapsulation efficiency,which were in line with the requirements of subsequent experiments.Percoll separation solution with a concentration of 65 percent had the best effect in separating infected erythrocytesat;uptaking of EM-ARM with a concentration of erythrocyte membrane of 0.6 mg/mL by merozoites was higher than that of 0.3 mg/mL and 0.9 mg/mL membrane protein,with a statistical difference(P<0.01),which indicated that there should be saturation in absorbing merozoites by erythrocyte membrane,and its optimal protein concentration was 0.6 mg/mL;when the infection rate was 5 percent,compared with P2EM-ARM and P3EM-ARM,infected erythrocytes had the best uptaking effect on P1EM-ARM(P<0.01),indicating that polypeptide 1 could target the everted PS on infected erythrocytes in the early stage of infection,so it was screened out for subsequent experiments.Laser confocal microscope observation showed that P1EM-ARM could bind to the everted PS on the surface of infected erythrocytes and enter the cell to target intracellular Plasmodium,and erythrocyte membrane on the surface of P1 EM-ARM could also effectively arrest free merozoites,which was consistent with the results of flow fluorescence detection.In the study of cellular uptaking mechanism,it was found that the uptaking of liposomes by normal or infected erythrocytes was significantly reduced after the intervention of low temperature or MβCD,and the difference was statistically significant(P<0.01),this result suggested that the uptaking of liposomes by erythrocytes should be mainly mediated by temperature and caveolin-mediated endocytosis.In vivo targeting of P1EM-ARM was studied by the HO/TO double labeling method of flow cytometry.and it was found that the uptaking of P1EM-ARM by trophozoites gradually increased,reaching a peak at 4h point.Compared with the f-NR and EM-ARM groups,there existed a significant difference(P<0.01).indicating that P1EM-ARM could target the exerted PS on infected erythrocytes and enter the cells to increase the concentration of artemether in trophozoites.In vivo drug efficacy research found that P1EM-ARM could significantly prolong the survival time of animals and reduce weight variation width.Compared with the model group(PBS),P1EM-ARM could enhance the content of reactive oxygen species in infected red blood cells(P<0.05)and reduce the mitochondrial activity of Plasmodium in infected erythrocytes(P<0.01),which were extremely benefit for anti-malarial effects.The numbers of apoptotic cells detected by tunel in the P1EM-ARM group was significantly less than that in the f-ARM and Lip-ARM groups(P<0.01),indicating that by inhibiting the adherence of infected erythrocytes to pulmonary epithelial cells,P1EM-ARM reduced the apoptosis of pulmonary epithelial cells.In addition,P1 EM-ARM effectively prevented the production of TNF and IL-6 and the deposition of hemozoin,thereby reducing inflammation in the lungs.Through flow cytometiy detection,it was found that P1EM-ARM group had a significant difference in reducing the proportion of the Q2 area(mixed fluorescent area)compared with the PBS,f-ARM and Lip-ARM groups(P<0.01),indicating that P1EM-ARM was capable of capturing merozoites,inhibiting their invasion to normal erythrocytes and prohibiting them from repeatedly infecting normal erythrocytes.Compared with the PBS group,the numbers of infected red blood cells on the lung tissue section of P1 EM-ARM group was significantly reduced,and the difference was statistically significant(P<0.05),indicating that P1EM-ARM could target the infected erythrocytes and inhibit their adherence to normal lung tissues,thereby improving the anti-malarial effect of the drugs.The hemolytic rate of P1EM-ARM w as less than 5%,which was similar to the negative group(PBS)and no hemolysis happened to P1EM-ARM.P1M-ARM was hypotoxic to MLE-12 cells and could reduce the accumulation of drugs in MLE-12 cells with ex cellent biological safety.In the hematoxylin-eosin staining experiment,it was found that P1EM-ARM not only did not cause pathological damage to normal important organs(heart,kidney,brain,etc.),but also played a role in protecting the important organs of the body(lungs,liver,spleen,etc.)against damage caused by Plasmodium infection.ConclusionP1EM-ARM constructed in this research could target the free merozoites and infected erythrocytes in the bloodstream dually.prolong the survival time of animals,effectively arrest the infection of Plasmodium,and had superior biological safety and expecting application prospect in anti-malarial effect.