Leveraging Thermosponge Nanoparticle For Reversing Artemisinin Resistance By Downregulating Glutathione Level

Objective:In order to enhance the susceptibility of Plasmodium to artemisinin drugs and reverse the drug resistance in drug-resistant strains,this study combined artemisinin（ART）and butthionine sulfoximine（BSO）,investigated the antimalarial activities and synergistic effects of the two drugs against artemisinin-sensitive strains（3D7）and drug-resistant strains（6233）in vitro,explored the oxidative stress in erythrocytes and malaria parasites after administration.Thermosponge nanoparticle（TSN）modified with choline analogs（Chos）co-loaded ART and BSO was prepared and the formulation was characterized.Then verified the targeting of TSN.Finally,the pharmacodynamics and safety of the preparations were evaluated in P.yoelii strain BY265 and P.berghei artemisinin-resistant strain K173.Provide new research ideas for artemisinin-based combination therapies（ACTs）and alleviating artemisinin resistance.Methods:1.Validation of the synergistic antimalarial effect of artemisinin and sulfoxideThe antimalarial activity of ART and BSO in the artemisinin-sensitive strain（3D7）and the drug-resistant strain（6233）of Plasmodium falciparum was determined by SYBR Green1 which were expressed as 50%inhibitory concentration(IC₅₀).5 concentrations(1/4 IC₅₀,1/2 IC₅₀,IC₅₀,2 IC₅₀,4 IC₅₀)were selected for mixing based on the IC₅₀ of ART and BSO.In vivo antimalarial activity studies were performed on 25 mixed drugs in different ratios to determine whether the two drugs had synergistic effects.2.Study on the mechanism of synergistic antimalarial effect of artemisinin and sulfoxide2’,7’-dichlorodihydrofluorescein diacetate（DCF-DA）fluorescent probe was used to evaluate the production of ROS by erythrocytes,using fluorescence spectrophotometer to detection total glutathione（t GSH）in Plasmodium and the ratio of reduced glutathione to oxidized glutathione（GSH/GSSG）,JC-1 fluorescent dye was applied to detect the damage in mitochondrial membrane potential,the DNA fragmentation was measured by TUNEL kit to compare the effects of single and combined use of two drugs on active substances in Plasmodium parasites.3.Preparation and characterization of thermosponge nanoformulations co-loaded with artemisinin and butionine sulfoximineThe in vitro analysis method of ART and BSO was es Table lished by high performance liquid chromatography（HPLC）,the artemisinin-butthionine sulfoxide thermosponge nanoparticles（ART+BSO-TSN）were prepared by nanoprecipitation and electrostatic adsorption;and the envelopment efficiency（EE）and drug loading（DL）of nanoparticles were measured by HPLC.TEM was used to observe the morphology of ART+BSO-TSN.The hemolysis of the nanocarriers was evaluated and the physical stablility of the nanoparticles was investigated by particle size and Zeta potential;the release behavior of ART and BSO in ART+BSO-TSN was investigated by high-speed centrifugation and the coumarin 6（C6）-encapsulated nanoparticles were used to investigate the targeting of the formulation.4.Pharmacodynamic and in vivo safety evaluation of thermosponge nanoformulationsThe malaria mouse model of ICR was injected with P.yoelii BY265 strain and the C57/6J was injected with P.berghei K173-resistant strain were es Table lished.The in vivo antimalarial activity of Chos-ART+BSO-TSN in two strains was investigated by Peter’s four-day inhibition assay.Construct negative control group:healthy mice;positive control group:saline;drug group:ART-Sol,ART+BSO-Sol,ART-TSN,ART+BSO-TSN,Chos-ART+BSO-TSN,To investigate the effects of drug combination,application of nanocarriers and modification of choline derivatives on body weight,infection rate,survival time and in vivo safety of malaria mice,and comprehensively evaluate the in vivo antimalarial effect of Chos-ART+BSO-TSN.Results:1.Validation of the synergistic antimalarial effect of artemisinin and sulfoxideThe results of in vitro antimalarial activity test of Plasmodium showed that both ART and BSO exhibited a good inhibitory effect on the growth of Plasmodium in a concentration-dependent manner.The IC50 values of ART-Sol and BSO-Sol in 3D7 strain were 10.2nmol/L and 59.8μmol/L,respectively.And the IC50 in 6233 strains were 26.7 nmol/L and64.3μmol/L.The two drugs were mixed with their respective IC50 concentrations to act on two Plasmodium parasites,and the lethality against Plasmodium in 3D7 strain reached 94%,and the combination index（CI）was 0.65,in 6233 strains the lethality against Plasmodium reached 99%with a CI of 0.26.2.Study on the mechanism of synergistic antimalarial effect of artemisinin and sulfoxideThe results of reactive oxygen species study showed that compared with ART-Sol and BSO-Sol,ART+BSO-Sol produced more ROS in erythrocytes infected with Plasmodium（P<0.05）,The results of glutathione content determination showed that ART+BSO-Sol significantly reduced t GSH and GSH/GSSG in Plasmodium（P<0.05）,And the results of the Plasmodium mitochondrial study showed that ART+BSO-Sol caused damage to mitochondrial membrane potential and destroyed the mitochondrial structure（P<0.05）.The results of DNA fragmentation study showed that ART+BSO-Sol significantly induced the apoptosis of Plasmodium（P<0.05）.3.Preparation and characterization of thermosponge nanoformulations co-loaded with artemisinin and butionine sulfoximineThe in vitro analysis method of ART and BSO was es Table lished and the optimal prescription was screened out by single factor optimization.The prepared nanoparticle dispersion was light blue,the particle size was 110.07±24 nm and the Zeta potential was20.47±0.46 m V.The encapsulation efficiency of ART and BSO were 48.4%and 34.8%,respectively.The nanoparticles were spherical,uniformly dispersed in water and have good physical s Table ility,the hemolysis rate of TSN was 3.1%.The cumulative release within72 h of ART was 84.85±0.60%and that of BSO within 12 h reached 75.55±3.32%in phosphate buffered saline（PBS）at p H 7.4.4.Pharmacodynamic and in vivo safety evaluation of thermosponge nanoformulationsThe results of the mice pharmacodynamics test showed that after injection of Chos-ART+BSO-TSN into the tail vein of two kinds of malaria-infected mice for 4 days,the growth of malaria parasites in vivo was effectively inhibited compared with other experimental groups,and the survival of mice was prolonged（P<0.05）.The inhibition of Yoelii-infected mice one day after drug withdrawal:ART（43.53±4.08%）<ART+BSO（53.69±3.10%）<ART-TSN（60.56±2.74%）<ART+BSO-TSN（79.32±2.28%）<Chos-ART+BSO-TSN（85.66±0.95%）,P.berghei-infected mice inhibition rate:ART（34.78±2.74%）<ART+BSO（49.38±2.59%）<ART-TSN（75.90±1.94%）<ART+BSO-TSN（86.18±1.29%）<Chos-ART+BSO-TSN（88.82±0.97%）.The results of H&E staining of pathological sections of important organs,hematological parameters and liver function indexes of mice showed that the prepared Chos-ART+BSO-TSN had no obvious in vivo toxicity and had good antimalarial effect.Conclusion:1.Validation of the synergistic antimalarial effect of artemisinin and sulfoxideBoth ART and BSO have good in vitro antimalarial activity,and the combination of the two drugs has better antimalarial effect in ART resistant strain（6233）and sensitive strain（3D7）.2.Study on the mechanism of synergistic antimalarial effect of artemisinin and sulfoxideCompared with ART or BSO used alone,the combined use of the two drugs can reduce the content of GSH in Plasmodium,generate more ROS in red blood cells,damage the mitochondrial structure of Plasmodium and cause DNA fragmentation,which significantly induce apoptosis in Plasmodium.3.Preparation and characterization of thermosponge nanoformulations co-loaded with artemisinin and butionine sulfoximineThe single-factor optimized nanoparticles have small particle size,high drug loading,temperature sensitivity,spherical appearance,uniform dispersion in water and good physical s Table ility.The nanoparticles can specifically target red blood cells infected with Plasmodium.In addition,ART and BSO in Chos-ART+BSO-TSN could be released slowly and continuously in PBS.4.Pharmacodynamic and in vivo safety evaluation of thermosponge nanoformulationsChos-ART+BSO-TSN has a certain antimalarial effect in mice infected with Plasmodium berghei and Plasmodium yoelii which can significantly reduce the infection rate of malaria mice,prolong the survival of mice,and no obvious toxicity to blood and organs.