Preparation Of Heparin-Modified Dihydroartemisinin-3-Methyladenine Prodrug Nanoliposomes And Evaluation Of Its Antimalarial Activity

Objective:Artemisinin drugs（ARTs）have been recommended by the World Health Organization as first-line drugs for the treatment of malaria.However,widespread use has led to the emergence of malignant plasmodium strains that are resistant to ARTs,resulting in decreased efficacy.Research has found that mutations in the Pfkelch13 gene of malignant Plasmodium strains are directly related to ARTs resistance,as the mutated Kelch13 protein leads to decreased binding with Plasmodium phosphatidylinositol 3-kinase（PfPI3K）,which in turn reduces PfPI3K ubiquitination and degradation,leading to an increase in levels of phosphatidylinositol 3-phosphate（PI3P）.PfPI3K and PI3P inhibit the transport of hemoglobin to the malaria parasite,causing developmental arrest during the trophozoite stage and reducing sensitivity to ARTs.In addition,decreased hemoglobin endocytosis reduces heme levels and blocks ARTs activation,leading to the emergence of resistance and decreased therapeutic efficacy.PfPI3K and human PI3K proteins exhibit high sequence homology,particularly with the structure of type III PI3K.As a broad-spectrum PI3K inhibitor,3-methyladenine（3-MA）can downregulate PI3K expression by inhibiting the PI3K/Akt pathway.Given the high sequence homology between PfPI3K and human PI3K,the inhibitory effect of 3-MA may potentially reverse ARTs resistance.Dihydroartemisinin（DHA）,a widely used first-line anti-malarial drug,also suffers from resistance.To address resistance of Plasmodium falciparum to ARTs and improve the anti-malarial efficacy of ARTs,a combination of 3-MA and DHA is proposed.Firstly,a drug-resistant animal model of Plasmodium falciparum K173 was established and resistance was induced.Then,the respective ED₅₀ values of the two drugs were determined following separate administration,and their combination index was obtained after co-administration,which showed a synergistic effect.Subsequently,DHA prodrugs were designed with 3-MA.Since the hydroxyl group of DHA is not easily reactive with the amino group of 3-MA,the prodrug of DHA artesunate（AS）,which has a carboxyl group,can undergo acylation reaction to produce AS-3-MA.After infecting red blood cells,Plasmodium falciparum expresses Plasmodium falciparum erythrocyte membrane protein 1（Pf EMP-1）on the surface of the red blood cell membrane,which can specifically bind to heparin.To enhance the targeting of subsequent preparations to infected red blood cells by Plasmodium falciparum,we have utilized heparin-modified nanoliposomes.Firstly,we prepared artesunate-3-methyladenine nanoliposomes（AMLs）and heparin-modified artesunate-3-methyladenine nanoliposomes（HAMLs）,and optimized the prescription of HAMLs.We investigated the formulation characteristics,phagocytosis resistance,in vitro targeting,and long circulation of HAMLs,which provided a basis for its subsequent use in anti-malaria treatment.Methods:1.Evaluation of combined administration of DHA and 3-MAFirst,a drug-resistant animal model of Plasmodium berghei K173 was constructed and resistance was induced in the animal model.After induction for ten generations,the resistance index showed moderate resistance.Then,the ED₅₀ values of DHA and 3-MA were obtained through separate drug administration in the drug-resistant animal model.Based on the obtained ED values,two methods of drug administration were set:administration in an equal molar ratio and administration in equal ED₅₀ doses.The doses administered in an equal molar ratio were:（0.55,1.1,2.2,4.4,8.8）μmol/kg,and the doses administered in equal ED₅₀doses were 1/4 ED₅₀,1/2 ED₅₀,ED₅₀,2 ED₅₀,and 4 ED₅₀.Using the animal model and based on the administered doses and inhibition rates after cessation of administration,the combination index（CI）for the synergistic effect of the two drugs was calculated.2.Synthesis of AS-3-MAAS and 3-MA were used as starting materials,HATU was used as a condensing agent,and DIPEA was used as a proton scavenger to synthesize AS-3-MA via an amidation reaction.The structure was confirmed using Fourier-transform infrared spectroscopy（FT-IR）,high-resolution mass spectrometry（HR-MS）,proton nuclear magnetic resonance spectroscopy（¹H-NMR）,and carbon spectroscopy(¹³C-NMR).3.Preparation and formulation characteristics of nanoliposomes of dihydroartemisinin-3-methyladenine prodrug.AMLs were prepared using a thin film dispersion method,and size and polydispersity index（PDI）were used as evaluation indicators.After single-factor optimization,response surface optimization was carried out to obtain the optimal AMLs formulation.AMLs were characterized by Fourier transform infrared spectroscopy（FT-IR）,and the dilution stability,physical stability in serum,storage stability,and in vitro release were investigated.4.Preparation and formulation investigation of heparin-modified nanoliposomes ofdihydroartemisinin-3-methyladenine prodrugThe nanoliposomes containing choline derivatives and AS-3-MA were prepared using a thin film dispersion method.Subsequently,they were co-incubated with a solution of heparin sodium at room temperature for 24 hours,and HAMLs were obtained by centrifugation to remove free heparin sodium.The morphology of HAMLs was observed by transmission electron microscopy.The size,PDI,and zeta potential of HAMLs were measured using a Malvern particle size analyzer.Dilution stability and physical stability in serum were investigated,and the in vitro release of AMLs and HAMLs was studied using dialysis.In addition,the hemolysis test of HAMLs was performed.5.In vitro targeting,anti-phagocytosis,and long circulation assessment of heparin-modified nanoliposomesCoumarin 6（C6）was used instead of AS-3-MA to prepare fluorescent nanoliposomes（C6Ls）and choline derivative-coupled C6 nanoliposomes by the thin film dispersion method.The latter was co-incubated with heparin sodium solution to obtain heparin-modified fluorescent nanoliposomes（HC6Ls）through electrostatic adsorption.The targeting effect of heparin-modified fluorescent nanoliposomes on infected red blood cells was analyzed using a high-content imaging system by co-incubating HC6Ls with red blood cells infected with Plasmodium berghei K173 resistant strain.The anti-phagocytosis effect of HC6Ls was investigated by incubating them with RAW264.7 cells in vitro,and the anti-phagocytosis status was observed using a high-content imaging system.HC6Ls were prepared with C6 as the fluorescent marker,and their metabolic situation was tracked and detected through the fluorescence signal of C6.C57 mice were injected with 0.15 m L of HC6Ls through the tail vein,and blood samples were collected at different time points.The fluorescence intensity of each well was measured using an enzyme immunoassay.This method was used to track the metabolic situation of nanoliposomes and evaluate their circulation time and stability in vivo to assess their long circulation.6.Efficacy of heparin-modified nanoliposomes on antimalarial activityUsing the previously induced drug-resistant K173 strain of Plasmodium berghei to establish a C57 mouse model of malaria,different doses of AS-3-MA solution,an estimated dose of DHA solution（8.8μmol/kg）,AMLs,and HAMLs nanoliposomes were injected via tail vein,and Pearson’s four-day suppression method was used for four consecutive days of administration.On the fourth day after cessation of treatment,blood was collected from the mice by tail snip,fixed with methanol,smeared,stained,and examined under a microscope to calculate the infection rate and percentage of inhibition of the malaria parasite in the mouse body,obtaining the ED₅₀ values for AMLs and HAMLs.The survival rates of the mice in each group were continuously monitored for 35 days,and survival curves were recorded and plotted for the malaria mouse group,AMLs group,and HAMLs group.On the fifteenth day after cessation of treatment,the liver,spleen,lung,and kidney tissues of normal mice,malaria mouse group,DHA solution group,AMLs group,and HAMLs group were collected,paraffin-embedded,and sliced,followed by H&E staining for histological analysis to preliminarily evaluate the safety of the formulation.Results:1.Synergistic antimalarial activity of DHA and 3-MAUsing the induced drug-resistant K173 strain of Plasmodium berghei to establish a C57mouse model of malaria,DHA and 3-MA were administered separately,and their ED₅₀values were obtained,which were（2.71±0.06）μmol/kg and（83.61±1.36）μmol/kg,respectively.They were then administered in equimolar ratios at increasing doses（0.55,1.1,2.2,4.4,8.8）μmol/kg.The inhibition rates of DHA and 3-MA on malaria mice were 35.6%,49.0%,70.0%,86.5%,and 94.0%,respectively.The synergy index values of the two drugs were 0.68,0.87,0.85,0.86,and 0.74,indicating weak synergistic effects.When administered in equi-ED₅₀ ratios at doses ranging from 1/4 ED₅₀ to 4 ED₅₀,the inhibition rates of the combined administration of the two drugs on malaria mice were 47.1%,60.0%,74.0%,85.3%,and 94.8%,respectively,and the synergy index values were 0.57,0.73,0.78,0.84,and 0.58,indicating synergistic effects and providing a basis for subsequent experiments.2.Synthesis of AS-3-MAIn the Fourier-transform infrared spectroscopy（FT-IR）spectrum,the absorption peak at 1720 cm^-1 corresponds to the stretching vibration of the N-H group on the carbonyl,while the absorption peak at 2928 cm^-1 represents the stretching vibration of the N-H group on the amide bond,and the absorption peak at 1253 cm^-1 corresponds to the stretching vibration of the C-N bond on the amide bond.The accurate mass measurement by HR-MS shows a relative error within one order of magnitude compared to the theoretical value.The ¹H-NMR and ¹³C-NMR spectra are consistent with the expected compound structure,indicating the successful synthesis of AS-3-MA.However,the yield of the amidation reaction for the synthesis of AS-3-MA is low,less than 10%,and the synthesis process needs to be optimized.3.Preparation and formulation characteristics of dihydroartemisinin-3-methyladenineprodrug nanoliposomesA successful HPLC method was established for the determination of AS-3-MA content and in vitro analysis.Within the concentration range of 8.0-600.0μg/m L,the AS-3-MA exhibited good linearity,with a detection limit of 4.0μg/m L and a quantification limit of 8.0μg/m L.The precision,specificity,and recovery rate of the established method met the requirements of methodology.By single-factor optimization and response surface optimization,an optimal formulation for the liposomes was obtained.The AMLs size prepared under this formulation was（75.95±0.78）nm,with a PDI of 0.30±0.02,and the particle size distribution was relatively uniform.The Zeta potential was（-21.63±1.59）m V,with a high absolute value.The measured encapsulation efficiency of AMLs was（80±0.03）%,and the prepared nanoliposome suspension appeared as a light blue milk.In the infrared spectrum,the AMLs lyophilized powder exhibited stretching vibration peaks of N-H groups on the carbonyl at1720 cm^-1 and on the amide bond at 2928 cm^-1,as well as characteristic peaks of m PEG-PLGA,soybean phospholipid,and cholesterol,without any new peaks appearing,indicating the successful preparation of the liposomes.In the dilution stability study,the change in Size and PDI of AMLs after dilution several times with ultrapure water at room temperature was not significant,indicating good dilution stability.In the serum stability test,after co-incubation of AMLs with fetal bovine serum for 24 hours,the absorbance of the suspension did not change significantly at different time points,indicating good serum stability.In the storage stability test,AMLs stored at 4℃were measured for size and PDI on the 1st,3rd,5th,7th,and 9th days,and no significant changes were observed,indicating good storage stability.In the release study,AMLs exhibited a slow release characteristic,with 44.59%released after 48 hours,while the AS-3-MA solution released nearly 75%,indicating the slow-release feature of AMLs.4.Preparation and formulation evaluation of heparin-modified dihydroartemisinin-3-methyladenine prodrug nanoliposomesHeparin-modified dihydroartemisinin-3-methyladenine prodrug nanoliposomes（HAMLs）were prepared by electrostatic adsorption.The size of HAMLs was（86.95±0.40）nm,the potential was（-24.60±0.25）m V,and the PDI was 0.29±0.002.The prepared nanoliposomes had a large potential absolute value,small PDI,and appeared as a light blue homogeneous milky liquid.The ratio of choline derivative to heparin sodium in the prescription was optimized,and the optimal prescription resulted in a particle size of（82.00±0.89）nm and a Zeta potential of（-24.0±0.55）m V.Within a 100-fold dilution range after dilution with ultrapure water,the size and PDI of HAMLs did not change significantly,indicating good dilution stability.After coincubation with fetal bovine serum for 24 hours,the absorbance did not change significantly,indicating good physical stability of the nanoliposomes in serum.The stability of HAMLs was evaluated during storage at 4°C for 9days,and the particle size and PDI did not change significantly,indicating good storage stability.When incubated with red blood cell suspension at different concentrations,HAMLs did not show obvious hemolysis,and the hemolysis rate of HAMLs at each concentration was less than 5%as determined by UV spectrophotometry,indicating good blood compatibility and safety of HAMLs.5.Heparin-modified dihydroartemisinin-3-methyladenine nanoliposomes:in vitrotargeting,anti-phagocytosis,and long circulation assessment.Two fluorescent nanoliposomes,C6Ls and HC6Ls,had uniform transparent appearances with a light greenish-yellow fluorescence.The size of C6Ls was（83.52±0.82）nm,and the PDI was 0.27±0.04.The size of HC6Ls was（92.22±2.23）nm,and the PDI was 0.26±0.01.The Zeta potentials of the two nanoliposomes were（-25.33±1.19）m V and（-17.00±0.40）m V,respectively.The encapsulation rates of the two liposomes were（86.03±1.03）%and（72.97±0.91）%,respectively.The cumulative release rates of C6Ls and HC6Ls after 9 h were（32.18±0.44）%and（23.90±0.26）%,respectively.The cumulative release rates of both nanoliposomes were less than 35%,while the cumulative release rate of C6 solution after 9 h of dialysis was（52.53±0.99）%,indicating that C6 was successfully encapsulated in the nanoliposomes and can be used in subsequent experiments.The high-content imaging system was used to investigate the in vitro targeting of HC6Ls,and the results showed that normal red blood cells had little C6 signal,but infected red blood cells showed strong fluorescence signals,indicating that heparin-modified nanoliposomes had targeted affinity to infected red blood cells.The high-content imaging system was used to observe the uptake of both fluorescent nanoliposomes by mouse macrophage RAW264.7.The results showed that the phagocytosis of HC6Ls was significantly lower than that of C6Ls,indicating that heparin-modified nanoliposomes can effectively evade phagocytosis.The long circulation of HC6Ls in vivo was assessed,and the half-life of HC6Ls in normal C57mice was（36.71±2.97）h,which was longer than that of C6Ls,indicating that heparin-modified nanoliposomes have a longer circulation time than unmodified nanoliposomes.6.Antimalarial effect of heparin-modified nanoliposomesIn an animal model constructed with the K173 resistant strain of Plasmodium berghei,a decreasing trend in infection rate was observed in each treatment group as the dosage increased.At the same dosage,the infection rate of the heparin-modified nanoliposomes（HAMLs）group was lower than that of the AMLs group and the AS-3-MA solution group.The ED₅₀ values of AMLs and HAMLs,measured at the infection rate on the 4th day after drug withdrawal,were（1.69±0.22）μmol/kg and（1.06±0.12）μmol/kg,respectively,which were lower than the ED₅₀ value of DHA administered alone,which was（2.71±0.06）μmol/kg.Through subsequent evaluation of mouse survival status,it was found that mice treated with the same dosage of HAMLs generally survived longer than those in the AMLs group and the AS-3-MA group.In the observation period of 30 days,half of the mice in the highest dosage group of AMLs survived,while all of the mice in the highest dosage group of HAMLs survived,but their physical condition was particularly weak,indicating that HAMLs have a good antimalarial effect.On the 15th day after drug withdrawal,normal mice,malaria-infected mice,mice treated with DHA solution,mice treated with AMLs,and mice treated with HAMLs were sacrificed,and their liver,spleen,lung,and kidney were paraffin-embedded for pathological analysis after H&E staining.Since the specimens were collected late,pathological damage such as hemozoin deposition and infiltration of inflammatory cells could be observed in all treatment groups.Compared with the other groups,the pathological damage in the HAMLs group was milder,indicating that its in vivo application is safe.Conclusion:1.Evaluation of the synergistic effect of DHA and 3-MAUsing the Plasmodium berghei K173-resistant strain of malaria parasite model that was developed,and based on the ED₅₀ obtained from separate administration of each drug,doses were set and the drugs were administered in two ways.The synergy index of the combined administration was calculated based on the infection rate on the first day after discontinuation of the drugs.It was found that the CI values were less than 1,indicating a synergistic effect between the two drugs.2.Synthesis of AS-3-MAAS-3-MA was synthesized through amide reaction,and its structure was confirmed by various techniques including FT-IR,HR-MS,¹H-NMR,and ¹³C-NMR,indicating the successful synthesis of the prodrug.3.Preparation and quality characterization of heparin-modified dihydroartemisinin-3-methyladenine prodrug nanoliposomesThe heparin-modified dihydroartemisinin-3-methyladenine prodrug nanoliposomes were spherical,with uniform particle size and no aggregation,and exhibited good dilution stability.After incubation with fetal bovine serum at（37±0.5）°C for 24 hours,there was no significant change in absorbance,indicating good physical stability in serum.The particle size and PDI did not change significantly after 9 days of storage at 4°C,indicating good initial storage stability.In vitro release experiments showed sustained release characteristics,and no hemolysis was observed or detected in the hemolysis assay,which met the experimental requirements.4.In vitro targeting,phagocytosis resistance,and long circulation of heparin-modifiednanoliposomesThe heparin-modified nanoliposomes could escape phagocytosis by macrophages,evade clearance by the immune system,and exhibited phagocytosis resistance.The heparin-modified fluorescent nanoliposomes had a longer apparent elimination half-life and longer residence time in vivo than the unmodified nanoliposomes,indicating long circulation characteristics.Additionally,the heparin-modified nanoliposomes exhibited good affinity targeting for infected red blood cells,enabling targeted drug delivery.5.Antimalarial efficacy of heparin-modified nanoliposomesIn the constructed P.berghei K173 mouse model,at the same dose,the infection rate of mice in the HAMLs group on the fourth day after drug cessation was lower than that of the AS-3-MA solution group and AMLs group.In the 30-day observation period,half of the mice in the highest dose AMLs group survived,while all mice in the highest dose HAMLs group survived.Overall,the antimalarial activity of HAMLs was superior to that of AMLs and AS-3-MA solution.H&E staining pathological analysis showed that the pathological damage of mice in the HAMLs group was lighter than that of the other groups.In general,HAMLs were safe for in vivo application.