Design Of Red Blood Cell Membrane-cloaked Dihydroartemisinin Nanoparticles With Enhanced Antimalarial Efficacy

Objective: In order to prolong action duration of dihydroartemisinin(DHA)in vivo,improve its antimalarial efficacy and develop affinity adhesion between host red blood cells infected with malaria parasites and normal red blood cells through "rosette" effect.Here,DHA loaded poly(lactic-co-glycolic acid)(PLGA)nanoparticles(PDNs)were prepared and further cloaked with red blood cell(RBC)membranes via electrostatic interactions to yield RBC membrane-cloaked PDNs(RPDNs).RPDNs were further prepared,in order to increase the delivery of red blood cell membrane biomimetic nanoparticles to target drugs of infected red blood cells through "rosette" effect,and optimize the preparation method of biomimetic nanoparticles.By investigating the formulation characteristics of nanoparticles,in vitro immune escaping,in vitro targeting of Plasmodium-infected RBCs,in vivo antimalarial activity,biosafety and in vivo blood circulation was investigated.The formulation were examined to provide a basis and guidance for the application of RBC membrane bionanoparticles in the treatment of malaria,improve the shortcomings of DHA and enhance antimalarial efficacy.Methods: 1.Preparation and quality characterization of PDNs PDNs were prepared by nanoprecipitation method,and the optimal prescriptions were selected by single-factor optimization and response surface optimization.The particle size,polydispersity(PDI),Zeta potential and entrapment efficiency(EE)as dependent variables.The drug-loading(DL)of PDNs was determined using high-speed centrifugation.Characterization of PDNs using Fourier transform infrared spectroscopy(FT-IR)and differential scanning calorimetry(DSC).Molecular dynamics simulations of PDNs formation process were simulated using the DPD module of Materials Studio software.2.Preparation of RPDNs and investigation of formulation properties The RBC membranes were extracted by hypotonic method,and red blood cell vesicles(RVs)of different particle sizes were prepared by ultrasonication and co-extrusion method.The RPDNs were prepared by co-extrusion of RVs and PDNs and the surface morphology of PDNs and RPDNs were observed by transmission electron microscopy(TEM).The particle size,PDI and Zeta potential of PDNs and RPDNs were characterized.Colocalization analysis of the fluorescently labeled RBC membrane cloaked-nanoparticles was performed using confocal laser scanning microscopy(CLSM).The release behavior of PDNs and RPDNs was investigated by dialysis,and the serum stability and 7-day preliminary stability of the two nanoparticles were examined.The surface-associated proteins of RPDNs were detected by SDS-PAGE protein electrophoresis and Western Blot.In vitro hemolysis of the two nanoparticles was examined.3.Examination of in vitro immune escaping and in vitro targeting of bio-nanoparticle Using coumarin-6(C6)as a fluorescent probe,fluorescently labeled nanoparticles PC6 Ns were prepared by nanoprecipitation and RPC6 Ns fluorescent bio-nanoparticles were prepared by co-extrusion method.In vitro immune escaping of the bio-nanoparticles was examined using the mouse mononuclear macrophages RAW264.7.Phagocytosis of RPC6 Ns by macrophages observed by high-inclusion cell imaging analysis system and CLSM.The phagocytosis of nanoparticles was examined quantitatively by flow cytometry.RPC6 Ns were incubated with Plasmodium-infected RBCs,and the fluorescence distribution and intensity were observed using CLSM.The Pearson co-localization coefficients was calculated to examine the affinity targeting of RPC6 Ns.4.In vivo antimalarial activity,biosafety and blood circulation of RPDNs To establish a model of Plasmodium-mice,five doses of PDNs and RPDNs nanoparticles were injected intravenously by Pearson four-day suppression administration method.On day 1 of drug withdrawal,blood was taken from the tail tip,stained,and examined microscopically to calculate the infection ratio,inhibition ratio,50% effective dose(ED50)and 90% effective dose(ED90).On day 7 of drug withdrawal,blood was again taken,stained,and examined microscopically to calculate the infection ratio.14 days after observation,blood was collected to test blood routine index and liver function index,and tissues were collected for pathological analysis of heart,liver,spleen,lung,and kidney.The body weight and survival number of mice were recorded daily.Normal ICR mice were injected with PDNs and RPDNs for 14 days,and blood was collected for blood routine tests to investigate the biosafety of nanoparticles.The fluorescent dye Di D was used as a probe to prepare Di D-labeled nanoparticles.The blood was collected at different time points after intravenous injection of the nanoparticles into mice,and the fluorescence intensity was measured using a fluorescence microplate reader to investigate the blood circulation of bionanoparticles in vivo.Results: 1.Preparation and quality characterization of PDNs The optimal prescription after single-factor and optimized formulation was prepared PDNs.The prepared PDNs suspension had a light blue opalescent appearance with a particle size of 71.23 ± 0.78 nm,PDI of 0.05 ± 0.01,Zeta potential of –21.63 ± 1.59 m V,EE of 70.10 ±0.79 %,and DL of 1.21 ± 0.09 %.In the infrared spectra,the lyophilized powder of PDNs had maximum absorption at 1750 cm-1 and 3370 cm-1,which were the characteristic peaks of PLGA and DHA,respectively,and no new peak shapes appeared.There is no melting peak of DHA in DSC analysis.The morphology of PDNs was obtained as spherical nanoparticles by DPD molecular dynamics simulations.2.Preparation of RPDNs and investigation of formulation properties The RVs were prepared by ultrasonication and co-extrusion method,and the final RVs were obtained sequentially through polycarbonate(PC)membranes of different pore sizes.The vesicle size of 85.62 ± 2.39 nm and zeta potential of approximately –29 m V.RPDNs were prepared by co-extrusion of RVs and PDNs,which had a pink-emulsion appearance,a particle size of 86.40 ± 2.54 nm,a PDI of 0.179 ± 0.011 and Zeta potential of –29.20 ± 4.19 m V.TEM showed a clear “core-shell” structure of RPDNs and two fluorescence colocalizations are observed,indicating that the RBC membrane was successfully encapsulated on the surface of PDNs.The cumulative release of DHA at 24 h was 39.23 % for RPDNs and 48.28 % for PDNs in 30 % ethanol-PBS release medium.Both nanoparticles were stable in fetal bovine serum as well as in water at 4 °C.SDS-PAGE protein electrophoresis showed that most of RBC membrane proteins in RPDNs were retained,and Western Blot results showed that the anti-phagocytic protein CD47 was retained.The in vitro hemolysis test showed no cell aggregation or hemolysis.3.Examination of in vitro immune escaping and in vitro targeting of bio-nanoparticle The results of high endocytosis cell imaging analysis system and CLSM observation showed that RPC6 Ns effectively reduced the uptake of nanoparticles by mononuclear macrophages relative to PC6 Ns.The quantitative analysis by the endocytosis module in the cell imaging analysis system showed that the average particle counts of RPC6 Ns was much lower than the average particle counts of PC6 Ns.Flow cytometry quantification results indicated that RPC6 Ns successfully evaded clearance by the immune system.Examining the in vitro targeting of RPC6 Ns to Plasmodium-infected RBCs,CLSM observation revealed that RPC6 Ns basically did not enter normal RBCs,but could enter Plasmodium-infected RBCs with affinity for i RBCs.The Pearson colocalization coefficient was calculated to be 0.7173,indicating that RBC membrane cloaked-nanoparticles had a targeted affinity for Plasmodium-infected RBCs,enabling the nanoparticles to be enriched around i RBCs.4.In vivo antimalarial activity,biosafety and blood circulation of RPDNs The ED50 values of PDNs and RPDNs were measured as 0.53 ± 0.01 μmol/kg and 0.13 ± 0.05 μmol/kg respectively,which were statistically different(P<0.05)compared with the ED50 values of DHA previously studied by the group(0.87 ± 0.27 μmol/kg)after 1 day of drug withdrawal.The infection ratio of each group increased after 1 week of drug withdrawal,and the infection ratio in RPDNs group was lower than that in PDNs group at the same concentration of 8.8 μmol/kg.The survival time of mice in the RPDNs group was longer than in PDNs group by 14 days observation,and RPDNs significantly improved the antimalarial activity.A dose-dependent manner in the infection ratio of treatment mice in the same formulation administration group as the dose administered increased.By analyzing the blood routine of treated mice and normal mice,the indicators of blood routine in the highdose RPDNs group and PDNs(35.2 μmol/kg)were within the normal threshold.H&E pathological analysis,no pathological tissue damage was found in RPDNs group and PDNs group.In vivo blood circulation study,the half-life of RPDi DNs in normal mice was 32.716 ± 0.611 h,which was significantly higher than that of PDi DNs(23.911 ± 1.464 h).It is well demonstrated that RBC membrane cloaked-nanoparticles could achieve long circulation in vivo.Conclusion: 1.Preparation of RPDNs and investigation of formulation properties RPDNs had distinct core-shell structure with uniform particle size,good EE and DL,the RBC membrane proteins were retained on the nanoparticle surface.RPDNs were more stable under serum and 4°C water,and with slow-release characteristic.The in vitro hemolysis assays all met the expected requirements.2.Examination of in vitro immune escaping and in vitro targeting of bio-nanoparticle The bio-nanoparticles can escape phagocytosis of macrophages,and avoiding drug clearance by the immune system and prolonging the duration of drug action.In vitro targeting studies showed that bio-nanoparticles have a certain affinity targeting effect on Plasmodium-infected RBCs and can achieve targeted drug delivery properties.3.In vivo antimalarial activity,biosafety and blood circulation of RPDNs Comparing different evaluation indexes,the antimalarial activity of bio-nanoparticles RPDNs was stronger than that of PDNs and DHA solution,and the bio-nanoparticles showed better antimalarial activity as well as biosafety,and were able to achieve long circulation in the blood of mice.