| Objective:Malaria parasite-infected red blood cells could express new permeability pathways(NPPs),and overexpress the erythrocyte choline carriers(ECCs)on erythrocyte membrane after the parasite invasion.To develop targeting carrier for ARM delivery,choline and polyethylene-glycol dually modified artemether lipid nanoparticles(CD-PEG-ARM-NLC)with particle size smaller than 80 nm were prepared by using CD-PEG-SA to extend circulation time and enhance targeted property to infected red blood cells.The CD-PEG-ARM-NLC characterization and its antimalarial activity in vitro and in vivo were investigated on Plasmodium falciparum 3D7(Pf3D7)and Plasmodium yoelii BY265(Py BY265),respectively.The targeting property of CD-PEG-NLC to infected red blood cells was evaluated by using coumarin-6 as fluorescent probe.Methods:1.The prescription processing of CD-PEG-ARM-NLC CD-PEG-ARM-NLC were prepared by using high-pressure homogenization method.The addition way and quantity of CD-PEG-SA were screened as single factor,based on our previous research.2.The characterization of CD-PEG-ARM-NLC The physicochemical property of CD-PEG-ARM-NLC was characterized using appearance,micro-morphology,particle size,Zeta potential,encapsulation efficiency and drug loading efficiency as evaluation indexes.Besides,the hemolysis test and release characteristics in vitro were investigated.3.The evaluation of antimalarial activity of CD-PEG-ARM-NLC in vitro Pf3D7 knob+,3D7 knob-were separated from conventional cultured Pf3D7 by gelatin deposition method.The antimalarial activity of the drugs was investigated by conventional culture Pf3D7 strain,3D7 knob+,3D7 knob-and with ARM solution as the positive control group.The growth inhibition to malaria parasites treated with ARM,ARM-NLC,PEG-ARM-NLC and CD-PEG-ARM-NLC of different concentrations were evaluated by SYBR Green I method.The value of IC50(IC50)was calculated as an indicator to examine the antimalarial activity of the drug in vitro.4.The preliminary study on the targeting property of CD-PEG-C6-NLC C6-NLC,PEG-C6-NLC,CD-PEG-C6-NLC were prepared by high-pressure homogenization method with coumarin-6(C6)as fluorescent probe and C6 solution as positive control.The nanoparticles were incubated with Pf3D7 in vitro incubated erythrocytes and Py BY265 in vivo infected mice red blood cells,respectively.The distribution and intensity of the fluorescence were observed under fluorescence microscope.The effects of NPP and ECC on nanoparticles entering erythrocytes were analyzed by NPP and ECC inhibition tests.5.The pharmacodynamics study in vivo of CD-PEG-ARM-NLC Mice malaria model of Py BY265 was employed to evaluate the antimalarial activity of ARM-NLC,PEG-ARM-NLC,CD-PEG-ARM-NLC using four days inhibition test and 28 days-cure test with ARM solution as the positive control group and solvent,three blank nanoparticles and no treatment(blank)as negative control groups.Results:1.The studied prescription of CD-PEG-ARM-NLC The adding way of CD-PEG-SA was plus ethanol method.The adding ratio of CD-PEG-SA to lipid was 19.24%(g/g).The studied formulation was GMS 1.9286 g,MCT 1.0714 g,RH40 1.2 g,ARM 0.768 g,CD-PEG-SA 0.5772 g,deionized water 100ml.2.The quality characterization of CD-PEG-ARM-NLC The CD-PEG-ARM-NLC was homogeneous translucent liquid preparation with light blue opalescence and with spherical particle equally distributed under transmission electron microscope.And the average particle size was 65.10 ± 0.64 nm,PDI was 0.186± 0.004,Zeta potential was-24.47 ± 1.01 m V.The encapsulation efficiency was 87.03%± 0.71%,and the drug loading efficiency was 11.62% ± 0.10%.The cumulative release rate of 72 h in vitro was 81.95% ± 0.85% in phosphate buffer midea(p H 7.4).Also,the drug release followed the Weibull equation with t50 of 10.36 h.In vitro hemolysis test showed no erythrocyte aggregation and hemolysis.3.The evaluation of antimalarial activity in vitro of CD-PEG-ARM-NLC The IC50 values of ARM,ARM-NLC,PEG-ARM-NLC,CD-PEG-ARM-NLC were4.28 ± 0.05,3.69 ± 0.07,3.22 ± 0.05,2.68 ± 0.05 nmol/L for normal incubated Pf3D7,and 4.32 ± 0.06,3.93 ± 0.03,3.58 ± 0.08,3.01 ± 0.05 nmol/L for Pf3D7 knob+,and 4.27± 0.06,3.34 ± 0.07,2.81 ± 0.08,2.12 ± 0.09 nmol/L for Pf3D7 knob-,respectively.4.The preliminary study on the targeting property of CD-PEG-C6-NLC The observation under fluorescent microscope showed that all C6-loaded NLCs seemed could not be intaked by the uninfected RBC,but be able to enter the infected RBC and mainly gathered around the parasite in RBC.The fluorescence intensity of CD-PEG-C6-NLC group was higher than that of C6-NLC group and PEG-C6-NLC group and the results showed that CD-PEG-C6-NLC could target the infected red blood cells.The addition of NPP inhibitor(furosemide)can block C6-NLC and PEG-C6-NLC into the infected RBC,but not CD-PEG-C6-NLC.Concomitantly adding of NPP inhibitor(furosemide)and ECC natural substrate(choline)could block the entry of CD-PEG-C6-NLC into the infected RBC.5.The pharmacodynamics study in vivo of CD-PEG-ARM-NLC The ED50 and ED90 of ARM administrated intravenously were 0.266 mg/(kg·day)and 0.945 mg/(kg·day),respectively.The ED50 values of ARM-NLC,PEG-ARM-NLC and CD-PEG-ARM-NLC were 0.245,0.233 and 0.28 mg/(kg·day),and the ED90 valueswere 0.927,0.905 and 0.872 mg/(kg·day),respectively,which indicated that the antimalarial activity of ARM was increased slightly after loading in the nanocarrier.Conclusion:CD-PEG-ARM-NLC was prepared using optimized formulation.The physicochemical properties including appearance,particle size,Zeta potential,the encapsulation efficiency,the drug loading efficiency,hemolysis test and release tests in vitro of CD-PEG-ARM-NLC were meet with respective requirement.CD-PEG-ARM-NLC exhibited enhanced antimalaria activity than ARM,ARM-NLC and PEG-ARM-NLC both in vitro and in vivo.Also the preliminary targeting observation indicated that CD-PEG-C6-NLC could target the parasite-infected erythrocytes,and further study was needed to get more validations. |
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