Artesunate Based Nanomedicines:Synthesis,Characterization And Evaluation For Malaria Therapy

Artemisinin and its derivatives are highly effective drugs in the treatment of P.falciparum malaria.However,their clinical applications face challenges because of short half-life,poor bioavailability and growing drug resistance.In order to address these shortcomings,three kinds novel nanoparticles based on artemisinin were developed as follows:In the second chapter,novel dimeric artesunate phospholipid-based?Di-ART-GPC?liposomes were developed by a combination of dimerization and self-assembly.Firstly,Di-ART-GPC conjugate was synthesized by a facile esterification of artesunate?ART?and glycerophosphorylcholine?GPC?and confirmed by MS,¹H-NMR and ¹³C-NMR.The conjugate was then assembled to form liposomes without excipient by thin film hydration method.The assembled Di-ART-GPC liposomes have spherical and typical multilamellar vesicle structure with bilayer morphology as determined by transmission electron microscopy?TEM?and cryogenic electron microscopy?cryo-EM?.Moreover,the liposomes displayed an average hydrodynamic diameter of 190 nm and negative zeta potential at?20.35 mV as determined by Zetasizer.The drug loading capacity of ART increased up to 77.6%by weight with this liposomal formulation after a simple calculation.In vitro drug release and degradation results showed that Di-ART-GPC liposomes were stable in neutral physiological conditions but effectively degraded to release parent ART in simulated weakly acidic microenvironment.In vivo pharmacokinetics study revealed that Di-ART-GPC liposomes and conjugate have longer retention half-life in bloodstream.Importantly,Di-ART-GPC liposomes(IC₅₀ 0.39 nM)and conjugate(IC₅₀ 1.90 nM)demonstrated excellent in vitro antiplasmodial activities without causing hemolysis of erythrocytes,which were superior to free ART(IC₅₀ 5.17 nM)and conventional ART-loaded liposomes(IC₅₀ 3.13 nM).Furthermore,the assembled liposomes resulted in enhanced parasites killing in P.berghei-infected mice in vivo with delayed recrudescence and improved survivability,compared to free ART administration.In the third chapter,artesunate-heparin conjugate polymer prodrug?ART-HEP?was developed for intracellular release of ART in malaria therapy.Owing to both hydrophobic and hydrophilic moieties,the conjugate was self-assembled into artesunate-heparin nanocapsules?ART-HEP-NCPs?with lower critical micelle concentrations?CMC?of about 20?g/mL.Dynamic light scattering?DLS?and transmission electron microscopy?TEM?revealed that ART-HEP-NCPs have a hydrodynamic diameter of 112.1 nm with negatively charged surface?-11 mV?and typical micellar nanostructure.Interestingly,such modification achieved high drug loading of ART 29.3 wt.%,which is significantly higher than conventional ART-loaded nanoparticles.The nanocapsules demonstrated lower in vitro ART release under neutral physiological environment?<24%?but higher release rate was observed in simulated acidic microenvironment?>80%?in 70h test.Storage and hemolytic studies exhibited that ART-based nanocapsules was stable and safe material for i.v injection.Notably,ART-HEP-NCPs displayed promising targeting and in vitro inhibitory effect against P.falciparum 3D7 with an IC₅₀ of 11.26 nM,which slightly higher than free ART(IC₅₀ 6.49 nM).This expected lower inhibitory effect of polymeric prodrug could be ascribed to the gradual release of ART from the polymer chain over time.More importantly,the in vivo pharmacokinetics study indicated that the nanoscale characteristics of nanocapsules substantially contributed to the extended circulation of ART in blood,which is desired for artemisinin polymer prodrug.In the fourth chapter,the amphiphilic dual-tailed artesunate cationic conjugate?di-ART-CC?was developed by a facile esterification and assembled to form dual-tailed artesunate cationic liposomes?di-ART-CLPs?without excipient through thin film hydration method.The conjugate was characterized by MS,¹H-NMR and ¹³C-NMR analysis.di-ART-CLPs displayed unique spherical bilayer morphological nanostructure as analyzed through transmission electron microscopy?TEM?.Additionally,di-ART-CLPs showed an average hydrodynamic diameter of141.7 nm with polydispersity index of 0.591 as determined by dynamic light scattering?DLS?.The liposomes also demonstrated positive zeta potential at+15.9 mV.Importantly,the ART loading contents was calculated as 89.1%by weight,which was significantly improved versus earlier reported conventional liposomes.Moreover,di-ART-CC conjugate and di-ART-CLPs liposomes demonstrated tremendous in vitro inhibitory effect against P.falciparum 3D7 with IC₅₀ values of 0.62 and 0.36 nM respectively,which were lower than free ART(IC₅₀ 6.49 nM).Based on the above-mentioned encouraging results,Di-ART-GPC liposomal formulation could be a replacement to parent ART in clinical malarial therapy after thorough investigation.Whereas,ART-HEP-NCPs nanocapsules with higher ART contents and extended half-life could be promising platform for targeted antimalarial drug delivery.Moreover,in vitro inhibitory potential of di-ART-CC conjugate and di-ART-CLPs liposomes against P.falciparum 3D7confirmed its excellent antimalarial effect,however,further detailed in vivo investigations are needed.