Preparation And Evaluation Of Brain Targeted Dihydroartemisinin/perillyl Alcohol Co-delivered Liposomes For Murine Cerebral Malaria

Objective:Malaria is a serious infectious disease that has serious harm to human beings,causing more than 600,000 people deaths every year.It is estimated that about 1%of children infected with Plasmodium falciparum will develop a more severe brain complication called cerebral malaria（CM）.At the same time,the main problem of drug therapy for CM is how to across the blood brain barrier（BBB）.Currently,there is a lack of effective means to deliver drugs to the brain tissue.In order to improve the effectiveness of CM treatments,and reduce the nervous system damage caused by CM,and deliver more drugs to the brain tissue,dihydroartemisinin（DHA）,acted as an artemisinin-like derivative with strong killing effective on plasmodium,was adopted in this study.In addition,combined with perillyl alcohol（POH）,which has neuroprotective effect on the brain and antimalarial activity,the synergistic effective of the two drugs combined was evaluated.Meanwhile,due to the high expression of LAT1 transporter on BBB,tyrosine（Tyr）was selected as the target head to modify DHA and POH co-delivered liposomes,and Tyr-modified brain targeted liposomes of DHA and POH co-delivery were obtained（DP@Tyr-Lips）.Single factor optimization method and response surface optimization method were used to optimize the formulation.Finally,a series of investigation and evaluation of DP@Tyr-Lips were conducted from the aspects of in vitro release,anti-phagocytic effect,in vivo anti-cerebral malaria effect,brain targeting and biological safety,etc.This provides research basis for the later application of DP@Tyr-Lips in the treatment of cerebral malaria,and reduces the damage of nervous system in cerebral malaria patients.Methods:1.Evaluation of the synergistic effect of dihydroartemisinin/perillyl alcohol on murine cerebral malariaCM model was constructed in C57BL/6J male mice using plasmodium berghei ANKA strain.With normal mice as Control group,malaria mice group（ANKA group）were intraperitoneally injected 0.2 ml erythrocyte suspension containing 1×10⁷ Pb.ANKA strains.From the first day of inoculation,the mice were subjected to tail tip blood sampling and smear every day,and the thin blood smear was stained with Jemsa dye solution to observe the infection rate of mice.The body weight and survival of mice were observed and recorded from the day of infection,and the curves were drawn.The BBB permeability of mice was evaluated by Evans Blue staining.According to the rapid mouse coma behavior scale（RMCBS）,the behavioral parameters of ANKA group mice were evaluated and determined to evaluate whether the brain malaria model was constructed successfully.The antimalarial activities of DHA and POH in Pb.ANKA-infected mice were measured by pearson four-day inhibitory administration method,And median effective dose(ED₅₀)was calculated.Five mixed concentration gradients(1/4 ED₅₀,1/2 ED₅₀,ED₅₀,2 ED₅₀and 4 ED₅₀)of the two drugs were set based on the half effective dose of DHA and POH to evaluate the antimalarial activity,and the synergistic index of the two drugs was calculated based on the inhibition rate.The synergistic antimalarial effect of different concentrations of DHA and POH was discussed.2.Preparation and evaluation of brain targeting liposomes delivered by dihydroartemisinin/perillyl alcoholTyrosine modified vitamin E polyethylene glycol succinate（Tyr-TPGS）was prepared by chemical synthesis method.The synthesis of Tyr-TPGS was verified by hydrogen magnetic resonance spectroscopy.The tyrosine-modified dihydroartemisinin/perillyl alcohol co-loaded liposome（DP@Tyr-Lips）was prepared by thin film dispersion method.The optimal formulation of the liposome was obtained by single factor optimization and response surface optimization method.The encapsulation efficiency（EE）and drug loading（DL）of POH and DHA in liposomes was determined by high performance liquid chromatography（HPLC）and HPLC-postcolumn derivatization,respectively.The morphology of DP@Tyr-Lips was observed by transmission electron microscopy（TEM）.The particle Size（Size）,polymer dispersity index（PDI）,and zeta potential of liposomes were measured by Malvern particle size analyzer.The dilution stability,long-term stability and serum stability of liposomes were evaluated by the change of Size and PDI.The release behaviors of DHA-sol,POH-sol,DP-Lips and DP@Tyr-Lips in vitro were investigated by dialysis.The effect of DP@Tyr-Lips on macrophage phagocytosis was investigated by image Xpress PICO.The hemolytic evaluation of DP@Tyr-Lips was performed in vitro with Varioskan Flash multi-function reading instrument.3.Evaluation of in vivo pharmacodynamics and long cycle effects of dihydroartemisinin/perillyl alcohol codelivery of brain-targeted liposomes.The anti-cerebral malaria effect of DP@Tyr-Lips in experimental mice was investigated by pearson four-day inhibition test.The normal group,malaria mouse group,DHA-sol group,POH-sol group,DP-Lips group and DP@Tyr-Lips group were established,And the infection rate,inhibition rate,weight change,survival and the contents of TNF-αand IFN-γin brain tissue of CM mice in each group were determined.coumarin 6（C6）-labeled liposomes（C6-DP@Tyr-Lips）were prepared.The fluorescent-labeled liposomes were injected into CM mice,and the C6 content in brain tissues was determined.Finally,C6-DP@Tyr-Lips was injected into CM mice,and the whole blood of mice was collected at a specific time point to measure the changes of C6 content in blood to evaluate the long-term circulation behavior in vivo.Results:1.Evaluation of the synergistic effect of dihydroartemisinin/perillyl alcohol on murine cerebral malaria.The experimental brain malaria model showed that the infection rate of Pb.ANKA infected mice increased daily,and reached more than 35%on the 12th day.The body weight and RMCBS score were decreased compared with the initial,and the BBB penetration capacity was greatly increased compared with that of normal mice,while the blank mice maintained zero infection rate,It has obvious characteristics of cerebral malaria infection,and the model is in line with the expected requirements.The anti-cerebral malaria activity of DHA and POH in vivo showed that DHA showed strong inhibitory effect on the growth of Pb.ANKA strain of malaria parasite,and also had a certain concentration-dependence;the anti-cerebral malaria activity of POH in vivo was weak.The ED₅₀ of DHA-sol and POH-sol in Pb.ANKA was 1.93μmol/kg/day and 58.07μmol/kg/day,respectively.The ratio of ED₅₀ of the two drugs was used as the combination ratio（the molar ratio of DHA and POH was 1:30）for the treatment of CM,and the combination index（CI）of DHA and POH was calculated,and the CI values of the five concentration gradients were 0.59,0.38,0.73,1.00 and 1.07,respectively,showing synergistic effects,therefore,the molar ratio of DHA and POH was selected as 1:30 as the proportion of combined application of two drugs.2.Preparation and evaluation of brain targeting liposomes delivered by dihydroartemisinin/perillyl alcohol.The methods of in vitro analysis of POH and DHA were established and confirmed.The optimal prescription of DP@Tyr-Lips was selected by single factor optimization and response surface optimization.The Size and PDI of liposome prepared by better prescription was 91.99 nm±2.67 nm and was 0.318±0.019,respectively,The EE of DHA and POH were 88.60±3.23%and 82.89±3.76%,The DL of DHA and POH were 14.7±1.06%and12.0±2.03%,respectively.Liposomes are light blue opalescent,spheroid,uniform in size,and have good physical stability.The percentage of cumulative release of DHA and POH reached 66.91±1.56%and 58.76±0.31%in vitro,respectively within 24 h,showing a certain sustained release effect.The liposome also has good anti-phagocytosis effect and Blood safety.3.Evaluation of in vivo pharmacodynamics and long cycle effects of dihydroartemisinin/perillyl alcohol codelivery of brain-targeted liposomes.In vivo pharmacodynamic results showed that DP@Tyr-Lips group could effectively inhibit the occurrence and development of CM and prolong the survival time of CM mice.On the first day of drug withdrawal,tail tip blood and smear microscopy were taken from mice in each experimental group to calculate the infection rate and inhibition rate.The inhibition rate of plasmodium was ranked from low to high in each administration group were:DHA-sol（71.70±6.20%）<DP-sol（78.10±1.70%）<DP-Lips（82.90±0.70%）<DP@Tyr-Lips（88.90±4.20%）.The results of H&E staining of brain,liver and spleen tissues,hematological parameters,ALT and AST in liver function,permeability of blood-brain barrier and the levels of TNF-αand IFN-γin brain tissue of mice in each group showed that DP@Tyr-Lips had high biological safety and good anti-cerebral malaria effect.The results of brain targeting experiment showed that the fluorescence intensity values of C6-DP-Lips group and C6-DP@Tyr-Lips group were 0.741±0.123 and 0.892±0.037,respectively,after 6 h of drug administration,indicating that tyrosine modification could enable liposome targeting to cross the BBB and act on brain tissue.The result of long cycle evaluation results showed that 2 h after drug injection,the accumulative concentration of C6in C6-DP-sol group was reduced to 13%,while the concentration of C6 in C6-DP-sol group was higher than 16.9%in animals at 12 h,and 24 h,The concentration of of C6 in C6-DP-Lips group was reduced to 14.2%,which was not different from that of C6 solution group.The concentration of of C6 in C6-DP@Tyr-Lips remained above 40%.It shows that DP@Tyr-Lips prolongs the retention time of preparation in the body and realizes long blood circulation.Conclusion:1.Evaluation of the synergistic effect of dihydroartemisinin/perillyl alcohol on murine cerebral malaria.The pharmacodynamic results of the combination of DHA and POH showed that the use of DHA and POH had a strong synergistic effect,which was in line with our expectation of the synergistic effect of the two drugs.2.Preparation and evaluation of brain targeting liposomes delivered by dihydroartemisinin/perillyl alcohol.DP@Tyr-Lips has uniform particle size,good PDI,EE and DL,good physical and serum stability,anti-macrophage phagocytosis and slow release properties,and good blood safety.3.Evaluation of in vivo pharmacodynamics and long cycle effects of dihydroartemisinin/perillyl alcohol codelivery of brain-targeted liposomes.After comparison of pharmacodynamics of different groups of mice,DP@Tyr-Lips administration group showed stronger anti-malaria effect and stronger brain targeting effect than DHA-sol,POH-sol and DP-Lips administration groups.DP@Tyr-Lips also showed good biosafety and long circulation in the body.