| Malaria is one of the major diseases that threaten human life safety caused by Plasmodium.Plasmodium has a wide variety of different genotypes,so it is easy to develop drug-resistant strains.Artemisinin(ART)has become the current first-line antimalarial drug due to its unique structure and antimalarial mechanism.However,resistance to artemisinin has also been gradually reported in recent years.In order to clarify the differences of macro-distribution and metabolism in the body and improve drug availability,This study from the angle of the pharmaceutics and pharmacokinetic,preparaing poly(lactic-co-glycolic acid)(PLGA)nanoparticles containing artemisinin,and their pharmacokinetic changes in mice infected with artemisinin-sensitive/resistant Plasmodium berghei were studied,expected to provide new ideas and theoretical basis for delaying and reversing artemisinin resistance.A liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the quantitative determination of artemisinin was established in this study.The method is easy to operate,quick to detect,and has highly sensitive and strong specificity,precision,recovery,repeatability meet the requirements.PLGA Nanoparticles Loaded with Artemisinin(PLGA-ART-NPs)were successfully prepared by emulsifying solvent volatilization method using PLGA as a nanocarrier.With the drug loading and encapsulation rate as evaluation indexes,the main influencing factors in the preparation process of PLGA-ART-NPs were screened by single factor analysis:the concentration of emulsifier,the ratio of water and oil two phases,and the ratio of carrier to drug.Taking the drug loading of PLGA-ART-NPs as the evaluation index,the formulation was further optimized by response surface methodology.The optimal formulation of PLGA-ARTNPs was obtained as follows:the concentration of emulsifier was 1.7(w/v)%,the ratio of water to oil was 18:1,the ratio of carrier to drug was 10:1,and the drug loading was(4.63±0.22)%,with good reproducibility.The particle size,Zeta potential,stability and in vitro release characteristics of PLGAART-NPs were evaluated by transmission electron microscopy,Malvin particle analyzer,liquid mass spectrometry and other detection instruments.The average particle size of PLGA-ARTNPs prepared according to the optimal formulation was(192.2±9.13)nm,the polydispersity index(PDI)was 0.256±0.28,and the Zeta potential was(-26.5±3.72)mV.The particle shape was round and smooth under transmission electron microscope.The determination of particle size and Zeta potential indicated that PLGA-ART-NPs had been successfully prepared.The stability study showed that PLGA-ART-NPs had good stability when incubated in artificial gastric juice,artificial intestinal juice and PBS solution containing 10%plasma for 8 h.In vitro release characteristics showed that artemisinin was mainly absorbed in the gastrointestinal tract,and PLGA-ART-NPs showed obvious sustained-release ability compared with the raw materials.In this study,the K173 genotype of Plasmodium berghei which showed sensitive/resistant to artemisinin was used to establish the mice model of sensitive/resistant Plasmodium berghei infection by intraperitoneal injection of Plasmodium berghei-containing blood.After infection for a period of time,the mice lost weight,the appearance changed significantly,the activity decreased,the ears and limbs had no color,the urine was yellow,and the stool was thin and sticky.Rick’s staining method for blood smears revealed significant changes in the infected red blood cells,with a large number of "ring-shaped" and "headset shaped" protozoa trophoblasts(dark purple substances).After twice blood transfusions,the infection rate of mice was observed and calculated by blood smear,and mice with an infection rate of 5%were selected as the model for administration.A rapid,sensitive and accurate LC-MS/MS method for quantitative analysis of artemisinin concentration in mice plasma was established in this study.Mice infected with artemisininsensitive/resistant Plasmodium berghei were traeted with oral raw materials(0.5%sodium carboxymethyl cellulose mixed)and PLGA-ART-NPs solution,respectively,and the pharmacokinetic characteristics and differences of the two groups were compared.Liquidliquid extraction method was used to extract artemisinin from plasma.The linear range of artemisinin in mice plasma was 1-200 ng·mL-1,and the lower limit of quantition was 1 ng·mL1.All the indicators of the methodological investigation met the requirements.The pharmacokinetic parameters of mice in the sensitive and resistant groups after an oral administration of raw materials were fitted by non-compartment model as follows:Cmax were 89.42±11.09 and 95.70±21.73 ng·mL-1;Tmax were 1.08±0.20 and 1.0 h;ti/2 were 3.11±1.02 and 3.29±2.72 h.AUC0-t were 199.42±12.18 and 208.53±35.65 h·ng·mL-1.MRT were 2.19±0.40 and 2.46±1.00 h;CL were 0.20±0.01 and 0.20±0.04 L·kg·h-1.Vd were 0.89±0.29 and 0.92±0.68 L·kg-1.The pharmacokinetic parameters of mice in the sensitive and resistant groups after an oral administration of PLGA-ART-NPs as follows:Cmax were 79.98±12.51 and 73.95±12.13 ng·mL-1,Tmax were 0.79±0.03 and 1.0 h;t1/2 were 12.54±3.09 and 11.87±1.93 h.AUC0-t were 580.07±106.75 and 593.56±120.09 h·ng·mL-1.MRT were 8.03±0.67 and 8.04±0.72 h;CL was 0.06±0.01 and 0.06±0.02 L·kg·h-1.Vd were 0.98±0.20 and 0.91±0.12 L·kg-1.Compared the pharmacokinetic parameters of raw materials and PLGA-ART-NPs,PLGAART-NPs significantly improved the pharmacokinetic process of artemisinin in model mice,and had a long-term antimalarial effect.Compared the pharmacokinetic parameters of raw materials and PLGA-ART-NPs in the resistant and sensitive groups,the absorption,distribution and metabolic process of artemisinin in the resistant and sensitive groups were not significantly different,suggesting that the possible mechanism of the occurrence of drug resistance and the method of reverse drug resistance can be elucidated from the heme related pathway in the future. |
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