[關(guān)鍵詞]
[摘要]
目的 為了改善丹參酮ⅡA(TanⅡA)的溶解性、增加腦組織中的藥物濃度,制備TanⅡA聚合物脂質(zhì)納米粒(TanⅡAPLNs)并進(jìn)行體外釋放、藥動(dòng)學(xué)、腦組織分布研究。方法 建立TanⅡA含量測(cè)定的高效液相色譜(HPLC)法,以納米制劑的包封率和粒徑為指標(biāo),優(yōu)化TanⅡA-PLNs的制備工藝,分別優(yōu)化TanⅡA與蛋黃磷脂(Egg-PC)的比例、Egg-PC與聚乳酸-羥基乙酸共聚物(PLGA)的比例、有機(jī)相與水相的比例;進(jìn)行TanⅡA-PLNs的處方驗(yàn)證、穩(wěn)定性考察、體外釋放考察。建立TanⅡA在血漿和腦勻漿中的液-質(zhì)聯(lián)用(LC-MS)檢測(cè)方法。SPF級(jí)雄性SD大鼠6只,隨機(jī)分為2組,給藥前12 h禁食,一組ig 25 mg·kg-1的TanⅡA混懸液,另一組尾iv 5 mg·kg-1的TanⅡA-PLNs溶液,于給藥后5、15、30、60、120、240、360、480、720 min眼眶取血約0.5 mL,LC-MS法進(jìn)行藥動(dòng)學(xué)檢測(cè)。取KM小鼠4只,尾iv DiR標(biāo)記的TanIIA-PLNs,給藥30、60、120、240 min后處死解剖,通過熒光成像觀察TanIIA-PLNs在組織中的分布情況。取KM小鼠12只,隨機(jī)分成4組,給藥前12 h禁食,尾iv 2.5 mg·kg-1的TanⅡA-PLNs溶液,于給藥后30、60、120、240 min處死小鼠,剝離完整大腦組織,稱質(zhì)量,加入2倍超純水勻漿,LC-MS法檢測(cè)TanⅡA水平。結(jié)果 TanⅡA-PLNs的最優(yōu)處方為TanⅡA∶Egg-PC為1∶4、Egg-PC∶PLGA為5∶2、有機(jī)分散體系∶水分散體系為1∶15。以Egg-PC/PLGA作為脂質(zhì)納米粒的膜材,采用納米粒沉淀法制備的TanⅡA-PLNs的平均粒徑為272 nm,Zeta電位為-4.93 mV,包封率為88.2%,載藥量為18%。在避光、干燥(室溫)的條件下TanIIA-PLNs凍干粉穩(wěn)定。TanIIA原料藥在48 h內(nèi)釋放完全,累積釋放率為(93.75±0.75)%,與Korsmeyer-Peppas釋放方程擬合程度較好; TanIIAPLNs在400 h左右釋放完全,累積釋放率為(89.44±2.22)%,與零級(jí)釋放方程擬合度最好。藥動(dòng)學(xué)結(jié)果表明,大鼠尾iv TanⅡA-PLNs給藥劑量是ig TanⅡA原料藥劑量的1/5,TanⅡA-PLNs的AUC0-t和t1/2ß是TanⅡA原料藥的2.8和1.5倍。組織分布實(shí)驗(yàn)結(jié)果顯示,30 min時(shí)肝組織顯示熒光; 60 min時(shí)腦組織顯示出熒光,120 min時(shí)腦組織熒光最強(qiáng),240 min時(shí)腦組織熒光變?nèi)?。TanⅡA-PLNs給藥2 h后,TanⅡA腦勻漿質(zhì)量濃度為235.5 ng·g-1。結(jié)論 制備的TanⅡA-PLNs均一穩(wěn)定,包封率較高,聚合物脂質(zhì)納米顆粒的應(yīng)用提高了TanⅡA的血藥濃度,可增加藥物在腦部的暴露。
[Key word]
[Abstract]
Objective Tanshinone IIA polymeric lipid nanoparticles (TanⅡA-PLNs) were prepared for improving the solubility and drug concentration in brain tissue to increase drug brain exposure. In vitro release, pharmacokinetics and brain tissue distribution were studied. Method A high performance liquid chromatography (HPLC) method was established to determine the content of TanⅡA, and the preparation process of TanⅡA-PLNs was optimized by taking the encapsulation rate and particle size of nano-preparations as indexes. The proportion of TanⅡA to Egg-PC, Egg-PC to polylactic acid - hydroxyacetic acid copolymer (PLGA) and organic phase to water phase were optimized respectively. The prescription validation, stability and in vitro release of Tan ⅡA-PLNs were investigated. To establish a method for the determination of TanⅡA in plasma and brain homogenates by liquid - mass spectrometry (LC-MS). Six SPF male SD rats were randomly divided into two groups, one group was given 25 mg·kg-1 TanⅡA suspension, the other group was given 5 mg·kg-1 TanⅡA-PLNs solution. At 5, 15, 30, 60, 120, 240, 360, 480, 720 min after administration, about 0.5 mL blood was collected from orbit, and pharmacokinetic detection was performed by LC-MS. Four KM mice were selected and TanⅡ A-PLNs labeled with iv DiR were sacrificed 30, 60, 120 and 240 min after administration, and the distribution of TanⅡA-PLNs in tissues was observed by fluorescence imaging. Twelve KM mice were randomly divided into four groups, fasted 12 h before administration, and treated with 2.5 mg·kg-1 TanⅡA-PLNs solution in tail iv. The mice were sacrificed 30, 60, 120 and 240 min after administration. The intact brain tissues were stripped, weighed, and the level of TanⅡA was detected by LC-MS. Results Egg-PC/PLGA (5:2) were used as membrane materials of TanIIA-PLNs nanoparticles, and the volume ratio of organic phase to water phase was 1: 15. The particle size, zeta potential, entrapment efficiency and drug loading capacity of TanIIA-PLNs were 272 nm, - 4.93 mV, 88.2%, 18% respectively. TanⅡA-PLNs freeze-dried powder is stable under dry conditions (room temperature) and away from light. Pharmacokinetics showed that the AUC (0-t) and t1/2ß of TanIIA-PLNs were 2.8 and 1.5 times higher than free TanIIA even the dose difference of administration was 1/5 times. The results of tissue distribution experiment showed that the liver tissue showed fluorescence at 30 min. The brain tissue showed fluorescence at 60 min, the fluorescence was the strongest at 120 min, and the fluorescence became weak at 240 min. The plasma concentration of TanIIA reached 235.5 ng/g after 2 h of TanIIA-PLNs administration. Conclusion TanIIA-PLNs were uniform and stable, and had a higher entrapment efficiency. In vivo pharmacokinetic parameters showed that the application of polymeric lipid nanoparticles improved the concentration of TanIIA in brain tissue and increased the exposure of the drug in the brain.
[中圖分類號(hào)]
R943;R969.1
[基金項(xiàng)目]
吉林省發(fā)展改革委員會(huì)項(xiàng)目(202000C032-4)