新型姜黄素纳米粒在小鼠体内组织分布的研究

Research on Tissue Distribution of Novel Curcumin Nanoparticles in Mice

关键词:  姜黄素油酸复合物  mPEG₅₀₀₀-PLGA  纳米粒  体内组织分布

Key words:  Cur(OA)₂  mPEG₅₀₀₀-PLGA  nanoparticles  tissue distribution in vivo

摘要:

目的 制备新型姜黄素油酸复合物肝靶向纳米粒[Cur（OA）₂-NPs]，并对其小鼠体内组织分布进行研究。方法 基于前期工作按最佳工艺，应用改良的溶剂挥发法制备Cur（OA）₂-NPs并进行表征；以25 mg·kg^-1小鼠尾静脉注射Cur（OA）₂-NPs，分别于给药后0.05，0.25，0.75，2，4，6，8，12 h，经眼球取血200 μL，小鼠解剖后取肝脏、心脏、脾脏、肺脏、肾脏和脑，经过液-液萃取法提取药物，HPLC测定姜黄素油酸[Cur（OA）₂]和姜黄素（Cur）的含量，分析Cur（OA）₂-NPs体内组织分布和药物释放特性；将Cur（OA）₂和DiR荧光染料包裹到mPEG₅₀₀₀-PLGA中制得纳米粒，按Cur剂量25 mg·kg^-1通过静脉注射给予正常小鼠和H22荷瘤小鼠，于不同时间点麻醉小鼠，将其置于785 nm激发波和820 nm发射波形成的活体红外成像系统中扫描成像，研究正常小鼠和H22荷瘤小鼠体内纳米粒的肿瘤靶向性特征。结果 纳米粒呈圆形，大小均匀，平均粒径为（93.39±1.71）nm，载药量为（19.35±0.12）%，包封率为（92.32±3.13）%。血浆中除脑组织外其余组织均可检测到Cur（OA）₂分布，分布迅速，Cur（OA）₂浓度4 h内从310.33 μg·mL^-1减少到28.94 μg·mL^-1，近90%从血管中被清除，肝脏中含量最高可达368.93 μg·g^-1；肝脏、脾脏和肾脏可检测到Cur，分别为125.72，33.60，16.81 μg·g^-1，而血浆、肺脏和脑中则无。纳米粒静脉注射后2 h左右出现峰值，其中肝脏Cur（OA）₂和Cur的浓度最高；活体成像结果也表明，小鼠体内纳米粒主要分布于肝脏和肿瘤部位，肝脏2 h左右达到峰值随后慢慢下降，而肿瘤组织8 h左右可见强烈荧光，并于12 h左右达到峰值，随后缓慢下降，且其荧光强度显著强于肝脏部位。结论 本研究完善了Cur（OA）₂-NPs的评价，为进一步研究其体内抗肿瘤作用奠定了良好的基础，也为难溶的、口服生物利用度低的药物开发提供了解决思路。

Abstract:

OBJECTIVE To prepare the novel liver-targeting Cur(OA)₂ nanoparticles (Cur(OA)₂-NPs) and investigate the tissue distribution in mice. METHODS Cur(OA)₂ loaded mPEG₅₀₀₀-PLGA nanoparticles were prepared by the modified emulsion solvent evaporation method according to the optimized technology and the Cur(OA)₂-NPs were characterized. 25 mg·kg^-1 of Cur(OA)₂-NPs was intravenously injected. At the 0.05, 0.25, 0.75, 2, 4, 6, 8, 12 h after administration, 200 μL of blood was taken through the eyeball. The organs(liver, heart, spleen, lung, kidney and brain) were harvested for the measurment of Cur(OA)₂ and curcumin(Cur) with HPLC method after extracting the drug by liquid-liquid extraction method, then analysized the tissue distribution and drug released in vivo. Nanoparticles were prepared by loading DiR and Cur(OA)₂ into mPEG₅₀₀₀-PLGA. Normal and H22 cancer model mice were injected at a dose of 25 mg·kg^-1 of Cur intravenously. At different time intervals, the mice were anesthetized and scanned by using an in vivo NIR optical imaging system with an excitation bandpass filter at 785 nm and an emission filter at 820 nm. Studied the tumor targeting characteristics of nanoparticles in normal mice and H22 cancer model mice. RESULTS The nanoparticles were spherical with uniform size. The average particle size, drug loading and encapsulation efficiency of nanoparticles were (93.39±1.71)nm, (19.35±0.12)% and (92.32±3.13)% respectively. After intravenous injection, the Cur(OA)₂ in the plasma reduced from 310.33 μg·mL^-1 to 28.94 μg·mL^-1 within 4 h, and 90% of Cur(OA)₂ was cleared from blood vessel rapidly. The results in the tissue distribution showed that Cur(OA)₂ was found in other organs besides brain. Cur(OA)₂ reached the highest level of 368.93 μg·g^-1 in liver. There was no Cur detected in plasma, lung and brain, but only detected in the liver(125.72 μg·g^-1), spleen(33.60 μg·g^-1) and kidney(16.81 μg·g^-1). The peak value was found at the 2nd hour after intravenous injection Cur(OA)₂-NPs, and the Cur(OA)₂ and Cur in the liver were greater than the other organs significantly. In vivo NIR optical imaging system provided evidence that Cur(OA)₂-NPs were accumulated in the liver and tumor, the NIR signal reached the climax at the 2nd hour and decreased slowly as time went by, and we observed a strong NIR signal in the tumor mass at the 8th hour after the administration of nanoparticles. The signal intensity reached the climax in the tumor mass at the 12th hour and decreased follow, and it was stronger than liver significantly. CONCLUSION This research completed the evaluation of Cur(OA)₂-NPs and laid the foundation for research on anti-tumor of Cur(OA)₂-NPs in vivo in future. Meanwhile, it can offer some clue for developing a new drug which is insoluble, easily metabolic or toxic in vivo.