黄芪多糖脂质体的制备及其在Caco-2细胞模型的转运特性研究

doi:10.16431/j.cnki.1671-7236.2024.10.041

摘要/Abstract

摘要： 【目的】研究黄芪多糖脂质体(Astragalus polysaccharide liposomes，APSL)制备及其在Caco-2细胞模型中的转运。【方法】采用薄膜分散法制备APSL，通过透射电镜观察APSL结构，鱼精蛋白法检测APSL的包封率及载药量；建立Caco-2单层细胞模型，通过透射电镜观察其结构，测量细胞电阻值及荧光素钠表观渗透系数(apparent permeability coefficient，Papp)来评价Caco-2细胞模型的致密性和完整性；分别进行肠腔侧(apical side，AP)到基底侧(basolateral side，BL)及BL到AP侧两个转运方向的跨膜转运试验，分析药物浓度及P-糖蛋白(P-gp)的抑制剂维拉帕米溶液(Verapamil，Ver)对转运的影响。【结果】 APSL包封率为71.74%±4.87%，载药量为2.92%，透射电镜下可见球状双层结构，粒径为(126.6±0.283)nm (n=3)，聚合物分散性指数(polymer dispersity index，PDI)为0.190±0.009；透射电镜下观察Caco-2细胞紧密结合，形成微绒毛结构，跨膜电阻值达到650 Ω·cm²，碱性磷酸酶(alkaline phosphatase，ALP)比值达到3.05，荧光素钠Papp为1.96×10^-7 cm/s，可满足转运试验的要求。在不同浓度下(75、150、300 μg/mL)，黄芪多糖(APS)的外排率(efflux ratio，ER)均＞1.5，APSL的ER均＜1.5。3个浓度上AP-BL吸收方向上APSL组Papp均极显著高于APS组(P＜0.01)，且ER均低于APS组。加入P-gp蛋白抑制剂后，与APS组相比，APS+Ver组AP-BL方向Papp极显著升高(P＜0.01)，ER降低；而APSL+Ver组两侧的Papp均没有明显变化(P＞0.05)。与APS组ER(3.995)相比，APSL组的ER(1.005)降低。【结论】 APS在体外Caco-2细胞单层模型上的转运较差，为低渗透性化合物，可能存在P-gp蛋白外排，而APSL能够增加APS的转运吸收，提高药物渗透性。

关键词: 黄芪多糖脂质体; Caco-2细胞模型; 吸收特性

Abstract: 【Objective】 The aim of this experiment was to study the preparation of Astragalus polysaccharide liposomes (APSL) and their transport in Caco-2 cell model. 【Method】 APSL was prepared by film dispersion method，the structure of APSL was observed by transmission electron microscopy，and the encapsulation rate and drug loading capacity of liposomes were detected by the method of fisetin. A Caco-2 monolayer cell model was established，and the structure was observed by transmission electron microscopy. The denseness and completeness of the Caco-2 cell model was evaluated by measuring the cell resistance value and apparent permeability coefficient (Papp) of sodium fluorescein，respectively. Transmembrane transport experiments were performed from the apical side (AP) to the basolateral side (BL) and from the BL to the AP side to analyze the effects of drug concentration，and P-gp inhibitor Verapamil (Ver) on the transport. 【Result】 The encapsulation rate of APSL was 71.74%±4.87%，the drug loading was 2.92%，the spherical bilayer structure was visible under transmission electron microscopy，the particle size was (126.6±0.283) nm (n=3)，and the polymer dispersity index (PDI) was 0.190±0.009. Caco-2 cells were observed to be tightly bound under transmission electron microscopy，forming a microvillus structure，and the transmembrane resistivity value reached 650 Ω·cm²，the alkaline phosphatase (ALP) ratio reached 3.05，and the Papp of sodium fluorescein was 1.96×10^-7 cm/s，which fulfilled the requirements of the transport experiments. At different concentrations (75，150 and 300 μg/mL)，the efflux ratio (ER) of APS was higher than 1.5，and the ER of APSL was lower than 1.5. The Papp of the APSL group was extremely significantly higher than that of APS group in the absorption direction of AP-BL at three concentrations (P＜0.01)，and the ER was lower than that of APS group. After adding P-gp protein inhibitors，compared with APS group，the Papp of APS+Ver group was extremely significantly increased in the AP-BL direction (P＜0.01)，while the ER was decreased. However，there was no significant change in Papp on both sides of APSL+Ver group (P＞0.05). Compared with the ER of APS group (3.995)，the ER of APSL group (1.005) was decreased.【Conclusion】 APS was poorly transported on the monolayer model of Caco-2 cells in vitro as a low-permeability compound，and there might be P-gp protein exocytosis，whereas APSL was able to increase the transport and absorption of APS，and improve the permeability of the drug.

Key words: Astragalus polysaccharide liposome; Caco-2 cell model; absorption characteristics

中图分类号:

S859.7

叶钰滢, 王鸿鑫, 刘栩沂, 韩梓杰, 邓桦, 张楠, 张德显, 杨鸿. 黄芪多糖脂质体的制备及其在Caco-2细胞模型的转运特性研究[J]. 中国畜牧兽医, 2024, 51(10): 4606-4615.

YE Yuying, WANG Hongxin, LIU Xuyi, HAN Zijie, DENG Hua, ZHANG Nan, ZHANG Dexian, YANG Hong. Preparation of Astragalus Polysaccharide Liposomes and Its Transport Characteristics in Caco-2 Cell Model[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4606-4615.

参考文献

[1] 禹博威,潘晓琼,陈君第霞,等.黄芪多糖对糖尿病动脉粥样硬化大鼠糖脂代谢的影响及血管内皮保护机制[J].浙江中医药大学学报,2021,45(5):447-453.YU B W，PAN X Q，CHEN J D X，et al. Effect of Astragalus polysaccharides on glucose and lipid metabolism in diabetic atherosclerosis rats and vascular endothelial protec-tion mechanism[J]. Journal of Zhejiang Chinese Medical University,2021,45(5):447-453.(in Chinese)
[2] 马艳春,胡建辉,吴文轩，等.黄芪化学成分及药理作用研究进展[J].中医药学报,2022,50(4):92-95.MA Y C，HU J H，WU W X，et al. Research progress on chemical constituents and pharmacological effects of Radix astragali[J]. Acta Chinese Medicine and Pharmacology,2022,50(4):92-95.(in Chinese)
[3] ZHENG Z M,PAN X L,LOU L，et al. Advances in oral absorption of polysaccharides: Mechanism，affecting factors，and improvement strategies[J].Carbohydrate Polymers,2022,282:119110.
[4] TORCHILIN VLADIMIR P. Multifunctional，stimuli-sensitive nanoparticulate systems for drug delivery[J]. Nature Reviews. Drug Discovery,2014,13(11):813-827.
[5] YU Q，YAN Z，YANG Y Y，et al. Yolk-shell cationic liposomes overcome mucus and epithelial barriers for enhanced oral drug delivery[J]. Giant,2024,17:100221.
[6] 李铭莹,林霖,王岩，等.人参皂苷抗肿瘤机制及其纳米药物递送系统的研究进展[J].中草药,2024,55(2):688-696.LI M Y，LIN L，WANG Y，et al. Research progress on antitumor mechanism and nano-drug delivery system for ginsenosides[J].Chinese Traditional and Herbal Drugs,2024,55(2):688-696.(in Chinese)
[7] MARAINE C T，GIULIA B，ICARO S P，et al. Predicting absorption of amphotericin B encapsulated in a new delivery system by an in vitro Caco-2 cell model[J].Journal of Drug Delivery Science and Technology,2022,71:103345.
[8] ZHAO M Z，CHENG D B，SHANG Z R，et al. An “in vivo self-assembly” strategy for constructing superstructures for biomedical applications[J].Chinese Journal of Polymer Science,2018,36(10):1103-1113.
[9] AGHEBATI-MALEKI A，DOLATI S，AHMADI M，et al. Nanoparticles and cancer therapy: Perspectives for application of nanoparticles in the treatment of cancers[J].Journal of Cellular Physiology,2020,235:1962-1972.
[10] 黄莹颖.基于白及多糖/纳米氧化锌复合载体的pH响应递送系统的构建及其负载白藜芦醇的抗癌活性评价[D].上海：上海海洋大学,2023.HUANG Y Y. Construction of pH-responsive delivery system based on white and polysaccharide/nano-zinc oxide composite carrier and evaluation of anticancer activity of resveratrol[D]. Shanghai: Shanghai Ocean University,2023.(in Chinese)
[11] SHAO Y Y,ZHAO Y N,SUN Y F，et al. Investigation of the internalization and transport mechanism of Codonopsis Radix polysaccharide both in mice and Caco-2 cells[J].International Journal of Biological Macromolecules,2022,215(31):23-35.
[12] 张勇军,巫辅达,徐志高,等.黄芪多糖脂质体的制备及表征研究[J].中国兽医杂志,2019,55(7):72-75.ZHANG Y J，WU F D，XU Z G，et al. Preparation and characterization of Astragalus polysaccharide liposome [J].Chinese Journal of Veterinary Medicine,2019,55(7):72-75.(in Chinese)
[13] SILVIA L，ANJA W. Evaluation of Caco-2 and human intestinal epithelial cells as in vitro models of colonic and small intestinal integrity[J].Biochemistry and Biophysics Reports,2022,31:101314.
[14] ARTURSSON P，KARLSSON J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells[J]. Biochemical and Biophysical Research Communications,1991,175(3):880-885.
[15] ZHANG M Y，SU Y J，LI J H，et al. Fabrication of phosphatidylcholine-EGCG nanoparticles with sustained release in simulated gastrointestinal digestion and their transcellular permeability in a Caco-2 monolayer model[J].Food Chemistry,2023,437(1):137580.
[16] VOLPE D A，FAUSTINO P J，CIAVARELLA A B，et al. Classification of drug permeability with a Caco-2 cell monolayer assay[J]. Clinical Research and Regulatory Affairs,2007,24(1)：39-47.
[17] ARTURSSON P，PALM K，LUTHMAN K. Caco-2 monolayers in experimental and theoretical predictions of drug transport[J]. Advanced Drug Delivery Reviews,2001,46(3):27-43.
[18] SANTOS R R D，RAMOS M C，FERREIRA J V，et al. Biopharmaceutical evaluation of kavain in Piper methysticum G. Forst dried extract: Equilibrium solubility and intestinal permeability in Caco-2 cell model[J].Journal of Ethnopharmacology,2022,296,115480.
[19] 舒婷,常艳波,杨蓉，等.靛玉红固体脂质纳米粒的制备及Caco-2单层细胞转运研究[J].中国药学杂志,2023,58(22):2085-2091.SHU T，CHANG Y B，YANG R，et al. Preparation of lndirubin solid lipid nanoparticles and transport of Caco-2 monolayer cells[J]. Chinese Pharmaceutical Journal,2023,58(22):2085-2091.(in Chinese)
[20] 李发鹏，王刚，蒋安蓉，等.槲皮素及其纳米脂质体在Caco-2细胞的吸收特性研究[J].安徽医药杂志,2011，15(10):1206-1208.LI F P，WANG G,JIANG A R，et al. Absorption characteristic of quercetin lipid nanoparticles across Caco-2 cell[J].Anhui Medical and Pharmaceutical Journal,2011,15(10):1206-1208.(in Chinese)
[21] BEIS K. Structural basis for the mechanism of ABC transporters[J]. Biochemical Society Transactions，2015，43:889-893.
[22] 沈晓玲，杨志宏.多药耐药相关ABC转运蛋白信号转导通路研究进展[J].中国药理学与毒理学杂志，2018，32(10):816-826.SHEN X L,YANG Z H. Advancement of multidrug resistance induced by ABC transporters[J]. Chinese Journal of Pharmacology and Toxicology，2018，32(10):816-826.(in Chinese)
[23] SCHINKEL A H，JONKER J W. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: An overview[J].Advanced Drug Delivery Reviews,2003,55(1):3-29.
[24] 邢艾雅,高迪,陈跃杰.影响姜黄素口服吸收的因素及药物递送策略[J].医药导报,2024,43(5):762-768.XING A Y，GAO D，CHEN Y J. Overview of factors affecting oral absorption of curcumin and its drug delivery strategies [J].Herald of Medicine,2024,43(5):762-768.(in Chinese)
[25] ATTIA M S，ELSEBAEY M T，YAHYA G，et al. Pharmaceutical polymers and P-glycoprotein: Current trends and possible outcomes in drug delivery[J].Materials Today Communications,2023,34:105318.
[26] MURAKAMI T，TAKANO M. Intestinal efflux transporters and drug absorption[J]. Expert Opinion on Drug Metabolism & Toxicology，2008，4(7):923-939.
[27] HUANG S，ZHAI B，FAN Y，et al. Development of paeonol liposomes: Design，optimization，in vitro and in vivo evaluation[J]. International Journal of Nanomedicine，2022，17:5027-5046.