两性离子聚合物链长对纳米粒穿黏液及细胞摄取能力的影响
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摘要
本研究旨在构建不同链长的聚磺酸甜菜碱甲基丙烯酸酯[poly(sulfobetaine methacrylate), pSBMA]修饰的纳米粒(pSBMAn NPs),以探究两性离子聚合物链长对纳米粒穿黏液及细胞摄取能力的影响。结合己内酯的开环聚合反应和原子转移自由基聚合反应(atom transfer radical polymerization, ATRP)合成不同链长的两嵌段聚合物——聚己内酯-聚磺酸甜菜碱甲基丙烯酸酯共聚物[poly(ε-caprolactone)-block-poly(sulfobetaine methacrylate), PCLpSBMA],并通过纳米沉淀法制备相应的纳米粒。采用黏蛋白吸附实验和Transwel小室实验考察纳米粒的穿黏液能力。以人源结肠癌Caco-2细胞和可分泌黏液的HT-MTX-E12细胞为模型,考察链长对纳米粒摄取及穿黏液能力的影响。研究结果表明:制得的pSBMAn NPs粒径相近,均约为100 nm,电位约为-7 mV。短链pSBMA修饰的纳米粒(pSBMA_(10)NPs)的表观渗透系数(apparent permeability coefficient, P_(app))仅是长链pSBMA纳米粒(pSBMA_(80)NPs)的42.83%,但细胞摄取是pSBMA_(80)NPs的2.44倍。有黏液存在时, pSBMAn NPs的摄取均降低,但pSBMA_(10)NPs的细胞摄取能力仍最强。体内实验结果表明, pSBMA_(20)NPs的口服生物利用度高于pSBMA_(10)NPs (动物实验根据四川大学关于实验动物的饲养和使用准则进行,并得到四川大学实验动物伦理委员会批准)。本文为两性离子纳米粒的口服研究提供了参考。
To investigate the influences of zwitterionic polymer chain length on mucus permeability and cellular uptake, the nanoparticles(NPs) were coated with poly(sulfobetaine methacrylate)(pSBMA) with different chain lengths. The di-block polymer poly(ε-caprolactone)-block-poly(sulfobetaine methacrylate)(PCL-pSBMA)with different chain lengths were synthesized via atom transfer radical polymerization(ATRP) combining with ring-opening polymerization of ε-caprolactone, and corresponding nanoparticles(pSBMAn NPs) were prepared by nanoprecipitation method. The sizes of different pSBMAn NPs were around 100 nm, and zeta potential were about-7 mV. Mucin interaction or mucus penetration study based on transwell systems were employed to evaluate mucus permeability of NPs. Caco-2 cells and mucus-producing HT-MTX-E12 cells were employed to illustrate the endocytosis efficiency of pSBMAn NPs. The results showed that the permeability coefficient of NPs coated with shorter chain length of pSBMA(pSBMA_(10) NPs) was only 42.83% of that coated with longer pSBMA(pSBMA_(80) NPs). On the contrary, the cellular uptake of pSBMA_(10) NPs was 2.44 fold higher compared to pSBMA_(80) NPs.Although the cellular uptake of pSBMAn NPs was reduced in the presence of mucus, pSBMA_(10) NPs still presented the highest cellular uptake. However, the in vivo results indicated that the oral bioavailability of pSBMA_(20) NPs was higher than that of pSBMA_(10) NPs. All animal procedures were performed in accordance with the Guidelines of the Sichuan University Animal Care and Use Committee and were approved by the Animal Ethics Committee of Sichuan University. This study provides a reference for oral delivery of zwitterionic nanoparticles.
To investigate the influences of zwitterionic polymer chain length on mucus permeability and cellular uptake, the nanoparticles(NPs) were coated with poly(sulfobetaine methacrylate)(pSBMA) with different chain lengths. The di-block polymer poly(ε-caprolactone)-block-poly(sulfobetaine methacrylate)(PCL-pSBMA)with different chain lengths were synthesized via atom transfer radical polymerization(ATRP) combining with ring-opening polymerization of ε-caprolactone, and corresponding nanoparticles(pSBMAn NPs) were prepared by nanoprecipitation method. The sizes of different pSBMAn NPs were around 100 nm, and zeta potential were about-7 mV. Mucin interaction or mucus penetration study based on transwell systems were employed to evaluate mucus permeability of NPs. Caco-2 cells and mucus-producing HT-MTX-E12 cells were employed to illustrate the endocytosis efficiency of pSBMAn NPs. The results showed that the permeability coefficient of NPs coated with shorter chain length of pSBMA(pSBMA_(10) NPs) was only 42.83% of that coated with longer pSBMA(pSBMA_(80) NPs). On the contrary, the cellular uptake of pSBMA_(10) NPs was 2.44 fold higher compared to pSBMA_(80) NPs.Although the cellular uptake of pSBMAn NPs was reduced in the presence of mucus, pSBMA_(10) NPs still presented the highest cellular uptake. However, the in vivo results indicated that the oral bioavailability of pSBMA_(20) NPs was higher than that of pSBMA_(10) NPs. All animal procedures were performed in accordance with the Guidelines of the Sichuan University Animal Care and Use Committee and were approved by the Animal Ethics Committee of Sichuan University. This study provides a reference for oral delivery of zwitterionic nanoparticles.
引文
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[18]Morimoto N,Wakamura M,Muramatsu K,et al.Membrane translocation and organelle-selective delivery steered by poly‐meric zwitterionic nanospheres[J].Biomacromolecules,2016,17:1523-1535.
[2]He Z,Santos JL,Tian H,et al.Scalable fabrication of sizecontrolled chitosan nanoparticles for oral delivery of insulin[J].Biomaterials,2017,130:28-41.
[3]Shan W,Zhu X,Liu M,et al.Overcoming the diffusion barrier of mucus and absorption barrier of epithelium by self-assembled nanoparticles for oral delivery of insulin[J].ACS Nano,2015,9:2345-2356.
[4]Xu Q,Ensign LM,Boylan NJ,et al.Impact of surface poly‐ethylene glycol(peg)density on biodegradable nanoparticle transport in mucus ex vivo and distribution in vivo[J].ACSNano,2015,9:9217-9227.
[5]Mao J,Li JM,Ding JS.The application of poly(2-ethyl-2-oxazo‐line)in drug delivery system[J].Acta Pharm Sin(药学学报),2017,52:1235-1240.
[6]Chang Y,Shih YJ,Lai CJ,et al.Blood-inert surfaces via ion-pair anchoring of zwitterionic copolymer brushes in human whole blood[J].Adv Funct Mater,2013,23:1100-1110.
[7]Cheng G,Zhang Z,Chen SF,et al.Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces[J].Biomaterials,2007,28:4192-4199.
[8]Sin MC,Chen SH,Chang Y.Hemocompatibility of zwitterionic interfaces and membranes[J].Polym J,2014,46:436-443.
[9]Chang Y,Chang WJ,Shih YJ,et al.Zwitterionic sulfobetainegrafted poly(vinylidene fluoride)membrane with highly effec‐tive blood compatibility via atmospheric plasma-induced surface copolymerization[J].ACS Appl Mater Interfaces,2011,3:1228-1237.
[10]Wang WL,Wang B,Liu SR,et al.Bioreducible polymer nanocarrier based on multivalent choline phosphate for enhanced cellular uptake and intracellular delivery of doxorubicin[J].ACSAppl Mater Interfaces,2017,9:15986-15994.
[11]Behrens I,Pena AIV,Alonso MJ,et al.Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats:the effect of mucus on particle adsorption and transport[J].Pharm Res,2002,19:1185-1193.
[12]Chater PI,Wilcox MD,Pearson JP.Efficacy and safety concerns over the use of mucus modulating agents for drug delivery using nanoscale systems[J].Adv Drug Deliv Rev,2018,124:184-192.
[13]Zhai SY,Ma YH,Chen YY,et al.Synthesis of an amphiphilic block copolymer containing zwitterionic sulfobetaine as a novel pH-sensitive drug carrier[J].Polym Chem,2014,5:1285-1297.
[14]Guo SS,Janczewski D,Zhu XY,et al.Surface charge control for zwitterionic polymer brushes:tailoring surface properties to antifouling applications[J].J Colloid Interface Sci,2015,452:43-53.
[15]Ren PF,Yang HC,Liang HQ,et al.Highly stable,proteinresistant surfaces via the layer-by-layer assembly of poly(sulfo‐betaine methacrylate)and tannic acid[J].Langmuir,2015,31:5851-5858.
[16]Polzer F,Heigl J,Schneider C,et al.Synthesis and analysis of zwitterionic spherical polyelectrolyte brushes in aqueous solution[J].Macromolecules,2011,44:1654-1660.
[17]Ye Y,Zheng Y,Ji C,et al.Self-assembly and disassembly of amphiphilic zwitterionic perylenediimide vesicles for cell membrane imaging[J].ACS Appl Mater Interfaces,2017,9:4534-4539.
[18]Morimoto N,Wakamura M,Muramatsu K,et al.Membrane translocation and organelle-selective delivery steered by poly‐meric zwitterionic nanospheres[J].Biomacromolecules,2016,17:1523-1535.