Comparative Study on Chitosan Modified with Different Hepatocyte Ligands as Scaffold Material for Liver Tissue Regeneration: Preparation, Characterization and Evaluation

Abstract

To achieve the ideal scaffolds for liver tissue regeneration, chitosan(CS) was modified with lactobionic acid(LA) or/and glycyrrhetinic acid(GA) to obtain LA-modified CS(LC), GA-modified CS(GC) and GA/LA-modified CS(GLC), and the composite nanofibrous scaffolds composed of silk fibroin(SF) and the above modified CS were fabricated by green electrospinning. Fourier transform infrared(FTIR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy and X-ray diffraction(XRD) patterns were used to characterize the chemical components and structures. The water contact angle was measured to evaluate the hydrophilicity, and thermal gravimetric analysis(TGA) was carried out to obtain the thermal properties. These scaffolds were hydrophilic, and their hydrophilicity and thermal stability decreased with the increase of the modified CS content, while their crystallinity increased. The scaffolds showed good performance in promoting the proliferation of the human hepatoma cell line(HepG2 cell) as well as their secretion of both albumin and urea. Furthermore, the scaffolds with LC had a better performance of hepatocellular compatibility than those with GC or GLC.

Keywords

glycyrrhetinic acid(GA) / galactose / chitosan(CS) / silk fibroin(SF) / liver tissue engineering

References

[1] ASRANI S K, DEVARBHAVI H, EATON J, et al. Burden of liver diseases in the world[J]. Journal of Hepatology, 2019, 70(1):151-171.

[2] LINARES I, HAMAR M, SELZNER N, et al. Steatosis in liver transplantation:current limitations and future strategies[J]. Transplantation, 2019, 103(1):78-90.

[3] LEE J S, SHIN J, PARK H M, et al. Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering[J]. Biomacromolecules, 2014, 15(1):206-218.

[4] KULIG K M, VACANTI J P. Hepatic tissue engineering[J]. Transplant Immunology, 2004, 12(3/4):303-310.

[5] OHASHI K, YOKOYAMA T, YAMATO M, et al. Engineering functional two-and three-dimensional liver systems in vivo using hepatic tissue sheets[J]. Nature Medicine, 2007,13:880-885.

[6] KASOJU N, BORA U. Silk fibroin based biomimetic artificial extracellular matrix for hepatic tissue engineering applications[J]. Biomedical Materials, 2012, 7(4):045004.

[7] JIANG W C, CHENG Y H, YEN M H, et al. Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering[J]. Biomaterials, 2014, 35(11):3607-3617.

[8] LIU H, WANG H, XU Y H, et al. Lactobionic acid-modified dendrimer-entrapped gold nanoparticles for targeted computed tomography imaging of human hepatocellular carcinoma[J]. ACS Applied Materials & Interfaces, 2014, 6(9):6944-6953.

[9] ZHAO R R, LI T, ZHENG G R, et al. Simultaneous inhibition of growth and metastasis of hepatocellular carcinoma by co-delivery of ursolic acid and sorafenib using lactobionic acid modified and pH-sensitive chitosan-conjugated mesoporous silica nanocomplex[J]. Biomaterials, 2017,143:1-16.

[10] ZHOU N, ZOU C Y, QIN M L, et al. A simple method for evaluation pharmacokinetics of glycyrrhetinic acid and potential drug-drug interaction between herbal ingredients[J]. Scientific Reports, 2019,9:11308.

[11] HUANG W, WANG W, WANG P, et al. Glycyrrhetinic acid-functionalized degradable micelles as liver-targeted drug carrier[J]. Journal of Materials Science:Materials in Medicine, 2011, 22(4):853-863.

[12] ZHANG C N, WANG W, LIU T, et al. Doxorubicin-loaded glycyrrhetinic acid-modified alginate nanoparticles for liver tumor chemotherapy[J]. Biomaterials, 2012, 33(7):2187-2196.

[13] CAI Y E, XU Y Q, CHAN H F, et al. Glycyrrhetinic acid mediated drug delivery carriers for hepatocellular carcinoma therapy[J]. Molecular Pharmaceutics, 2016, 13(3):699-709.

[14] ALONSO S. Exploiting the bioengineering versatility of lactobionic acid in targeted nanosystems and biomaterials[J]. Journal of Controlled Release, 2018,287:216-234.

[15] ROHILLA R, GARG T, BARIWAL J, et al. Development,optimization and characterization of glycyrrhetinic acid-chitosan nanoparticles of atorvastatin for liver targeting[J]. Drug Delivery, 2016, 23(7):2290-2297.

[16] AHSAN S M, THOMAS M, REDDY K K, et al. Chitosan as biomaterial in drug delivery and tissue engineering[J]. International Journal of Biological Macromolecules, 2018,110:97-109.

[17] LEVENGOOD S L, ZHANG M Q. Chitosan-based scaffolds for bone tissue engineering[J]. Journal of Materials Chemistry B, 2014, 2(21):3161-3184.

[18] CROISIER F, JÉRÔME C. Chitosan-based biomaterials for tissue engineering[J]. European Polymer Journal, 2013, 49(4):780-792.

[19] TIAN Q, ZHANG C N, WANG X H, et al. Glycyrrhetinic acid-modified chitosan/poly(ethylene glycol) nanoparticles for liver-targeted delivery[J]. Biomaterials, 2010, 31(17):4748-4756.

[20] LI M, WANG Y, JIANG S, et al. Biodistribution and biocompatibility of glycyrrhetinic acid and galactose-modified chitosan nanoparticles as a novel targeting vehicle for hepatocellular carcinoma[J]. Nanomedicine, 2020, 15(2):145-161.

[21] CHEN H X, LI M, WAN T, et al. Design and synthesis of dual-ligand modified chitosan as a liver targeting vector[J]. Journal of Materials Science:Materials in Medicine, 2012, 23(2):431-441.

[22] YU F, YANG X X, ZHOU X F, et al. Green electrospun silk fibroin/galactose chitosan composite nanofibrous scaffolds for hepatic tissue engineering[J]. Journal of Donghua University (English Edition), 2017, 34(1):142-146.

[23] MA X X, ZHANG J, HUANG Z Y, et al. Hydrophilic composite polybutylene terephthalate/polyvinyl alcohol membranes prepared by electrospinning[J]. Journal of Donghua University (English Edition), 2022, 39(6):549-556.

[24] WU T, HUANG C, CHEN J F, et al. Fabrication of multi-layered composite scaffolds by Bi-directional electrospinning method[J]. Journal of Donghua University (English Edition), 2014, 31(5):625-629.

[25] GAO Z, LIU Y, WANG C, et al. Hippocampal neuron-derived extracellular matrix coated nanofibrous scaffold for neural tissue engineering[J]. Journal of Donghua University (English Edition), 2019, 36(5):431-436.

[26] HAN H Y, NING H Y, LIU S S, et al. Silk biomaterials with vascularization capacity[J]. Advanced Functional Materials, 2016, 26(3):421-432.

[27] HUANG H Y, TIAN Z F, YI H G, et al. Designing and cloning of the gene sequence encoding silk fibroin amorphous domain[J]. Journal of Donghua University (English Edition), 2012, 29(6):489-492.

[28] XIAO B, WAN Y, ZHAO M Q, et al. Preparation and characterization of antimicrobial chitosan-N-arginine with different degrees of substitution[J]. Carbohydrate Polymers, 2011, 83(1):144-150.

[29] ZHENG D D, DUAN C X, ZHANG D R, et al. Galactosylated chitosan nanoparticles for hepatocyte-targeted delivery of oridonin[J]. International Journal of Pharmaceutics, 2012, 436(1/2):379-386.

[30] VALIZADEH A, MUSSA FARKHANI S. Electrospinning and electrospun nanofibres[J]. IET Nanobiotechnology, 2014, 8(2):83-92.

[31] CHEN Y, LIU Y H, ZHANG P H. Fabrication and performance of electrospinning fiber membrane for bile duct stents[J]. Journal of Donghua University (Natural Science), 2016, 42(6):800-808. (in Chinese)

[32] DEITZEL J M, KLEINMEYER J, HARRIS D, et al. The effect of processing variables on the morphology of electrospun nanofibers and textiles[J]. Polymer, 2001, 42(1):261-272.

[33] SUBBIAH T, BHAT G S, TOCK R W, et al. Electrospinning of nanofibers[J]. Journal of Applied Polymer Science, 2005, 96(2):557-569.

[34] YI G H, CHENG X Y, GENG M X, et al. Construction of oriented structure in inner surface of small-diameter artificial blood vessels:a review[J]. Journal of Donghua University (English Edition), 2023, 40(2):149-163.

[35] ZHANG F, ZUO B Q, BAI L. Study on the structure of SF fiber mats electrospun with HFIP and FA and cells behavior[J]. Journal of Materials Science, 2009, 44(20):5682-5687.

[36] TASIC-KOSTOV M, PAVLOVIC D, LUKIC M, et al. Lactobionic acid as antioxidant and moisturizing active in alkyl polyglucoside-based topical emulsions:the colloidal structure,stability and efficacy evaluation[J]. International Journal of Cosmetic Science, 2012, 34(5):424-434.

[37] HOLLISTER S J. Porous scaffold design for tissue engineering[J]. Nature Materials, 2005,4:518-524.

[38] BIHARI S, BANNARD-SMITH J, BELLOMO R. Albumin as a drug:its biological effects beyond volume expansion[J]. Critical Care and Resuscitation, 2020, 22(3):257-265.

[39] TAN E H, MA F J, GOPINADHAN S, et al. C/EBPα knock-in hepatocytes exhibit increased albumin secretion and urea production[J]. Cell and Tissue Research, 2007, 330(3):427-435.

[40] WILKENING S, STAHL F, BADER A. Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties[J]. Drug Metabolism and Disposition:the Biological Fate of Chemicals, 2003, 31(8):1035-1042.

Funding

Science & Technology Commission of Shanghai Municipality(20DZ2254900)

Municipal Public Welfare Research Project from Jiaxing, Zhejiang Province(2022AY10001)

Open Project Program of Jiaxing Key Laboratory of Virus-Related Infectious Diseases.