Antifibrotic effects of specific siRNA targeting connective tissue growth factor delivered by polyethyleneimine‑functionalized magnetic iron oxide nanoparticles on LX‑2 cells

Yu,Qin; Xiong,Xiaoqin; Zhao,Lei; Xu,Tingting; Wang,Qianhua

doi:10.3892/mmr.2019.10834

Antifibrotic effects of specific siRNA targeting connective tissue growth factor delivered by polyethyleneimine‑functionalized magnetic iron oxide nanoparticles on LX‑2 cells

Authors:
- Qin Yu
- Xiaoqin Xiong
- Lei Zhao
- Tingting Xu
- Qianhua Wang
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Affiliations: Department of Clinical Laboratory, Wuhan Blood Center, Wuhan, Hubei 430000, P.R. China, Hubei Key Laboratory of Purification and Application of Plant Anticancer Active Ingredients, School of Chemistry and Life Sciences, Hubei University of Education, Wuhan, Hubei 430205, P.R. China, Department of Obstetrics and Gynecology, Union Hospital, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China
Published online on: November 20, 2019 https://doi.org/10.3892/mmr.2019.10834
Pages: 181-190
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Connective tissue growth factor (CTGF) is a possible key determinant of progressive fibrosis. Nanotechnology has been considered as a potential tool for developing novel drug delivery systems for various diseases, including liver fibrosis. The present study aimed to investigate the potential antifibrotic activity of CTGF small interfering RNA (siRNA) mediated by polyethyleneimine (PEI)‑functionalized magnetic iron oxide (Fe3O4) nanoparticles (NPs) in LX‑2 cells. PEI‑Fe3O4/siRNA complexes were synthesized to facilitate siRNA delivery and were transfected into LX‑2 cells. Laser confocal microscopy was employed to investigate the cell uptake of PEI‑Fe3O4/siRNA complexes. Reverse transcription‑quantitative PCR (RT‑qPCR) and western blotting were used to verify the effect of gene silencing. The results showed that siRNA‑loaded PEI‑Fe3O4 exhibited low cytotoxicity. The transfection efficiency of PEI‑Fe3O4/siRNA reached 73.8%, and RT‑qPCR and western blotting demonstrated effective gene silencing. These results indicated that CTGF siRNA delivered by PEI‑Fe3O4 NPs significantly reduces CTGF expression and collagen production in activated LX‑2 cells, providing a basis for future in vivo studies.

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1	Atta HM: Reversibility and heritability of liver fibrosis: Implications for research and therapy. World J Gastroenterol. 21:5138–5148. 2015. View Article : Google Scholar : PubMed/NCBI
2	Kisseleva T and Brenner DA: Hepatic stellate cells and the reversal of fibrosis. J Gastroenterol Hepatol. 21 (Suppl 3):S84–S87. 2006. View Article : Google Scholar : PubMed/NCBI
3	Gressner OA and Gressner AM: Connective tissue growth factor: A fibrogenic master switch in fibrotic liver diseases. Liver Int. 28:1065–1079. 2008. View Article : Google Scholar : PubMed/NCBI
4	Salazar-Montes AM, Hernández-Ortega LD, Lucano-Landeros MS and Armendariz-Borunda J: New gene therapy strategies for hepatic fibrosis. World J Gastroenterol. 21:3813–3825. 2015. View Article : Google Scholar : PubMed/NCBI
5	Gonzalez-Rodriguez A and Valverde AM: RNA interference as a therapeutic strategy for the treatment of liver diseases. Curr Pharm Des. 21:4574–4586. 2015. View Article : Google Scholar : PubMed/NCBI
6	Cheng K, Yang N and Mahato RI: TGF-beta1 gene silencing for treating liver fibrosis. Mol Pharm. 6:772–779. 2009. View Article : Google Scholar : PubMed/NCBI
7	Omar R, Yang J, Liu H, Davies NM and Gong Y: Hepatic stellate cells in liver fibrosis and siRNA-based therapy. Rev Physiol Biochem Pharmacol. 172:1–37. 2016. View Article : Google Scholar : PubMed/NCBI
8	Motoyama H, Ogawa S, Kubo A, Miwa S, Nakayama J, Tagawa Y and Miyagawa S: In vitro reprogramming of adult hepatocytes into insulin-producing cells without viral vectors. Biochem Biophys Res Commun. 385:123–128. 2009. View Article : Google Scholar : PubMed/NCBI
9	Zhou J, Shum KT, Burnett JC and Rossi JJ: Nanoparticle-based delivery of RNAi therapeutics: Progress and challenges. Pharmaceuticals (Basel). 6:85–107. 2013. View Article : Google Scholar : PubMed/NCBI
10	Poilil Surendran SP, Thomas RG, Moon MJ and Jeong YY: Nanoparticles for the treatment of liver fibrosis. Int J Nanomedicine. 12:6997–7006. 2017. View Article : Google Scholar : PubMed/NCBI
11	Li TT, Shen X, Chen Y, Zhang CC, Yan J, Yang H, Wu CH, Zeng HJ and Liu YY: Polyetherimide-grafted Fe₃O₄@SiO₂ nanoparticles as theranostic agents for simultaneous VEGF siRNA delivery and magnetic resonance cell imaging. Int J Nanomedicine. 10:4279–4291. 2015. View Article : Google Scholar : PubMed/NCBI
12	Shirazi AN, Paquin KL, Howlett NG, Mandal D and Parang K: Cyclic peptide-capped gold nanoparticles for enhanced siRNA delivery. Molecules. 19:13319–13331. 2014. View Article : Google Scholar : PubMed/NCBI
13	Wang Y, Zhao Q, Han N, Bai L, Li J, Liu J, Che E, Hu L, Zhang Q, Jiang T and Wang S: Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine. 11:313–327. 2015. View Article : Google Scholar : PubMed/NCBI
14	Reina G, Gismondi A, Carcione R, Nanni V, Peruzzi C, Angjellari M, Chau NDQ, Canini A, Terranova ML and Tamburri E: Oxidized and amino-functionalized nanodiamonds as shuttle for delivery of plant secondary metabolites: Interplay between chemical affinity and bioactivity. Appl Surface Sci. 470:744–754. 2019. View Article : Google Scholar
15	Getz T, Qin J, Medintz IL, Delehanty JB, Susumu K, Dawson PE and Dawson G: Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells. J Neurochem. 139:872–885. 2016. View Article : Google Scholar : PubMed/NCBI
16	Gupta AK and Gupta M: Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials. 26:3995–4021. 2005. View Article : Google Scholar : PubMed/NCBI
17	Wu W, He QG and Jiang CZ: Magnetic iron oxide nanoparticles: Synthesis and surface functionalization strategies. Nanoscale Res Lett. 3:397–415. 2008. View Article : Google Scholar : PubMed/NCBI
18	Xiao S, Castro R, Rodrigues J, Shi X and Tomás H: PAMAM dendrimer/pDNA functionalized-magnetic iron oxide nanoparticles for gene delivery. J Biomed Nanotechnol. 11:1370–1384. 2015. View Article : Google Scholar : PubMed/NCBI
19	Saeed M, Ren W and Wu A: Therapeutic applications of iron oxide based nanoparticles in cancer: Basic concepts and recent advances. Biomater Sci. 6:708–725. 2018. View Article : Google Scholar : PubMed/NCBI
20	Li J, Zou S, Gao J, Liang J, Zhou H, Liang L and Wu W: Block copolymer conjugated Au-coated Fe₃O₄ nanoparticles as vectors for enhancing colloidal stability and cellular uptake. J Nanobiotechnology. 15:562017. View Article : Google Scholar : PubMed/NCBI
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
22	Friedman SL: Cytokines and fibrogenesis. Semin Liver Dis. 19:129–140. 1999. View Article : Google Scholar : PubMed/NCBI
23	Bataller R and Brenner DA: Liver fibrosis. J Clin Invest. 115:209–218. 2005. View Article : Google Scholar : PubMed/NCBI
24	Elpek GÖ: Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: An update. World J Gastroenterol. 20:7260–7276. 2014. View Article : Google Scholar : PubMed/NCBI
25	Trautwein C, Friedman SL, Schuppan D and Pinzani M: Hepatic fibrosis: Concept to treatment. J Hepatol. 62 (1 Suppl):S15–S24. 2015. View Article : Google Scholar : PubMed/NCBI
26	Zhang CY, Yuan WG, He P, Lei JH and Wang CX: Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J Gastroenterol. 22:10512–10522. 2016. View Article : Google Scholar : PubMed/NCBI
27	Schuppan D, Surabattula R and Wang XY: Determinants of fibrosis progression and regression in NASH. J Hepatol. 68:238–250. 2018. View Article : Google Scholar : PubMed/NCBI
28	Lipson KE, Wong C, Teng Y and Spong S: CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair. 5 (Suppl 1):S242012. View Article : Google Scholar : PubMed/NCBI
29	Hao C, Xie Y, Peng M, Ma L, Zhou Y, Zhang Y, Kang W, Wang J, Bai X, Wang P and Jia Z: Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo. Clin Exp Med. 14:141–150. 2014. View Article : Google Scholar : PubMed/NCBI
30	Li G, Xie Q, Shi Y, Li D, Zhang M, Jiang S, Zhou H, Lu H and Jin Y: Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats. J Gene Med. 8:889–900. 2006. View Article : Google Scholar : PubMed/NCBI
31	Taymouri S and Taheri A: Use of nanotechnology in diagnosis and treatment of hepatic fibrosis: A review. Curr Drug Deliv. 13:662–672. 2016. View Article : Google Scholar : PubMed/NCBI
32	Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 98:503–517. 1975. View Article : Google Scholar : PubMed/NCBI
33	Wu J and Zern MA: Hepatic stellate cells: A target for the treatment of liver fibrosis. J Gastroenterol. 35:665–672. 2000. View Article : Google Scholar : PubMed/NCBI
34	Gressner AM, Weiskirchen R, Breitkopf K and Dooley S: Roles of TGF-beta in hepatic fibrosis. Front Biosci. 7:d793–d807. 2002. View Article : Google Scholar : PubMed/NCBI
35	Yang N, Dang S, Shi J, Wu F, Li M, Zhang X, Li Y, Jia X and Zhai S: Caffeic acid phenethyl ester attenuates liver fibrosis via inhibition of TGF-β1/Smad3 pathway and induction of autophagy pathway. Biochem Biophys Res Commun. 486:22–28. 2017. View Article : Google Scholar : PubMed/NCBI
36	Lee UE and Friedman SL: Mechanisms of hepatic fibrogenesis. Best Pract Res Clin Gastroenterol. 25:195–206. 2011. View Article : Google Scholar : PubMed/NCBI
37	Huang G and Brigstock DR: Regulation of hepatic stellate cells by connective tissue growth factor. Front Biosci (Landmark Ed). 17:2495–2507. 2012. View Article : Google Scholar : PubMed/NCBI
38	Yuhua Z, Wanhua R, Chenggang S, Jun S, Yanjun W and Chunqing Z: Disruption of connective tissue growth factor by short hairpin RNA inhibits collagen synthesis and extracellular matrix secretion in hepatic stellate cells. Liver Int. 28:632–639. 2008. View Article : Google Scholar : PubMed/NCBI