Cyclam-Modified Polyethyleneimine for Simultaneous TGFβ siRNA Delivery and CXCR4 Inhibition for the Treatment of CCl4-Induced Liver Fibrosis

Abstract

Aftab Ullah,1 Gang Chen,2 Abid Hussain,3,4 Hanif Khan,1 Azar Abbas,5 Zhanwei Zhou,5 Muhammad Shafiq,6 Saleem Ahmad,7 Usman Ali,8 Muhammad Usman,6 Faisal Raza,8 Abrar Ahmed,8 Zijie Qiu,5 Maochao Zheng,1 Daojun Liu1 1Department of Pharmacy, Shantou University Medical College, Shantou, 515041, Guangdong, People’s Republic of China; 2Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, People’s Republic of China; 3School of Life Science, Key Laboratory of Molecular Medicine and Biotherapy, Beijing Institute of Technology, Beijing, 100081, People’s Republic of China; 4Chinese Academy of Sciences (CAS) Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, 100190, People’s Republic of China; 5School of Pharmacy, China Pharmaceutical University, Nanjing, 210028, Jiangsu, People’s Republic of China; 6Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong, 515041, people’s Republic of China; 7Department of Medicine, Shantou University Medical College Cancer Hospital, Shantou, People’s Republic of China; 8School of Pharmacy, Shanghai Jiaotong University, Shanghai, 200240, Shanghai, People’s Republic of ChinaCorrespondence: Aftab Ullah; Daojun Liu Email [email protected]; [email protected]: Liver fibrosis is a chronic liver disease with excessive production of extracellular matrix proteins, leading to cirrhosis, hepatocellular carcinoma, and death.Purpose: This study aimed at the development of a novel derivative of polyethyleneimine (PEI) that can effectively deliver transforming growth factor β (TGFβ) siRNA and inhibit chemokine receptor 4 (CXCR4) for TGFβ silencing and CXCR4 Inhibition, respectively, to treat CCl4-induced liver fibrosis in a mouse model.Methods: Cyclam-modified PEI (PEI-Cyclam) was synthesized by incorporating cyclam moiety into PEI by nucleophilic substitution reaction. Gel electrophoresis confirmed the PEI-Cyclam polyplex formation and stability against RNAase and serum degradation. Transmission electron microscopy and zeta sizer were employed for the morphology, particle size, and zeta potential, respectively. The gene silencing and CXCR4 targeting abilities of PEI-Cyclam polyplex were evaluated by luciferase and CXCR4 redistribution assays, respectively. The histological and immunohistochemical staining determined the anti-fibrotic activity of PEI-Cyclam polyplex. The TGFβ silencing of PEI-Cyclam polyplex was authenticated by Western blotting.Results: The 1H NMR of PEI-Cyclam exhibited successful incorporation of cyclam content onto PEI. The PEI-Cyclam polyplex displayed spherical morphology, positive surface charge, and stability against RNAse and serum degradation. Cyclam modification decreased the cytotoxicity and demonstrated CXCR4 antagonistic and luciferase gene silencing efficiency. PEI-Cyclam/siTGFβ polyplexes decreased inflammation, collagen deposition, apoptosis, and cell proliferation, thus ameliorating liver fibrosis. Also, PEI-Cyclam/siTGFβ polyplex significantly downregulated α-smooth muscle actin, TGFβ, and collagen type III.Conclusion: Our findings validate the feasibility of using PEI-Cyclam as a siRNA delivery vector for simultaneous TGFβ siRNA delivery and CXCR4 inhibition for the combined anti-fibrotic effects in a setting of CCl4-induced liver fibrosis.Keywords: polyethyleneimine, cyclam, gene delivery, liver fibrosis, CXCR4

Keywords

• polyethyleneimine
• cyclam
• gene delivery
• liver fibrosis
• cxcr4