Cell-free synthesis of connexin 43-integrated exosome-mimetic nanoparticles for siRNA delivery. (15th September 2019)

• Record Type: Journal Article
• Authors: Lu, Mei; Zhao, Xiaoyun; Xing, Haonan; Liu, Hui; Lang, Lang; Yang, Tianzhi; Xun, Zhe; Wang, Dongkai; Ding, Pingtian
• Is Part Of: Acta biomaterialia. Volume 96(2019)
• Page Start: 517
• Page End: 536
• Publication Date: 2019-09-15
• Subjects: Exosomes -- siRNA delivery -- Connexin 43 -- Cell-free protein synthesis -- Lipid bilayers -- Chitosan nanoparticles; BCA bicinchoninic acid -- CF cell-free -- Chol cholesterol -- CLSM confocal laser scanning microscopy -- CS NPs chitosan nanoparticles -- CTX-B cholera toxin subunit b -- Cx43 connexin 43 -- Cx43/L/CS NPs Cx43-integrated lipid-coated chitosan nanoparticles -- DMEM Dulbecco's modified Eagle's Medium -- DLS dynamic light scattering -- DOPC 1, 2-dioleoyl-sn-glycero-3-phosphocholine -- DOPE 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine -- DOPS 1, 2-dioleoyl-sn-glycero-3-phosphoserine -- EE encapsulation efficiency -- ELISA enzyme-linked immunosorbent assay -- EXO exosomes -- FAM-siNC FAM-labeled negative siRNA -- FBS fetal bovine serum -- FCM flow cytometry -- GA 18β-glycyrrhetinic acid -- GAPDH glyceraldehyde phosphatedehydrogenase -- GJ gap junction -- L/CS NPs lipid coated chitosan nanoparticles -- Lipo 2000 Lipofectamine 2000 -- MPs membrane proteins -- MTT thiazolyl blue tetrazolium bromide -- PBS phosphate buffer saline -- RNAi RNA interference -- qRT-PCR quantitative real-time polymerase chain reaction -- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis -- siVEGF anti-VEGF siRNA -- SM sphingomyelin -- TEM transmission electron microscope -- TPP sodium tripolyphosphate -- VEGF vascular endothelial growth factor -- WF five times washing and ultra-filtration; Biomedical materials -- Periodicals; 610.28
• DOI: 10.1016/j.actbio.2019.07.006
• Languages: English
• ISSNs: 1742-7061

Abstract

Graphical abstract: Abstract: Exosomes are naturally secreted nanovesicles that have emerged as a promising therapeutic nanodelivery platform, due to their specific composition and biological properties. However, challenges like considerable complexity, low isolation yield, drug payload, and potential safety concerns substantially reduce their pharmaceutical acceptability. Given that the nano-bio-interface is a crucial factor for nanocarrier behavior and function, modification of synthetic nanoparticles with the intrinsic hallmarks of exosomes' membrane to create exosome mimetics could allow for siRNA delivery in a safer and more efficient manner. Herein, connexin 43 (Cx43)-embedded, exosome-mimicking lipid bilayers coated chitosan nanoparticles (Cx43/L/CS NPs) were constructed by using cell-free (CF) synthesis systems with plasmids encoding Cx43 in the presence of lipid-coated CS NPs (L/CS NPs). The integration of de novo synthesized Cx43 into the lipid bilayers of L/CS NPs occurred cotranslationally during one-pot reaction and, more importantly, the integrated Cx43 was functionally active in transport. In addition to considerably lower cytotoxicity (<four-fold) than cationic Lipo 2000, the obtained Cx43/L/CS-siRNA NPs showed feasible cellular uptake and silencing efficacy that was significantly higher than free siRNA and CS-siRNA NPs. By using a gap junction (GJ) inhibitor, 18β-glycyrrhetinic acid, we demonstrated that Cx43 facilitated the delivery of siRNA into Cx43-expressing U87 MG cells. Additionally, the cellular entry of Cx43/L/CS-siRNA NPs may rely on different endocytic mechanisms, depending on the types of recipient cells. However, Cx43/L/CS-siRNA NPs still exhibited far from adequate delivery efficiency compared with transfection reagent Lipo 2000. Taken together, our study provides a brand new strategy to construct Cx43-functionalized, exosome-mimetic nanoparticles, which may further encourage the establishment of more biomimetic nanocarriers with higher biocompatibility and delivery efficiency. Significance of statement: The major issue to move RNA interference (RNAi) therapy from bench to bedside is the lack of safe and efficient delivery vehicles. Given the certain advantages and limitations of exosomes and synthetic nanocarriers, a promising strategy is to facilitate positive feedbacks between the two fields, in which the superiority of exosomes regarding special membrane composition beneficial for cytoplasmic delivery and the better pharmaceutical acceptance of synthetic nanocarriers could be combined. In this study, we reported to construct Cx43-integrated, exosome-mimetic lipid bilayers coated nanoparticles by using CF synthesis technique. The obtained Cx43/L/CS-siRNA NPs were characterized by desirable cytotoxicity profile and feasible delivery efficiency. This study provides a new avenue and insights for the synthesis of more biocompatible and effective bio-mimetic siRNA delivery platforms.