title: Conjugates of HA2 with octaarginine-grafted HPMA copolymer offer effective siRNA delivery and gene silencing in cancer cells.
abstract: The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4-9.5mol-%) to define the optimal R8 content that allowed the polymer to serve both as a siRNA-binding domain and as an intracellular transduction moiety mediating improved cellular delivery. A subunit of the influenza virus hemagglutinin (HA2), known for its ability to disrupt endosomal membranes, was further conjugated to P-R8-FITC copolymer to promote endosomal escape. Of the different P-(R8)-FITC conjugates considered, only that polymer containing the highest mol-% of R8 (P-(R8)9.5-FITC) was able to encapsulate siRNA molecules into nano-sized polyion complexes (PICs) presenting positive surface charge, low in vitro cytotoxicity, and high serum stability. P-(R8)9.5-FITC/cy5-siRNA complexes can efficiently deliver siRNA molecules into cells, while naked siRNA or siRNA encapsulated within polymers with lower R8mol-% were unable to transfect the same cells. Conjugation of HA2 fusogenic peptide to P-(R8)-FITC significantly decreased the oncogenic RAC1 mRNA levels in cancer cells. This indicates that P-(R8)-(HA2)-FITC can deliver siRNA into target cells, and that the siRNA can reach the perinuclear region where it interacts with the RNA-induced silencing complex.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
authors.forename: Moran
authors.initials: M
authors.lastname: Golan
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
authors.forename: Valeria
authors.initials: V
authors.lastname: Feinshtein
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. Electronic address: ayeletda@bgu.ac.il.
authors.forename: Ayelet
authors.initials: A
authors.lastname: David
mesh_terms: D000072283:A549 Cells; D000178:Acrylamides / Q000737:chemistry*; D045744:Cell Line, Tumor / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000235:genetics*; D009842:Oligopeptides / Q000737:chemistry*; D010051:Ovarian Neoplasms / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D013499:Surface Properties
chemical_list: D000178:Acrylamides; D009842:Oligopeptides; D010455:Peptides; D011108:Polymers; D034741:RNA, Small Interfering; C448619:octaarginine; D012313:RNA; D016650:Fluorescein-5-isothiocyanate; C032976:N-(2-hydroxypropyl)methacrylamide
keywords: Cancer therapy; Cell penetrating peptides; Endosomal escape motif; HPMA copolymer; Octaarginine; siRNA delivery