title: A novel tyrosine-modified low molecular weight polyethylenimine (P10Y) for efficient siRNA delivery in vitro and in vivo.
abstract: The delivery of nucleic acids, particularly of small RNA molecules like siRNAs for the induction of RNA interference (RNAi), still represents a major hurdle with regard to their application in vivo. Possible therapeutic applications thus rely on the development of efficient non-viral gene delivery vectors. While low molecular weight polyethylenimines (PEIs) have been successfully explored, the introduction of chemical modifications offers an avenue towards the development of more efficient vectors. In this paper, we describe the synthesis of a novel tyrosine-modified low-molecular weight polyethylenimine (P10Y) for efficient siRNA complexation and delivery. The comparison with the respective parent PEI reveals that knockdown efficacies are considerably enhanced by the tyrosine modification, as determined in different reporter cell lines, without appreciable cytotoxicity. We furthermore identify optimal conditions for complex preparation as well as for storing or lyophilization of the complexes without loss of biological activity. Beyond reporter cell lines, P10Y/siRNA complexes mediate the efficient knockdown of endogenous target genes and, upon knockdown of the anti-apoptotic oncogene survivin, tumor cell inhibitory effects in different carcinoma cell lines. Pushing the system further towards its therapeutic in vivo application, we demonstrate in mice the delivery of intact siRNAs and distinct biodistribution profiles upon systemic (intravenous or intraperitoneal) injection. No adverse effects (hepatotoxicity, immunostimulation/alterations in immunophenotype, weight loss) are observed. More importantly, profound tumor-inhibitory effects in a melanoma xenograft mouse model are observed upon systemic application of P10Y/siRNA complexes for survivin knockdown, indicating the therapeutic efficacy of P10Y/siRNA complexes. Taken together, we (i) establish tyrosine-modified PEI (P10Y) as efficient platform for siRNA delivery in vitro and in vivo, (ii) identify optimal preparation and storage conditions as well as (iii) physicochemical and biological properties of P10Y complexes, and (iv) demonstrate their applicability as siRNA therapeutic in vivo (v) in the absence of adverse effects.
authors.affiliation: Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ewe
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Przybylski
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Burkhardt
authors.affiliation: Leibniz-Institut für Polymerforschung, Dresden, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Janke
authors.affiliation: Leibniz-Institut für Polymerforschung, Dresden, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Appelhans
authors.affiliation: Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Germany. Electronic address: achim.aigner@medizin.uni-leipzig.de.
authors.forename: Achim
authors.initials: A
authors.lastname: Aigner
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics; D008819:Mice, Nude; D008970:Molecular Weight; D011094:Polyethyleneimine / Q000008:administration & dosage* / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry; D012097:Repressor Proteins / Q000235:genetics*; D000077022:Survivin; D014443:Tyrosine / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: C117749:Birc5 protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000077022:Survivin; D014443:Tyrosine; D011094:Polyethyleneimine
keywords: P10Y; Polyethlyenimine; RNAi in vivo; Therapeutic siRNA delivery; Tyrosine modification; siRNA transfection