Solid-phase-assisted synthesis of targeting peptide–PEG–oligo(ethane amino)amides for receptor-mediated gene delivery
Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park (PCB), Baldiri Reixac 10, 08028 Barcelona, Spain.Organic & Biomolecular Chemistry (Impact Factor: 3.56). 03/2012; 10(16):3258-68. DOI: 10.1039/c2ob06907e
In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.
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- "This has nicely been shown by the innovating work of Wagner et al. and Kataoka and co-workers.     A biocompatible corona of the polyplexes, e.g. from PEG or p[HPMA] for stealth like properties, is thereby indispensable for systemic in vivo applications to (i) avoid unspecific interactions with serum proteins, to (ii) prevent extra-and intracellular degradation of the pDNA and to (iii) prevent unspecific immune response due to stimulation of toll like receptors.    A DC specific uptake can be achieved by the incorporation of targeting ligands to the polyplex' surface to induce receptor mediated uptake. "
ABSTRACT: Polylysine-b-p[HPMA] block copolymers containing a redox-responsive disulfide bond between both blocks are synthesized by RAFT polymerization of pentafluorphenyl-methacrylate with a macro-CTA from Nϵ -benzyloxycarbonyl (Cbz) protected polylysine (synthesized by NCA polymerization). This polylysine-b-p[PFMA] precursor block copolymer is converted to polylysine(Cbz)-b-p[HPMA] by postpolymerization modification with 2-hydroxypropylamine. After removal of the Cbz protecting group, cationic polylysine-b-p[HPMA] copolymers with a biosplittable disulfide moiety became available, which can be used as polymeric transfection vectors. These disulfide linked polylysine-S-S-b-p[HPMA] block copolymers show low cytotoxicity and increased transfection efficiencies (HEK-293T cells) compared to analogous blockcopolymers without disulfide group making them interesting for the transfection of sensitive immune cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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ABSTRACT: Two antitumoral siRNAs (directed against target genes Eg5 and Ran) complexed with one of three sequence-defined cationic oligomers were compared in gene silencing in vitro and antitumoral in vivo efficacy upon intratumoral injection. Two lipo-oligomers (T-shape 49, i-shape 229) and the three-arm oligomer 386 were chosen because of their high efficiency in previous marker gene silencing screens. The oligomers showed very similar target-specific gene knockdown in murine neuroblastoma cells. Silencing persisted only for a short period (maximum on day 1 at mRNA and day 2 at protein level) triggering siRNA specific in vitro tumor cell killing. The fastest onset of protein knockdown and strongest antitumoral effect was mediated by oligomer 386. Tumor growth reduction in vivo was evaluated in the subcutaneous Neuro2A mouse model. Intratumoral injections of either Eg5 or Ran siRNA/oligomer 49 polyplexes led to reduced tumor growth and prolonged survival of mice compared to control siRNA and buffer treatment. Target knockdown was evidenced in tumors by mitotic Aster formation for Eg5 knockdown and apoptotic TUNEL stain for Ran knockdown. Ran siRNA displayed better antitumoral efficacy and was chosen for in vivo comparison of the oligomers. A very clear order of antitumoral activity (oligomer 386 > 49 > 229) was observed. In summary, the similar in vitro gene silencing efficiencies on mRNA level by the tested oligomers did not correlate with the observed therapeutic effects in vivo. Oligomer 386 with the fastest onset of protein knockdown and best in vitro cell killing mediated the best in vivo antitumoral efficacy.
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