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PEGylation Improves Nanoparticle Formation and Transfection Efficiency of Messenger RNA

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ABSTRACT

Purpose

Cationic polymers have been intensively investigated for plasmid-DNA (pDNA), but few studies addressed their use for messenger-RNA (mRNA) delivery. We analyzed two types of polymers, linear polyethylenimine (l-PEI) and poly-N,N-dimethylaminoethylmethacrylate P(DMAEMA), to highlight specific requirements for the design of mRNA delivery reagents. The effect of PEGylation was investigated using P(DMAEMA-co-OEGMA) copolymer.

Methods

The influence of polymer structure on mRNA binding and particle formation was assessed in a side-by-side comparison with pDNA by methods such as agarose-retardation assay and scanning probe microscopy. Transfection studies were performed on bronchial epithelial cells.

Results

Binding of cationic polymers inversely correlated with type of nucleic acid. Whereas P(DMAEMA) bound strongly to pDNA, only weak mRNA binding was observed, which was vice versa for l-PEI. Both polymers resulted in self-assembled nanoparticles forming pDNA complexes of irregular round shape; mRNA particles were significantly smaller and more distinct. Surprisingly, PEGylation improved mRNA binding and transfection efficiency contrary to observations made with pDNA. Co-transfections with free polymer improved mRNA transfection.

Conclusions

Gene delivery requires tailor-made design for each type of nucleic acid. PEGylation influenced mRNA-polymer binding efficiency and transfection and may provide a method of further improving mRNA delivery.

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REFERENCES

  1. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000;288:669–72.

    Article  PubMed  CAS  Google Scholar 

  2. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science. 2002;296:2410–3.

    Article  PubMed  CAS  Google Scholar 

  3. Maguire AM, High KA, Auricchio A, Wright JF, Pierce EA, Testa F, et al. Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet. 2009;374:1597–605.

    Article  PubMed  CAS  Google Scholar 

  4. McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2004;350:913–22.

    Article  PubMed  CAS  Google Scholar 

  5. Mueller C, Flotte TR. Gene therapy for cystic fibrosis. Clin Rev Allergy Immunol. 2008;35:164–78.

    Article  PubMed  CAS  Google Scholar 

  6. Calos MP. The phiC31 integrase system for gene therapy. Curr Gene Ther. 2006;6:633–45.

    Article  PubMed  CAS  Google Scholar 

  7. Ivics Z, Izsvak Z. Transposons for gene therapy! Curr Gene Ther. 2006;6:593–607.

    Article  PubMed  CAS  Google Scholar 

  8. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 2007;25:786–93.

    Article  PubMed  CAS  Google Scholar 

  9. Dauty E, Verkman AS. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. J Biol Chem. 2005;280:7823–8.

    Article  PubMed  CAS  Google Scholar 

  10. Munkonge FM, Dean DA, Hillery E, Griesenbach U, Alton EW. Emerging significance of plasmid DNA nuclear import in gene therapy. Adv Drug Deliv Rev. 2003;55:749–60.

    Article  PubMed  CAS  Google Scholar 

  11. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem. 1998;273:7507–11.

    Article  PubMed  CAS  Google Scholar 

  12. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem. 1995;270:18997–9007.

    Article  PubMed  CAS  Google Scholar 

  13. Yamamoto A, Kormann M, Rosenecker J, Rudolph C. Current prospects for mRNA gene delivery. Eur J Pharm Biopharm. 2009;71:484–9.

    Article  PubMed  CAS  Google Scholar 

  14. Van Tendeloo VF, Ponsaerts P, Berneman ZN. mRNA-based gene transfer as a tool for gene and cell therapy. Curr Opin Mol Ther. 2007;9:423–31.

    PubMed  Google Scholar 

  15. Van Driessche A, Van de Velde AL, Nijs G, Braeckman T, Stein B, De Vries JM, et al. Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial. Cytotherapy. 2009;11:653–68.

    Article  PubMed  Google Scholar 

  16. Probst J, Weide B, Scheel B, Pichler BJ, Hoerr I, Rammensee HG, et al. Spontaneous cellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent. Gene Ther. 2007;14:1175–80.

    Article  PubMed  CAS  Google Scholar 

  17. Scheel B, Aulwurm S, Probst J, Stitz L, Hoerr I, Rammensee HG, et al. Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA. Eur J Immunol. 2006;36:2807–16.

    Article  PubMed  CAS  Google Scholar 

  18. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247:1465–8.

    Article  PubMed  CAS  Google Scholar 

  19. Kormann MS, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-Jonat S, Huppmann M, Mays LE, Illenyi M, Schams A, Griese M, Bittmann I, Handgretinger R, Hartl D, Rosenecker J, Rudolph C. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol. 29:154–7

  20. Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood. 2001;98:49–56.

    Article  PubMed  Google Scholar 

  21. Bettinger T, Carlisle RC, Read ML, Ogris M, Seymour LW. Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells. Nucleic Acids Res. 2001;29:3882–91.

    Article  PubMed  CAS  Google Scholar 

  22. Read ML, Singh S, Ahmed Z, Stevenson M, Briggs SS, Oupicky D, et al. A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids. Nucleic Acids Res. 2005;33:e86.

    Article  PubMed  Google Scholar 

  23. Zohra FT, Chowdhury EH, Akaike T. High performance mRNA transfection through carbonate apatite-cationic liposome conjugates. Biomaterials. 2009;30:4006–13.

    Article  PubMed  CAS  Google Scholar 

  24. Orgis M, Wagner E. Linear polyethylenimine: synthesis and transfection procedures for in vitro and in vivo. In: Friedman T, Rossi J, editors. Gene transfer: delivery and expression of cDNA and RNA, a laboratory manual. New York: Cold Spring Habor Laboratory Press; 2007. p. 521–8.

    Google Scholar 

  25. Ungaro F, De Rosa G, Miro A, Quaglia F. Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations. J Pharm Biomed Anal. 2003;31:143–9.

    Article  PubMed  CAS  Google Scholar 

  26. Üzgün S, Akdemir Ö, Hsenpusch G, Maucksch C, Golas MM, Sander B, Stark H, Imker R, Lutz JF, Rudolph C. Characterization of tailor-made copolymers of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and N,N-dimethylaminoethyl methacrylate (DMAEMA) as nonviral gene transfer agents—influence of macromolecular structure on gene vector particle properties and transfection efficiency. Biomacromolecules. in press

  27. Wagner E, Plank C, Zatloukal K, Cotten M, Birnstiel ML. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc Natl Acad Sci USA. 1992;89:7934–8.

    Article  PubMed  CAS  Google Scholar 

  28. Cherng JY, van de Wetering P, Talsma H, Crommelin DJ, Hennink WE. Effect of size and serum proteins on transfection efficiency of poly ((2-dimethylamino) ethyl methacrylate)-plasmid nanoparticles. Pharm Res. 1996;13:1038–42.

    Article  PubMed  CAS  Google Scholar 

  29. Convertine AJ, Benoit DS, Duvall CL, Hoffman AS, Stayton PS. Development of a novel endosomolytic diblock copolymer for siRNA delivery. J Control Release. 2009;133:221–9.

    Article  PubMed  CAS  Google Scholar 

  30. Severin N, Zhuang W, Ecker C, Kalachev AA, Sokolov IM, Rabe JP. Blowing DNA bubbles. Nano Lett. 2006;6:2561–6.

    Article  PubMed  CAS  Google Scholar 

  31. Hansma HG, Vesenka J, Siegerist C, Kelderman G, Morrett H, Sinsheimer RL, et al. Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science. 1992;256:1180–4.

    Article  PubMed  CAS  Google Scholar 

  32. Chernov KG, Curmi PA, Hamon L, Mechulam A, Ovchinnikov LP, Pastre D. Atomic force microscopy reveals binding of mRNA to microtubules mediated by two major mRNP proteins YB-1 and PABP. FEBS Lett. 2008;582:2875–81.

    Article  PubMed  CAS  Google Scholar 

  33. Harada A, Kataoka K. Chain length recognition: core-shell supramolecular assembly from oppositely charged block copolymers. Science. 1999;283:65–7.

    Article  PubMed  CAS  Google Scholar 

  34. Boeckle S, von Gersdorff K, van der Piepen S, Culmsee C, Wagner E, Ogris M. Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med. 2004;6:1102–11.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Molecular Pulmonology, Department of Pediatrics, Ludwig Maximilians University of Munich, Lindwurmstr. 2a, 80337, Munich, Germany

    Senta Üzgün, Gabriela Nica, Corinna Pfeifer, Kai Michaelis, Joseph Rosenecker & Carsten Rudolph

  2. Fraunhofer Institute for Applied Polymer Research, 14476, Potsdam-Golm, Germany

    Jean-François Lutz

  3. Department of Pharmaceutical Technology, Free University of Berlin, 12169, Berlin, Germany

    Senta Üzgün, Corinna Pfeifer & Carsten Rudolph

  4. Pharmaceutical Nanotechnology, Saarland University, Campus A4 1, 66123, Saarbrücken, Germany

    Michele Bosinco & Marc Schneider

Authors
  1. Senta Üzgün
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  2. Gabriela Nica
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  3. Corinna Pfeifer
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  4. Michele Bosinco
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  5. Kai Michaelis
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  6. Jean-François Lutz
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  7. Marc Schneider
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  8. Joseph Rosenecker
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  9. Carsten Rudolph
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Correspondence to Carsten Rudolph.

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Üzgün, S., Nica, G., Pfeifer, C. et al. PEGylation Improves Nanoparticle Formation and Transfection Efficiency of Messenger RNA. Pharm Res 28, 2223–2232 (2011). https://doi.org/10.1007/s11095-011-0464-z

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  • DOI: https://doi.org/10.1007/s11095-011-0464-z

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