Abstract
Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of proteinous or peptidic ligands can form nanosized polyplexes with plasmid DNA or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of linear PEI (LPEI) from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and plasmid DNA.
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References
Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 92:7297–7301
Tang MX, Szoka FC (1997) The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther 4:823–832
Kopatz I, Remy JS, Behr JP (2004) A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 6:769–776
Sonawane ND, Szoka FC Jr, Verkman AS (2003) Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem 278:44826–44831
Ferrari S, Moro E, Pettenazzo A, Behr JP, Zacchello F, Scarpa M (1997) ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther 4:1100–1106
Goula D, Benoist C, Mantero S, Merlo G, Levi G, Demeneix BA (1998) Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther 5:1291–1295
Wightman L, Kircheis R, Rossler V, Carotta S, Ruzicka R, Kursa M, Wagner E (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 3:362–372
Brissault B, Kichler A, Guis C, Leborgne C, Danos O, Cheradame H (2003) Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjug Chem 14:581–587
Ogris M, Wagner E (2008) Linear polyethylenimine: synthesis and transfection procedures for in vitro and in vivo. In: Friedmann T, Rossi J (eds) Gene transfer: delivery and expression of DNA and RNA, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 521–526
Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci U S A 102:5679–5684
Boeckle S, von Gersdorff K, van der Piepen S, Culmsee C, Wagner E, Ogris M (2004) Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med 6:1102–1111
Goula D, Becker N, Lemkine GF, Normandie P, Rodrigues J, Mantero S, Levi G, Demeneix BA (2000) Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes 965. Gene Ther 7:499–504
Chollet P, Favrot MC, Hurbin A, Coll JL (2002) Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. J Gene Med 4:84–91
Itaka K, Harada A, Yamasaki Y, Nakamura K, Kawaguchi H, Kataoka K (2004) In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J Gene Med 6:76–84
de Bruin KG, Fella C, Ogris M, Wagner E, Ruthardt N, Brauchle C (2008) Dynamics of photoinduced endosomal release of polyplexes. J Control Release 130:175–182
Zintchenko A, Susha AS, Concia M, Feldmann J, Wagner E, Rogach AL, Ogris M (2009) Drug nanocarriers labeled with near-infrared-emitting quantum dots (quantoplexes): imaging fast dynamics of distribution in living animals. Mol Ther 17:1849–1856
Kursa M, Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R, Wagner E (2003) Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer. Bioconjug Chem 14:222–231
Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6:595–605
Schwerdt A, Zintchenko A, Concia M, Roesen N, Fisher KD, Lindner LH, Issels RD, Wagner E, Ogris M (2008) Hyperthermia induced targeting of thermosensitive gene carriers to tumors. Hum Gene Ther 19:1283–1292
Smrekar B, Wightman L, Wolschek MF, Lichtenberger C, Ruzicka R, Ogris M, Rodl W, Kursa M, Wagner E, Kircheis R (2003) Tissue-dependent factors affect gene delivery to tumors in vivo. Gene Ther 10:1079–1088
Fella C, Walker GF, Ogris M, Wagner E (2008) Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding. Eur J Pharm Sci 34:309–320
Walker GF, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M, Wagner E (2005) Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol Ther 11:418–425
Klutz K, Schaffert D, Willhauck MJ, Grunwald GK, Haase R, Wunderlich N, Zach C, Gildehaus FJ, Senekowitsch-Schmidtke R, Goke B, Wagner E, Ogris M, Spitzweg C (2011) Epidermal growth factor receptor-targeted (131)I-therapy of liver cancer following systemic delivery of the sodium iodide symporter gene. Mol Ther 19:676–685
Kircheis R, Wightman L, Schreiber A, Robitza B, Rossler V, Kursa M, Wagner E (2001) Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Ther 8:28–40
Ogris M, Steinlein P, Carotta S, Brunner S, Wagner E (2001) DNA/polyethylenimine transfection particles: Influence of ligands, polymer size, and PEGylation on internalization and gene expression. AAPS Pharm Sci 3:E21
Ogris M, Walker G, Blessing T, Kircheis R, Wolschek M, Wagner E (2003) Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes. J Control Release 91:173–181
Schafer A, Pahnke A, Schaffert D, Van Weerden WM, de Ridder CM, Rodl W, Vetter A, Spitzweg C, Kraaij R, Wagner E, Ogris M (2011) Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR) mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation. Hum Gene Ther 22:1463–1473
Sidi AA, Ohana P, Benjamin S, Shalev M, Ransom JH, Lamm D, Hochberg A, Leibovitch I (2008) Phase I/II marker lesion study of intravesical BC-819 DNA plasmid in H19 over expressing superficial bladder cancer refractory to bacillus calmette-guerin. J Urol 180:2379–2383
Ungaro F, De Rosa G, Miro A, Quaglia F (2003) Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations. J Pharm Biomed Anal 31:143–149
Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, Simeon-Rudolf V, Reiner E (2003) Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal Biochem 312:224–227
Jeong JH, Song SH, Lim DW, Lee H, Park TG (2001) DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline). J Control Release 73:391–399
Schleef M, Schmidt T (2004) Animal-free production of ccc-supercoiled plasmids for research and clinical applications. J Gene Med 6(Suppl 1):S45–S53
Magnusson T, Haase R, Schleef M, Wagner E, Ogris M (2011) Sustained, high transgene expression in liver with plasmid vectors using optimized promoter-enhancer combinations. J Gene Med 13:382–391
Kasper JC, Schaffert D, Ogris M, Wagner E, Friess W (2011) The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes. Eur J Pharm Biopharm 77:182–185
Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E (1998) The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther 5:1425–1433
Acknowledgments
This work was supported by the Center for Nanoscience (CeNS) and the German Research Foundation (SFB824) to M.O., and the Nanosystems Initiative Munich (NIM) to E.W.
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Rödl, W., Schaffert, D., Wagner, E., Ogris, M. (2013). Synthesis of Polyethylenimine-Based Nanocarriers for Systemic Tumor Targeting of Nucleic Acids. In: Ogris, M., Oupicky, D. (eds) Nanotechnology for Nucleic Acid Delivery. Methods in Molecular Biology, vol 948. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-140-0_8
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DOI: https://doi.org/10.1007/978-1-62703-140-0_8
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