Abstract
Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency.
Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system.
Results. LMW-PEI (Mw 11′900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l′616′OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition.
Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.
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REFERENCES
F. D. Ledley. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum. Gene Ther. 6:1129-1144 (1995).
R. C. Mulligan. The basic science of gene therapy. Science 260:926-932 (1993).
H. Schreier. The new frontier: gene and oligonucleotide therapy. Pharm. Acta Helv. 68:145-159 (1994).
E. Tomlinson and A. Rolland. Controllable gene therapy: pharmaceutics of non-viral gene delivery systems. J. Contr. Rel. 39:357-371 (1996).
J. P. Behr. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 51:34-36 (1997).
J. S. Remy, B. Abdallah, M. A. Zanta, O. Boussif, J. P. Behr, and B. A. Demeneix. Gene transfer with lipospermines and polyethy-lenimines. Adv. Drug Del. Rev. 30:85-95 (1998).
O. Boussif, F. Lezoulac'h, M. A. Zanta, M. Mergny, D. Scherman, B. Demeneix, and J. P. Behr. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethy-lenimine. Proc. Natl. Acad. Sci. USA 92:7297-7303 (1995).
A. Baker and M. Cotten. Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier. Nucl. Acid Res. 25:1950-1956 (1997).
C. Meunier-Durmont, H. Grimal, L. M. Sachs, B. A. Demeneix, and C. Forest. Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells. Gene Therapy 4:808-814 (1997).
A. Baker, M. Salik, H. Lehmann, I. Killisch, V. Mautner, G. Lamm, and C. Christofori. Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery. Gene Ther. 4:773-782 (1997).
R. Kircheis, A. Kichler, G. Wallner, M. Kursa, M. Ogris, T. Felzmann, M. Buchberger, and E. Wagner. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4:409-418 (1997).
O. Boussif, M. A. Zanta, and J. P. Behr. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 3:1074-1080 (1996).
B. Abdallah, A. Hassan, C. Benoist, D. Goula, J. P. Behr, and B. A. Demeneix. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7:1947-1954 (1997).
A. Boletta, A. Benigni, J. Lutz, G. Remuzzi, M. S. Soria, and L. Monaco. Nonviral gene delivery to the rat kidney with polyethy-lenimine. Hum. Gene Ther. 8:1243-1251 (1997).
W. T. Godbey, K. K. Wu, and A. G. Mikos. Size matters: molecular weight affects the efficiency of poly(ethylenimine) as gene delivery vehicle. Proceed. Int. Symp. Control. Rel. Bioact. Mater. 25:230-231 (1998).
B. H. Zimm. The normal-coordinate method for polymer chains in dilute solution. In F. R. Eirich (ed.) Rheology. Academic Press, New York, 1960, chapter 1.
T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods 65:55-63 (1983).
S. Ito and M. J. Karnovsky. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168-169 (1968).
J. H. Luft. Improvements in epoxin resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414 (1961).
C. R. Dick and G. E. Ham. Characterization of polyethylenimine. J. Macromol. Sci. A4:1301-1314 (1997).
D. Fischer, R. Zange, and T. Kissel. Comparative in vitro cytotoxicity studies of polycations for gene therapy. Proc. Intern. Symp. Control. Rel. Bioact. Mater. 24:527-528 (1997).
D. Fischer, T. Bieber, H. P. Elsässer, and T. Kissel. Polyethylenimine: synthesis and in vitro cytotoxicity of a low molecular weight polycation for gene transfer. Eur. J. Cell. Biol. 75(suppl. 48):108 (1998).
S. Choksakulnimitr, S. Masuda, H. Tokuda, Y. Takakura, and M. Hashida. In vitro cytotoxicity of macromolecules in different cell culture systems. J. Contr. Rel. 34:233-241 (1995).
H. J. P. Ryser. A membrane effect of basic polymers dependent on molecular size. Nature 215:934-936 (1967).
S. Ferrari, E. Moro, A. Pettenazzo, J. P. Behr, F. Zacchello, and M. Scarpa. ExGene 500 is an effective vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 4:1100-1106 (1997).
D. Goulda, C. Benoist, S. Mantero, G. Merlo, G. Levi, and B. A. Demeneix. Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther. 5:1291-1295 (1998).
D. Goulda, J. S. Remy, J. S. Erbacher, M. Wasowicz, G. Levi, B. Abdallah, and B. A. Demeneix. Size, diffusability and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther. 5:712-717 (1998).
X. Zhou, A. L. Klibanov, and L. Huang. Lipophilic polylysines mediate efficient DNA transfection in mammalian cells. Biochim. Biophys. Acta 1065:8-14 (1991).
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Fischer, D., Bieber, T., Li, Y. et al. A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity. Pharm Res 16, 1273–1279 (1999). https://doi.org/10.1023/A:1014861900478
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DOI: https://doi.org/10.1023/A:1014861900478