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. 2005 Mar 21;22(3):373–380. doi: 10.1007/s11095-004-1874-y

Cross-linked Small Polyethylenimines: While Still Nontoxic, Deliver DNA Efficiently to Mammalian Cells in Vitro and in Vivo

Mini Thomas 1, Qing Ge 2, James J Lu 2, Jianzhu Chen 2, Alexander Klibanov 1,✉
PMCID: PMC7089549  PMID: 15835742

No Heading

Purpose.

Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation’s effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.

Methods.

The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing β-galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs.

Results.

Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI.

Conclusions.

Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.

Key Words: biodegradability, COS-7, cells, cross-linking, cytotoxicity, in vitro gene delivery, in vivo gene delivery, plasmid DNA, polyethylenimine

References

  • 1.Schwiebert L. M. Cystic fibrosis, gene therapy, and lung inflammation: for better or worse? Am. J. Physiol. 2004;286:L715–L716. doi: 10.1152/ajplung.00363.2003. [DOI] [PubMed] [Google Scholar]
  • 2.Kingdon H. S., Lundblad R. L. An adventure in biotechnology: the development of haemophilia A therapeutics - from whole-blood transfusion to recombinant DNA to gene therapy. Biotechnol. Appl. Biochem. 2002;35:141–148. doi: 10.1042/ba20010082. [DOI] [PubMed] [Google Scholar]
  • 3.Kuo C. J., Farnebo F., Yu E. Y., Christofferson R., Swearingen R. A., Carter R., von Recum H. A., Yuan J., Kamihara J., Flynn E., D’Amato R., Folkman J., Mulligan R. C. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer. Proc. Natl. Acad. Sci. USA. 2001;98:4605–4610. doi: 10.1073/pnas.081615298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Lecellier C.-H., Voinnet O. RNA silencing: no mercy for viruses? Immunol. Revs. 2004;198:285–303. doi: 10.1111/j.0105-2896.2004.00128.x. [DOI] [PubMed] [Google Scholar]
  • 5.Frater A. J., Fidler S. J., McClure M. O. Gene therapy for AIDS and other infectious diseases. Gene Ther. 2002;9:189–213. [Google Scholar]
  • 6.Ge Q., Filip L., Bai A., Tam N., Eisen H. N., Chen J. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc. Natl. Acad. Sci. USA. 2004;101:8676–8681. doi: 10.1073/pnas.0402486101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.7. Gene therapy clinical trials worldwide provided by the Journal of Gene Medicine. http://www.wiley.co.uk/genmed/clinical/.
  • 8.Davis M. E. Non-viral gene delivery systems. Curr. Opin. Biotechnol. 2002;13:128–131. doi: 10.1016/s0958-1669(02)00294-x. [DOI] [PubMed] [Google Scholar]
  • 9.Thomas M., Klibanov A. M. Non-viral gene therapy: polycation-mediated DNA delivery. Appl. Microbiol. Biotechnol. 2003;62:27–34. doi: 10.1007/s00253-003-1321-8. [DOI] [PubMed] [Google Scholar]
  • 10.Wiethoff C. M., Middaugh R. C. Barriers to nonviral gene delivery. J. Pharm. Sci. 2003;92:203–217. doi: 10.1002/jps.10286. [DOI] [PubMed] [Google Scholar]
  • 11.Check E. Gene therapy: a tragic setback. Nature. 2002;420:116–118. doi: 10.1038/420116a. [DOI] [PubMed] [Google Scholar]
  • 12.Hacein-Bey-Abina S., Von Kalle C., Schmidt M., McCormack M. P., Wulffraat N., Leboulch P., Lim A., Osborne C. S., Pawliuk R., Morillon E., Sorensen R., Forster A., Fraser P., Cohen J.I., de Saint Basile G., Alexander I., Wintergerst U., Frebourg T., Aurias A., Stoppa-Lyonnet D., Romana S., Radford-Weiss I., Gross F., Valensi F., Delabesse E., Macintyre E., Siqaux F., Soulier J., Leiva L. E., Wissler M., Prinz C., Rabbitts T. H., Le Deist F., Fischer A., Cavazzana-Calvo M. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419. doi: 10.1126/science.1088547. [DOI] [PubMed] [Google Scholar]
  • 13.Boyce N. Trial halted after gene shows up in semen. Nature. 2001;414:677–678. doi: 10.1038/414677a. [DOI] [PubMed] [Google Scholar]
  • 14.Wu G. Y., Wu C. H. Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 1988;263:14621–14624. [PubMed] [Google Scholar]
  • 15.Boussif O., Lezoualc’h F., Zanta M. A., Mergny M. D., Scherman D., Demeneix B., Behr J.-P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo polyethylenimine. Proc. Natl. Acad. Sci. USA. 1995;92:7297–7301. doi: 10.1073/pnas.92.16.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Godbey W. T., Wu K. K., Mikos A. G. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. USA. 1999;96:5177–5181. doi: 10.1073/pnas.96.9.5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Thomas M., Klibanov A. M. Enhancing polyethylenimine’s delivery of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. USA. 2002;99:14640–14645. doi: 10.1073/pnas.192581499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Suh J., Wirtz D., Hanes J. Efficient active transport of gene nanocarriers to the cell nucleus. Proc. Natl. Acad. Sci. USA. 2003;100:3878–3882. doi: 10.1073/pnas.0636277100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Sonawane N. D., Szoka F. C., Jr., Verkman A. S. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J. Biol. Chem. 2003;278:44826–44831. doi: 10.1074/jbc.M308643200. [DOI] [PubMed] [Google Scholar]
  • 20.Wagner E. Strategies to improve DNA polyplexes for in vivo gene transfer: will “artificial viruses” be the answer? Pharm. Res. 2004;21:8–14. doi: 10.1023/b:pham.0000012146.04068.56. [DOI] [PubMed] [Google Scholar]
  • 21.Kirchler A. Gene transfer with modified polyethylenimines. J. Gene Med. 2004;6:S3–S10. doi: 10.1002/jgm.507. [DOI] [PubMed] [Google Scholar]
  • 22.Forrest M. L., Meister G. E., Koerber J. T., Pack D. W. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm. Res. 2004;21:365–371. doi: 10.1023/b:pham.0000016251.42392.1e. [DOI] [PubMed] [Google Scholar]
  • 23.Lim Y.-B., Kim S.-M., Suh H., Park J.-S. Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and non-toxic gene delivery carrier. Bioconjug. Chem. 2002;13:952–957. doi: 10.1021/bc025541n. [DOI] [PubMed] [Google Scholar]
  • 24.Akinc A., Lynn D. M., Anderson D. G., Langer R. Parallel synthesis and characterization of a degradable polymer library for gene delivery. J. Am. Chem. Soc. 2003;125:5316–5323. doi: 10.1021/ja034429c. [DOI] [PubMed] [Google Scholar]
  • 25.Kichler A., Leborgne C., Marz J., Danos O., Bechinger B. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc. Natl. Acad. Sci. USA. 2003;100:1564–1568. doi: 10.1073/pnas.0337677100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Liu Y., Wenning L., Linch M., Reineke T. M. New poly(D-glucaramidoamine)s induce DNA nanoparticle formation and efficient gene delivery into mammalian cells. J. Am. Chem. Soc. 2004;126:7422–7423. doi: 10.1021/ja049831l. [DOI] [PubMed] [Google Scholar]
  • 27.Han S.-O., Mahato R. I., Kim S. W. Water-soluble lipopolymer for gene delivery. Bioconjug. Chem. 2001;12:337–345. doi: 10.1021/bc000120w. [DOI] [PubMed] [Google Scholar]
  • 28.Kim S., Choi J. S., Jang H. S., Suh H., Park J. Hydrophobic modification of polyethyleneimine for gene transfectants. Bull. Korean Chem. Soc. 2001;22:1069–1075. [Google Scholar]
  • 29.Gebhart C. L., Sriadibhatla S., Vinogradov S., Lemieux P., Alakhov V., Kabanov A. V. Design and formulation of polyplexes based on pluronic-polyethylenimine conjugates for gene transfer. Bioconjug. Chem. 2002;13:937–944. doi: 10.1021/bc025504w. [DOI] [PubMed] [Google Scholar]
  • 30.Oku N., Yamazaki Y., Matsuura M., Sugiyama M., Hasegawa M., Nango M. A novel non-viral gene transfer system, polycation liposomes. Adv. Drug Deliv. Revs. 2001;52:209–218. doi: 10.1016/s0169-409x(01)00212-5. [DOI] [PubMed] [Google Scholar]
  • 31.Gosselin M. A., Guo W., Lee R. J. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjug. Chem. 2001;12:989–994. doi: 10.1021/bc0100455. [DOI] [PubMed] [Google Scholar]
  • 32.Petersen H., Kunath K., Martin A. L., Stolnik S., Roberts C. J., Davies M. C., Kissel T. Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines. Biomacromolecules. 2002;3:926–936. doi: 10.1021/bm025539z. [DOI] [PubMed] [Google Scholar]
  • 33.Thomas M., Klibanov A. M. Conjugation to gold nanoparticles enhances polyethylenimine’s transfer of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. USA. 2003;100:9138–9143. doi: 10.1073/pnas.1233634100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Panyam J., Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 2003;55:329–347. doi: 10.1016/s0169-409x(02)00228-4. [DOI] [PubMed] [Google Scholar]
  • 35.Wang J., Mao H.-Q., Leong K. W. A novel biodegradable gene carrier based on polyphosphoester. J. Am. Chem. Soc. 2001;123:9480–9481. doi: 10.1021/ja016062m. [DOI] [PubMed] [Google Scholar]
  • 36.Peterson H., Merdan T., Kunath K., Fisher D., Kissel T. Poly(ethylenimine-co-L-lactamide-co-succinimide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery. Bioconjug. Chem. 2002;13:812–821. doi: 10.1021/bc0255135. [DOI] [PubMed] [Google Scholar]
  • 37.Ahn C.-H., Chae S. Y., Bae Y. H., Kim S. W. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J. Control. Rel. 2002;80:273–282. doi: 10.1016/s0168-3659(01)00547-8. [DOI] [PubMed] [Google Scholar]
  • 38.Lim Y.-B., Han S.-O., Kong H.-U., Lee Y., Park J.-S., Jeong B., Kim S. W. Biodegradable polyester, poly[(α-(4-aminobutyl)-L-glycolic acid], as a non-toxic gene carrier. Pharm. Res. 2000;17:811–816. doi: 10.1023/a:1007552007765. [DOI] [PubMed] [Google Scholar]
  • 39.Lim Y.-B., Choi Y. H., Park J.-S. A self-destroying polycationic polymer: biodegradable poly(4-hydroxy-L-proline ester) J. Am. Chem. Soc. 1999;121:5633–5639. [Google Scholar]
  • 40.Forrest M. L., Koerber J. T., Pack D. W. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug. Chem. 2003;14:934–940. doi: 10.1021/bc034014g. [DOI] [PubMed] [Google Scholar]
  • 41.von Harpe A., Petersen H., Li Y., Kissel T. Characterization of commercially available and synthesized polyethylenimines for gene delivery. J. Control. Rel. 2000;69:309–322. doi: 10.1016/s0168-3659(00)00317-5. [DOI] [PubMed] [Google Scholar]
  • 42.Tan Y., Huang L. Overcoming the inflammatory toxicity of cationic gene vectors. J. Drug Target. 2002;10:153–160. doi: 10.1080/10611860290016757. [DOI] [PubMed] [Google Scholar]
  • 43.Schaffer D. V., Fidelman N. A., Dan N., Lauffenburger D. A. Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol. Bioeng. 2000;67:598–606. doi: 10.1002/(sici)1097-0290(20000305)67:5<598::aid-bit10>3.0.co;2-g. [DOI] [PubMed] [Google Scholar]
  • 44.Moon C., Oh Y., Roth J. A. Current status of gene therapy for lung cancer and head and neck cancer. Clin. Cancer Res. 2003;9:5055–5067. [PubMed] [Google Scholar]
  • 45.Willis A. C., Chen X. The promise and obstacle of p53 as a cancer therapeutic agent. Curr. Mol. Med. 2002;2:329–345. doi: 10.2174/1566524023362474. [DOI] [PubMed] [Google Scholar]
  • 46.Zhang Y., Li T., Fu L., Yu C., Li Y., Xu X., Wang Y., Ning H., Zhang S., Chen W., Babiuk L. A., Chang Z. Silencing SARS-CoV spike protein expression in cultured cells by RNA interference. FEBS Lett. 2004;560:141–146. doi: 10.1016/S0014-5793(04)00087-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Weiss D. J. Delivery of gene transfer vectors to lung obstacles and the role of adjunct techniques for airway administration. Mol. Ther. 2002;6:148–152. doi: 10.1006/mthe.2002.0662. [DOI] [PubMed] [Google Scholar]

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