Polyplex Evolution: Understanding Biology, Optimizing Performance

Polyethylenimine (PEI) is a gold standard polycationic transfectant. However, the highly efficient transfecting activity of PEI and many of its derivatives is accompanied by serious cytotoxic complications and safety concerns at innate immune levels, which impedes the development of therapeutic polycationic nucleic acid carriers in general and their clinical applications. In recent years, the dilemma between transfection efficacy and adverse PEI activities has been addressed from in-depth investigations of cellular processes during transfection and elucidation of molecular mechanisms of PEI-mediated toxicity and translation of these integrated events to chemical engineering of novel PEI derivatives with an improved benefit-to-risk ratio. This review addresses these perspectives and discusses molecular events pertaining to dynamic and multifaceted PEI-mediated cytotoxicity, including membrane destabilization, mitochondrial dysfunction, and perturbations of glycolytic flux and redox homeostasis as well as chemical strategies for the generation of better tolerated polycations. We further examine the effect of PEI and its derivatives on complement activation and interaction with Toll-like receptors. These perspectives are intended to lay the foundation for an improved understanding of interlinked mechanisms controlling transfection and toxicity and their translation for improved engineering of polycation-based transfectants. Polyethylenimine (PEI) is a gold standard polycationic transfectant. However, the highly efficient transfecting activity of PEI and many of its derivatives is accompanied by serious cytotoxic complications and safety concerns at innate immune levels, which impedes the development of therapeutic polycationic nucleic acid carriers in general and their clinical applications. In recent years, the dilemma between transfection efficacy and adverse PEI activities has been addressed from in-depth investigations of cellular processes during transfection and elucidation of molecular mechanisms of PEI-mediated toxicity and translation of these integrated events to chemical engineering of novel PEI derivatives with an improved benefit-to-risk ratio. This review addresses these perspectives and discusses molecular events pertaining to dynamic and multifaceted PEI-mediated cytotoxicity, including membrane destabilization, mitochondrial dysfunction, and perturbations of glycolytic flux and redox homeostasis as well as chemical strategies for the generation of better tolerated polycations. We further examine the effect of PEI and its derivatives on complement activation and interaction with Toll-like receptors. These perspectives are intended to lay the foundation for an improved understanding of interlinked mechanisms controlling transfection and toxicity and their translation for improved engineering of polycation-based transfectants. Gene therapy, the transfer of nucleic acids to a target cell, has been endorsed optimistically as an advanced therapeutic treatment/management strategy against otherwise difficult or incurable diseases.1Al-Dosari M.S. Gao X. 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Polyethylenimine (PEI) is among the most intensively investigated polycations for nucleic acid delivery. Because of its highly efficient transfection capabilities, PEI has served as a gold standard transfectant polymer.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar PEI is structured from repeating units of two aliphatic carbon groups and amino nitrogen and is commercially available in both linear and branched morphologies, with the molecular weight ranging between 200 Da and 1,500 kDa.13Boussif 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. 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Favrot M. In vivo delivery to tumors of DNA complexed with linear polyethylenimine.Hum. Gene Ther. 1999; 10: 1659-1666Crossref PubMed Scopus (0) Google Scholar The branched PEI contains primary, secondary, and tertiary amines, whereas the linear form has only secondary amines. The protonable amine groups provide PEIs with the unique features of high cationic charge density and buffering capacity at extracellular and endo-lysosomal pH levels.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar This trait makes PEI exceptionally effective in compacting nucleic acids into polyplexes that effectively shield nucleic acids against degradation by nucleases. Additionally, PEI-nucleic acid complexes (PEI polyplexes) with a net positive charge are favorably taken up by mammalian cells through different endocytic mechanisms as well as plasma membrane destabilization.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar, 14Neu M. Fischer D. Kissel T. Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives.J. Gene Med. 2005; 7: 992-1009Crossref PubMed Scopus (0) Google Scholar, 22Godbey W.T. Wu K.K. Mikos A.G. Poly(ethylenimine) and its role in gene delivery.J. Control. Release. 1999; 60: 149-160Crossref PubMed Scopus (892) Google Scholar, 23Godbey W.T. Wu K.K. Hirasaki G.J. Mikos A.G. 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Ther. 2013; 21: 149-157Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Regardless of the internalization pathways, the effectiveness of PEI transfection procedures is well known to be both architecture- and molecular weight-dependent and directly correlates with the positive charge of the polyplexes, which, in turn, is associated with cytotoxic complications.19Wightman L. Kircheis R. Rössler V. Carotta S. Ruzicka R. Kursa M. Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 2001; 3: 362-372Crossref PubMed Scopus (469) Google Scholar, 24Godbey W.T. Wu K.K. Mikos A.G. Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.J. Biomed. Mater. Res. 1999; 45: 268-275Crossref PubMed Scopus (557) Google Scholar, 32Godbey W.T. Wu K.K. Mikos A.G. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability.Biomaterials. 2001; 22: 471-480Crossref PubMed Scopus (271) Google Scholar, 33Kunath K. von Harpe A. Fischer D. Petersen H. Bickel U. Voigt K. Kissel T. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.J. Control. Release. 2003; 89: 113-125Crossref PubMed Scopus (0) Google Scholar, 34Fischer D. Li Y. Ahlemeyer B. Krieglstein J. Kissel T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.Biomaterials. 2003; 24: 1121-1131Crossref PubMed Scopus (1258) Google Scholar In particular, higher molecular weight (HMW) PEIs do exhibit superior transfection efficiencies in vitro and in vivo but are, unfortunately, also linked with greater cytotoxicity.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar, 19Wightman L. Kircheis R. Rössler V. Carotta S. Ruzicka R. Kursa M. Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 2001; 3: 362-372Crossref PubMed Scopus (469) Google Scholar, 20Ferrari S. Moro E. Pettenazzo A. Behr J.P. Zacchello F. Scarpa M. ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo.Gene Ther. 1997; 4: 1100-1106Crossref PubMed Google Scholar, 21Coll J.L. Chollet P. Brambilla E. Desplanques D. Behr J.P. Favrot M. In vivo delivery to tumors of DNA complexed with linear polyethylenimine.Hum. Gene Ther. 1999; 10: 1659-1666Crossref PubMed Scopus (0) Google Scholar, 35Ahn C.H. Chae S.Y. Bae Y.H. Kim S.W. Biodegradable poly(ethylenimine) for plasmid DNA delivery.J. Control. Release. 2002; 80: 273-282Crossref PubMed Scopus (245) Google Scholar In contrast, lower molecular weight (LMW) PEIs display very few cytotoxic effects but are also known for their inferior transfection abilities.33Kunath K. von Harpe A. Fischer D. Petersen H. Bickel U. Voigt K. Kissel T. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.J. Control. Release. 2003; 89: 113-125Crossref PubMed Scopus (0) Google Scholar, 34Fischer D. Li Y. Ahlemeyer B. Krieglstein J. Kissel T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.Biomaterials. 2003; 24: 1121-1131Crossref PubMed Scopus (1258) Google Scholar Hence, there exists a fine-tuned intrinsic balance with increasing molecular weight, better transfection efficiency, and higher cytotoxic complications that has to be taken into account when selecting/designing the optimal PEI-based delivery system. Although both linear and branched PEI morphologies have achieved excellent transfection efficiencies in a wide range of clinically relevant cell lines, the structural configuration of PEI appears to influence the transfection abilities.19Wightman L. Kircheis R. Rössler V. Carotta S. Ruzicka R. Kursa M. Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 2001; 3: 362-372Crossref PubMed Scopus (469) Google Scholar Although some studies have promoted the branched morphology, it appears that the linear structure is a superior transfection agent both in vitro and in vivo.19Wightman L. Kircheis R. Rössler V. Carotta S. Ruzicka R. Kursa M. Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 2001; 3: 362-372Crossref PubMed Scopus (469) Google Scholar, 20Ferrari S. Moro E. Pettenazzo A. Behr J.P. Zacchello F. Scarpa M. ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo.Gene Ther. 1997; 4: 1100-1106Crossref PubMed Google Scholar, 36Wiseman J.W. Goddard C.A. McLelland D. Colledge W.H. A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo.Gene Ther. 2003; 10: 1654-1662Crossref PubMed Scopus (0) Google Scholar, 37Bragonzi A. 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Furthermore, it is still unclear whether these variations in transfection abilities, amid diverse PEI structures in different studies, represent actual beneficial effects from structural differences or whether the superior transfection occurs as a consequence of fewer cytotoxic complications and cell death associated with architecture-dependent cytotoxicity in a cell/tissue-specific manner. Regardless of which structural configuration is superior as a transfection agent, accumulating evidence shows that, although cationic polymers, and PEI in particular, comprise many prominent features required for effective nucleic acid delivery, it remains a great concern that those cationic polymers containing the best transfection abilities are also those generally found to be most cytotoxic.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar, 19Wightman L. Kircheis R. Rössler V. Carotta S. Ruzicka R. Kursa M. Wagner E. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.J. Gene Med. 2001; 3: 362-372Crossref PubMed Scopus (469) Google Scholar, 21Coll J.L. Chollet P. Brambilla E. Desplanques D. Behr J.P. Favrot M. In vivo delivery to tumors of DNA complexed with linear polyethylenimine.Hum. Gene Ther. 1999; 10: 1659-1666Crossref PubMed Scopus (0) Google Scholar, 33Kunath K. von Harpe A. Fischer D. Petersen H. Bickel U. Voigt K. Kissel T. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.J. Control. Release. 2003; 89: 113-125Crossref PubMed Scopus (0) Google Scholar, 34Fischer D. Li Y. Ahlemeyer B. Krieglstein J. Kissel T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.Biomaterials. 2003; 24: 1121-1131Crossref PubMed Scopus (1258) Google Scholar To counteract this dilemma, chemical engineering and advanced polymer technologies have, in conjunction with comprehensive cellular and molecular studies, detected important structure-toxicity associations that have aided the design of safer cationic polymer-based vectors for nucleic acid delivery. Accordingly, several strategies have been followed to decrease PEI-mediated cytotoxicity while maintaining the intended efficiency. Important strategies can be classified as follows: control of size and topology, biodegradable cross-linking of LMW PEI, statistical surface modification, synthesis of block co-polymers, and oligoamine segment conjugation. This review aims to provide much needed insight into the principal mechanistic aspects underlying the cytotoxic complications manifested upon exposure to cationic polymers and to address the most promising approaches utilized for chemical engineering of new PEI derivatives that have lowered cytotoxic complications but preserved or even improved transfection capabilities. During the last decade, a number of studies have shown that the PEI-mediated cytotoxic responses are highly interlinked with perturbations in cellular membranes and bioenergetic processes in conjunction with polymer concentration, molecular weight, electric charge, and structure.13Boussif 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: 7297-7301Crossref PubMed Scopus (4469) Google Scholar, 24Godbey W.T. Wu K.K. Mikos A.G. 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