Gene Therapy and Molecular Biology Vol 8, page 369 369 Gene Ther Mol Biol Vol 8, 369-384, 2004 Transfection pathways of nonspecific and targeted PEI-polyplexes Review Article Vicent M. Guillem 1 and Salvador F. Aliño 2 1 Servei d’ Hematologia i Oncologia. Hospital Clínic Universitari. Facultat de Medicina. Universitat de València. Avda. Blasco Ibañez 17, 46010 – València (Spain) 2 Grup de Teràpia Gènica. Departament de Farmacologia. Facultat de Medicina. Universitat de València. Avda. Blasco Ibañez 15, 46010 – València (Spain) __________________________________________________________________________________ *Correspondence: Salvador F. Aliño, Departament de Farmacologia. Facultat de Medicina, Universitat de València, Blasco Ibañez 15, 46010 – Valencia (Spain); Phone: (+34) 96 386 46 21; Fax: (+34) 96 386 49 72; E-mail: Salvador.Alino@uv.es Key words: PEI-polyplexes, transfection, DNase degradation, Interactions, cell surface, cell culture medium, specificity, efficacy, cell internalization, Endosome trafficking, proton-sponge effect, Cytoplasm transport, nuclear accession, dissociation Abbreviations: Polyethyleneimine, (PEI); polylysine, (PLL); polyamidoamine dendrimers, (PAMAM dendrimers); epithelial growth factor, (EGF); basic fibroblast growth factor, (bFGF); 2-(dimethylamino)ethylmethacrylate, (pDMAEMA); transferrin-polylysine polyplexes, (Tf-pLL); poly-[N-(2-hydroxypropyl)methacrylamide], (pHPMA) Received: 30 April 2004; Accepted: 24 June 2004; electronically published: September 2004 Summary Polyethyleneimine (PEI) based vectors have become in an important vehicle for nonviral gene transfer. However, despite their extensive use and efficacy in the transfection of several cellular models both in vitro and in vivo , the mechanism by which they transfect cells has not been fully elucidated, and controversy remains over the interpretation of some apparently contradictory findings. A review is made of the studies on PEI polyplexes, focusing on PEI polyplex transfection properties (as physico-chemical characteristics important for transfection) and the mechanistic findings of PEI polyplex transfection comprising cell membrane binding with nonspecific and targeted–PEI polyplexes, the putative internalization pathways (such as the proton sponge hypothesis), the nuclear bioavailability of the transported nucleic acid, and other relevant issues such as the influence of polyplex size in vitro upon transfection activity. I. Introduction Specific and efficient delivery of nucleic acid into targeted cells is a priority objective of gene therapy. To achieve successful modification of the gene expression pattern, the exogenous nucleic acid must overcome a series of obstacles to gain access first to the cell and posteriorly to the intracellular compartments, where the nucleic acid exerts its function. Since nucleic acid uptake by cells is an inefficient process, it has been necessary to develop several strategies to increase nucleic acid delivery. One of the approaches is based on the use of nonviral vehicles such as liposomes (Wong et al, 1980; Alino et al, 1993), lipoplexes or nucleic acid-cationic lipid complexes (Felgner et al, 1997), and polyplexes (Gebhart and Kabanov 2001) - complexes of nucleic acids and cationic polymers such as polyethyleneimine (PEI) (Boussif et al, 1995). Due to its intrinsic transfection properties, PEI has been used to conform the backbone of a great number of vector formulations. Despite their widespread use and demonstrated efficacy in the transfection of several cellular models both in vitro and in vivo, the mechanism by which they transfect cells has not been fully elucidated, and controversy remains over the interpretation of some apparently contradictory findings. The present review discusses the hypothetical transfection pathways of PEI-polyplexes - from vector binding to the cell membrane to nucleic acid arrival in the nucleus, the influence of physico-chemical properties of PEI in transfection activity and other relevant issues such as the influence of polyplex size and cell type upon transfection activity, and the most relevant differences or similarities between PEI and other polymers used in transfection (fundamentally polylysine polyplexes). II. Characteristics of PEI-polyplexes A. PEI physico-chemical properties of importance for transfection PEI is a synthetic polymer with a nitrogen-carbon base (32.5% nitrogen). Ethanolamine, the monomeric unit of PEI (CH 2 -CH 2 -NH-), confers great PEI solubility in