Under physiological salt concentration, plasmid DNA compossible reason for this effect. Application of the small parplexed with transferrin-conjugated or unmodified polyethyticles in more concentrated form and over extended perlenimine (PEI, 800 kDa) forms huge (up to Ͼ1000 nm) iods of time improves transfection activity. Reduced intraaggregates, unless the individual components are mixed cellular release may be another explanation for the at a highly positive nitrogen/phosphate (N/P) charge ratio.decreased transfection efficiency; incubation with chloroAt low ionic strengths, however, small particles with an quine or incorporation of the endosomolytic peptide INF5 average size of 40 nm are formed over a broad range of into the small complexes enhances gene expression N/P ratios. Interestingly, in transfection experiments these approximately 10-fold. Analysis of gene expression at the small particles result in a 10-fold (B16F10 cells) to more cellular level using a green fluorescence protein reporter than 100-fold (Neuro2A cells, K562 cells) reduced lucifergene and flow cytometry revealed that the differences in ase gene expression efficiency in comparison to the large overall gene expression largely result from different intencomplexes formed in physiological salt solutions. Limited sities per expressing cell, while the difference in the pertransport of the small particles to the cell surfaces is one centage of expressing cells is less substantial.

Branched and linear PEI/DNA complexes differ in their ability to transfect cells. The greater efficiency of linear PEI might be due to an inherent kinetic instability under salt conditions. Understanding how to employ this kinetic instability of linear PEI could help in designing future vectors with greater flexibility and transfection efficiency in vivo.

Systemic application of positively charged polycation/DNA complexes has been shown to result in predominant gene expression in the lungs. Targeting gene expression to other sites, eg distant tumors, is hampered by nonspecific interactions largely due to the positive surface charge of transfection complexes. In the present study we show that the positive surface charge of PEI (25 kDa branched or 22 kDa linear)/DNA complexes can be efficiently shielded by covalently incorporating transferrin at sufficiently high densities in the complex, resulting in a dramatic decrease in nonspecific interactions, eg with erythrocytes, and decreased gene expression in the lung. Systemic application of transferrinshielded PEI/DNA complexes into A/J mice bearing subcut-

Recently the high transfection potential of the cationic polyfold increased transfection efficiency. This activity depends mer polyethylenimine (PEI) was described (Boussif O et al. on ligand-receptor interaction and was observed also at Proc Natl Acad Sci USA 1995; 92: 7297-7301

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.