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.

In many cases, nonviral particle-mediated gene delivery is highly dependent on the cell cycle status of transfected cells. Here we compare particle-mediated delivery with linear polyethylenimine (PEI) and physical transfer of DNA by electroporation with branched PEI and lipofection for their ability to transfect cells at different stages of the cell cycle. In contrast to other particle-mediated delivery methods (using Lipofectamine or branched PEI) linear PEI led to only small differences (within 1 log unit) in gene transfer between HeLa cells transfected in G1 and those in S/G2. Parallel transfections (lipofection or branched PEI) resulted in 2 to > 3 log-unit differences in luciferase expression between cells transfected in G1 and S/G2. Gene transfer by electroporation also revealed hardly any cell cycle dependence and displayed completely different expression kinetics. Reporter gene expression is already very high 3 hours after electroporation with roughly the same level of reporter gene expression in all cell cycle phases. We suggest that DNA electroporation and DNA transfection with linear PEI particles have improved nuclear import characteristics relative to the other tested DNA delivery systems.

Targeted gene delivery capitalizes on the presence of specific cell surface receptors for DNA uptake into cells by receptormediated endocytosis (1-3). Therefore, receptor binding ligands are coupled to polycationic compounds like polylysine (pL) 1 that bind and condense DNA. Following this concept, transferrin polylysine (TfpL)-based gene transfer systems were developed to target transferrin receptor for DNA delivery into cells (1, 4 -7). Binding of TfpL/DNA complexes to transferrin receptor causes internalization and DNA uptake into the endosomal compartment (8). To facilitate DNA release from this compartment, endosomolytic agents (such as inactivated adenoviruses) were included in transfection complexes and were demonstrated to effectively enhance gene transfer efficiency (8 -10). More recently, transferrin polyethylenimine (TfPEI) conjugates have been synthesized and used for DNA delivery, thereby combining the high intrinsic transfection efficacy of polyethylenimine (PEI) with receptor-targeted gene transfer (11,12). PEI possesses DNA binding and condensing activity together with a high pH buffering capacity that is believed to protect DNA from degradation and to enhance exit from the endosomal compartment. Accordingly, PEI is effective in gene delivery into a variety of cell types even without the addition of cell binding ligands or endosomolytic agents (13, 14). Here we investigated whether the mannose receptor that is abundantly expressed on dendritic cells (DC) represents a suitable entry site for targeted gene delivery into DC using mannosylated PEI (ManPEI).DC are professional antigen-presenting cells that occur in peripheral organs like skin, where these cells are exposed to antigens, which they capture and process (15-18). Upon inflammatory stimuli, DC migrate to lymphoid tissue and present processed antigens on major histocompatibility complex (MHC) class I and II molecules to T cells, to elicit an antigenspecific T cell response. Because of their central role in the initiation of primary immune responses, there is high interest in employing DC for immunotherapy of diseases, such as cancer (19 -21). Following such approaches, gene-modified DC offer several potential advantages over peptide/protein-pulsed DC. For example, gene-modified DC can be expected to induce T cell responses against multiple and/or undefined epitopes of tumor antigens, possibly in the context of both MHC class I and II, and with any MHC allele. Furthermore, the expression of chemokines and cytokines in DC simultaneously with tumorspecific and/or associated antigens would additionally allow modulation of the immune response. DC and T cell functions are effectively regulated by a variety of cytokines, and local cytokine production by DC might represent an important adjunct for T cell activation in medical therapy, for example in cancer patients who are often immunosuppressed. However, so far the generation of gene-modified immunocompetent DC has remained difficult mainly due to limitations in DNA delivery techniques (12,21,22)...

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.