Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins.

Nose-to-brain delivery presents a promising alternative route compared to classical blood-brain barrier passage, especially for the delivery of high molecular weight drugs. In general, macromolecules are rapidly degraded in physiological environment. Therefore, nanoparticulate systems can be used to protect biomolecules from premature degradation. Furthermore, targeting ligands on the surface of nanoparticles are able to improve bioavailability by enhancing cellular uptake due to specific binding and longer residence time. In this work, transferrin-decorated chitosan nanoparticles are used to evaluate the passage of a model protein through the nasal epithelial barrier in vitro. It was demonstrated that strain-promoted azide-alkyne cycloaddition reaction can be utilized to attach a functional group to both transferrin and chitosan enabling a rapid covalent surface-conjugation under mild reaction conditions after chitosan nanoparticle preparation. The intactness of transferrin and its binding efficiency were confirmed via SDS-PAGE and SPR measurements. Resulting transferrin-decorated nanoparticles exhibited a size of about 110-150 nm with a positive surface potential. Nanoparticles with the highest amount of surface bound targeting ligand also displayed the highest cellular uptake into a human nasal epithelial cell line (RPMI 2650). In an air-liquid interface co-culture model with glioblastoma cells (U87), transferrin-decorated nanoparticles showed a faster passage through the epithelial cell layer as well as increased cellular uptake into glioblastoma cells. These findings demonstrate the beneficial characteristics of a specific targeting ligand. With this chemical and technological formulation concept, a variety of targeting ligands can be attached to the surface after nanoparticle formation while maintaining cargo integrity.

Microfluidic mixing as platform technology for production of chitosan nanoparticles loaded with different macromolecules.

In recent decades, biotechnological drugs have emerged as relevant therapeutic tools. However, therapeutic molecules can exert their activity only if properly formulated and delivered into the body. In this regard, nano-sized drug delivery systems have been shown to provide protection, stability, and controlled release of payloads, increasing their therapeutic efficacy. In this work, a microfluidic mixing technique for the preparation of chitosan-based nanoparticles was established with the capability of easily exchanging macromolecular biological cargos such as the model protein ß-Galactosidase, mRNA, and siRNA. The nanoparticles obtained showed hydrodynamic diameters ranging from 75 nm to 105 nm, low polydispersity of 0.15 to 0.22 and positive zeta potentials of 6 mV to 17 mV. All payloads were efficiently encapsulated (>80 %) and the well-known cytocompatibility of chitosan-based nanoparticles was confirmed. Cell culture studies demonstrated increased cellular internalization of loaded nano-formulations compared to free molecules as well as successful gene silencing with nano-formulated siRNA, suggesting the ability of these nanoparticles to escape the endosome.

Integrin alphaVbeta3 targeted gene delivery using RGD peptidomimetic conjugates with copolymers of PEGylated poly(ethylene imine).

This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.

Synthesis and characterization of chemically condensed oligoethylenimine containing beta-aminopropionamide linkages for siRNA delivery.

Polyethylenimines (PEI) are often inefficient in gene knockdown experiments with small interfering RNA (siRNA), presumably due to the strong complexing properties. A more efficient and potentially degradable oligoethylenimine-based carrier was synthesized by the condensation of 800 molecular weight PEI oligomers with hexanedioldiacrylate. Reaction conditions were chosen such that Michael reaction occurs followed by complete N-acylation of all residual ester bonds resulting in beta-aminopropionamide linkage sites and an average molecular weight of 30,000. Based on NMR analysis, these conditions produced 38% tertiary amides and 62% secondary amides, with about 2% residual carboxylate, presumably from hydrolysis. The ionizable equivalent weight of the carrier increased to 51, compared to a value of 43 for standard PEI. Sensible in vitro knockdown of the luciferase gene in stably transfected HUH7 cells, up to 80% in comparison to non-specific siRNA, demonstrated its suitability for siRNA delivery.

Targeting of immunoliposomes to endothelial cells using a single-chain Fv fragment directed against human endoglin (CD105).

We generated immunoliposomes targeting proliferating endothelial cells by chemically coupling a single-chain Fv fragment (scFv A5) directed against human endoglin to the liposomal surface. For this purpose, we introduced an additional cysteine residue at the C-terminus of the scFv fragment. This scFv' fragment was expressed in soluble form in bacteria and allowed for a site-directed coupling to sulfhydryl-reactive lipids incorporated into the lipid bilayer. The immunoliposomes (ILA5) showed rapid and strong binding to human endoglin-expressing endothelial cells (HUVEC, HDMEC), while no binding was observed with various endoglin-negative cell lines and blood lymphocytes. In vitro, ILA5 were stable for several hours in serum- or plasma-containing medium. Incubation of endothelial cells with ILA5 at 37 degrees C led to increased binding and internalisation of the liposomes as evidenced by a perinuclear accumulation. In vitro, doxorubicin-loaded ILA5 showed an increased cytotoxicity towards endothelial cells compared to untargeted liposomes and free doxorubicin. Since the vasculature of tumours is easily accessible to drug carrier systems, the described endothelial cell-specific immunoliposomes may be useful for the development of efficacious and safe vascular targeting agents in cancer therapy.

Prospects for cationic polymers in gene and oligonucleotide therapy against cancer.

Gene and antisense/ribozyme therapy possesses tremendous potential for the successful treatment of genetically based diseases, such as cancer. Several cancer gene therapy strategies have already been realized in vitro, as well as in vivo. A few have even reached the stage of clinical trials, most of them phase I, while some antisense strategies have advanced to phase II and III studies. Despite this progress, a major problem in exploiting the full potential of cancer gene therapy is the lack of a safe and efficient delivery system for nucleic acids. As viral vectors possess toxicity and immunogenicity, non-viral strategies are becoming more and more attractive. They demonstrate adequate safety profiles, but their rather low transfection efficiency remains a major drawback. This review will introduce the most important cationic polymers used as non-viral vectors for gene and oligonucleotide delivery and will summarize strategies for the targeting of these agents to cancer tissues. Since the low efficiency of this group of vectors can be attributed to specific systemic and subcellular obstacles, these hurdles, as well as strategies to circumvent them, will be discussed. Local delivery approaches of vector/DNA complexes will be summarized and an overview of the principles of anticancer gene and antisense/ribozyme therapy as well as an outline of ongoing clinical trials will be presented.

Recent advances in intravenous delivery of poorly water-soluble compounds.

In recent years, with the widespread application of high-throughput screening technologies in drug discovery, an increasing number of new chemical entities with extremely poor aqueous solubility have been generated. Their poor solubility represents a major challenge for formulation of these compounds for both oral and parenteral administration. Formulations for intravenous (i.v.) application are of significant importance because they are frequently used in several key therapeutic areas, such as oncology and anesthesia. Furthermore, i.v. formulations of new compounds are often needed to determine basic biopharmaceutical parameters and to obtain proof of concept results in the early phase of product development. This review provides an overview of the recent advances in formulation approaches and drug delivery technologies for poorly water-soluble compounds applicable to i.v. administration. The advantages and disadvantages of different strategies are highlighted and an expert opinion on each technical field is presented.

Induction of apoptosis in murine neuroblastoma by systemic delivery of transferrin-shielded siRNA polyplexes for downregulation of Ran.

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.

Poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) nanoparticles for local delivery of paclitaxel for restenosis treatment.

Catheter-based local delivery of biodegradable nanoparticles (NP) with sustained release characteristics represents a therapeutic approach to reduce restenosis. Paclitaxel-loaded NP consisting of poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) (PVA-g-PLGA) with varying PLGA chain length as well as poly(lactide-co-glycolide) (PLGA), were prepared by a solvent evaporation technique. NP of <180 nm in diameter characterized by photon correlation spectroscopy (PCS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) are spherical and show smooth surfaces. Yields typically range from 80 to 95% with encapsulation efficiencies between 77 and 87%. The extent of initial in vitro paclitaxel release was affected by the PVA-g-PLGA composition. Blank nanoparticles from PVA(300)-g-PLGA(30) and PVA(300)-g-PLGA(15) showed excellent biocompatibility in rabbit vascular smooth muscle cells (RbVSMC) at polymer concentrations of 0.37 mg/ml. Paclitaxel-loaded NP have an increased antiproliferative effect on cells in comparison to free drug. Confocal laser scanning microscopy of RbVSMC confirmed cellular uptake of nanoparticles composed of fluorescently labeled PVA(300)-g-PLGA(15) loaded with Oregon Green labeled paclitaxel. Cells showed a clearly increased fluorescence activity with a co-localization of paclitaxel and polymer nanoparticles during incubation with particle suspension. To evaluate the antirestenotic effect in vivo, paclitaxel-loaded nanoparticles were administered locally to the wall of balloon-injured rabbit iliac arteries using a porous balloon catheter. As a result a 50% reduction in neointimal area in vessel segments treated with paclitaxel-loaded nanoparticles compared to control vessel segments could be observed (local paclitaxel nanoparticle treated segments 0.80+/-0.19 mm(2), control segments 1.58+/-0.6 mm(2); p<0.05).

Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors.

In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2,000 Da)(10)-graft-poly(ethylene imine 25 kDa) (PEG-PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG-PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130-180 nm. zeta potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab-PEI-PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG-PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG-PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab-PEI-PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.

Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility.

Soluble supramolecular inclusion complexes were formed by threading alpha-cyclodextrin (alpha-CD) molecules over poly(ethylene glycol) (PEG) and poly(epsilon-caprolactone) (PCL) chains of ternary block copolymers of PEG, PCL and polyethylenimine (PEI). Characteristic shifts of PCL absorptions in FTIR, (1)H NMR and UV spectra strongly suggest that alpha-CD is threaded over PEG and PCL blocks. Due to the reduced hydrophobic interaction between PCL blocks, the resulting supramolecular complexes displayed a dramatically increased solubility, in comparison with the ternary block copolymers. Their ability to complex DNA was almost as efficient as that of branched PEI 25 kDa, as shown in the ethidium bromide fluorescence quenching experiments. Resulting DNA polyplexes displayed a size of around 200 nm and a neutral surface charge. Microscopy studies in 3T3 fibroblasts revealed an efficient cellular uptake. Transfection efficiencies of inclusion complexes were in the same order of magnitude as PEI. In contrast to PEI a 100x lower toxicity was observed by MTT-assay, allowing the administration of nitrogen-to-phosphate ratios of up to 20. These new gene delivery systems merit further characterization under in vivo conditions.

PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice.

The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 microg and 25 microg of pDNA per mouse. Our studies demonstrated that both PEI and PEG-PEI form stable polyplexes with DNA with similar sizes of 100-130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG-PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and pDNA blood-level time curves from polyplexes at both doses indicative for significant stability in the bloodstream. A very rapid clearance from the bloodstream was observed and as major organs of accumulation liver and spleen were identified. PEG-PEI/pDNA complexes at a dose of approximately 25 microg exhibit similar profiles except a significantly lower deposition in the lung. At the lower dose of approximately 1.5 microg pDNA, however, for polyplexes from PEG-PEI, significant differences in blood level curves and organ accumulation of polymer and pDNA were found. In this case PEG-PEI shows a greatly enhanced circulation time in the bloodstream. By contrast, pDNA was rapidly cleared from circulation and significant amounts of radioactivity were found in the urine, suggesting a rapid degradation possibly by serum nucleases after complex separation. Regarding in vivo gene expression, no luciferase expression could be detected at approximately 1.5 microg dose in any organ using both types of complexes. At 25 microg only in the case of PEI/pDNA complexes were significant levels of the reporter gene detected in lung, liver, and spleen. This coincided with high initial accumulation of pDNA complexed with PEI and a high acute in vivo toxicity. For PEG-PEI, initial accumulation was much lower and no gene expression as well as a low acute toxicity was found. In summary, our data demonstrate that PEG-PEI used in this study is not suitable for low dose gene delivery. At a higher dose of approximately 25 microg, however, polyplex stability is similar to PEI/pDNA combined with a more favorable organ deposition and significantly lower acute in vivo toxicity. These findings have consequences for the design of PEG-PEI-based gene delivery systems for in vivo application.

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PURPOSE: Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. METHODS: Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. RESULTS: Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. CONCLUSIONS: Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Integrin targeting using RGD-PEI conjugates for in vitro gene transfer.

BACKGROUND: Targeting to integrin receptor alpha(nu)beta(3) by RGD peptides seems to be a promising approach for gene delivery to proliferating endothelial cells of tumor metastases. PEGylation of cationic polymers offers a reduction of non-specific binding to cell surfaces. However, little knowledge exists on the influence of charge shielding by PEGylation on targeted gene delivery. Therefore, a variety of RGD peptide-polyethylenimine (PEI) conjugates with different degrees of substitution, with or without poly(ethylene glycol) (PEG) spacer, were synthesized. Influence of degree of substitution and PEG spacer on physicochemical properties as well as on integrin targeting of DNA/polymer complexes was evaluated. METHODS: The tetrapeptide RGDC was coupled to PEI with or without a PEG spacer. Complex formation with DNA was monitored by ethidium bromide (EtBr) fluorescence quenching. Hydrodynamic diameters of complexes and zeta-potential were assessed using a Zetasizer. Fluorescence correlation spectroscopy (FCS) was used to determine peptide binding to living cells. Transfection efficiency was evaluated employing a luciferase reporter gene. Binding of complexes to Mewo cells was monitored by flow cytometry. RESULTS: Polyplexes of RGD-PEI or RGD-PEG-PEI and DNA showed reduced quenching of EtBr fluorescence compared with PEI. All RGD conjugates formed small polyplexes (approximately 100 nm in diameter at a nitrogen/phosphate (N/P) ratio of 6.7). At N/P = 6.7, the zeta-potentials of RGD-PEI complexes were similar to PEI complexes (25-30 mV), while RGD-PEG-PEI formed neutral complexes. FCS showed saturable binding of RGD peptide to Mewo human melanoma cells and only low binding to A549 human lung carcinoma cells. A degree of substitution of 4.6% with SPDP as coupling reagent yielded a conjugate showing 50 times higher luciferase expression in Mewo cells than unmodified PEI at low N/P ratios around 3.3, while a degree of substitution of 1.6% only led to a moderately increased transfection efficiency. Flow cytometry experiments suggest that this effect is partly caused by increased attachment of complexes to cell surfaces. No improvement in transfection efficiency was found in alpha(nu)beta(3)-negative A549 cells. RGD-PEG-PEI complexes showed reasonable transfection efficiencies at high N/P ratios; however, no targeting effect could be found. CONCLUSIONS: Coupling of the tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin-expressing Mewo cells by 1-2 orders of magnitude, especially at low N/P ratios. The use of a PEG spacer seems to impair targeting, possibly by not only shielding PEI, but also the RGD ligand.

Core-cross-linked polymeric micelles as paclitaxel carriers.

Cross-linkable di- and triblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized. These amphiphilic copolymers self-assembled into nanoscale micelles capable of encapsulating hydrophobic paclitaxel in their hydrophobic cores in aqueous solutions. To further enhance their thermodynamic stability, the micelles were cross-linked by radical polymerization of the double bonds introduced into the PCL blocks. Reaction conditions were found to significantly affect both the cross-linking efficiency and the micelle size. The encapsulation of paclitaxel into the micelles was confirmed by the proton nuclear magnetic resonance (1H NMR) spectroscopy. Encouragingly, paclitaxel-loading efficiency of micelles was enhanced significantly upon micelle core-cross-linking. Both the micelle size and the drug loading efficiency increased markedly with increasing the PCL block lengths, no matter if the micelles were core-cross-linked or not. However, paclitaxel-loading did not obviously affect the micelle size or size distribution. The cross-linked micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents. The efficient cellular uptake of paclitaxel loaded in the nanomicelles was demonstrated by confocal laser scanning microscopy (CLSM) imaging. This new biodegradable nanoscale carrier system merits further investigations for parenteral drug delivery.

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery.

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.

Pegylated polyethylenimine-Fab' antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells.

Specific targeting of ovarian carcinoma cells using pegylated polyethylenimine (PEG-PEI) conjugated to the antigen binding fragment (Fab') of the OV-TL16 antibody, which is directed to the OA3 surface antigen, was the objective of this study. OA3 is expressed by a majority of human ovarian carcinoma cell lines. To demonstrate the ability of the PEG-PEI-Fab' to efficiently complex DNA, an ethidium bromide exclusion assay was performed. Comparison with PEG-PEI or PEI 25 kDa showed only minor differences in the ability to condense DNA. Since conjugation of Fab' to PEG-PEI might influence complex stability, this issue was addressed by incubating the complexes with increasing amounts of heparin. This assay revealed stability similar to that of unmodified PEG-PEI/DNA or PEI 25 kDa/DNA complexes. Complexes displayed a size of approximately 150 nm with a zeta potential close to neutral. The latter property is of particular interest for potential in vivo use, since a neutral surface charge reduces nonspecific interactions. Binding studies using flow cytometry and fluorescently labeled DNA revealed a more than 6-fold higher degree of binding of PEG-PEI-Fab'/DNA complexes to epitope-expressing cell lines compared to unmodified PEG-PEI/DNA complexes. In OA3-expressing OVCAR-3 cells, luciferase reporter gene expression was elevated up to 80-fold compared to PEG-PEI and was even higher than that of PEI 25 kDa. The advantage of this system is its specificity, which was demonstrated by competition experiments with free Fab' in the cell culture media during transfection experiments and by using OA3-negative cells. In the latter case, only a low level of reporter gene expression could be achieved with PEG-PEI-Fab'.