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Due to the polyanionic nature of DNA, typically cationic or neutral delivery vehicles have been used for gene delivery. As a new approach, this study focuses on the design, development, and validation of nonviral polypeptide-based carriers for oligonucleotide delivery based on a negatively charged poly-l-glutamic acid (PGA) backbone partly derivatized with oligoaminoamide residues. To this end, PGA-derivatives modified with different pentameric succinyl tetraethylene pentamines (Stp 5 ) are designed. Optionally, histidines for modulation of endosomal buffer capacity and cysteines for pDNA complex stabilization are included, followed by characterization of biophysical properties and gene transfer efficiency in N2a neuroblastoma or 4T1 breast cancer cells., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As an option for genetic disease treatment and an alternative for traditional cancer chemotherapy, gene therapy achieves significant attention. Nucleic acid delivery, however, remains a main challenge in human gene therapy. Polymer-based delivery systems offer a safer and promising route for therapeutic gene delivery. Over the past five decades, various cationic polymers have been optimized for increasingly effective nucleic acid transfer. This resulted in a chemical evolution of cationic polymers from the first-generation polycations towards bioinspired multifunctional sequence-defined polymers and nanocomposites. With the increasing of knowledge in molecular biological processes and rapid progress of macromolecular chemistry, further improvement of polymeric nucleic acid delivery systems will provide effective tool for gene-based therapy in the near future.

We report novel pH-reversibly surface-shielded polyplexes with enhanced gene transfer activity upon systemic administration. A four-arm-structured sequence-defined cationic oligomer KK[HK[(H-Sph-K)3HC]2]2 was designed and synthesized on solid-phase, containing additional lysine residues not only for improved pDNA polyplex stability, but also providing attachment points for subsequent polyplex functionalization with amine-reactive shielding polymers. Herein, the surface of polyplexes was shielded with hydrophilic polymers, monovalent PEG or monovalent and multivalent pHPMA, optionally attached to the polyplex via the acid-labile linker AzMMMan. Overall, surface modification with PEG or pHPMA resulted in a decrease in the zeta potential of polyplexes, consistent with the degree of surface shielding. At pH 6.0, only polyplexes modified via the acid-labile linkage showed an increase in zeta potential, consistent with a "deshielding" in acidic environment, expected as beneficial for endosomal escape. Shielding was more efficient for multivalent pHPMA (20kDa, 30kDa) as compared to monovalent pHPMA (10kDa, 20kDa, 30kDa) or PEG (5kDa). In vitro transfection studies revealed higher gene expression by the polyplexes with the acid-labile shield as compared to their irreversibly shielded counterparts. Intravenous administration of AzMMMan-pHPMA modified polyplexes in an in vivo tumor mouse model mediated enhanced gene expression in the subcutaneous tumor and reduced undesirable expression in the liver., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cationic polymers present a versatile platform for the nonviral delivery of therapeutic nucleic acids. In order to achieve effective nucleic acid transfer, polymeric carriers ought to comprise multiple functionalities. Precise chemistries for site-specific placements of the different delivery modules within the carriers present the basis for uncovering structure-activity relationships required for further optimization. Here we present the design and systematic evaluation of a library of 42 sequence-defined oligo(ethanamino)amides generated by solid-phase assisted syntheses. The carriers contained two- or four-arm topologies of different artificial oligoamino acid domains for nucleic acid complexation, terminated by cysteines for disulfide-triggered polyplex stabilization, linked with monodisperse polyethylene glycol (PEG) for surface shielding and terminal folic acid for receptor specific cellular uptake. Additional functional elements included histidines for endosomal escape and/or tyrosine trimers for enhanced hydrophobic polyplex stabilization. In vitro screening of the oligomer library identified a folate-PEG-linked two-arm oligocation structure comprising histidines and tyrosine trimers as the most effective class of carriers for the delivery of pDNA and siRNA.

Identifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Artificial oligoamino acids with appropriate protecting groups can be used for the sequential assembly of oligoaminoamides on solid-phase. With the help of these oligoamino acids multifunctional nucleic acid (NA) carriers can be designed and produced in highly defined topologies. Here we describe the synthesis of the artificial oligoamino acid Fmoc-Stp(Boc3)-OH, the subsequent assembly into sequence-defined oligomers and the formulation of tumor-targeted plasmid DNA (pDNA) polyplexes.

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.

For active cell targeting, viruses frequently capitalize on dual-receptor binding. With the intention to mimic this natural process, a dual peptide-based approach for targeting cancer cells was evaluated. For this purpose, sequence-defined pDNA binding oligo (ethane amino) amides containing a PEG chain with a peptidic targeting ligand at its distal end were applied. Integrin receptor-directed cyclic peptide cRGDfk, transferrin receptor-addressing peptide B6, and epidermal growth factor receptor-targeting peptide GE11 were used in the study in DU145 prostate cancer cells that express all three receptors. Dual-receptor targeted pDNA polyplexes were designed by combining two of the above ligands at various ratios, in order to find an optimal ligand combination. Two polycation/pDNA ratios of nitrogen/phosphate (N/P) 6 and 12 were tested. Dual targeting effects were most pronounced at the lower N/P ratio and found for all three combinations. Cell binding studies and pDNA transfections revealed GE11 plus B6 as the most potent combination. In general, a good correlation of cell binding with gene transfer was observed. Interestingly, GE11 peptide-based polyplexes-mediated bimodal cell association profiles. In contrast, B6 ligand, cRGD ligand, and dual-targeted polyplexes triggered more homogenous monomodal cell binding characteristics., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The role of cell free DNA (cfDNA) has been intensively discussed under various pathological conditions and after acute bouts of exercise. To date, there is still no conclusive evidence concerning the cellular origin of cfDNA and the entire mechanism leading to elevated cfDNA concentrations in human plasma and serum. Here, we investigated the cellular origin of cfDNA in sex-mismatched haematopoietic stem cell transplantation (HSCT) and liver transplantation (LT) patients by determining the relative proportion of Y-chromosomal to total nuclear cfDNA. Total nuclear cfDNA and Y-chromosomal cfDNA concentrations were determined in blood plasma before and after an incremental exercise test via quantitative real-time PCR (qPCR). Female HSCT patients showed high proportions of Y-chromosomal cfDNA. Both total nuclear and Y-chromosomal cfDNA increased significantly and in a highly correlated fashion due to exercise. In male HSCT patients with female donors less than 10% of the cfDNA was of Y-chromosomal origin at any point in time and even though the total amount of cfDNA increased during exercise, no increases in Y-chromosomal DNA could be detected. The percentage of Y-chromosomal cfDNA in female LT patients with male donors was very low and levels remained unchanged during exercise. This indicates that cells not derived from the bone marrow, in this case transplanted liver cells, represented only a minor fraction of cfDNA in blood plasma and were not released during acute physical exercise. Even though many physiological conditions may be altered in transplant patients versus healthy people, our results strongly suggest that cells from the haematopoietic lineage are the main source of cfDNA released during acute bouts of exercise., (Copyright © 2015 International Society of Exercise and Immunology. All rights reserved.)

Despite their great potential, gene delivery polyplexes have a number of limitations, including their tendency for aggregation in vivo or upon storage. In previous studies, we could show that hydroxyethyl starch (HES)-decoration of polyplexes reduces aggregation in vitro and in vivo. The current study investigates the ability of HES-decoration to improve the stability of polyplexes upon storage as frozen-liquid or lyophilizate, and uses naked polyplexes or PEGylated ones as controls. For this purpose, freeze-thaw (FT) experiments of the polyplexes were conducted in the presence of standard excipients (glucose, sucrose or trehalose). Dynamic light scattering (DLS) measurements showed that HES-decoration imparted better stability when glucose was used, while both HES and PEG were effective in inhibiting aggregation in the presence of trehalose or sucrose. In contrast, the lyophilized HES-coated polyplexes were more stable than the PEGylated ones as shown by DLS, even after storage for 10 weeks at an elevated temperature. Evaluation of the gene transfer efficiency of the stored samples showed no negative effect of storage, except for the lyophilized naked polyplexes. In general, this study shows that, while both HES- or PEG-coats could prevent aggregation under frozen-liquid storage, the HES-coat resulted in superior protective effect upon lyophilization, with possible advantages for in vivo application. In summary, our developed HES-coats provided effective cryo- and lyoprotection to the DNA polyplexes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Establishing precise structure-activity relationships is important for the optimization of synthetic carriers for gene delivery. Sequence-defined oligomers with branched or linear shapes were synthesized to investigate the influence of topology on their biophysical properties and biological performance. Comb-like structures were synthesized consisting of an oligolysine peptide backbone modified at the ε-amino groups with four different artificial oligoamino acids, succinyl-diethylene triamine (Sdt), succinyl-triethylene tetramine (Stt), succinyl-tetraethylene pentamine (Stp), and succinyl-pentaethylene hexamine (Sph). Optionally the amino acids histidine and alanine were inserted into the oligolysine backbone to assess a possible buffer or spacer effect. After the evaluation of biophysical properties, the best performing oligomers, containing the Stp or Sph building blocks, were compared to corresponding linear oligomers where Stp or Sph are directly integrated into the linear oligolysine row. Clear differences between the comb and linear carriers were observed in the comparison of properties such as DNA complexation ability, buffer capacity, cellular association and internalization, and gene transfer. For the Stp containing structures, the comb topology mediated an increased buffer capacity at endosomal pH. For the Sph containing structures, in sharp contrast, the linear topology displayed advantageous endosomal buffering. Interestingly, for both Stp and Sph carriers, the comb in comparison to the linear topologies mediated a higher overall cellular uptake despite a lower cell association. For Stp combs, the combined advantage in both buffering and cellular uptake resulted in a strong (10- to >100-fold) increase in DNA transfection efficiency. In the case of Sph carriers, comb topology mediated only moderately (maximum 4-fold) enhanced gene transfer over the linear topology.

Molecular medicine opens into a space of novel specific therapeutic agents: intracellularly active drugs such as peptides, proteins or nucleic acids, which are not able to cross cell membranes and enter the intracellular space on their own. Through the development of cell-targeted shuttles for specific delivery, this restriction in delivery has the potential to be converted into an advantage. On the one hand, due to the multiple extra- and intracellular barriers, such carrier systems need to be multifunctional. On the other hand, they must be precise and reproducibly manufactured due to pharmaceutical reasons. Here we review the design of precise sequence-defined delivery carriers, including solid-phase synthesized peptides and nonpeptidic oligomers, or nucleotide-based carriers such as aptamers and origami nanoboxes.

Hydroxyethyl starch (HES) has been proposed as a biodegradable polymer for shielding of DNA polyplexes, where the feasibility of this approach was shown both in vitro and in vivo. In this study, we report on the physicochemical characterization, the in vitro cytocompatibility and hemotoxicity of HES-decorated polyplexes. For this purpose, various HES molecules were coupled to a 22 kDa linear polyethylenimine (LPEI22) to produce a library of nine different HES-PEI conjugates. Particle analysis using dynamic light scattering showed that, neither the molar mass of HES nor the amount of HES in the polyplexes affected the particle diameter, as it was consistently around 70-80 nm. Imaging using atomic force microscopy and transmission electron microscopy showed that, both naked and HESylated polyplexes were in the same size range and had a spherical morphology. Meanwhile, the HES-mediated particle-shielding effect, manifested as reduction in the surface charge, strongly correlated with the molar mass of HES, where the charge decreased linearly with the increase in molar mass. Ethidium bromide binding assay showed that HES-PEI did not negatively affect DNA condensation at N/P ratios higher than 4. HES conjugation also showed a stabilizing effect against salt-induced particle disassembly, and particle aggregation in protein-containing media. Compatibility tests included cellular viability, as well as erythrocyte aggregation and hemolysis assays. HES-PEI conjugates showed lower cytotoxicity, no aggregation, and much lower hemolysis compared to unmodified PEI. In conclusion, these results show that the HES-PEI conjugates are promising gene delivery polymers with favorable physicochemical properties and compatibility profile.

Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of proteinous or peptidic ligands can form nanosized polyplexes with plasmid DNA or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of linear PEI (LPEI) from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and plasmid DNA.

Cationic oligomers were assembled by solid-phase supported synthesis in few coupling steps based on C-terminal alanine and two lysine branchings, followed by elongation of the four arms with two to five repeats of artificial oligoamino acids containing the 1,2-diaminoethane motif, and ended by N-terminal cysteines or alanines. These sequence-defined oligomers, containing between 28 and 68 protonatable nitrogens, were evaluated for complex formation with plasmid DNA (pDNA) and short interfering RNA (siRNA), followed by reporter gene transfer and gene silencing experiments in Neuro2A cells. By two simple variations, the pDNA gene transfer activity could be thousand-fold improved, exceeding the gold standard linear PEI up to >50-fold. Firstly, the N-terminal cysteines introduced for bioreversible stabilization of polyplexes by internal disulfide links after complex formation greatly enhanced gene transfer. Secondly, variation of the artificial oligoamino acid building blocks containing either triethylene tetramine (Gtt), tetraethylene pentamine (Stp), or pentaethylene hexamine (Sph) disclosed a clear ranking in the order of Sph>Stp>>Gtt for both pDNA compaction and transfection activity. Extending the chain lengths of the arms beyond three building blocks had marginal impact on the performance. For the much smaller siRNA cargo, polyplex stabilization by cysteine disulfides presents a strict requirement. Sph and Stp based cysteine-ended four-arms displayed similar binding activity, with Stp providing best gene silencing efficiency., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Heterogeneity of polymeric carriers is one of the most elusive obstacles in the development of nonviral gene delivery systems, concealing interaction mechanisms and limiting the use of structure-activity relationship studies. In this report, novel sequence-defined polyaminoamides, prepared by solid-phase assisted synthesis, were used to establish first structure-activity relationships for polymer-based plasmid DNA delivery. By combining a cationic building block with hydrophobic modifications and bioreversible disulfide cross-linking sites, transfection polymers with tailored lytic and DNA binding properties were designed. These polymers demonstrated clear correlation between structure and performance in lysis and DNA binding assays. In vitro studies showed negligible toxicity and highly efficient gene transfer, demonstrating the potential of this platform in the fast, combinatorial development of new transfection polymers.

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer., (This journal is © The Royal Society of Chemistry 2012)

The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using (1)H NMR, colorimetric copper-assay, and SEC. HES-PEI conjugates were used to generate polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of α-amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-lines±AA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for the HES-shielded polyplexes. This study shows that decorating nanoparticles with HES can be a promising tool for the controlled shielding/deshielding of polyplexes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken.

Cell-penetrating peptides (CPPs) are a very interesting class of molecules to be introduced in gene and siRNA vectors. They can be used to overcome one of the biggest hurdles in gene and siRNA delivery in vitro and in vivo, the transfer across cell membranes. This chapter describes protocols for the synthesis and biological evaluation of a polylysine-based polymer. In this carrier system, melittin is used as CPP with a high activity to disrupt membranes. pH-Labile masking is applied to render the lytic activity specific for intracellular acidic endolysosomal organelles.

Background: HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations., Methods: HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP-linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by periodate oxidation of the glycosylation sites., Results: Crosslinked DNA polyplexes showed an increased stability against exchange reaction by salt or heparin. Disulfide bond containing DSP-linked polyplexes were susceptible to reducing conditions. These polyplexes displayed the highest gene expression levels in vitro and in vivo (upon intratumoral application in mice), and these were significantly elevated and prolonged over standard or DSS-stabilized HD O formulations. DSP-stabilized HD O polyplexes with or without Tf coating were well-tolerated after intravenous application. High gene expression levels were found in tumor tissue, with negligible gene expression in any other organ., Conclusions: Lateral stabilization of HD O polyplexes with DSP crosslinker enhanced gene transfer efficacy and was essential for the incorporation of a ligand (Tf) into a stable particle formulation.

Plasmid DNA and siRNA have a large potential for use as therapeutic nucleic acids in medicine. The way to the target cell and its proper compartment is full of obstacles. Polymeric carriers help to overcome the encountered barriers. Cationic polymers can interact with the nucleic acid in a nondamaging way but still require optimization with regard to transfer efficiency and biocompatibility. Aiming at virus-like features, as viruses are the most efficient natural gene carriers, the design of bioresponsive polymers shows promising results regarding DNA and siRNA delivery. By specific chemical modifications dynamic structures are created, programmed to respond towards changing demands on the delivery pathway by cleavage of labile bonds or conformational changes, thus enhancing biocompatible gene delivery.

Locoregional hyperthermia (HT) can be used for site-directed activation of macromolecular drug delivery systems. We have developed a gene delivery system based on thermosensitive block copolymers (TSCs) with a phase transition temperature of 42 degrees C [Zintchenko, A., Ogris, M., and Wagner, E. (2006). Bioconjug. Chem. 17, 766-772], in which the statistical copolymer of vinylpyrrolidinone and N-isopropylacryamide is grafted on polyethylenimine (PEI). Here we applied polyplexes consisting of plasmid DNA and TSCs systemically in A/J mice bearing a syngeneic Neuro2A neuroblastoma tumor subcutaneously in each hind limb. One limb was selectively treated by HT at 42 degrees C, at the same time that polyplexes were injected via the tail vein. Hyperthermia led to increased accumulation of thermosensitive polymer and aggregation of thermosensitive polyplexes in HT-treated tumors, resulting in up to 10-fold increased DNA deposition compared with non-HT-treated tumor. The level of transgene expression induced by TSC polyplexes in HT-treated tumors was significantly higher and selective for tumor tissue. With nonthermosensitive PEI polyplexes HT did not influence transgene deposition or expression in tumor.

Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS(2a)) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS(2a) has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.

PEGylation which is reversed after the therapeutic agent reaches the target cell presents an attractive feature for drug, protein or nucleic acid delivery. Amine-reactive, endosomal pH cleavable polyethylene glycol aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters (PEG-HZN-NHS) were synthesized and applied for bioreversible surface shielding of DNA polyplexes. Monofunctional mPEG-HZN-NHS was synthesized by reacting succinimidyl hydraziniumnicotinate with mPEG-butyraldehyde (20 kDa). Bifunctional OPSS-PEG-HZN-NHS was synthesized analogously via a omega-2-pyridyldithio-PEG (10 kDa) propionaldehyde intermediate. Polyethylenimine (PEI) polyplexes were reacted with the pH-sensitive (mPEG-HZN-NHS) or the corresponding stable (mPEG-NHS) reagent. Both types of polyplexes remained shielded at pH 7.4 as demonstrated by particle size and zeta potential measurements after 4h of incubation at 37 degrees C. Polyplex deshielding at endosomal pH 5 was observed only with the mPEG-HZN-NHS shielded particles. This was confirmed by fluorescence correlation spectroscopy using the analogous Alexa-488 fluorescently labeled bifunctional PEGylation reagents. Luciferase gene transfections with epidermal growth factor (EGF) containing polyplexes using EGF-receptor overexpressing hepatoma HUH7 cells showed an up to 16-fold enhancement in gene expression with the reversibly shielded polyplexes as compared to stably shielded polyplexes. Consistently, the reversibly shielded polyplexes mediated also an enhanced tumor specific in vivo transgene expression after intravenous administration in a subcutaneous HUH7 tumor model in SCID mice.

Two types of acid-degradable nonviral gene carriers, OEI-MK and OEI-BAA, were synthesized by polymerizing oligoethylenimine of 800 Da (OEI800) with the pH-sensitive acetone ketal cross-linker 2,2-bis(N-maleimidoethyloxy) propane (MK) or the 4-methoxybenzaldehyde bisacrylate acetal cross-linker 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). Corresponding acid-insensitive counterparts (OEI-BM and LT-OEI-HD) were synthesized as well, representing control polymers. Kinetics of hydrolysis were measured and confirmed the pH-dependent degradation profile of the acetal functions, with short half-lives of 3 min at pH 5.0, and 5 h (OEI-MK) or 3.5 h (OEI-BAA) at physiological pH 7.4 and 37 degrees C. DNA polyplexes of a luciferase expression plasmid were tested for gene transfer efficiency and biocompatibility in two cell lines (B16F10 and Neuro2A). Polyplexes with acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low cation/plasmid (c/p) w/w ratios the transfection efficiency of pH-sensitive polymers was slightly reduced, but it became similar or superior to the efficiency of acid-stable polymers at higher c/p ratios. An improved in vivo biocompatibility of the acid-degradable polymers over the stable control polymers was confirmed by liver histology after systemic administration of polymers in Balb/c mice.

Photochemical internalization (PCI) is a physico-chemical targeting method that enables light directed delivery of nucleic acids into cells. The technology is based on photosensitizers that localize in the membranes of endocytic vesicles. A light activation of the photosensitizers induces photochemical reactions that lead to rupture of the vesicular membranes. This results in the release of endocytosed compounds (e.g., nucleic acids) into the cell cytosol. Physico-chemical and biological targeting techniques can be combined to promote efficient and specific gene delivery to target cells. The present protocol describes PCI of epidermal growth factor receptor (EGFR)-targeted DNA polyplexes. The DNA polyplexes made are small (50-100 nm in diameter), and they contain polyethylenimine (PEI) conjugated with the EGF protein as a cell-binding ligand for EGFR-mediated endocytosis and polyethylene glycol (PEG) for masking the polyplex surface charge. PCI of such targeted PEG-PEI/DNA polyplexes enables high and EGFR-specific gene transfer activity in cells. Although describing in detail PCI of DNA polyplexes, the methodology presented in this protocol is also applicable for PCI of other gene therapy vectors (e.g. viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), and for vectors targeted to alternate cell surface receptors. Generally, PCI can be applied whenever 100% survival of the treated cell population is not required.

Introduction: The reaction between alpha,beta-poly(aspartylhydrazide) (PAHy), a water soluble synthetic polymer and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTACl) produced copolymers bearing permanent positive charges (PAHy-CPTA) with molecular weight of 10 kDa and PAHy-CPTA copolymers differing in positive charge amount (18-58%) were chosen for biological investigations., Materials and Methods: Biophysical properties of DNA/PAHy-CPTA polyplexes were evaluated in terms of DNA condensation, zeta potential and size distribution. Cytotoxicity studies on Neuro2A murine neuroblastoma cells evidenced absence of toxicity of these copolymers up to 300 microg/ml unlike linear polyethylenimine (LPEI) that was highly toxic already at 20 microg/ml., Results and Discussion: PAHy-CPTA copolymers did not induce any erythrocyte aggregation up to 1 mg/ml. Cellular interaction studies of PAHy-CPTA polyplexes evidenced a faster binding of these polyplexes with cells compared to DNA/LPEI polyplexes. The in vitro transfection ability of PAHy-CPTA polyplexes was strongly affected by experimental conditions reaching about 10% of the transfection efficiency of optimized LPEI polyplexes., Conclusions: Finally, in vivo application studies confirmed the biocompatibility of PAHy-CPTA copolymers. With LPEI, clear signs of microvesicular fatty liver were observed and with LPEI polyplexes significant weight loss. In strong contrast, PAHy-CPTA did not induce histopathological changes or weight loss.

p-Piperazinobenzaldehyde methoxy poly(ethylene glycol) (mPEG, 5 kDa) acetal was synthesized by the Buchwald-Hartwig coupling reaction from piperazine and p-bromobenzaldehyde mPEG acetal. Introduction of a maleimide moiety yielded a novel acetal-based PEGylation reagent (PEG-acetal-MAL) for pH-sensitive conjugation of PEG to thiol-functionalized biomolecules. For reversible shielding of polyplexes, PEG-acetal-MAL was conjugated to polyethylenimine (PEI). At 37 degrees C, the PEG-acetal-PEI conjugate had a half-life of 3 min at endosomal pH 5.5 and 2 h at physiological pH 7.4, respectively. PEI polyplexes containing PEG-acetal-PEI had a zeta potential of +3 mV and were stable to salt-induced aggregation for 2 h at pH 7.4. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. Epidermal growth factor or transferrin receptor-targeted polyplexes shielded with the pH-sensitive PEG-acetal mediated enhanced luciferase gene expression in receptor-expressing target cells (Renca-EGFR or K562) as compared to stably shielded control polyplexes. Thus, the novel PEG-acetal-MAL reagent may present a versatile tool for drug and gene delivery formulations when pH-sensitive PEGylation is preferred.

Combination of the degradable polymeric gene carriers OEI-HD-1 and LT- OEI-HD-1 with an EGF targeting conjugate resulted in strongly (up to 900-fold) enhanced polyplex activity in EGF-receptor rich HUH7 hepatocellular carcinoma cells. The targeting ligand effect was DNA dose dependent, could be blocked by competitive receptor binding with unbound EGF ligand, and was not observed in receptor-negative control cells. Measures which enhance intracellular endosomal escape, either photochemically enhanced intracellular release (PCI) or the incorporation of a novel membrane-active melittin analog NMA-3, further enhanced gene transfer activity of EGF/OEI-HD-1 polyplexes.

Melittin-polyethylenimine (PEI) conjugates have been shown to enhance gene transfer efficiency of polyplexes due to their membrane-destabilizing properties. Inherent lytic activity at neutral pH however also provokes high cytotoxicity due to plasma membrane damage. In order to shift the lytic activity towards the endosomal membrane, several melittin analogs were designed. Acidic modification of melittin by replacing neutral glutamines (Gln-25 and Gln-26) with glutamic acid residues greatly improved the lytic activity of C-terminally linked PEI conjugates at the endosomal pH of 5. This activity correlated well with the gene transfer efficiency of polyplexes in four different cell lines. Melittin-PEI conjugates with high lytic activities at endosomal pH were then incorporated into EGF receptor-targeted and polyethylene glycol-shielded polyplexes. The resulting particles had virus-like dimension (150 nm) with a neutral surface charge and were subsequently purified by size exclusion chromatography to remove unbound toxic PEI conjugate. These purified polyplexes mediated EGF-receptor-specific gene transfer with up to 70-fold higher activity compared to the corresponding PEI polyplexes without melittin.

Fluorescence correlation spectroscopy (FCS) and gel electrophoresis measurements are performed to investigate both the number and size of complexes of linear double-stranded DNA (dsDNA) fragments with 1:1 diblock copolymers consisting of a cationic moiety, branched polyethyleneimine (bPEI) of 2, 10, or 25 kDa, covalently bound to a neutral shielding moiety, poly(ethylene glycol) (PEG; 20 kDa). By systematically decreasing the bPEI length, the PEG grafting density along the DNA chain can be directly controlled. For 25 and 10 kDa bPEI-PEG copolymers, severe aggregation is observed despite the presence of the shielding PEG. Upon decreasing the bPEI length to 2 kDa, controlled self-assembly of monomolecular DNA nanoparticles is observed. The resulting complexes are in quantitative agreement with a theoretical model based on a single DNA encased in a dense PEG polymer brush layer. The resulting PEGylated complexes show high stability against both salt and protein and hence are of potential use for in vivo gene delivery studies.

Background: One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency., Methods: The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH., Results: All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines., Conclusions: The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model., (2005 John Wiley & Sons, Ltd.)

Recently, cryoconservable polyethylene glycol (PEG)-shielded and epidermal growth factor receptor (EGFR)-targeted polyplexes (EGF+ polyplexes) were engineered in our laboratory for tumor-directed transfer and expression of DNA. Here, we further analyzed specificity and kinetics of EGFR-mediated cellular uptake of these polyplexes. Similar to our previous results, EGF+ polyplexes significantly enhanced the transfection efficiency as compared to polyplexes without EGF (EGF- polyplexes) in HUH-7 hepatoma cells and Renca-EGFR renal carcinoma cells. EGF+ polyplexes rapidly associated with the cells within 30 min of exposure, and binding of EGF+ polyplexes to the cells after 4 h was significantly higher than that of EGF- polyplexes. In the presence of free EGF, both cell association and transfection efficiency of EGF+ polyplexes were markedly reduced indicating that these effects were primarily mediated via ligand receptor interaction. Fluorescence microscopy revealed that the cell-associated EGF+ polyplexes aggregated to micrometer sized clusters, resembling typical clustering of receptors upon ligand binding. In conclusion, EGFR-targeting enhances transfection efficiency due to accelerated and increased cell association followed by aggregation of the bound EGF+ polyplexes.

Nonviral vectors should undergo "virus-like" changes compatible with the steps of gene delivery. Poly(ethylene) glycol (PEG) shielding of DNA/polycation polyplexes protects from nonspecific interactions with the extracellular environment. pH-triggered removal of the shield within the endosome may be advantageous. Polycation and PEG were linked via acylhydrazides or pyridylhydrazines. The pyridylhydrazone prepared from polylysine and propionaldehyde-PEG showed the greatest acid-dependent hydrolysis; at pH 5, 37 degrees C for 10 min, 90% hydrolyzed, while at pH 7.4 the half-life was 1.5 h. Particle size and zeta potential measurements of the polyplexes showed complete deshielding within 1 h at pH 5, while at pH 7.4 the shield remained at 4 h, 37 degrees C. For gene transfection a targeting conjugate was also included in the polyplex, transferrin as ligand for K562 and Neuro2A cells and epidermal growth factor for HUH-7 and Renca-EGFR cells. Marker gene expression showed that the reversibly shielded polyplexes exhibited up to 2 log orders of magnitude higher gene expression in vitro and 1 log magnitude higher gene expression in an in vivo mouse model, compared to the stably shielded control polyplexes. Engineering of polyplexes with more dynamic domains is an encouraging new direction in nonviral vector design.

Epidermal growth factor receptor (EGFR) targeted DNA polyplexes, containing polyethylenimine (PEI) conjugated with EGF protein as cell-binding ligand for endocytosis and polyethylene glycol (PEG) for masking the polyplex surface charge, mediated a 3- to 30-fold higher luciferase gene expression in HUH7, HepG2 and A431 cell transfections than analogous untargeted PEG-PEI polyplexes. Transfection levels can be further enhanced by treatment of cells with amphiphilic photosensitizers followed by illumination. In this process photosensitizers localized in membranes of endocytic vesicles are activated by light, resulting in the destruction of endocytic membrane structures and releasing co-endocytosed polyplexes into the cell cytosol. Photochemical enhanced gene expression was observed in all cell lines, with the magnitude of enhancement depending on the particular PEI polyplex formulation and cell line, ranging between 2- and 600-fold. Importantly, improved gene transfer retained EGF receptor specificity, as demonstrated by comparison with ligand-free polyplexes and by receptor antibody or ligand competition experiments. These results suggest that this combined procedure enables a dual mode of targeting polyplexes: biological targeting via EGFR interaction, combined with physical targeting with light to direct a photochemical delivery of therapeutic genes to a desired location.

For the purpose of introducing nucleic acids into cells, cationic polymers have been steadily improved as gene carriers. This has resulted in improved polymer-based gene transfer formulations, termed polyplexes, which efficiently transfect cell cultures and also have shown encouraging gene transfer potential in in vivo administration. Targeted delivery to liver, lung, tumor, or other tissues has been achieved in experimental animals by localized or systemic application. Therapeutic effect has been demonstrated, although efficiencies are still too low to justify clinical use. The limitations of first-generation polymeric carriers (modest activity and significant toxicity) have been addressed by developments of new biodegradable polycations, incorporation of targeting and intracellular transport functions, and polyplex formulations that avoid unspecific adverse interactions with the host. A key future step will be the development of polyplexes into artificial viruses, with virus-like entry functions presented by smart polymers and polymer conjugates. These polymers have to sense their biologic microenvironment, respond in a more dynamic manner to alterations in pH, ionic or redox environment, undergoing programmed structural changes compatible with the different gene delivery steps.

Surface-shielded DNA delivery systems have been synthesized with virus-like characteristics that target gene expression into distant tumor tissues. Polyethylenimine (PEI)/DNA complexes ('polyplexes') conjugated with the cell-binding ligand transferrin (Tf) or epidermal growth factor (EGF) were used to achieve receptor-mediated endocytosis. The surface charge of the complexes was masked by covalently linking PEI to polyethylene glycol (PEG). Three alternatives for generating these surface-shielded formulations were utilized, attaching ligand and PEG molecules to PEI either before or after DNA complex formation. The stabilized formulations could be ultra-concentrated, stored frozen, and applied systemically after thawing. Intravenous injection of Tf-PEG-coated polyplexes resulted in gene transfer to subcutaneous Neuro2a neuroblastoma tumors of syngeneic A/J mice; EGF-PEG-coated polyplexes were intravenously applied for targeting human hepatocellular carcinoma xenografts in SCID mice. In these models, luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor alpha (TNF-alpha) into tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origin (Neuro2a, M-3 or B16 melanoma).

We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/DNA complexes (containing 3 microg DNA) with varying PEI-nitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 x 10(3) relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using beta-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 microg naked DNA resulted in an 11-fold lower transgene expression value (0.9 x 10(3) RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 microg) into fetal livers yielded expression levels of 3.2 x 10(4) RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 x 10(3) RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA.

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.

Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.

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.

Incorporation of the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) into DNA/polyethylenimine (PEI) complexes was found to enhance gene transfer into tumor cell lines in a receptor-dependent manner. In systemic applications, the surface charge of DNA complexes dominated the in vivo characteristics of gene transfer. Administration of surface-shielded Tf-polycation/DNA complexes into the tail vein of A/J mice resulted in preferential gene delivery into distantly growing subcutaneous Neuro2a tumors. In contrast, application of positively charged DNA/PEI complexes directed gene transfer primarily to the lung. Two alternatives of masking the surface charge of complexes were accomplished. In the first case, shielding was obtained by covalently coating of DNA/Tf-PEI complexes with polyethylene glycol (PEG). Alternatively, incorporation of sufficient Tf protein into the DNA complexes resulted in charge shielding even without PEGylation. In the latter case lower-molecular weight polycations (25 kDa PEI for Tf-PEI complexes, or 32 kDa polylysine for AVET complexes) were used.

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.

We describe the comparison of non-viral polycation transfection reagents, adenovirus-enhanced transferrinfection (AVET), polyethylenimine (PEI800) and transferrin-conjugated PEI800 (Tf-PEI800) in their ability to transfect murine and primary human melanoma cell lines. Expression of a reporter gene, cell surface marker and secreted protein (interleukin-2) was assessed for each vector system. Testing for luciferase reporter gene expression in murine and primary human cell lines, AVET and Tf-PEI800, both showed high levels of expression and comparable activity. Furthermore, when the melanoma cell line B16F10 was transfected with a cell surface marker up to approximately 97% of the cells expressed the protein on the cell surface. Assessing the levels of secreted IL-2 in murine cell lines, AVET/IL-2, Tf-PEI800/IL-2 and PEI800/IL-2 all expressed high levels of the cytokine (up to 20 microg IL-2/10(6) cells/24 h). In primary human melanoma cell lines, AVET/IL-2 transfected cells secreted more IL-2 than cells transfected with either Tf-PEI800/IL-2 or PEI800/IL-2. In murine melanoma cell culture experiments, positively charged PEI800/DNA and Tf-PEI800/DNA complexes gave similar transfection efficiencies. However, when subcutaneous tumors in mice were injected with the luciferase reporter gene complexed with either Tf-PEI800 or AVET, higher transfection activity was measured in the tumors as compared to ligand free PEI800/DNA complexes.