Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

A pH-sensitive polymer that enhances cationic lipid-mediated gene transfer.

The efficient release of nonviral gene carriers from endosomes is an important step for the successful delivery of DNA into the cell nucleus. A synthetic pH-sensitive anionic polymer, poly(propylacrylic acid) (PPAA), was designed to aid in endosomal escape of nonviral vectors and improve the transfection efficiencies with these vectors. Transfection of NIH3T3 fibroblasts with ternary physical mixtures of the cationic lipid DOTAP, pCMVbeta plasmid DNA, and PPAA showed marked enhancement of both gene expression levels and fraction of cells transfected compared to binary control mixtures of DOTAP and DNA. PPAA also significantly improved the serum-stability of DOTAP/DNA vectors. The DOTAP/DNA/PPAA vectors maintained high levels of transfection in media containing up to 50% serum. The striking enhancement of transfection efficiency with cationic lipid/DNA/PPAA mixtures, along with the enhanced serum-stability, suggests that PPAA may provide significant improvements for the in vivo intracellular delivery of drugs such as DNA, oligonucleotides, proteins, and peptides.     

Lysine dendrimers as vectors for delivery of genetic constructs to eukaryotic cells

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for DNA delivery to eukaryotic cells and provide a basis for constructing more refined nonviral module carriers.     

New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA, and application for gene delivery

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ～100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.     

New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid-DNA complexes for gene delivery

BACKGROUND: Gene carriers based on lipids or polymers-rather than on engineered viruses-constitute the latest technique for delivering genes into cells for gene therapy. Cationic liposome-DNA (CL-DNA) complexes have emerged as leading nonviral vectors in worldwide gene therapy clinical trials. To arrive at therapeutic dosages, however, their efficiency requires substantial further improvement. METHODS: Newly synthesized multivalent lipids (MVLs) enable control of headgroup charge and size. Complexes comprised of MVLs and DNA have been characterized by X-ray diffraction and ethidium bromide displacement assays. Their transfection efficiency (TE) in L-cells was measured with a luciferase assay. RESULTS: Plots of TE versus the membrane charge density (sigmaM, average charge/unit area of membrane) for the MVLs and monovalent 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) merge onto a universal, bell-shaped curve. This bell curve leads to the identification of three distinct regimes, related to interactions between complexes and cells: at low sigmaM, TE increases with increasing sigmaM; at intermediate sigmaM, TE exhibits saturated behavior; and unexpectedly, at high sigmaM, TE decreases with increasing sigmaM. CONCLUSIONS: Complexes with low sigmaM remain trapped in the endosome. In the high sigmaM regime, accessible for the first time with the new MVLs, complexes escape by overcoming a kinetic barrier to fusion with the endosomal membrane (activated fusion), yet they exhibit a reduced level of efficiency, presumably due to the inability of the DNA to dissociate from the highly charged membranes in the cytosol. The intermediate, optimal regime reflects a compromise between the opposing demands on sigmaM for endosomal escape and dissociation in the cytosol.     

Enhancement of transfection by physical concentration of DNA at the cell surface

Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.     

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.     

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection

Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer‐based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra‐ and extracellular barriers. The recent progress has shown that stimuli‐responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA‐polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 363–375, 2015.     

Polymer‐coated viral vectors: hybrid nanosystems for gene therapy

The advantages and critical aspects of nanodimensional polymer‐coated viral vector systems potentially applicable for gene delivery are reviewed. Various viral and nonviral vectors have been explored for gene therapy. Viral gene transfer methods, although highly efficient, are limited by their immunogenicity. Nonviral vectors have a lower transfection efficiency as a result of their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology‐mediated interventions that involve the coating or modification of virus using polymers have emerged as a new paradigm in gene therapy. These alterations not only modify the tropism of the virus, but also reduce their undesirable interactions with the biological system. Also, co‐encapsulation of other therapeutic agents in the polymeric coating may serve to augment the treatment efficacy. The viral particles can aid endosomal escape, as well as nuclear targeting, thereby enhancing the transfection efficiency. The integration of the desirable properties of both viral and nonviral vectors has been found beneficial for gene therapy by enhancing the transduction efficiency and minimizing the immune response. However, it is essential to ensure that these attempts should not compromise on the inherent ability of viruses to target and internalize into the cells and escape the endosomes.     

Biophysical properties and supramolecular structure of self-assembled liposome/ε-peptide/DNA nanoparticles: correlation with gene delivery

Using solid-phase synthesis, lysine can be oligomerized by a reaction of the peptide carboxylate with the ε-amino group to produce nontoxic, biodegradable cationic peptides, ε-oligo(L-lysines). Here α-substituted derivatives of such ε-oligo(L-lysines) containing arginine and histidine in the side chain were tested as vectors for in vitro gene delivery. Combination of ε-oligolysines with the cationic lipid DOTAP and plasmid DNA resulted in transfection efficiency exceeding that of DOTAP alone, without significant increase in cytotoxicity. Synchrotron small-angle X-ray scattering studies revealed self-assembly of the DOTAP, ε-oligolysines, and DNA to ordered lamellar complexes. High transfection efficiency of the nanoparticles correlates with increase in zeta potential above +20 mV and requires particle size to be below 500 nm. The synergistic effect of branched ε-oligolysines and DOTAP in gene delivery can be explained by the increase in surface charge and by the supramolecular structure of the DOTAP/ε-oligolysine/DNA nanoparticles.     

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.     

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ("peptiplexes") enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the "chelate effect" and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.     

Dendritic poly(ether imine) based gene delivery vector

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Biophysical and transfection studies of an amine-modified poly(vinyl alcohol) for gene delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.     

Biodegradable poly(vinyl alcohol)-polyethylenimine nanocomposites for enhanced gene expression in vitro and in vivo

Use of cationic polymers as nonviral gene vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. In this study, varying amounts of low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) were introduced on to a neutral polymer, poly(vinyl alcohol) (PVA), to bring in cationic charge on the resulting PVA-PEI (PP) nanocomposites. We rationalized that by introducing bPEI 1.8, buffering and condensation properties of the proposed nanocomposites would result in improved gene transfer capability. A series of PVA-PEI (PP) nanocomposites was synthesized using well-established epoxide chemistry and characterized by IR and NMR. Particle size of the PP/DNA complexes ranged between 120 to 135 nm, as determined by dynamic light scattering (DLS), and DNA retardation assay revealed efficient binding capability of PP nanocomposites to negatively charged nucleic acids. In vitro transfection of PP/DNA complexes in HEK293, HeLa, and CHO cells revealed that the best working formulation in the synthesized series, PP-3/DNA complex, displayed ~2-50-fold higher transfection efficiency than bPEIs (1.8 and 25 kDa) and commercial transfection reagents. More importantly, the PP/DNA complexes were stable over a period of time, along with their superior transfection efficiency in the presence of serum compared to serum-free conditions, retaining the nontoxic property of low molecular weight bPEI. The in vivo administration of PP-3/DNA complex in Balb/c mice showed maximum gene expression in their spleen. The study demonstrates the potential of PP nanocomposites as promising nonviral gene vectors for in vivo applications.     

Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes ("polyplexes") from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH ("proton sponge"). We synthesized a new polymer with two tertiary amine groups in each monomeric unit [poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA]. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis.