Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.     

Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility

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

Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis-based method and produced small, stable, and negatively charged DNA-containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 +/- 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty-fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA-containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo.     

Delivery of electric pulses for DNA electrotransfer to mouse muscle does not induce the expression of stress related genes

In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer offers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low field strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have profiled gene expression patterns in muscles at different times after delivery of electric pulses using Stress/Toxicology microarrays. No significant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, fine-tuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.     

Physicochemical optimisation of plasmid delivery by cationic lipids

Non-viral gene therapy is based on the use of plasmid expression vectors and chemical or physical plasmid DNA delivery systems. This review discusses the roles of cationic lipids as vectors for gene transfection, reviews different strategies employed to improve cationic lipids for in vivo use, and provides original results on the physicochemistry of lipoplexes. Cationic lipid/DNA delivery vehicles have evolved considerably since their initial gene transfection experiments. Much work has been carried out to investigate their structure/activity relationships, methods of formulation and physicochemical properties. Further work has also focused on enhancing and prolonging their stability in a physiological environment as well as increasing their site-specific and tissue-specific interactions. Original data presented in this report confirm that cationic lipids associated to DNA form supramolecular lamellar structures, which protect DNA from serum DNAse degradation. The effect of formulation (and hence the size of the particles) on lipoplex in vivo circulation half-life and biodistribution is also discussed. A list of abbreviations can be found at the end of the review.     

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.     

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.     

Efficient gene transfer by transferrin lipoplexes in the presence of serum

Cationic lipids are being used increasingly as reagents for gene delivery both in vitro and in vivo. One of the limitations to the application of cationic lipid-DNA complexes (lipoplexes) in vivo is the inhibition of gene delivery by serum. In this study, we have shown that transferrin (Tf)-lipoplexes, which had transferrin adsorbed at their surface via electrostatic interactions, are much more effective than plain lipoplexes in transfecting cells in the presence of relatively high concentrations (up to 60%) of fetal bovine serum (FBS). Serum even enhanced transfection by Tf-lipoplexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/dioleoylphosphatidylethanolamine (DOPE)/pCMVLacZ at high lipid/DNA (+/-) charge ratios, and inhibited lipofection for those with low charge ratios when they were added to the cells immediately after the preparation of complexes. The effect of serum on lipofection was dose-dependent. Preincubation of the complexes at 20 degrees C for 6 h led to serum resistance, even for the negatively charged transferrin-lipoplexes. A similar tendency was observed for DOTAP/cholesterol and DOTAP/DOPE/cholesterol liposomes. The percentage of cells transfected, measured by beta-galactosidase expression, also increased with the serum concentration. Cell viability was not affected significantly when the cells were incubated with the complexes for 4 h at 37 degrees C, followed by a 48-h incubation. Our findings extend the scope of previous studies where transferrin-lipoplexes were used to introduce DNA into cells, rendering these complexes and their future derivatives potential alternatives to viral vectors for gene delivery in vivo.     

Trypanosoma cruzi: aberrant expression of class II major histocompatibility complex molecules in skeletal and heart muscle cells of chronically infected mice

In isolated skeletal, heart, and smooth muscle cells from BALB/c and C3H/HeJ mice infected with different strains of Trypanosoma cruzi the presence of class II MHC molecules was investigated by immunocytochemical techniques. We employed single muscle fibers instead of conventional cryostat sections to obtain a more accurate antigen localization. Approximately half of the skeletal muscle cells isolated from the rectus femoris expressed Ia antigens on their surface, irrespective of the mouse or parasite strain combination. Ia expression was apparent only at 30 days postinfection and thereafter. The heart muscle cells expressed class II molecules only at 1 and 3 months postinfection. In no case did the smooth muscle cells from infected mice express Ia antigens. Studies of the same molecules in the noninfected animals gave constantly negative results. We conclude that in the course of the chronic infection of mice with T. cruzi, ectopic expression of class II MHC molecules occurs at the surface of skeletal and heart muscle cells, providing a possible mechanism for explaining the anti-striped muscle autoreactivity present in Chagas' disease.     

Size reduction of galactosylated PEI/DNA complexes improves lectin-mediated gene transfer into hepatocytes.

Hepatocytes are interesting targets for gene therapy applications. Several hepatocyte-directed gene delivery vectors have been described. For example, simple galactosyl residues coupled to polyethylenimine (PEI) gave an efficient vector which selectively transfected hepatocytes via the asialoglycoprotein receptor-mediated endocytosis [Zanta, M. A., et al. (1997) Bioconjugate Chem. 8, 839-844]. However, the large size of these galactosylated PEI/DNA complexes prevented their use in vivo. We have investigated the role of the saccharide length on the size of glycosylated-PEI/DNA particles. When 5% of the PEI nitrogens were grafted with a linear tetragalactose structure (lGal4), small and stable particles were formed upon complexation with plasmid DNA. These particles were essentially toroids having a size of 50-80 nm and a zeta-potential close to neutrality. Moreover, these slightly charged PEI-lGal4/DNA complexes were as selective as the previously described galactosylated-PEI vector to transfect hepatocytes, but in addition, they were more efficient. It is expected that the properties of the PEI-lGal4/DNA complexes may increase their diffusion into the liver and their efficiency to transfect hepatocytes.     

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.     

Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos

ABSTRACT: BACKGROUND: Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications. RESULTS: We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes. CONCLUSIONS: PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.     

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

Prominent mitochondrial DNA recombination intermediates in human heart muscle

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.     

Effects of the incorporation of a hydrophobic middle block into a PEG-polycation diblock copolymer on the physicochemical and cell interaction properties of the polymer-DNA complexes

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for the formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly( n-butyl acrylate) (PnBA), and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer that had comparable molecular weights for individual blocks. In aqueous solution, the PEG-PnBA-PDMAEMA polymer forms positively charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-sized DNA particles that are coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance ( (1)H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named "micelleplex" for convenience) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that compared with PDMAEMA-DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations and show reduced interactions with negatively charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable to the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and the PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.