DNA complexing with polyamidoamine dendrimers: implications for transfection.

DNA and polyamidamine (PAMAM) dendrimers form complexes on the basis of the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and protonated (positively charged) amino groups of the polymers. Charge neutralization of both components and subsequent increases of the net positive charge of the complex result in changes in the physicochemistry and biological properties of the complexes. The formation of soluble, low-density and insoluble, high-density complexes was analyzed using UV light absorption and measurements of radioactive labeled DNA. Formation of high molecular weight and high-density complexes depended mainly on the DNA concentration and was enhanced by increasing the dendrimer-DNA charge ratio. Electrostatic charge related effects (attraction or repulsion of charged particles) appeared to be modulated by the generation of dendrimer (size of the polymer). With the progressive increases in the dendrimer-DNA charge ratio (above 20), an increase in the amount of low-density, soluble complexes was observed. Functional analysis revealed that the great majority (>90%) of transfection is carried by low-density, soluble, complexes which only represent approximately 10-20% of total complexed DNA. The ability of the dendrimer to complex and form aggregates with DNA is crucial for efficient transfection and the function of the complexed DNA.     

High-generation polycationic dendrimers are unusually effective at disrupting anionic vesicles: membrane bending model

The membrane disruption properties of high generation (G4 to G7) poly(amidoamine) (PAMAM) dendrimers are evaluated and compared to linear poly(lysine). The G6 and G7 dendrimers are unusually effective at inducing leaky fusion of anionic, large unilamellar vesicles, as determined by standard fluorescence assays for lipid mixing, leakage, and contents mixing. Both G7 dendrimer and poly(lysine) are able to disrupt sterically stabilized vesicles that are coated with poly(ethylene glycol). A G7 dendrimer/DNA complex with a 1:1 concentration ratio of dendrimer surface amines to DNA phosphate groups is unable to induce leakage of 3:7 POPA-PE vesicles; however, extensive leakage is observed when the surface amine to phosphate stoichiometry is >/=3:1. Thus, the DNA/dendrimer complexes that typically induce high levels of cell transfection are also able to induce high levels of vesicle leakage. The G7 dendrimer does not induce membrane phase separation in 3:7 POPA-PE vesicles, but an inverse hexagonal phase is observed by (31)P NMR. The enhanced membrane disruption is interpreted in terms of a membrane bending model. A rigid, polycationic dendrimer sphere uses electrostatic forces to bend a malleable, anionic membrane and induce bilayer packing stresses. This bending model is biomimetic in the sense that protein-induced membrane bending is currently thought to be an important factor in the fusion mechanism of influenza virus.     

Substituted beta-cyclodextrins interact with PAMAM dendrimer-DNA complexes and modify transfection efficiency

The efficiency of PAMAM dendrimer-mediated DNA transfer can be improved by the addition of substituted beta-cyclodextrins (beta-CDs) as formulation excipients. In vitro CAT expression increased approximately 200-fold when dendrimer/DNA/beta-CD formulations were applied on the surface of collagen membranes. The inclusion of beta-CD into the formulations resulted in particles that were smaller and more evenly distributed on the surface of the solid support. The average size of the complex formed at 50 microg/ml and at charge ratio of 1 decreased from 156 nm to 5.8 nm and 21.2 nm in 0.025-0.1% w/vol beta-CDs. Sulfonated beta-CDs bind to dendrimer and in the increased concentration may displace DNA in the dendrimer/DNA complex. High concentrations of amphoteric beta-CD do not dissociate dendrimer/DNA complexes; however, they may decrease their ability to transfect cells. At the optimized formulations the surface-modified beta-CDs may enhance solid support-based transfection in vitro, through modification of dendrimer/DNA complex composition and improved surface distribution.     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Two modes of aggregation of artificial H1-DNA complexes depending on the rate of decrease of ionic strength.

The properties of H1-DNA artificial complexes, formed at different rates of decrease of NaCl concentration from 0.9 to 0.15 M, were investigated. It was found that two distinct processes, both depending on the rate of the concentration decrease, lead to the formation of aggregates differing in: the ability to form sediments, the distribution of sedimentation constants, the initial turbidity and its changes during trypsin and DNAase I digestion, and the H1/DNA ratio in the sediments. The accessibility of DNA in the complexes to DNAase I and the properties of nonaccessible DNA fragments led us to the conclusion that, at the H1/DNA ratio equal 0.2, the H1 molecules are clustered along the DNA chain independently of the rate of complex formation.     

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.     

Interaction of histone H1 with superhelical DNA. Sedimentation and electron microscopical studies at low salt concentration

Complexes of histones H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na+. Depending on the molar input ratio H1/DNA three classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical DNA with a sedimentation coefficient s2o,w of 25 S observable up to 335 Mol H1/Mol DNA (w/w = 2); (2) a component with s2o,w = 120 S appearing at 135 Mol H1/Mol DNA and (3) growing amounts of heterogeneous aggregates greater than 1000 S. Electron micrographs revealed the 25 S component to consist of double-fibers formed from one DNA molecule and the 120 S component to consist of bundles of several such double-fibers. The aggregates represent cable-like structures. The addition of ethidium bromide to 25 S complexes induces the formation of bundles, if H1 is present in a quantity which alone is not sufficient to bring about this effect. This result indicates that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

Cross-linked hydroxypropyl-β-cyclodextrin and γ-cyclodextrin nanogels for drug delivery: Physicochemical and loading/release properties

Due to their size and high surface-to-volume ratio, nanogels can give some unique drug delivery opportunities. A novel technique to prepare cyclodextrin (CD) nanogels, in which the cross-linking takes place simultaneously with an emulsification/solvent evaporation process, has been implemented. The aqueous phase consisted of γ-cyclodextrin (γCD) or hydroxypropyl-β-cyclodextrin (HPβCD) at a fix concentration of 20% (w/w) with or without hydroxypropyl methylcellulose (HPMC) or agar at various concentrations. The incorporation of the cross-linking agent, ethyleneglycol diglycidyl ether (EGDE), was essential for the nanogel formation. By contrast, nanogels could be formed in the absence of surfactant such as Span 80, which can be attributed to the emulsion stabilizing effect of CDs by forming inclusion complexes with the organic solvent at the interface. Gas chromatography-mass spectrometry (GC-MS) analysis of the nanogels confirmed that dichloromethane levels were below the safety limit and, therefore, that these conditions of the organic solvent evaporation (60 °C for 180 min) led to nanogels that satisfy residual solvent requirements. Infrared analysis (IR), transmission electron microscopy (TEM) and dynamic light scattering (DLS) provided information about the cross-linking degree, the size and the size distribution of the nanogels. The ability of the nanogels to host a molecule that can form inclusion complexes and to sustain its release was tested using 3-methylbenzoic acid (3-MBA) as a probe with a high affinity for both β-cyclodextrin (βCD) and γCD. Permeability tests confirmed that 3-MBA was indeed taken up by the nanogels and then slowly released.     

PEG-assisted DNA solubilization in organic solvents for preparing cytosol specifically degradable PEG/DNA nanogels

DNA was dissolved in selected organic solvents in the presence of poly(ethylene glycol) (PEG). Nanoscale PEG/DNA complex (approximately 100 nm) was produced in dimethylsulfoxide (DMSO) phase. Using a thiol-functionalized six-arm branched PEG for DNA solubilization, the PEG/DNA nanocomplex was cross-linked through the formation of disulfide linkages between the thiol groups, resulting in the production of stable PEG/DNA nanogels in aqueous solution. DNA release from the nanogels could be modulated by changing the concentration of an external reducing agent. The released plasmid DNA from the nanogels maintained intact structural integrity and exhibited appreciable gene transfection efficiency. The PEG/DNA nanogels could be potentially applied for gene therapy including DNA vaccination.     

Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study

This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.     

Improved DNA chip with poly(amidoamine) dendrimer peripherally modified with biotin and avidin

Important properties for a biosensor are the sensitive detection of target DNA at low concentration, the specific and accurate distinction of the target and other DNA having a similar sequence, and measurement capability over a wide range of target concentrations. To these ends, generation 3 polyamidoamine (PAMAM) dendrimer was used to improve DNA chip properties. PAMAM dendrimer surface amine moieties were modified to biotin and immobilized on glass slides using biotin-avidin conjugation. The surface morphologies of the avidin-biotin-dendrimer complexes were observed using atomic force microscopy and scanning electron microscopy. Detection sensitivity for fluorescence-labeled target DNA increased approximately 4-fold by the dendrimer coating. Dendrimer coating also markedly improved the dynamic range and detection of single nucleotide polymorphisms. Dendrimer complex morphology had little effect on the sensitivity.     

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.     

Self-assembled ternary complex of cationic dendrimer,cucurbituril, and DNA: noncovalent strategy in developing a gene delivery carrier

A ternary complex of PPI-DAB dendrimer [(1,4-diaminobutane); Gen = N; dendri-poly(propyleneimine); -[NHC(=O)CH(2)NH(2)(+)(CH(2))(4)NH(3)(+)](z)()], DNA, and cucurbituril (CB) was evaluated as an example of a totally self-assembled gene delivery carrier. The complex was formed in a noncovalent way in which DNA interacts with PPI-DAB electrostatistically and CB with PPI-DAB through multiple noncovalent interactions. Dynamic light scattering data indicated that the diameter and size distributions of the complexes were dependent upon the sequence of mixing of each component with unimodal distribution ranging from 150.8 to 210.2 nm under favorable conditions. Fluorescence studies showed the quantitative binding of CB to PPI-DAB after ternary complex formation. The complex was able to transfect mammalian cells with high efficiency and the cytotoxicity of the PPI-DAB/CB complex was relatively low.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

Enhancement of gene expression by polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins.

To improve the transfection efficiency of nonviral vector, we synthesized the starburst polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins (CDE conjugates), expecting the synergistic effect of dendrimer and cyclodextrins (CyDs). The (1)H NMR spectroscopic data indicated that alpha-, beta-, and gamma-CyDs are covalently bound to dendrimer in a molar ratio of 1:1. The agarose gel electrophoretic studies revealed that CDE conjugates formed the complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I in the same manner as dendrimer. CDE conjugates showed a potent luciferase gene expression, especially in the dendrimer conjugate with alpha-CyD (alpha-CDE conjugate) which provided the greatest transfection activity (approximately 100 times higher than those of dendrimer alone and of the physical mixture of dendrimer and alpha-CyD) in NIH3T3 and RAW264.7 cells. In addition, the gene transfer activity of alpha-CDE conjugate was superior to that of Lipofectin. The enhancing gene transfer effect of alpha-CDE conjugate may be attributable to not only increasing the cellular association, but also changing the intracellular trafficking of pDNA. These findings suggest that alpha-CDE conjugate could be a new preferable nonviral vector of pDNA.     

Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis

Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.     

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic ε-oligolysine with the pendant α-amino acids Leu-Tyr-Arg (LYR), ε-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA-DNA distance in the complexes varies as a function of the net positive to negative (lipid+peptide)/DNA charge ratio. An explanation for the observed dependence of DNA-DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation-DNA mixtures.