Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.     

Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA–chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA–chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from ∼10 to ∼1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Glycosaminoglycan destabilization of DNA-chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA-chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA-chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from approximately 10 to approximately 1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Synergistic effects in semidilute mixed solutions of alginate and lactose-modified chitosan (chitlac)

The present study specifically aimed at preparing and characterizing semidilute binary polymer mixtures of alginate and chitlac which might find an application in the field of cell encapsulation. A polyanion, alginate, and a polycation, a lactose-modified chitosan, were mixed under physiological conditions (pH 7.4 and NaCl 0.15) and at a semidilute concentration avoiding associative phase separation. The mutual solubility was found to be dependent on the charge screening effect of the added NaCl salt, being prevented below 0.05 M NaCl. A comparison with the behavior of the polyanion (alginate) under the same experimental conditions revealed that both the viscosity and the relaxation times of the binary polymer solutions are strongly affected by the presence of the polycation. In particular, the occurrence of electrostatic interactions between the two oppositely charged polysaccharides led to a synergistic effect on the zero-shear viscosity of the solution, which showed a 4.2-fold increase with respect to that of the main component of the solution, i.e., alginate. Moreover, the relaxation time, calculated as the reciprocal of the critical share rate, markedly increased upon reducing the alginate fraction in the binary polysaccharide solution. However, the formation of the soluble complexes and the synergistic effect are reduced upon increasing the concentration of the 1:1 electrolyte. By containing a gel-forming polyanion (alginate, e.g., with Ca(2+) ions) and a bioactive polycation (chitlac, bearing a beta-linked D-galactose), the present system can be regarded as a first step toward the development of biologically active scaffold from polysaccharide mixtures.     

The influence of charge density of chitosan in the compaction of the polyanions DNA and xanthan

In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.     

Monitoring DNA/poly-L-lysine polyplex formation with time-resolved multiangle laser light scattering

Nonviral DNA complexes show promise as alternative and attractive gene delivery vectors for treating genetic diseases. Nonviral DNA complexes are typically formed by combining DNA with various condensing/complexing agents such as lipids, polyelectrolytes, polymers, polypeptides, and surfactants in solution. DNA/poly-L-lysine polyplex formation kinetics are probed by time-resolved multiangle laser light scattering (TR-MALLS), which yields the time evolution of the supramolecular complex mass and geometric size. Primary polyplexes whose geometric size is smaller than individual DNA molecules in solution are formed very rapidly upon mixing DNA and poly-L-lysine. Over time, these primary polyplexes aggregate into larger structures whose ultimate size is determined primarily by the relative concentrations of DNA and poly-L-lysine. This final polyplex size varies with the DNA/poly-L-lysine mass ratio in a non-monotonic fashion, with the maximum polyplex size occurring at a DNA/poly-L-lysine mass ratio of approximately two to three (charge ratio near unity). The utility of TR-MALLS for monitoring the temporal evolution of DNA loading and supramolecular complex size growth (mean square radius and molar mass) throughout the DNA/poly-L-lysine polyplex formation process is demonstrated. The polyplex DNA loading and size, both geometric and molar mass, are key to understanding the transfection process and for developing optimal gene therapy vectors.     

Environmental responsiveness of polygalacturonic acid-based multilayers to variation of pH

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pK(a) of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pK(a).     

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Colloidal structure and stability of DNA/polycations polyplexes investigated by small angle scattering

Polyplexes of short DNA-fragments (300 b.p., 100 nm) with tailor-made amine-based polycations of different architectures (linear and hyperbranched) were investigated in buffer solution as a function of the mixing ratio with DNA. The resulting dispersed polyplexes were characterized using small-angle neutron and X-ray scattering (SANS, SAXS) as well as cryo-TEM with respect to their mesoscopic structure and their colloidal stability. The linear polyimines form rather compact structures that have a high tendency for precipitation. In contrast, the hyperbranched polycation with enzymatic-labile pentaethylenehexamine arms (PEHA) yields polyplexes colloidally stable for months. Here the polycation coating of DNA results in a homogeneous dispersion based on a fractal network with low structural organization at low polycation amount. With increasing polycation, bundles of tens of aligned DNA rods appear that are interconnected in a fractal network with a typical correlation distance on the order of 100 nm, the average length of the DNA used. With higher organization comes a decrease in stability. The 3D network built by these beams can still exhibit some stability as long as the material concentration is large enough, but the structure collapses upon dilution. SAXS shows that the complexation does not affect the local DNA structure. Interestingly, the structural findings on the DNA polyplexes apparently correlate with the transfection efficiency of corresponding siRNA complexes. In general, these finding not only show systematic trends for the colloid stability, but may allow for rational approaches to design effective transfection carriers.     

Effect of polycation length on its complexation with DNA and with poly(oxyethylene-block-sodium methacrylate)

Polyelectrolyte complexes of a synthetic polycation with either a genomic DNA or a synthetic poly(oxyethylene-block-sodium methacrylate), POE-b-PMANa, have been studied in aqueous solutions as a function of cation:anion ratio, the degree of polymerization of the polycation, the ionic strength, and temperature using dynamic light scattering and turbidity measurements. The polycation was a copolymer of methacryl oxyethyl trimethylammonium chloride and poly(oxyethylene) monomethyl ether monomethacrylate with 4-5 oxyethylene repeating units, PMOTAC-g-POE. The molar masses of the polycations in a homological series were 0.3, 0.9, and 2.1 x 10(6) g/ mol. The amount of comonomers with poly(oxyethylene) tails in the copolymers was 15 mol %. The molar mass of the POE-b-PMANa was 75000 g/mol and that of the POE-block was 5000 g/mol. The molar mass of the polycation was shown to have a dramatic effect on the stability and size of the complexes formed by either of the polyanions. An increase in the polycation molar mass shifts the cloud point toward the lower polycation content in the complexes, and a macro phase separation occurs in the solutions with the cation to anion molar ratios much below than 1:1. Increasing the ionic strength has a similar effect. Further addition of salt to turbid and phase-separated solutions results in dissociation of the complexes, and the polyions dissolve as individual macromolecules. The effect of POE on the stability of polyelectrolyte complexes is discussed as well.     

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.     

Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.     

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.     

Development of a nonviral gene delivery vehicle for systemic application

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, beta-cyclodextrin-containing polycations (betaCDPs). Here, a new method of betaCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated betaCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated betaCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated betaCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of betaCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.     

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor alpha, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.     

Permeation regulation of charged species by the component change of polyion complex membranes

Three kinds of polyion complex membranes were prepared on a glassy carbon electrode: polycation (poly-L-lysine)-rich membrane, polyanion (DNA)-rich membrane, and equivalent membrane. The permeation of electroactive species (e.g., hydrogen peroxide, L-ascorbate, urate, dopamine) through the membrane was measured by the oxidation current of species at base electrode. Permeation of the anionic species can be depressed through the anion-rich membrane, and permeation of the cation can also be regulated through the cation-rich membrane. It is obvious that the charge exclusion can be controlled by changing the component ratio of polycation and polyanion during preparation.     

Influence of alkali cation nature on structural transitions and reactions of biopolyelectrolytes

A general thermodynamic analysis is presented, describing how counterion species of different nature, but the same valency, influence polyelectrolyte transformations and reactions of the general form: PA1.B1-M(+)-->PA2.B2M+ + (B1 - B2)M+. Here PA1 and PA2 are two different states or structural forms of a polyanion, B1 and B2 are the number of M+ ions thermodynamically bound to the polyanions PA1 and PA2, respectively. The specific effects of the two counterions, M1+ and M2+, on this equilibrium can be simply related to the quotient of their selectivity constants, D2M2M1/D1M2M1, for the polyion states 1 and 2. We analyze how different monovalent counterions (particularly, sodium and potassium) affect polyelectrolyte reactions and transformations such as, e.g., the DNA helix-coil transition. Previous experimental results on the competition between DNA and the synthetic polyanion, poly(methacrylic acid), for binding to the synthetic polycation, poly(N-ethylvinylpyridinium), has been investigated with respect to sodium and potassium ion specificity, using our model. We also discuss the DNA-histone disassembly/assembly reaction modeled as a competition of two polyanions for binding to a polycation.     

Electrostatic interactions in the early events of VSV infection

The inportance of electrostatic interactions in the early phases of vesicular stomatitis virus (VSV) infection has been investigated in susceptible cells of different origin, human (HeLa) and avian (CER), by using some polyanions (heparin, polygalacturonic acid and mucin) and polycations (polymyxin B sulphate, poly-L-lysine, protamine, histone and polybrene). In HeLa cells, the attachment of VSV was enhanced by polymers having a positive charge and inhibited by those having a negative charge. In CER cells, all the polyanions tested reduced virus infection. Among the polycations, histone, polymyxin B sulphate and poly-L-lysine enhanced virus plaque forle protamine and polybrene reduced virus attachment. The effect of polyions on VSV particles and on cell membrane receptors has also been investigated. The analysis of the results obtained suggest that, although electrostatic interactions play an essential role in the binding of VSV to the cell membrane, more specific structural features appear to be required for viral attachment to occur.