Controllable stability of DNA-containing polyelectrolyte complexes in water-salt solutions

Destruction of polyelectrolyte complexes (PECs) formed by DNA and synthetic polyamines of different structures was carried out by addition of low molecular weight electrolyte to PEC solution at different pHs. The dissociation was studied by the fluorescence quenching technique using the ability of cationic dye ethidium bromide to intercalate into free sites of DNA double helix followed by ignition of ethidium fluorescence. Structure of amine groups of the polycation was shown to be a decisive factor of PEC stability. PECs formed by polycations with quaternary amine groups, i.e., poly(N-alkyl-4-vinylpyridinium) bromides, poly(N, N-dimethyldiallylammonium) chloride, and ionene bromide, were pH independent and the least tolerant to destruction by the added salt. Primary amine groups of basic polypeptides poly-L-lysine hydrobromide and poly-L-arginine hydrochloride as well as synthetic polycation poly(vinyl-2-aminoethyl ether) provided the best stability of PECs in water-salt solutions under wide pH range. Moderate and pH-dependent stability was revealed for PECs included poly(N,N-dimethylaminoethylmethacrylate) with tertiary amine groups in the chain or branched poly(ethylenimine) with primary, secondary, and tertiary amine groups in the molecule. The data obtained appear to be the basis for design of DNA-containing PECs with given and controllable stability. The design may be accomplished not only by proper choice of polyamine of one or another type, but by using of tailor-made polycations with given composition of amine groups of different structure in the chain as well. Thus, quaternization of a part of tertiary amine groups of poly(N, N-dimethylaminoethylmethacrylate) resulted in expected decrease of stability of DNA-containing PECs in water-salt solutions. The destruction of PEC formed by random copolymer of 4-vinylpyridine and N-ethyl-4-vinylpyridinium bromide was pH sensitive and could be performed under pH and ionic strength closed to the physiological conditions. This result appears to be particularly promising for addressing DNA packed in PEC species to the target cell.     

Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production.     

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation.

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.     

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.     

Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.     

DNA-polycation complexation and polyplex stability in the presence of competing polyanions

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.     

Structural effects of carbohydrate-containing polycations on gene delivery. 1. Carbohydrate size and its distance from charge centers

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and beta-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to beta-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression.     

Complexation of DNA with poly(methacryl oxyethyl trimethylammonium chloride) and Its poly(oxyethylene) grafted analogue

Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and Mw of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (Mw) of DNA was 10.4 x 10(6) g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.     

DNA complexes with polycations used for delivery of DNA into cells

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.     

Cationic carriers of genetic material and cell death: A mitochondrial tale

Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(l-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum.     

Fractional precipitation of plasmid DNA from lysate by CTAB

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.     

Observation of the three-dimensional structure of actin bundles formed with polycations

Three-dimensional structures of actin bundles formed with polycations were observed by using transmission electron microtomography and atomic force microscopy. We found, for the first time, that the cross-sectional morphology of actin bundles depends on the polycation species and ionic strength, while it is insensitive to the degree of polymerization and concentration of polycation. Actin bundles formed with poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary show a ribbon-like cross-sectional morphology in low salt concentrations that changes to cylindrical cross-sectional morphology with hexagonal packing of the actin filaments in high salt concentrations. Contrastingly, actin bundles formed with poly-L-lysine show triangular cross-sectional morphology with hexagonal packing of the actin filaments. These variations in cross-sectional morphology are discussed in terms of anisotropy in the electrostatic energy barrier.     

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.     

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.     

The induction of large unilamellar vesicle fusion by cationic polypeptides: the effects of mannitol,size, charge density and hydrophobicity of the cationic polypeptides

Polylysine and lysine-based copolymers induced fusion of large unilamellar vesicles only in media containing at least 0.4 M mannitol. In the absence of mannitol, polylysine and certain lysine-based copolymers also containing acidic amino acids were not able to induce fusion. Fusion, in the presence of mannitol, was induced at nanomolar concentrations of the polycations. Excess polymer caused reduced rate and extent of fusion. In the presence of 100 mM NaCl the effective concentration range of the polycations was narrower. Kinetic analysis determined that salt increased the aggregation constant C₁₁ while reducing the fusion constant f₁₁. Addition of polylysine in excess resulted in smaller C₁₁. Short polylysine (3500) was less effective on a molar basis than a long one (37 000). Copolymers were more effective than polylysine due to higher aggregation potential. Copolymers were also more effective in promoting Ca²⁺-induced fusion in the absence of mannitol, their greater efficiency resulting from substantially larger fusion potential, without a greater rate of leakage. Preincubation of the vesicles with the polycations for less than 20 s resulted in faster fusion rates, while longer preincubations caused slower fusion rates. Addition of polycations to the preincubated mixture enhanced the fusion rates, indicating that the polycations were not available, rather than the vesicles being not susceptible to fusion. The effect of preincubation suggests two phases in the binding of the polycations to the vesicles; a fast phase of partial binding and a slower phase resulting in complete binding. The addition of millimolar concentrations of pyrophosphate or sulphate provided a fine control of the effective polycation concentration and its effect on fusion.     

Extrinsic cotton effect and helix-coil transition in a DNA-polycation complex

A strong, positive, extrinsic CD band ([θ]_242.5 = ～2 × 10^?3 deg cm²/dmole) has been observed for a α-bromo-poly[methylene-1,4-phenylenecarbonyloxyethylene(dimethylamino) bromide] (I). The extrinsic Cotton effect is attributed to the ordered arrangement of the aromatic chromophores along the DNA helix. The extrinsic band had a linear dependence on the amount of polycation I added from r ≤ 0.3 to r = ～0.5, but decreased thereafter. Addition of the polycation decreased the positive CD band of DNA at 275 nm. The transformation of B → C form in the presence of salts or other polycations caused similar changes. The decrease in [θ]₂₇₅ was reversed at higher concentrations of the polycation (r > 0.4). Thermal denaturation studies indicated both stabilization of the helix conformation (Δt_m = 21°C) and a high degree of cooperativity in the melting of DNA-polycation complex as compared to native calf thymus DNA. Using the linear relationship between r (polycation residue/DNA phosphate) and F (fraction of bound base pairs), a value of 0.6 was derived for β (number of monomer residues of polycation/nucleotide). Both electrostatic and hydrophobic effects probably influence the stability of the DNA-polycation complex, since the strength of the 242.5 nm CD band is a function of both salt and urea concentrations.     

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.     

Effects of polycations on ion channels formed by neutral and negatively charged alamethicins

The effects of the peptide polycations salmon protamine (M _r = 4332,z = + 21) and poly-l-lysine (M _r 100,00,z + 775) on ion channels formed by synthetic alamethicin Alm-F30 (one negative charge), natural Alm-F50 (neutral) and phosphorylated Alm-F50 (two negative charges) reconstituted in planar lipid bilayers have been studied at the single channel level. It was observed that both polycations in micromolar concentrations transiently block ion permeation through the channels formed by each alamethicin analogue, although in case of the neutral Alm-F50 to a significantly lesser extent. Poly-l-lysine showed to be more effective than protamine in blocking these channels. If either polycation is present in the cis-compartment, blockade occurs only at cis positive membrane voltages. At constant polycation concentration, dwell times in the blocked state increase when salt concentration is lowered, and decrease at acidic pH with an apparent pK of 4.8. Mean lifetime of blockade events shortens when membrane voltage is increased, which suggests that both polycations may permeate through the oligomeric alamethicin channels if conductance levels are > 2. We suggest that blockade is caused by electrostatic binding of a single polycation molecule to the C-terminal channel mouth; in case of Alm-F30, Glu18 has to be considered as the putative binding site. Our results provide further evidence for the barrel-stave model and a parallel orientation of dipole monomers in the channel aggregate, the C-termini facing the membrane side with the more positive membrane potential.     

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.     

Molecular Dynamics Simulations of DNA-Polycation Complex Formation

Complexes formed from DNA and polycations are of interest because of their potential use in gene therapy; however, there remains a lack of understanding of the structure and formation of DNA-polycation complexes at atomic scale. In this work, molecular dynamics simulations of the DNA duplex d(CGCGAATTCGCG) in the presence of polycation chains are carried out to shed light on the specific atomic interaction that result in complex formation. The structures of complexes formed from DNA with polyethylenimine, which is considered one of the most promising DNA vector candidates, and a second polycation, poly-L-lysine, are compared. After an initial separation of ∼50 Å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes on complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, with polycation intrusion into the major and minor grooves dependent on the identity and charge state of the polycation. The ability of the polycation to effectively neutralize the charge of the DNA phosphate groups and the resulting influence on the DNA helix interaction are discussed.