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Nonviral gene delivery systems based on DNA complexes with polycations have recently been extensively studied. An important consideration in the design of such therapeutic systems is the understanding of forces governing the DNA−polycation interaction and the complex stabilization. In this work, the interaction of a cationic polymer from the poly(amidoamine) family (poly(bis-acryloylpiperazine-2-methyl-piperazine), p(BAP-2MP)) with DNA at different salt concentrations and pHs and in different buffers was studied using isothermal titration microcalorimetry as a principal tool. The binding isotherms were analyzed using a proposed complexation−condensation model, to calculate the observed binding constant, Kobs, enthalpies of the complex formation and condensation process, %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%com%@sbx@%%@mx@% and %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%con%@sbx@%%@mx@% , and complexation site and condensation site exclusion sizes, ncom and ncon, respectively. Kobs, %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%com%@sbx@%%@mx@% , and %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%con%@sbx@%%@mx@% decreased with increasing salt concentration, indicating involvement of electrostatic interactions. However, ncom was rather independent of salt concentration and nearly equal to the average number of ionized charges at the polycation, indicating that the ratio of nucleotide to p(BAP-2MP) monomer units is close to 1. Dissection of the free energy of interaction showed that electrostatic contributions dominate the p(BAP-2MP)−DNA interaction. The use of buffers with different protonation enthalpies demonstrated that the p(BAP-2MP) interaction with DNA is coupled with a proton extraction from the buffer resulting in an enthalpy change and enabling the polymer ionization and interaction at a stoichiometric ratio. Obtained intrinsic binding parameters (in a buffer with ΔHion = 0) reveal that p(BAP-2MP) interaction with DNA is nonspecific with a moderate binding constant and that it contains the enthalpic contribution (probably due to the formation of hydrogen bonding). The values for the condensation process, ncon and %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%con%@sbx@%%@mx@% , indicate that there is no further ionization of the p(BAP-2MP) polymer to interact with DNA and that the stoichiometric ratio is preserved.

The optimization of DNA−cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA−cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, Kobs increased from 7.8 × 10⁵ to 20.4 × 10⁵ M^-1. More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.

A series of poly(lactic acid)−poly(ethylene oxide) (PLA−PEG) AB block copolymers have been synthesized and used to produce aqueous dispersions of micellar-like nanoparticles. The hydrodynamic radius of the PLA core plus the stabilizing 5 kDa PEG layer, as determined by photon correlation spectroscopy, increased with the molecular weight of the PLA block. PLA−PEG particles have possible application as drug carriers for targeted drug delivery. In addition to particle size, the layer thickness and mobility of the hydrated PEG chains will be important in determining the particle's ability to avoid uptake by the defense system of the body, the phagocytic cells of the reticuloendothelial system. Viscoelastic measurements were used to investigate interparticle interactions in concentrated dispersions of the PLA−PEG nanoparticles. This approach has not previously been used to study how the interactions in dispersions of micellar-like assemblies depend on the copolymer geometry. When the volume fraction of the dispersions, φcore, was increased, the storage modulus, G‘, became greater than the loss modulus, G‘ ‘, indicating steric interaction between the PEG chains. At higher φcore values, compression of the grafted polymer chains was observed. The critical volume fraction at which overlap of the PEG chains commences, φcore^crit (i.e., G‘ = G‘ ‘), was used in conjunction with the overall hydrodynamic radius to determine the grafted PEG chain layer thickness. Micellar-like nanoparticles assembled from a PLA−PEG copolymer with a 5 kDa PEG block and a relatively low molecular weight 3 kDa PLA chain had an appreciable steric layer thickness of 6.3 nm. Hence, interparticle interactions in the concentrated dispersion were “soft” in nature as a result of the long-range interactions, as shown by the weak scaling dependence of the storage modulus on the volume fraction. The layer became more extended as the molecular weight of the PLA block was increased from 3 to 6 kDa, and as a consequence the PEG chains became less compressible. By using this method to characterize the PEG layer, it should be possible to rationalize the in vivo performance of the PLA−PEG nanoparticles and therefore aid the design of particulate drug carriers.

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (Mw 19−26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 μm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80−110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology:  PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 ± 59 to 61 ± 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 ± 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 ± 60 nm). The ζ-potential of complexes was reduced with increasing degree of PEG grafting if MW ≥ 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution:  all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.

The morphology of condensates formed between plasmid DNA and a PEG−poly(amidoamine) triblock copolymer were investigated using TEM and AFM in aqueous conditions. Both techniques revealed the presence of ring and extended linear structures; however, a greater proportion of linear structures was obtained with the PEG copolymer than was seen with the poly(amidoamine) homopolymer, indicating that the PEG was interfering with condensation. Differences in the ring diameters between the PEG copolymer and the homopolymer were more marked with TEM than AFM, suggesting that compact toroidal structures are largely due to constriction on dehydration and that this is prevented from occurring in the presence of PEG. AFM revealed an increase in condensate size with increasing polymer/DNA ratio, which can be attributed to the association of more polymer with the complex. With TEM, the proportion of linear structures increased with increasing polymer/DNA ratio, whereas it remained relatively constant for AFM across all ratios. It is suggested that an increased amount of PEG associated with the complexes promotes disruption of the ring structure upon dehydration. This supports the hypothesis that rings are formed via the bending of linear structures, with an equilibrium existing between the two, that is altered in favor of the linear structure by PEG.