Search

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.

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.

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.