Three different categories of blend interfaces are examined systematically in order to isolate the role of the interface in the development of cocontinuous morphologies during melt mixing. They are Type I, compatible binary blends based on high-density polyethylene (HDPE)/styrene-ethylene-butylenestyrene (SEBS) and HDPE/styrene-ethylene-butylene (SEB); Type II, an incompatible binary system comprised of HDPE/polystyrene (PS); and Type III, compatible ternary systems comprised of HDPE/PS compatibilized by SEBS in one case and by SEB in another. The Type I and Type III systems represent conventional approaches to preparing blend systems of low interfacial tension. The cocontinuous morphology is analyzed using three techniques: microscopy/image analysis, solvent extraction/gravimetric analysis, and BET characterization of surface area and pore size. A mechanism for the formation of dualphase continuity based on droplet and fiber lifetimes during melt mixing has been proposed. For the Type I compatible binary systems continuity development and microstructural features are dominated by thread-thread coalescence. In the Type II incompatible HDPE/PS binary system and Type III compatibilized ternary systems, continuity development and microstructural features are controlled by droplet-droplet coalescence and reduced droplet-droplet coalescence, respectively. In the latter case, the generation of fresh interface during droplet deformation results in a system that is only partially emulsified. The relative presence of fibers or droplets during dynamic mixing is analyzed quantitatively using a matrix dissolution/image analysis technique. A thread frequency ratio (TFR) is proposed as a basic general parameter to classify the relative presence of fibers to droplets during mixing and hence the type of continuity development for a given system.

The interaction of chitosan with plasmid DNA was investigated as a function of pH, buffer composition, degree of deacetylation (DDA), and molecular weight (M(n)) of chitosan, using isothermal titration microcalorimetry (ITC). The Single Set of Identical Sites model was used to obtain the enthalpy of interaction, the binding constant, and the stoichiometry of binding. The chitosan-DNA interaction was shown to be coupled with proton transfer from the buffer to chitosan, as revealed by the dependence of the measured heat release on the ionization enthalpy of the buffer. The measured enthalpy of binding was almost entirely due to proton transfer, because it was accounted for by the enthalpy of ionization of the buffer and of chitosan once the number of protons transferred was calculated. This proton transfer during binding resulted in the protonation of an additional 17, 37, and 58% of total glucosamine units at pH 5.5, 6.5, and 7.4, respectively. The strong polyanionic nature of DNA facilitates the ionization of glucosamines of chitosan upon complexation and is responsible for proton transfer. Interestingly, using the chitosan-DNA stoichiometry provided by ITC and the calculated degree of ionization of chitosan in the complex, the charge ratio of protonated amines to negative phosphate groups in the complex was nearly constant at 0.50-0.75 after saturation and was independent of the pH, buffer type and chitosan molecular characteristics. The chitosan-DNA binding constant was in the range of 10(9)-10(10) M(-1). The binding constant was pH-dependent and was greater at lower pH due to increased electrostatic attraction to DNA when chitosan is highly charged. Furthermore, the DDA and molecular weight of chitosan exerted a great influence on binding affinity which increased by almost an order of magnitude with an increase of the latter from 7 to 153 kDa. The binding affinity did not change significantly with DDA from 72 to 80% when the M(n) was kept constant near 80 kDa, but it increased substantially with DDA from 80 to 93% to reach a value similar to that obtained with chitosan of M(n) = 153 kDa and 80% DDA. These results provide insight into the previously reported dependence of the transfection efficiency of DNA/chitosan complexes on chitosan DDA and molecular weight, where complex stability and chitosan-DNA binding strength play a critical role.

The composition of samples obtained upon complexation of DNA with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV-visible, multiangle light scattering (MALS), and dynamic light scattering (DLS) detectors. A chitosan labeled with rhodamine B to facilitate UV-vis detection of the polycation was complexed with DNA under conditions commonly used for transfection (chitosan glucosamine to DNA phosphate molar ratio of 5). AF4 analysis revealed that 73% of the chitosan-rhodamine remained free in the dispersion and that the DNA/chitosan complexes had a broad size distribution ranging from 20 to 160 nm in hydrodynamic radius. The accuracy of the data was assessed by comparison with data from batch-mode DLS and scanning electron microscopy. The AF4 combined with DLS allowed the characterization of small particles that were not detected by conventional batch-mode DLS. The AF4 analysis will prove to be an important tool in the field of gene therapy because it readily provides, in a single measurement, three important physicochemical parameters of the complexes: the amount of unbound polycation, the hydrodynamic size of the complexes, and their size distribution.

Asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis spectrophotometry, multiangle light scattering (MALS), and dynamic light scattering (DLS) detection was used to analyze dispersions of DNA/rhodamine B labeled chitosan (Ch-rho) complexes frequently used as gene delivery vectors. The method yielded, in a single experiment, important characteristics of the complexes, such as their hydrodynamic radius, size distribution, conformation, composition, and the amount of free Ch-rho in the dispersions. Samples for analysis were obtained by varying experimental parameters known to influence the transfection efficiency of DNA/chitosan complexes, including the DNA concentration at mixing (82-164 μg/mL), the ratio of chitosan amino groups to DNA phosphate groups (3 ≤ N/P ratio ≤ 15), the chitosan molecular weight (10-76 kDa), and its degree of deacetylation. In all preparations, DNA/Ch-rho complexes had hydrodynamic radii ranging from 15 to 160 nm. Both the DNA concentration and the Ch-rho molecular weight influence the size distribution of the complexes: a greater fraction of large particles was detected in dispersions prepared with the most concentrated DNA solution or the Ch-rho of highest molar mass. All dispersions contained free Ch-rho in solution. The free Ch-rho content ranged from 53 to 92% of the total Ch-rho concentration in dispersions prepared with N/P ratios from 3 to 15, respectively, implying that the N/P ratio of the complexes ranged from 1.3 to 1.6 in all samples. The accuracy of the free Ch-rho determination by AF4/UV-vis/MALS+DLS was confirmed by an independent method involving (1) ultracentrifugation of the dispersions prepared with unlabeled chitosan and (2) analysis of the supernatant by the Orange II dye depletion method. This study demonstrates the ability of AF4/UV-vis/MALS+DLS to provide a complete physicochemical characterization of DNA/polycation complexes used in nonviral gene delivery.