Multiple Scale Reorganization of Electrostatic Complexes of PolyStyrene Sulfonate and Lysozyme

We report on a SANS investigation into the potential for these structural reorganization of complexes composed of lysozyme and small PSS chains of opposite charge if the physicochemical conditions of the solutions are changed after their formation. Mixtures of solutions of lysozyme and PSS with high matter content and with an introduced charge ratio [-]/[+]intro close to the electrostatic stoichiometry, lead to suspensions that are macroscopically stable. They are composed at local scale of dense globular primary complexes of radius ~ 100 {\AA}; at a higher scale they are organized fractally with a dimension 2.1. We first show that the dilution of the solution of complexes, all other physicochemical parameters remaining constant, induces a macroscopic destabilization of the solutions but does not modify the structure of the complexes at submicronic scales. This suggests that the colloidal stability of the complexes can be explained by the interlocking of the fractal aggregates in a network at high concentration: dilution does not break the local aggregate structure but it does destroy the network. We show, secondly, that the addition of salt does not change the almost frozen inner structure of the cores of the primary complexes, although it does encourage growth of the complexes; these coalesce into larger complexes as salt has partially screened the electrostatic repulsions between two primary complexes. These larger primary complexes remain aggregated with a fractal dimension of 2.1. Thirdly, we show that the addition of PSS chains up to [-]/[+]intro ~ 20, after the formation of the primary complex with a [-]/[+]intro close to 1, only slightly changes the inner structure of the primary complexes. Moreover, in contrast to the synthesis achieved in the one-step mixing procedure where the proteins are unfolded for a range of [-]/[+]intro, the native conformation of the proteins is preserved inside the frozen core

Secondary structure of rhBMP-2 in a protective biopolymeric carrier material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.This work was supported by the French Ministry of Research through an ANR-EmergenceBIO grant (ANR-09-EBIO-012-01), by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370), and by GRAVIT (081012_FIBIOS). C.P. is grafetul to IUF for financial support

Interaction of Astramol Poly(propyleneimine) Dendrimers with DNA and Poly(methacrylate) Anion in Water and Water–Salt Solutions

Interaction of poly­(propyleneimine) dendrimers DAB-<i>dendr-</i>(NH₂)_<i>x</i> of five generations (<i>x</i> = 4, 8, 16, 32, and 64) with either calf thymus DNA or tagged by pyrenyl groups poly­(methacrylate) anion (PMA*) as well as destruction of formed polyelectrolyte complexes by the added sodium chloride were studied by fluorescence quenching techniques. DNA-containing complexes (dendriplexes) were investigated by ethidium bromide assay, whereas formation of PMA* complexes was estimated by fluorescence of the pyrenyl groups that remained free of contact with the dendrimers-quenchers. The ion pairing with DNA phosphate groups was pH-sensitive and accompanied by inaccessibility of a part of the dendrimer amino groups even in slightly acidic media. The growth of the generation number resulted in successive stabilization of the dendriplexes against the added salt. The dendriplexes of all dendrimers except DAB-<i>dendr-</i>(NH₂)₄ were stable at physiological ionic strength. In contrast to the highly charged cationic polymer poly­(<i>N</i>-ethyl-4-vinylpyridinium) bromide of different degrees of polymerization, the dendrimers formed more stable complexes with flexible PMA* rather than with DNA, proving the inaccessibility of a part of the amino groups for the rigid double helix. The revealed regularities appear to be a platform for design of dendriplexes with controllable stability, in particular fulfilling the requirements imposed for gene delivery vehicles