Ivo B. Rietveld

Silica-collagen bionanocomposites as three-dimensional scaffolds for fibroblast immobilization.

Silica-collagen bionanocomposite hydrogels were obtained by addition of silica nanoparticles to a protein suspension followed by neutralization. Electron microscopy studies indicated that larger silica nanoparticles (80 nm) do not interact strongly with collagen, whereas smaller ones (12 nm) form rosaries along the protein fibers. However, the composite network structurally evolved with time due to the contraction of the cells and the dissolution of the silica nanoparticles. When compared to classical collagen hydrogels, these bionanocomposite materials showed lower surface contraction in the short term (1 week) and higher viability of entrapped cells in the long term (3 weeks). A low level of gelatinase MMP2 enzyme expression was also found after this period. Several proteins involved in the catabolic and anabolic activity of the cells could also be observed by immunodetection techniques. All these data suggest that the bionanocomposite matrices constitute a suitable environment for fibroblast adhesion, proliferation and biological activity and therefore constitute an original three-dimensional environment for in vitro cell culture and in vivo applications, in particular as biological dressings.

In vitro Studies and Preliminary In vivo Evaluation of Silicified Concentrated Collagen Hydrogels

Hybrid and nanocomposite silica-collagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblasts while decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silica-collagen biomaterials as biological dressings.

Benzocaine polymorphism: Pressure-temperature phase diagram involving forms II and III

Understanding the phase behavior of an active pharmaceutical ingredient in a drug formulation is required to avoid the occurrence of sudden phase changes resulting in decrease of bioavailability in a marketed product. Benzocaine is known to possess three crystalline polymorphs, but their stability hierarchy has so far not been determined. A topological method and direct calorimetric measurements under pressure have been used to construct the topological pressure-temperature diagram of the phase relationships between the solid phases II and III, the liquid, and the vapor phase. In the process, the transition temperature between solid phases III and II and its enthalpy change have been determined. Solid phase II, which has the highest melting point, is the more stable phase under ambient conditions in this phase diagram. Surprisingly, solid phase I has not been observed during the study, even though the scarce literature data on its thermal behavior appear to indicate that it might be the most stable one of the three solid phases.

Enantiomer resolution by pressure increase: inferences from experimental and topological results for the binary enantiomer system (R)- and (S)-mandelic acid.

In pharmacy, racemic compounds are often problematic, because generally only one of the enantiomers possesses therapeutic activity and it is often difficult to separate them. Even though this problem is likely as old as the pharmaceutical industry, one thermodynamically obvious way of separating racemic crystals has never been studied experimentally, which is by using pressure. Data have been obtained on the equilibria of the (R)- and (S)-mandelic acid system as a function of pressure and temperature. With the use of thermodynamic arguments including the Clapeyron, Schröder, and Prigogine-Defay equations, it has been demonstrated that the conglomerate (crystals of separated enantiomers) becomes more stable than the racemic compound at approximately 0.64 GPa and 460 K. Even though this pressure is still higher than at the bottom of the Mariana Trench, there are no technical obstacles to produce such conditions, making pressure a viable option for separating enantiomers.

Dimorphism of the prodrug l‐tyrosine ethyl ester: Pressure–temperature state diagram and crystal structure of phase II

Polymorphism is important in the field of solid-state behavior of drug molecules because of the continuous drive for complete control over drug properties. By comparing different structures of a series of L-tyrosine alkyl esters, it became apparent that the ethyl ester possesses dimorphism. Its structure was determined by powder diffraction and verified by density functional theory calculations; it is orthorhombic, P2(1) 2(1) 2(1) with a = 12.8679(8) Å, b = 14.7345(7) Å, c = 5.8333 (4) Å, V = 1106.01(11) Å, and Z = 4. The density of phase II is in line with other tyrosine alkyl esters and its conformation is similar to that of l-tyrosine methyl ester. The hydrogen bonds exhibit similar geometries for phase I and phase II, but the H-bonds in phase I are stronger. The solid II-solid I transition temperature is heating-rate dependent; it levels off at heating rates below 0.5 K min(-1), leading to a transition temperature of 306 ± 4 K. Application of the Clapeyron equation in combination with calorimetric and X-ray data has led to a topological diagram providing the relative stabilities of the two solid phases as a function of pressure and temperature; phase II is stable under ambient conditions.

Overall stability for the ibuprofen racemate: experimental and topological results leading to the pressure-temperature phase relationships between its racemate and conglomerate.

Enantiomer resolution is much sought after for pharmaceutical applications, because many optically active drug molecules have only one pharmaceutically active enantiomer. Although it is always possible to force separation, it will come at a cost. The present method, based on thermodynamics, provides a relatively easy approach to investigate whether separation can be thermodynamically spontaneous. A topological phase diagram of the binary enantiomer system at 0.5 mol-fraction is constructed as a function of temperature and pressure after analysis of pressure and heat related quantities. It is demonstrated that for ibuprofen, an optically active analgesic, the racemate is the only stable solid form; the phase relationship between the racemate and the conglomerate is analogous to dimorphism with overall monotropy in pure chemical compounds.

Electrospray deposition producing ultra-thin polymer films with a regular surface structure

Poly(vinylidene fluoride) films about 100 nm thick and with a highly reproducible surface structure were prepared by electrospray deposition. The films consist of dome-shaped domains, which exhibit short-range order in a liquid-like fashion. No special requirements for the electrospray setup are necessary for the preparation of these films. Electrostatic repulsion creates the domes and it controls their position with respect to other domes, similar to colloidal crystal formation. Infrared, X-ray and piezoresponse measurements indicate that the internal film structure is independent of the dome-shape structure and mainly consists of a ferroelectric β-phase. The film does not have a pronounced ferroelectric dipole moment.

Phenomenology of crystalline polymorphism

The stability hierarchy of crystalline polymorphs is often determined on the basis of limited calorimetric data even when other useful data such as specific volumes may be available. This may lead to wrong conclusions or an incomplete picture of the stability behavior of a compound and may be problematic in many applications, for example for pharmaceuticals. Therefore, the topological approach has been applied to the pharmaceutical FK664, which exhibits crystalline dimorphism, using heat- and work-related thermodynamic data to obtain a pressure–temperature phase diagram and elucidate its phase behavior. The approach leads to the conclusion that FK664 is overall monotropic with form B the most stable solid phase. In other words, form A does not have a stable domain for any pressure–temperature coordinate. The case of FK664 demonstrates that making use of the full available thermodynamic data set in combination with statistical information of the phase behavior of small organic molecules and classical thermodynamics leads to a sound evaluation of the stability hierarchy for crystalline dimorphism.

Rimonabant Dimorphism and Its Pressure–Temperature Phase Diagram: A Delicate Case of Overall Monotropic Behavior

Crystalline polymorphism occurs frequently in the solid state of active pharmaceutical ingredients, and this is problematic for the development of a suitable dose form. Rimonabant, an active pharmaceutical ingredient developed by Sanofi and discontinued because of side effects, exhibits dimorphism; both solid forms have nearly the same melting temperatures, melting enthalpies, and specific volumes. Although the problem may well be academic from an industrial point of view, the present case demonstrates the usefulness of constructing pressure-temperature phase diagrams by direct measurement as well as by topological approach. The system is overall monotropic and form II is the more stable solid form. Interestingly, the more stable form does not possess any hydrogen bonds, whereas the less stable one does.

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd‐mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a “fluctuating entry point,” as suggested by Zwanzig (J Chem Phys 1992;97:3587–3589), properly describes the diffusion of oxygen through the protein matrix. Proteins 2003;53:000–000.