Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates.

Traditional methods to analyze interactions and conformational changes of proteins adsorbed onto biomaterials are limited by the protein's associations with the substrate material and the complexity of the surrounding media. We have used EPR spectroscopy in combination with site-directed spin labeling (SDSL) to investigate single protein and competitive adsorption kinetics of horse hemoglobin (Hgb) and bovine serum albumin (BSA) on a silica-calcium-phosphate bioceramic substrate. Combined continuous wave and pulsed (DEER) EPR techniques were employed to monitor local mobility/flexibility changes within the proteins and tertiary structure dynamics upon adsorption. An alternate labeling technique was introduced to allow for specific quantification of each protein adsorbed to the bioceramic surface. We show that at buffer pH 7.4 and 4.7 the amount of adsorbed hemoglobin was increased by a factor of 4-5 compared with BSA. The tertiary structure of hemoglobin was strongly affected upon adsorption, leading to a dissociation of the tetrameric molecule into monomers or αß dimers. When the bioceramic substrate was previously functionalized with a layer of BSA, dissociation was reduced by 71 % compared with the untreated surface, indicating a "primer" effect of BSA for better adhesion of the globular hemoglobin.

Freeze-dried and spray-dried zinc-containing silica microparticles entrapping insulin.

New approaches for oral administration of insulin are strongly related to novel insulin carriers. The aim of this study was the insulin microencapsulation in a new zinc-silica matrix for drug protection and controlled release. Zinc-silica microparticles loaded with insulin were obtained by sol-gel process via spray drying and freeze drying methods. Inorganic silica matrix isolates and constrains the movement of the biomolecules preventing their aggregation and denaturation, while the zinc oxide improves the system stability. Moreover, formation of insulin hexamers in the presence of zinc ions leads to an increased stability of the insulin three-dimensional structure during preparation, storage and release. The particles were characterized with respect to average size, specific surface area, porosity and morphology. In vitro behavior of insulin-loaded particles together with protein structural conformation was also evaluated. The release profile can be adapted by synthesis route of microparticles.

Evaluation of the dose distribution gradient in the close vicinity of brachytherapy seeds using electron paramagnetic resonance imaging.

Electron paramagnetic resonance (EPR) spectroscopy has been successfully employed to determine radiation dose using alanine. The EPR signal intensity reflects the number of stable free radicals produced, and provides a quantitative measurement of the absorbed dose. The aim of the present study was to explore whether this principle can be extended to provide information on spatial dose distribution using EPR imaging (EPRI). Lithium formate was selected because irradiation induces a single EPR line, a characteristic that is particularly convenient for imaging purposes. (125)I-brachytherapy seeds were inserted in tablets made of lithium formate. Images were acquired at 1.1 GHz. Monte Carlo (MC) calculations were used for comparison. The dose gradient can be determined using two-dimensional (2D) EPR images. Quantitative data correlated with the dose estimated by the MC simulations, although differences were observed. This study provides a first proof-of-concept that EPRI can be used to estimate the gradient dose distribution in phantoms after irradiation.

Molecular electron paramagnetic resonance imaging of melanin in melanomas: a proof-of-concept.

The incidence of malignant melanoma is increasing at an alarming rate. As the clinical outcome of the disease strongly depends on the localization of the lesion, early detection at the initial stages of development is critical. Here, we suggest spatial characterization of melanoma based on the presence of endogenous stable free radicals in melanin pigments. Taking into account the abundance of these naturally occurring free radicals in proliferating melanocytes and their localization pattern, we hypothesized that electron paramagnetic resonance (EPR) imaging could be a unique tool for mapping melanomas with high sensitivity and high resolution. The potential of EPR to image melanoma samples was demonstrated in vitro in animal and human samples. Using EPR systems operating at low frequency, we were also able to record in vivo EPR spectra and images from the melanin present in a subcutaneous melanoma implanted in a mouse. In addition to the proof-of-concept and the achievement of providing the first non-invasive image of an endogenous radical, this technology may represent a key advance in improving the diagnosis of suspected melanoma lesions.