Controlled association and delivery of nanoparticles from jet-sprayed hybrid microfibrillar matrices.

To develop bioactive scaffolds of targeted properties for tissue repair or biomedical applications, hybrid microfiber-nanoparticle (MF-NP) matrices capable of controlled nanoparticle (NP) delivery were prepared through two novel approaches. In a first strategy, the suppleness of the jet-spraying method to produce polymer microfibers (MF) was used to deposit poly(d,l-lactide) (PLA) NP on poly(lactic-co-glycolic acid) (PLGA) MF by direct co-projection. The second approach relied on the post-incubation of PLA NP aqueous dispersion with MF preliminarily prepared by jet-spraying. NP coverage density onto MF and NP release was assessed by scanning electron microscopy and fluorescence measurements using coumarin-6 loaded NP. The first process was shown to allow high coverage density of NP onto MF (300 µg/mg MF) and strong association, with no NP release observed over time. In the second approach, direct incubation of PLA NP with PLA MF led to lower NP coverage density (40 µg/mg MF) with very fast release of NP from MF. The pre-coating of MF with poly-l-lysine (PLL) or the one of NP with lysozyme as a model protein drug afforded a higher coverage density and stronger association, coupled with a more sustained release of NP from MF over time. These results show the possibility to control the immobilization density and release of NP through appropriate preparation process and surface modification, and are of prime interest for the development of complex scaffolds with orchestrated bioactivity.

Direct determination of phosphatase activity from physiological substrates in cells.

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.