References of "De Pauw, Marie-Claire"
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See detailGold nanorods coated with a thermo-responsive poly(ethylene glycol)-b-poly(N-vinylcaprolactam) corona as drug delivery systems for remotely near infrared-triggered release
Liu, Ji ULg; Detrembleur, Christophe ULg; De Pauw, Marie-Claire ULg et al

in Polymer Chemistry (2014), 5(3), 799-813

Poly(ethylene glycol)-b-poly(N-vinylcaprolactam) (PEG-b-PNVCL) copolymers are prepared from a PEG macro-chain transfer agent in DMF at 65 °C via reversible addition-fragmentation chain transfer (RAFT ... [more ▼]

Poly(ethylene glycol)-b-poly(N-vinylcaprolactam) (PEG-b-PNVCL) copolymers are prepared from a PEG macro-chain transfer agent in DMF at 65 °C via reversible addition-fragmentation chain transfer (RAFT) polymerization. The well-defined PEG114-b-PNVCL237 copolymer with a cloud point temperature of 39 °C is used for the formation of a thermo-responsive polymer corona on the surface of gold nanorods (GNRs) via a “grafting-to” approach. Thermo-responsiveness and thermo-dependent optical properties of the as-obtained GNR@PEG-b-PNVCL nanoparticles are studied with dynamic light scattering and UV/vis spectroscopy techniques. Near infrared (NIR)-induced heating of GNR@PEG-b-PNVCL is also explored in aqueous suspension under NIR laser irradiation (802 nm, up to 250 mW). The potential of these GNR@PEG-b-PNVCL nanoparticles to be used as smart drug delivery systems (DDS) is then studied. A hydrophilic drug model, Rhodamine ® B, is used to assess the guest loading capacity, and triggered release behaviours are then evaluated under conventional external heating or internal heating induced by remote NIR irradiation. Cytotoxicity evaluation of the GNR@PEG-b-PNVCL against the fibroblast-like L929 cell line is carried out via the MTS assay in order to confirm the improved biocompatibility of the GNRs after polymer coating. These thermo-responsive GNR@PEG-b-PNVCL nanoparticles are promising DDS that combine the chemotherapeutic and phototherapeutic functions. [less ▲]

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See detailReversibly crosslinked thermo- and redox-responsive nanogels for controlled drug release
Liu, Ji ULg; Detrembleur, Christophe ULg; Hurtgen, Marie ULg et al

in Polymer Chemistry (2014), 5(1), 77-88

Reversibly crosslinked poly(vinyl alcohol)-b-poly(N-vinylcaprolactam) PVOH-b-PNVCL nanogels were prepared by using a redox-responsive crosslinking agent, 3,30-dithiodipropionic acid (DPA), to crosslink ... [more ▼]

Reversibly crosslinked poly(vinyl alcohol)-b-poly(N-vinylcaprolactam) PVOH-b-PNVCL nanogels were prepared by using a redox-responsive crosslinking agent, 3,30-dithiodipropionic acid (DPA), to crosslink the PVOH corona, above the lower critical solution temperature (LCST) of the PNVCL block. The stability of the as-prepared nanogels against heating and diluting with water was studied by dynamic light scattering (DLS) to follow the evolution of the hydrodynamic diameter and size distribution. Stability under reductive conditions was also studied by DLS and transmission electron microscopy (TEM) after exposure to dithiothreitol (DTT) buffer solutions at different pH. The reversibility of the crosslinking was evaluated by treating the de-crosslinked nanogels with hydrogen peroxide (H2O2) above the LCST. As a hydrophobic drug model, Nile red (NR) was loaded into the nanogels, and triggered release behaviours were studied after exposure to the same DTT buffer solutions. Moreover, two PVOH-b-PNVCL copolymers with different compositions and LCST were used to evaluate the effect of the LCST on the release behaviours of the nanogels. The cytotoxicity of the nanogels against a mouse fibroblast-like L929 cell line was assessed via the MTS assay, and preliminary studies on cellular uptake of the nanogels within human melanoma MEL-5 cells were also carried out by fluorescence microscopy and fluorescence-activated cell sorting. [less ▲]

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See detailPoly(acrylic acid)-block-poly(vinyl alcohol) anchored maghemite nanoparticles designed for multi-stimuli triggered drug release
Liu, Ji ULg; Detrembleur, Christophe ULg; Debuigne, Antoine ULg et al

in Nanoscale (2013), 5(23), 11464-11477

Original core/corona nanoparticles composed of amaghemite core and a stimuli-responsive polymer coating made of poly(acrylic acid)-block-poly(vinyl alcohol) macromolecules were fabricated for drug ... [more ▼]

Original core/corona nanoparticles composed of amaghemite core and a stimuli-responsive polymer coating made of poly(acrylic acid)-block-poly(vinyl alcohol) macromolecules were fabricated for drug delivery system (DDS) application. This kind of DDS aims to combine the advantage of stimuli-responsive polymer coating, in order to regulate the drug release behaviours under different conditions and furthermore, improve the biocompatibility and in vivo circulation half-time of the maghemite nanoparticles. Drug loading capacity was evaluated with methylene blue (MB), a cationic model drug. The triggered release of MB was studied under various stimuli such as pH, ionic strength and temperature. Local heating generated under alternating magnetic field (AMF) application was studied, and remotely AMF-triggered release was also confirmed, while a mild heating-up of the release medium was observed. Furthermore, their potential application as magnetic resonance imaging (MRI) contrast agents was explored via relaxivity measurements and acquisition of T2-weighted images. Preliminary studies on the cytotoxicity against mouse fibroblast-like L929 cell line and also their cellular uptake within human melanoma MEL-5 cell line were carried out. In conclusion, this kind of stimuli-responsive nanoparticles appears to be promising carriers for delivering drugs to some tumour sites or into cellular compartments with an acidic environment. [less ▲]

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See detailIntraocular lenses with functionalized surfaces by biomolecules in relation with lens epithelial cell adhesion
Huang, Yi-Shiang ULg; Alexandre, Michaël ULg; Bozukova, Dimitriya et al

Poster (2013, April 25)

A cataract is pathology opacity of the lens or capsule of the eye, causing impairment of vision or even blindness. Surgery, with lens extraction and intraocular lens implantation, is still the only ... [more ▼]

A cataract is pathology opacity of the lens or capsule of the eye, causing impairment of vision or even blindness. Surgery, with lens extraction and intraocular lens implantation, is still the only currently available treatment. The most common complication after implantation of intraocular lenses (IOLs) is the posterior capsular opacification (PCO) or secondary cataract. This is the result of lens epithelial cells (LECs) proliferation and their transition to mesenchymal cells. In 1997, a Sandwich theory was proposed to elucidate the developmental process of PCO. [1] According to this model, an IOL with higher affinity to LECs will induce a less PCO. In our research, the pHEMA (Poly(2-hydroxyethyl methacrylate)) based acrylic hydrophilic polymer is subjected to the surface modification by conjugating with the bioactive peptides. The RGD sequence, known for its excellent biocompatibility, is designed to stimulate the biointegration between the LECs and the polymer implant. [2]. From our research, The RGD peptide immobilized onto pHEMA surfaces significantly facilitates the adhesion of the porcine LEC. The peptide immobilized surface retains its biological function even after 10 times of autoclave. On the other hand, the immobilized peptide does not alter the hydrophobicity of the surface, the light transmission, as well as the cytotoxicity of the material. This functionalized biomaterial would possibly prevent the formation of PCO. [1] J Cataract Refract Surg. 1997 Dec;23(10):1539-42 [2] Trends Biotechnol. 2008 Jul;26(7):382-92 [less ▲]

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See detailIntraocular Lens Adsorbome: a Proteomic Study of Adsorbed Proteins onto Acrylic Materials and Its Implication in Secondary Cataract
Huang, Yi-Shiang ULg; Bertrand, Virginie ULg; Mazzucchelli, Gabriel ULg et al

Poster (2012, September 17)

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the ... [more ▼]

The intraocular lens (IOL) is a polymer implant designed to replace the natural lens after cataract surgery. When the implant is introduced into the lens capsule, the polymer starts to interact with the aqueous humour and the exchange of molecules between the solid and the liquid begins. The nature of exchange in water, ions, and biomolecules may result in several postoperative complications including glistening, calcification, and posterior capsular opacification. The posterior capsular opacification (PCO, also called “Secondary Cataract”) is raised from the over-growth of residual lens epithelial cells. The first step of the over-growth process of the cells is their adhesion to the deposited biomolecules, such as proteins involved in extra-cellular matrices. The purpose of this study is to identify the principal proteins adsorbed onto the acrylic polymers by mass spectrometry. The concept of adsorbome is to generate a list of adsorbed proteins to the hydrophilic and hydrophobic polymers, and then compare the difference to the original component of aqueous humour in order to see the affinity of individual protein to each material. Two kinds of hydrophilic and two kinds of hydrophobic acrylic polymers were tested for their adsorbomes by treating them with an aqueous humour analogue and the major adsorbed proteins were identified by mass spectrometry. Interestingly, the hydrophilic acrylic polymer shows a relative lower protein adsorption rate but shows a higher incidence of secondary cataract. This phenomenon implies the adsorbed proteins play a crucial role in progress of secondary cataract. [less ▲]

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See detailAnalysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging
Bertrand, Virginie ULg; Debois, Delphine ULg; Calligaris, David ULg et al

Conference (2012, September 04)

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract ... [more ▼]

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract. The surgical intervention consists in removing the cloudy lens and to replace it by an artificial intraocular lens (IOL). The in vivo implantation of these synthetic lenses involves the evaluation of several factors as their physico-chemical properties, their capacities to interact with lens epithelial cells and proteins, as well as their biocompatibility. During a previous study, we demonstrated major differences concerning the tackiness (atomic force microscopy), the cellular adhesion and the protein adsorption of various polymer disks intended for the manufacturing of intraocular lenses. The aim of this work was to correlate a histological analysis to a mass spectrometry imaging analysis performed on the same sample. To estimate the biocompatibility of the biomaterials, an animal testing was realized in rabbits. The various polymers were implanted subcutaneously. After one month, the 2 cm x 3 cm pieces of rabbit skin and underlying muscle with a 2 cm thickness were removed, fixed with formaldehyde 10% during six days, treated for the paraffin inclusion and stored at room temperature until use. Slices of 5 µm thickness were performed using a microtome. Paraffin was removed and tissue sections were washed in graded ethanol baths. The slices were then stained with the hematoxylin and eosin dyes. The analysis of stained sections showed different histo-morphological features according to the implanted polymer. For MALDI MSI purposes, on tissue protein digestion was performed using trypsin (1) and the MALDI matrix (α-cyanohydroxycinnamic acid, 5 mg/mL in ACN/0.2% TFA 70:30) was deposited using an ImagePrep automated sprayer (Bruker Daltonics, Bremen, Germany). Experiments were carried out using an UltraFlex II TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). MALDI imaging can show the detection of different proteomic profiles according to the tested biomaterials, which may be considered as biocompatibility markers. The MALDI images of these markers are then correlated with the histo-morphological profiles. Consequently, mass spectrometry imaging can become a powerful tool in the evaluation of the biocompatibility of artificial implants in biomedical application. [less ▲]

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See detailBioactive Intraocular Lens - A New Concept to Control Secondary Cataract
Huang, Yi-Shiang ULg; Alexandre, Michaël ULg; Bozukova, Dimitriya et al

Poster (2012, August 29)

A cataract is pathology opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant ... [more ▼]

A cataract is pathology opacity of the lens, causing impairment of vision or even blindness. Today, a surgery is still the only available treatment. The intraocular lens (IOL) is a polymer implant designed to replace the natural lens in the cataract surgery. The materials for IOL require excellent optical properties for light transmission, mechanical properties for folding injection during surgery, and biological properties for preventing body rejection. The biocompatibility - or more specified, bio-inert - seems to be the prerequisite in selecting the materials. [1] However, the bioinert materials could not satisfy the unmet need in the secondary cataract control. Posterior capsular opacification (PCO, or Secondary Cataract), characterized by a thick and cloudy layer of lens epithelial cells (LECs), is the most common postoperative complication. In 1997, a “Sandwich Theory” model was proposed to elucidate the developmental process of PCO. [2] In this model, the residual LECs between the lens capsular bag and the IOL undergo proliferation, migration, as well as transdifferentiation and finally induce PCO if the affinity to the IOL material is low. In our research, a bioactive molecule is introduced to the conventional acrylic hydrophilic polymer pHEMA(Poly(2-hydroxyethyl methacrylate)) by covalent conjugation. The RGD peptide sequence, being well-known for its tissue integration ability, is designed to stimulate the biointegration between the LECs and the IOL. [3]. Our data have shown the peptide grafted biomaterial not only exhibits similar optical and mechanical properties, but also reveals enhanced biological properties in cell adhesion and cell morphology maintenance. By means of surface functionalization of IOL to stabilize and restore LECs, the secondary cataract could be controlled in a regenerative medicine way. References [1] Dimitriya Bozukova (2010) Materials Science and Engineering R, 69: 63-83. [2] Reijo Linnola (1997) J Cataract Refract Surg., 10: 1539–42. [3] Ruoslahti E (1986) Cell, 44(4): 517-8. [less ▲]

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See detailSurface and bio-adhesion properties of new hydrophobic and current materials for artificial intraocular lens
Bertrand, Virginie ULg; Svaldo Lanero, Tiziana ULg; Duwez, Anne-Sophie ULg et al

Poster (2012)

A high bio-adhesion appears to be one of the key factor for posterior capsular opacification (PCO) prevention. Indeed, the proteins adsorption and the lens epithelial cells (LEC) adhesion both contribute ... [more ▼]

A high bio-adhesion appears to be one of the key factor for posterior capsular opacification (PCO) prevention. Indeed, the proteins adsorption and the lens epithelial cells (LEC) adhesion both contribute to PCO development. We present in this work the comparison of a new glistening free hydrophobic material (GF® from Physiol) with benchmark hydrophobic and hydrophilic materials regarding their chemicophysical properties and their respective ability to interact with lens epithelial cells and proteins. For this purpose, we determined the hydrophobicity by contact angle measurement (assessed by water drop and air bubble methods), the surface adhesiveness by atomic force microscopy (AFM), the proteins adsorption by fluorescent measurement and the LEC adhesion by the determination of cell density. The new hydrophobic material presents comparable hydrophobicity, proteins adsorption and LEC adhesion to current commercial hydrophobic material. Its adhesiveness, measured with the AFM, is intermediate between hydrophilic and hydrophobic materials. In conclusion, the bio-adhesion properties of this new glistening free hydrophobic IOL material are similar to generic hydrophobic acrylic materials and therefore should to the same extent prevent PCO. [less ▲]

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See detailDesign of reversibly disulfide core cross-linked polymer micelles
Cajot, Sébastien ULg; Schol, Daureen ULg; Danhier, F. et al

Poster (2011, December 07)

Detailed reference viewed: 31 (10 ULg)
See detailSmart block copolymers for biomedical applications
Sibret, Pierre ULg; De Pauw, Marie-Claire ULg; Jérôme, Christine ULg

Poster (2011, November 21)

Smart polymers are polymers that respond with rapid changes to external stimuli such as pH, temperature, light or ionic strength. Responses to the stimuli may manifest themselves as changes in solubility ... [more ▼]

Smart polymers are polymers that respond with rapid changes to external stimuli such as pH, temperature, light or ionic strength. Responses to the stimuli may manifest themselves as changes in solubility, shape or surface characteristics. These materials are very intersesting for different biomedical applications such as drug delivery systems, tissue engineering or sensors. In this work, we focused on two separate systems: on the one hand, micelles and, on the other hand, iron oxide nanoparticles. These nanoparticles are generally synthesized in a one-step process by alkaline coprecipitation of iron (II) and iron (III) precursors. However, iron oxide nanoparticles are not stable enough in physiological conditions to be used as is. A stabiliser coating is needed to avoid aggregation and precipitation of the colloids in body fluids. For this coating, the polymer blocks chosen are PEO (to confer stealthiness), PAA (pH-responsive) and PNIPAM (thermoresponsive). This triblock copolymer was synthesized by RAFT polymerization and was used alone to form micelles and with iron oxide to make magnetic stabilized nanoparticles. The behaviour of micelles and coated nanoparticles was investigated by a combination of DLS, TEM and zeta potential measurements. To highlight the potentiality of these nanomaterials, their cytotoxicity will be studied and compared. [less ▲]

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See detailDesign of reversibly disulfide core cross-linked polymer micelles
Cajot, Sébastien ULg; Schol, Daureen ULg; Danhier, F. et al

Poster (2011, November 21)

Over the last decade, polymer micelles attracted an increasing interest in drug pharmaceutical research because they could be used as efficient drug delivery systems. Micelles of amphiphilic block ... [more ▼]

Over the last decade, polymer micelles attracted an increasing interest in drug pharmaceutical research because they could be used as efficient drug delivery systems. Micelles of amphiphilic block copolymers are supramolecular core-shell type assemblies of tens of nanometers in diameter. An accumulation of polymer nanocarriers to solid tumours is possible due to the EPR effect. Even if micelles get a high stability in aqueous media, the dissociation of micelles is not always preserved when they are injected in the blood compartment. This work aims at reporting on the design of reversibly cross-linked micelles based on PEO-b-PCL copolymers by introducing disulfide bridges in the micelle core to provide higher stability. Different kinds of macromolecular architectures are employed to study their impact on the micelles and their biological behavior. These new functional copolymers were all successfully micellized, reversibly cross-linked and are stealthy, which show the efficiency of the developed cross-linking process and offer a set of nanocarriers to be tested further, as shown on the first biological tests. [less ▲]

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See detailSmart block copolymers for biomedical applications
Sibret, Pierre ULg; Schol, D; De Pauw, Marie-Claire ULg et al

Poster (2011, September 03)

Stimuli-responsive polymers are polymers that respond with rapid changes to external stimuli such as pH, temperature, light or ionic strength. Responses to the stimuli may manifest themselves as changes ... [more ▼]

Stimuli-responsive polymers are polymers that respond with rapid changes to external stimuli such as pH, temperature, light or ionic strength. Responses to the stimuli may manifest themselves as changes in solubility, shape or surface characteristics. They can also lead to the fomation of micelles or a sol-gel transition. These materials are very intersesting for different biomedical applications such as drug delivery systems, tissue engineering or sensors. In this work, we focused on two separate systems: on the one hand, micelles and, on the other hand, iron oxide nanoparticles. These nanoparticles are generally synthesized in a one-step process by alkaline coprecipitation of iron (II) and iron (III) precursors in aqueous solutions (Massart process). However, iron oxide nanoparticle suspensions produced by Massart process are not stable enough in physiological conditions to be used as is. A stabiliser coating is needed to avoid aggregation and consequent precipitation of the colloids in body fluids. For this coating, the polymer blocks chosen are: the poly(ethylene oxide) (PEO), the poly(acrylic acid) (PAA) and the poly(N-isopropyl acrylamide) (PNIPAM). The high flexibility and hydrophilicity of PEO chains make it an outstanding candidate for confering stealthiness to micelles and nanoparticles in order to avoid their rapid removal from the body by the opsonization process. The PAA is the pH-responsive block and the anchoring block. The PNIPAM is the thermoresponsive block with a thermal transition close to 37°C (99°F). Triblock copolymer was synthesized by a Reversible Addition Fragmentation Transfer Polymerization (RAFT) process combining poly(acrylic acid) PAA, poly(N-isopropylacrylamide) and poly(ethylene oxide) or poly[acrylate methoxy poly(ethylene oxide)]. This triblock copolymer was used alone to form micelles and with iron oxide to make magnetic stabilized nanoparticles. The behaviour of micelles and coated nanoparticles was investigated in different conditions by a combination of dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta potential measurements. Moreover, PAA-b-PNIPAM-b-PAMPEO nanofibers were obtained using electrospinning technique. These nanofibers present interesting prospects in the field of biomaterials and biomedical applications as they mimic the extracellular matrix of the skin. [less ▲]

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See detailDisulfide bridges, new prospect in drug delivery systems?
Cajot, Sébastien ULg; Danhier, F.; Schol, Daureen ULg et al

Poster (2011, September 03)

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See detailReversibly core-cross-linked micelles sensitive to reductive environment for the design of drug delivery systems
Cajot, Sébastien ULg; Danhier, F.; Collodoro, M. et al

Poster (2011, June 16)

Detailed reference viewed: 75 (16 ULg)
See detailSTUDY OF SELENITE AND SELENOMETHIONINE EFFECT ON METHYLMERCURY IN VITRO TOXICITY
Dupont, Aurélie ULg; Siebert, Ursula; Rosenberger, Tanja et al

Conference (2011, May 16)

Methylmercury (MeHg) and selenium (Se) can be found at elevated concentrations in blood of marine mammals and both display modulatory effects on the immune system. Whereas mercury (Hg)-Se antagonism in ... [more ▼]

Methylmercury (MeHg) and selenium (Se) can be found at elevated concentrations in blood of marine mammals and both display modulatory effects on the immune system. Whereas mercury (Hg)-Se antagonism in liver of marine mammals is well known, the protective role of Se against Hg immunotoxicity in marine mammals has been poorly described. We propose here an in vitro approach using combined Hg and Se in vitro exposure of peripheral blood mononuclear cells (PBMCs) of the harbor seal (Phoca vitulina). PBMCs were isolated from the blood of 10 harbor seals and exposed to environmental concentrations of MeHg (1µM) and selenite or selenomethionine (5µM), respectively inorganic and organic forms of Se. MeHg leaded to a decrease of lymphocyte proliferation, to an increase of cells with compromised mitochondrial membrane potentials and cell death. Preliminary results evidenced that none of the two Se forms had a protective effect against MeHg toxicity, although cells were slightly stimulated by Se alone. Therefore MeHg expresses its toxicity among blood circulating lymphocytes in presence or absence of selenite or selenomethionine. [less ▲]

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See detailSynthesis and characterization of reversibly core cross-linked micelles sensitive to reductive environment
Cajot, Sébastien ULg; Danhier, F.; Collodoro, M. et al

Poster (2011, May 12)

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See detailHydrogel nanocomposites: a potential UV/blue light filtering material for ophthalmic lenses
Bozukova, Dimitriya; Pagnoulle, Christophe; De Pauw, Marie-Claire ULg et al

in Journal of Biomaterials Science. Polymer Edition (2011), 22

Poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (poly(HEMA-co-MMA)) and ZnS hydrogel nanocomposites were prepared and characterized. The chemical composition of the inorganic nanoparticles was ... [more ▼]

Poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (poly(HEMA-co-MMA)) and ZnS hydrogel nanocomposites were prepared and characterized. The chemical composition of the inorganic nanoparticles was confirmed by X-ray diffraction, and the homogeneity of their distribution within the hydrogel was assessed by transmission electron microscopy. The influence of the content of ZnS nanoparticles on the optical performances of the nanocomposites was investigated by UV-Vis spectroscopy. The ability of the hydrogel nanocomposites to filter the hazardous UV light and part of the blue light was reported, which makes them valuable candidates for ophthalmic lens application. In contrast to the optical properties, the thermo-mechanical properties of neat poly(HEMA-co-MMA) hydrogels were found to be largely independent of filling by ZnS nanoparticles ( 2 mg/ml co-monomer mixture). Finally, in vitro cell adhesion test with lens epithelial cells (LECs), extracted from porcine lens crystalline capsule, showed that ZnS had no deleterious effect on the biocompatibility of neat hydrogels, at least at low content. [less ▲]

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See detailNovel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis
Fleron, Maximilien ULg; Greffe, Yannick ULg; Musmeci, Davide ULg et al

in Journal of Proteomics (2010), 73(10), 1986-2005

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy ... [more ▼]

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins. [less ▲]

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See detailMALDI-In Source Decay Applied to Mass Spectrometry Imaging: A New Tool for Protein Identification.
Debois, Delphine ULg; Bertrand, Virginie ULg; Quinton, Loïc ULg et al

in Analytical Chemistry (2010), 82(10), 3969-4304

Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section ... [more ▼]

Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section, allowing, for example, the discovery of new pathological biomarkers. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-in source decay (ISD) is used to fragment ions directly in the mass spectrometer ion source. This technique does not require any special sample treatment but only the use of a specific MALDI matrix such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene. MALDI-ISD is generally employed on classical, purified samples, but here we demonstrate that ISD can also be performed directly on mixtures and on a tissue slice leading to fragment ions, allowing the identification of major proteins without any further treatment. On a porcine eye lens slice, de novo sequencing was even performed. Crystallins not yet referenced in databases were identified by sequence homology with other mammalian species. On a mouse brain slice, we demonstrate that results obtained with ISD are comparable and even better than those obtained with a classical in situ digestion. [less ▲]

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See detailMALDI MS Tissue Imaging of Crystallins using an original metyhod to direct protein identification on lens slices
Bertrand, Virginie ULg; Debois, Delphine ULg; Quinton, Loïc ULg et al

Poster (2010, April 16)

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins ... [more ▼]

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins in lens. They constitute 90% of water soluble proteins and contribute to its transparency and refractive properties by a uniform concentration gradient in the lens. Nevertheless, if these crystallins undergo post translational modifications, they become less soluble and the opacity of eye lens increases. This phenomenon defines cataract. Yet, the nature and the mechanism of occurring of these modifications and how they happen are not fully understood. MALDI mass spectrometry imaging is a recent technique allowing examining proteins in their native location without the need for traditional processing methods such as extraction, homogenization, and separation. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-In Source Decay (MALDI-ISD) is a fragmentation process occurring in the mass spectrometer ion source. When the analyzed sample is a protein, ISD fragmentation leads to b-, c- and z-ions series, which allows for some sequencing of the protein. One great advantage of ISD is its fastness and easiness to be implemented since there is no need for a special treatment of the sample. The only requirement is the use of “ISD-favourable MALDI matrix” such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphtalene. 18 µm-thick equatorial sections of frozen porcine eye lenses were realized with a cryostat. 1,5-DAN matrix was either manually deposited or sprayed with an ImagePrep automated device (Bruker Daltonics). Data were acquired with an UltraFlex II MALDI-TOF/TOF mass spectrometer (BD) in positive reflector mode. For imaging experiments, the surface of the sample was divided into 100-µm-wide pixels and 500 shots were averaged on each. Based on calculated mass differences between consecutive ISD fragments peaks, tags of amino acids were established and submitted to a search in protein databases using a BLAST algorithm (search by sequence homology). Imaging experiments showed that the localization information may be very useful to associate fragments which exhibit close distributions, suggesting they are originating from the same protein. It is thus possible to arrange fragments in groups of probable origin and to extract the mass spectrum of a high-intensity pixel. This allows to work with a “purified” ISD mass spectrum where fragments of only one protein are present and potentially exhibiting a higher number of peaks, leading to a longer tag and to an easier identification. With this imaging strategy, we were able to identify (by homology) the Beta-Crystallins S and B2, the Gamma-Crystallin B, the Alpha-Crystallin A. [less ▲]

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