Lymphatic Transport and Lymph Node Location of Microspheres Subcutaneously Injected in the Vicinity of Tumors in a Rabbit Model of Breast Cancer.

PURPOSE: To assess the lymphatic transport of microparticles of 100 nm, 1 µm and 10 µm subcutaneously injected into the breast area of healthy and tumor-bearing rabbits, and to analyze their location in lymph node (LN) in relation to malignant cells. METHODS: Female rabbits (n = 9) bearing a VX2 tumor in one thoracic mammary gland were subcutaneously injected at D15 with polystyrene fluorescent particles around the nipple, on the tumor and on the healthy sides. The tumor and the LN measured by ultrasound at D9, D15 and D20 were explanted at D20. The LN metastases were evaluated by cytokeratin staining. LN uptake of the particles was measured by quantifying the green fluorescence surface in hot spot regions of healthy and pathologic LN. RESULTS: All animals developed mammary tumors. Metastases were found in 39% of LN from the tumor side. LN invasion was significantly lower for the 10 µm group versus the 100 nm group (p < 0.0348). The fully invaded area of metastatic LN contained significantly less 100 nm and 1 µm particles compared to the low and non-invaded regions and to the healthy LN. In the invaded LN, the 1 µm MS occupied more surface than the 100 nm particles. CONCLUSIONS: 1 µm MS arrived numerously into the areas low-invaded and non-invaded by the tumoral cells of the pathologic LN, but they were very rare in the fully invaded regions. Compared to the 100 nm nanospheres, the 1 µm were better retained (20 times) into the sentinel LN, showing the advantage of micrometric particles for lymph-targeted chemotherapy when injected before complete invasion by metastases.

Tunable delivery of niflumic acid from resorbable embolization microspheres for uterine fibroid embolization.

Uterine arteries embolization (UAE) is a recent technique that aims, by means of particles injected percutaneously, to stifle fibroids (leiomyomas). This treatment is non-invasive, compared with uterine ablation, but generates pelvic pain for a few days. A strategy to reduce the post-embolization pain would be to use calibrated embolization microspheres preloaded with a non-steroidal inflammatory drug (NSAID). In this study, we first compared four drugs, all active at low concentration on cyclooxygenase-2, i.e. ketoprofen, sodium diclofenac, flurbiprofen and niflumic acid (NFA), for their capacity to be loaded on resorbable embolization microspheres (REM) 500-700µm. NFA had the highest capacity of loading (5mg/mL) on resorbable microspheres. Then, we evaluated in vitro the NFA release profiles from REM having various degradation times of one, two or five days. NFA release was biphasic, with an initial burst (about 60% of the loading) followed by a sustained release that correlated significantly to REM's hydrolysis (rho=0.761, p<0.0001). For each group of beads, the size distribution was not modified by the loading of NFA and their delivery through microcatheter was not impaired by the drug. NFA eluted from REM inhibited the synthesis of prostaglandin E2 from rabbit uterus explants. In summary, NFA is loadable on REM in significant amount and its delivery can be tuned according to the degradation rate of REM to provide an antalgic effect for a few days after UAE.

bFGF Up-regulation Reduces Spontaneous Necrosis of VX2 Tumors Without Increasing Tumoral Microvascular Density.

AIM: To determine whether up-regulation of basic fibroblast growth factor (bFGF) in VX2 cells reduces tumor necrosis. MATERIALS AND METHODS: VX2 cells were transfected with expression vector containing cDNA of rabbit bFGF. Stable clones producing rabbit bFGF (bFGF-VX2) were selected. bFGF-VX2 (n=5) or non-transfected VX2 (control) (n=5) cells were implanted into leg muscle of 10 rabbits. The tumors were characterized 21 days after grafting. RESULTS: Overexpression of bFGF by VX2 tumors significantly reduced necrosis (p<0.0223) and increased cell viability (p<0.0223), without effect on the mean vascular density. bFGF concentration was significantly higher in bFGF-VX2 tumors (p<0.0062) and negatively correlated with tumor volume at day 21 (ρ=-0.927, p<0.0034). Vascular endothelial growth factor concentration was significantly lower in bFGF-VX2 tumors (p<0.0105) and negatively correlated with the bFGF concentration of tumors (ρ=-0.903, p<0.0067). CONCLUSION: The overexpression of bFGF in VX2 cells increased tumor viability and reduced necrosis, making the evaluation of long-term anticancer therapies possible in this model.

Comparative Chemosensitivity of VX2 and HCC Cell Lines to Drugs Used in TACE.

AIM: To compare the cytotoxic effects of 11 anticancer agents against VX2 and HepG2 cells in order to establish candidate drugs that can be tested preclinically on VX2 tumor model for transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: VX2 and HepG2 cells were incubated with different drug concentrations. The half-maximal inhibitory concentration (IC50) values were determined by total cell protein assay for anthracyclines, platins, irinotecan, mytomicin-C (MMC), 5-fluorouracil (5-FU) and antiangiogenics. RESULTS: IC50 values for VX2 and HepG2 were found close for doxorubicin (0.8 µM vs. 1.1 µM), MMC (13.9 µM vs. 8.7 µM), sunitinib (32.7 vs. 33.7 µM), sorafenib (10.3 vs. 8.9 µM), lapatinib (30 vs. 18.3 µM) and different for platins and irinotecan. Oxaliplatin was less active against VX2 than HepG2 (IC50=41 µM vs. 2.7 µM), cisplatin was more active against VX2 than HepG2 (IC50=8.0 µM vs. 15.9 µM), whereas carboplatin had a low toxicity against both cell lines (70.4 µM vs. 538.3 µM). The toxicity of 5-FU against VX2 and HepG2 was low (IC50=560.6 µM vs. 323.2 µM). Irinotecan was less active against VX2 vs. HepG2 (IC50=44.5 µM vs. 15.3 µM). Bevacizumab had no effect on either of the cell lines up to 6.7 µM. CONCLUSION: Drugs recommended for pre-clinical trials of TACE in the VX2 model are doxorubicin, sunitinib, sorafenib, MMC, lapatinib and 5-FU.

Anti-angiogenic drug delivery from hydrophilic resorbable embolization microspheres: an in vitro study with sunitinib and bevacizumab.

Anti-angiogenic (AA) drugs are proposed as novel agents for targeted therapies in hepatocellular carcinoma (HCC). Loading of AA drugs into drug delivery systems for local delivery would reduce their side effects. The present study investigated the loading and the delivery of two AA drugs, sunitinib and bevacizumab, from one day-resorbable embolization microspheres (REM). REM were prepared with 10 or 20% of methacrylic acid (MA) as active drug binding monomer. Sterilized beads (100-300 µm) were analyzed for cytotoxicity, AA loading and in vitro release. REM modified with MA were not cytotoxic and extemporaneous drug loading was significantly higher on REM containing 20% of MA. The drug release in saline buffer was sustained for several hours before complete REM degradation. MA content had low effect on drug release profile. When eluted from REM, sunitinib and bevacizumab reduced viability of tumoral VX2 cells, and proliferation of human endothelial cells, respectively. Deliverability of REM via microcatheter was not impaired by the loaded drugs. As conclusion, the loading values of sunitinib and bevacizumab on REM were close to those achieved for cytotoxic drugs onto non-degradable MS used in chemoembolization of HCC. Transcatheter delivery to liver tumors of anti-angiogenics could be achieved with REM.

In vitro evaluation of S-(+)-ibuprofen as drug candidate for intra-articular drug delivery system.

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the efficacy of S-(+)-ibuprofen on cartilage degradation as drug candidate for DDS loading. Humeral cartilage and joint capsule explants were collected from healthy sheep shoulder joints and they were cultured in mono- or in co-culture for 13 days with LPS in combination with S-(+)-ibuprofen at 50 µM and 1 mM. S-(+)-ibuprofen (50 µM) blocked prostaglandins production in LPS-activated explants but did not reduce cartilage degradation. By contrast, 1 mM S-(+)-ibuprofen treatment of cartilage explants reduced nitric oxide synthesis by 51% (p = 0.0072), proteoglycans degradation by 35% (p = 0.0114) and expression of serum amyloid protein - the main protein induced upon LPS challenge - by 44% (p < 0.0001). On contrary, in presence of synovial membrane, the protective effects of S-(+)-ibuprofen on cartilage damages were significantly diminished. At 1mM, S-(+)-ibuprofen reduced the cell lysis during culture of cartilage and joint capsule either in mono- or in co-culture. This study performed on sheep explants shows that 1 mM S-(+)-ibuprofen inhibited cartilage degradation via a mechanism independent of cyclooxygenase inhibition. Reduction of prostaglandins synthesis at 50 µM in all treatment groups and reduction of cartilage degradation observed at 1 mM suggest that S-(+)-ibuprofen could be considered as a promising drug candidate for the loading of intra-articular DDS.

A novel resorbable embolization microsphere for transient uterine artery occlusion: a comparative study with trisacryl-gelatin microspheres in the sheep model.

PURPOSE: To evaluate angiographic recanalization, inflammatory reaction, and uterine damage after sheep uterine artery embolization (UAE) with a novel calibrated resorbable embolization microsphere (REM) and compare the results with control nonresorbable microspheres. MATERIALS AND METHODS: Six hormonally artificially cycled sheep underwent bilateral UAE until stasis with either REM or trisacryl-gelatin microspheres (TGMS). At 7 days, control angiograms were obtained to assess the residual vascularization at arterial and parenchymal phases. The animals were then sacrificed for analysis of the presence of microspheres, inflammatory foreign body reaction, and surface areas of uterine damage. RESULTS: Mean volume of microspheres injected per uterine artery (UA) or per animal did not differ between groups. At day 7, the flow was normal for six of six UAs that received embolization with REM versus only three of six UAs with TGMS (P = .0455, χ(2) test). Uterine parenchymography showed no defects in six UAs in the REM group versus five defects in six UAs in the TGMS group (P = .0060, χ(2) test). No REM or residual fragments of microspheres were observed on histologic analysis. TGMS were observed in tissues and accompanied by a mild inflammatory response. Necrosis rates were not significantly different between the two products, either in endometrium (REM 23.5% ± 28.8% [median 8.1%] vs TGMS 21.8% ± 23.7% [median 14.6%]) or in myometrium (REM 8.2% ± 22.7% [median 0.0%] vs TGMS 8.8% ± 20.8% [median 0.9%]). Endometrium alteration rate was lower with REM than with TGMS (39.7% ± 25.7% [median 34%] vs 60.6% ± 27.1% [median 71%]; P = .0060, Mann-Whitney test). Myometrium alteration rates were not significantly different between REM (45.7% ± 37.1% [median 63.0%]) and TGMS (37.8% ± 34.0% [median 19.1%]). CONCLUSIONS: At 1 week after sheep UAE with REM, the recanalization was complete, the microspheres were completely degraded, and there was no remnant inflammatory response.

Synthesis of hydrophilic intra-articular microspheres conjugated to ibuprofen and evaluation of anti-inflammatory activity on articular explants.

The main limitation of current microspheres for intra-articular delivery of non-steroidal anti-inflammatory drugs (NSAIDs) is a significant initial burst release, which prevents a long-term drug delivery. In order to get a sustained delivery of NSAIDs without burst, hydrogel degradable microspheres were prepared by co-polymerization of a methacrylic derivative of ibuprofen with oligo(ethylene-glycol) methacrylate and poly(PLGA-PEG) dimethacrylate as degradable crosslinker. Microspheres (40-100 µm) gave a low yield of ibuprofen release in saline buffer (≈2% after 3 months). Mass spectrometry analysis confirmed that intact ibuprofen was regenerated indicating that ester hydrolysis occurred at the carboxylic acid position of ibuprofen. Dialysis of release medium followed by alkaline hydrolysis show that in saline buffer ester hydrolysis occurred at other positions in the polymer matrix leading to the release of water-soluble polymers (>6-8000 Da) conjugated with ibuprofen showing that degradation and drug release are simultaneous. By considering the free and conjugated ibuprofen, 13% of the drug is released in 3 months. In vitro, ibuprofen-loaded MS inhibited the synthesis of prostaglandin E2 in articular cartilage and capsule explants challenged with lipopolysaccharides. Covalent attachment of ibuprofen to PEG-hydrogel MS suppresses the burst release and allows a slow drug delivery for months and the cyclooxygenase-inhibition property of regenerated ibuprofen is preserved.

Poly(ethylene glycol) methacrylate hydrolyzable microspheres for transient vascular embolization.

Poly(ethylene glycol) methacrylate (PEGMA) hydrolyzable microspheres intended for biomedical applications were readily prepared from poly(lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-PLGA crosslinker and PEGMA as a monomer using a suspension polymerization process. Additional co-monomers, methacrylic acid and 2-methylene-1,3-dioxepane (MDO), were incorporated into the initial formulation to improve the properties of the microspheres. All synthesized microspheres were spherical in shape, calibrated in the 300-500 µm range, swelled in phosphate-buffered saline (PBS) and easily injectable through a microcatheter. Hydrolytic degradation experiments performed in PBS at 37 °C showed that all of the formulations tested were totally degraded in less than 2 days. The resulting degradation products were a mixture of low-molecular-weight compounds (PEG, lactic and glycolic acids) and water-soluble polymethacrylate chains having molecular weights below the threshold for renal filtration of 50 kg mol(-1) for the microspheres containing MDO. Both the microspheres and the degradation products were determined to exhibit minimal cytotoxicity against L929 fibroblasts. Additionally, in vivo implantation in a subcutaneous rabbit model supported the in vitro results of a rapid degradation rate of microspheres and provided only a mild and transient inflammatory reaction comparable to that of the control group.

Soil calcium availability influences shell ecophenotype formation in the sub-antarctic land snail, Notodiscus hookeri.

Ecophenotypes reflect local matches between organisms and their environment, and show plasticity across generations in response to current living conditions. Plastic responses in shell morphology and shell growth have been widely studied in gastropods and are often related to environmental calcium availability, which influences shell biomineralisation. To date, all of these studies have overlooked micro-scale structure of the shell, in addition to how it is related to species responses in the context of environmental pressure. This study is the first to demonstrate that environmental factors induce a bi-modal variation in the shell micro-scale structure of a land gastropod. Notodiscus hookeri is the only native land snail present in the Crozet Archipelago (sub-Antarctic region). The adults have evolved into two ecophenotypes, which are referred to here as MS (mineral shell) and OS (organic shell). The MS-ecophenotype is characterised by a thick mineralised shell. It is primarily distributed along the coastline, and could be associated to the presence of exchangeable calcium in the clay minerals of the soils. The Os-ecophenotype is characterised by a thin organic shell. It is primarily distributed at high altitudes in the mesic and xeric fell-fields in soils with large particles that lack clay and exchangeable calcium. Snails of the Os-ecophenotype are characterised by thinner and larger shell sizes compared to snails of the MS-ecophenotype, indicating a trade-off between mineral thickness and shell size. This pattern increased along a temporal scale; whereby, older adult snails were more clearly separated into two clusters compared to the younger adult snails. The prevalence of glycine-rich proteins in the organic shell layer of N. hookeri, along with the absence of chitin, differs to the organic scaffolds of molluscan biominerals. The present study provides new insights for testing the adaptive value of phenotypic plasticity in response to spatial and temporal environmental variations.

Intra-articular fate of degradable poly(ethyleneglycol)-hydrogel microspheres as carriers for sustained drug delivery.

A novel degradable microsphere (MS) for intra-articular drug delivery, composed of a polyethylene glycol (PEG) core containing degradable regions made of short poly-(lactic-co-glycolic acid) (PLGA) sequences - named PEG-hydrogel MS - was injected into the cavity of sheep shoulder joint, and compared to non-degradable MS devoid of hydrolysable crosslinker in terms of location, degradation and inflammation. One week after intra-articular injection both groups of MS were localized beneath the synovial lining of the synovial fringes located at bottom of the shoulder joint, while a fraction of particles remained in synovial fluid. Histological analyses made one and 4 weeks after intra-articular injection showed cell proliferation around the non-degradable MS entrapped within the synovium. By contrast, degradable PEG-hydrogel MS were surrounded by few cells. The degradation of degradable PEG-hydrogel MS within the synovium was slow and was not fully complete after four weeks. Our findings indicate that the tissue entrapment of MS below the synovial lining was independent of the material degradability, while degradable PEG-hydrogel MS are less inflammatory than the non-degradable one. Degradable PEG-hydrogel MS offer several advantages over the non-degradable MS as carriers for a sustained drug delivery in synovial tissue according to the low intensity of inflammatory reaction triggered in synovium.

What is the difference in organic matrix of aragonite vs. vaterite polymorph in natural shell and pearl? Study of the pearl-forming freshwater bivalve mollusc Hyriopsis cumingii.

Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140 kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55 kDa components, including a discrete band in vaterite extract.

Targeting and recanalization after embolization with calibrated resorbable microspheres versus hand-cut gelatin sponge particles in a porcine kidney model.

PURPOSE: To report on polyethylene glycol hydrogel-based resorbable embolization microspheres (REM) that were synthesized to resorb in < 24 hours, before inflammation and vascular remodeling, to achieve a complete arterial recanalization and to compare targeting and recanalization of REM of 300-500 µm, 500-700 µm, and 700-900 µm with hand-cut gelatin sponge particles (GSP). MATERIALS AND METHODS: Eight pigs underwent polar renal artery embolization with REM or GSP. Angiograms were obtained before embolization and 10 minutes and 7 days after embolization before pigs were sacrificed to determine the occlusion level, the percentage of occlusion, and the recanalization rate for each product. The distribution of embolic material was assessed in pathology, and infarction rate of the kidneys was measured. RESULTS: REM of 300-500 µm occluded more distal vessels than REM of 500-700 µm and 700-900 µm. At day 7, the recanalization rate was complete for the larger REM, whereas it was about 60% for the two smaller sizes. REM were completely degraded, with no residual material or inflammation. GSP occluded more proximal arteries than REM of 700-900 µm, were partly degraded at day 7, and were accompanied by a foreign body reaction in proximal and distal arteries. GSP recanalized at 79%. The infarction rate was higher with the two smaller sizes of REM and with GSP than with the largest REM. CONCLUSIONS: REM of different sizes targeted different occlusion levels in kidney arteries. GSP provided an extended occlusion level without actual targeting. Regardless of embolic material used, angiographic recanalization of renal arteries depended on the extent of necrosis. REM of 700-900 µm demonstrated the lowest infarction rate and the best recanalization rate.

Proteomic and profile analysis of the proteins laced with aragonite and vaterite in the freshwater mussel Hyriopsis cumingii shell biominerals.

Hyriopsis cumingii (Lea, Unionidae), a freshwater bivalve species widely distributed in China and commercially exploited for freshwater pearl production, was chosen as the reference model to investigate the protein signature in the organic scaffold matching calcium carbonate crystallization mode. This study takes advantage of different calcium carbonate habits production by the organism: aragonite in shell and pearl and vaterite in alternative pearl formation. Amino acid global composition and proteomics analysis have been undertaken to study the amino acid imbalance with respect to biominerals and microstructures. Forty peptides sequences were obtained by proteomics, of which ten are shared by all the different samples, nine are laced with aragonite; another nine with vaterite and twelve are related to pearls. Bioinformatics analysis allowed the peptides to be matched to the deduced protein sequences from EST databases and allowed functional assignment (e.g. scaffolding, strain strength, chitin binding or carbonic anhydrase function) to the proteins found in the different materials. Such panel of motifs tailored in vaterite and aragonite habits produced in a freshwater mollusk gives food for thought about organic control of the biomineralization processes.

Analysis of low complex region peptides derived from mollusk shell matrix proteins using CID, high-energy collisional dissociation, and electron transfer dissociation on an LTQ-orbitrap: implications for peptide to spectrum match.

Identification of proteins involved in mollusk biomineralization by proteomics approach is gaining importance. These proteins are often characterized by low-complexity regions (LCRs) made of repeating motifs that are constituted by few amino acids (e.g. IGG, DD, KK, and GGG). In this work, we have analyzed the fragmentation of model LCR peptides under different fragmentation regimes (CID, high-energy collisional dissociation [HCD], and electron transfer dissociation [ETD]) and its consequences on peptide to spectrum matches (PSMs) using two search algorithms (Mascot and PEAKS DB). For both search tools, higher number of PSMs was obtained using CID spectra, followed by HCD and ETD. Intense fragment ions present in the lower m/z region of HCD led to lower PSM scores and absence of low mass cut off seems to offer little advantage for the identification of LCR peptides. Generally, doubly charged peptides under ETD conditions did not fragment to yield sequence information rich spectra. The spectral quality is affected by the nature of the repeating motifs in the peptide. The performance of both Mascot and PEAKS DB (de novo based search tool) vary according to the fragment regime employed to acquire MS/MS spectra.

Modified model of VX2 tumor overexpressing vascular endothelial growth factor.

PURPOSE: To determine whether upregulated expression of vascular endothelial growth factor (VEGF) in VX2 cells can increase vessel density (VD) and reduce tumor necrosis. MATERIALS AND METHODS: The VX2 cell line was transfected with expression vectors containing cDNA for rabbit VEGF. Stable clones producing rabbit VEGF (VEGF-VX2) were selected. VEGF-VX2 cells (n = 5 rabbits) or nontransfected VX2 cells (controls; n = 5 rabbits) were implanted into leg muscle of 10 rabbits. The animals were sacrificed at day 21. Tumor volume, percentage of necrosis, VD, and VEGF concentration in tumor protein extract were quantified. RESULTS: Overexpression of VEGF by VX2 cells augmented tumor implantation efficiency 100% and favored cyst formation. The tumor volume was significantly larger for VEGF-VX2 transfected tumors versus controls (P = .0143). Overexpression of VEGF in VX2 cells significantly increased the VD of the tumors (P = .0138). The percentage of necrosis was reduced in VEGF-VX2 tumors versus controls (19.5% vs 38.5 %; P = .002). VEGF concentration in VEGF-VX2 tumors was significantly higher than in control tumors (P = .041) and was correlated with tumor volume (ρ = .883, P = .012). CONCLUSIONS: The overexpression of VEGF increased tumor growth and vascularization, favored cyst formation, and reduced tumor necrosis. This new phenotype of the VX2 tumor may offer some advantages over classic models of VX2 tumor for evaluating anticancer therapies.

Proteomic strategy for identifying mollusc shell proteins using mild chemical degradation and trypsin digestion of insoluble organic shell matrix: a pilot study on Haliotis tuberculata.

A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins.

In vitro evaluation of (S)-ibuprofen toxicity on joint cells and explants of cartilage and synovial membrane.

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the toxicity of (S)-ibuprofen on chondrocytes and synoviocytes isolated from sheep shoulder joint and cultured in monolayers during 72 h, and on joint explants (cartilage and capsule) cultured in mono- or in co-culture for 13 days. (S)-ibuprofen (5 µM up to 1 mM) did not reduce the cell viability and protein content when added on chondrocyte monolayers, while at 1 mM (S)-ibuprofen reduced (by 8%, p=0.01) the synoviocytes viability compared to untreated cells. During co-culture of joint explants, (S)-ibuprofen at 50 µM significantly reduced by 35% the spontaneous release of glycosaminoglycans (GAGs) from cartilage (p=0.0065) whereas in monoculture, (S)-ibuprofen was inactive on GAG metabolism. (S)-ibuprofen at 1 mM significantly reduced cell lysis (lactate dehydrogenase leakage) by 74% during monoculture of capsule explants (p=0.0136) and by 35% during co-culture of explants (p=0.0013). Our findings demonstrate that the active isomer of ibuprofen at micro- and millimolar levels was not toxic for chondrocytes and synoviocytes and may reduce at 1mM the cell lysis during culture of joint explants. The limited toxicity of (S)-ibuprofen at low and high concentration in sheep joint shoulder makes this enantiomer a promising drug candidate for the loading of intra-articular DDS.

Coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins: a study with P. margaritifera.

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

IL6 and TNF expression in vessels and surrounding tissues after embolization with ibuprofen-loaded beads confirms diffusion of ibuprofen.

PURPOSE: In the treatment of uterine fibroid embolization related pain, the use of embolics loaded with non-steroidal anti-inflammatory drugs (NSAID) relies on an efficient delivery and impregnation of the embolized tissue. Immuno-labelling and spectroscopic techniques have demonstrated the release of ibuprofen from drug eluting beads (Wassef et al., 2008; Namur et al., 2009) but failed to demonstrate diffusion of the drug beyond the vascular wall (VW). We investigated whether ibuprofen diffused beyond the VW in surrounding tissues (ST), by tracking its biological effects through the modulation of expression of two main inflammatory cytokines. MATERIALS AND METHODS: Uterine arteries of 6 sheep were embolized with ibuprofen loaded beads (IBU-BB) or non-loaded beads (BB) and sacrificed at one week. On frozen tissue slices, VWs of occluded arteries were isolated from ST using laser capture microdissection. RNA was extracted from VW and ST samples. Gene expression of IL6 and TNFα genes was measured by quantitative real-time PCR (qPCR). RESULTS: IL6 expression was significantly increased in IBU-BB compared to BB group both in VW (VW: fold-change (FC)=4.9, p=0.0009) and ST (ST: FC=8.7, p=0.0003). In IBU-BB, IL6 was significantly more expressed in VW than in ST (FC=4.4; p=0.0009). TNFα expression was not significantly different between IBU-BB and BB groups. CONCLUSION: Using qPCR+microdissection was useful to evaluate the spread of the biological effects of drug-loaded systems which attest of the tissular release. This approach can be considered when other drug detection techniques are unsuccessful or difficult to achieve. IL6 can be used as a marker of ibuprofen released by drug eluting beads in uterus. Gradient of expression of IL6 suggests diffusion of ibuprofen across the VW into the ST.