Biomedical subjects

C Vigneron

Publications and source records attributed to C Vigneron.

At least 19 recordsLinked to original sources

PubMed2001

Protein C-loaded monomethoxypoly (ethylene oxide)-poly(lactic acid) nanoparticles.

This paper deals with the preparation and characterization of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO-PLA) nanoparticles containing protein C, a plasma inhibitor which regulates the mechanism of blood coagulation. Protein C was entrapped in MPEO-PLA nanoparticles using the double emulsion method. The influence of MPEO-PLA copolymers on the different parameters was evaluated: characteristics of protein C-loaded nanoparticles, in vitro release of the protein, evolution of the particle size with incubation time and MPEO release. The nanoparticle size does not depend on copolymer characteristics (MPEO and/or PLA block molecular weight). On the other hand, the efficiency of protein C entrapment is affected by the copolymer characteristics. The burst effect during the protein C release is increased with the hydrophilic character of the copolymer. Moreover, protein C adsorption on the particle surface during its release may be related to MPEO release. Only 25% of the released protein C is active, which clearly illustrates that it is altered during its encapsulation. The optimization of the experimental parameters which disturbed entrapped protein C activity, i.e. sonication time and organic solvent was investigated and has led to a preservation of protein C activity. Then, to optimize its entrapment efficiency, a blend PLA/MPEO-PLA (25/75) was used to prepare nanoparticles. This blend limited burst effect of protein C and its adsorption. However, protein C is only partially released which implicates further investigation for a potential therapeutic use.

Lactates↗

PubMed2001

Effects of three Hb-based oxygen-carrying solutions on neutrophil activation in vitro: quantitative measurement of the expression of adherence receptors.

BACKGROUND: Hb-based oxygen carriers (HbOCs) are currently under investigation as RBC substitutes. These solutions have vasoactive properties and may modify the behavior of the RBC in vitro. The effects of these agents on other blood cells have not been extensively investigated. The aim of this study was to evaluate the potential effect(s) of three differently modified HbOCs on the activation of PMNs. STUDY DESIGN AND METHODS: Whole blood from humans was incubated with Hb dextran-benzene-tetracarboxylate, alphaalpha-Hb, or o-raffinose-poly-Hb for 15, 30, and 60 minutes. The expression of adherence receptors CD62L, CD18, and CD11b, which reflects the activation state of the neutrophils, was assessed in vitro by a direct immunofluorescence method using calibration beads measured by flow cytometry. RESULTS: The observed expression of each of the three adherence receptors for each solution at each time point was similar to that noted with the RPMI control. Decreases in CD62L and increases in CD18 expression were noted at 15 minutes of incubation but, when those values were compared to the values obtained with TNFalpha as the positive control of PMN activation, the profile of expression of adherence receptors obtained with HbOC solutions did not conform to the expected activation profile of PMNs. CONCLUSION: Hb solutions modified by a variety of methods, as obtained and employed in this study, did not appear to activate neutrophils in vitro.

Aspirin↗

PubMed2000

Involvement of neutrophilic granulocytes in the uptake of biodegradable non-stealth and stealth nanoparticles in guinea pig.

The in vivo behavior of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO20-PLA45/PLA (75/25)) nanoparticles in comparison with PLA ones was studied in guinea pig. Indeed, the aim of this study was to bring to the fore the in vivo stealth character of these copolymer nanoparticles and to identify the phagocytic circulating cells involved in their uptake. After the intravascular administration of fluorescent nanoparticles (rubrene), their phagocytosis by granulocytes and monocytes was assayed by flow cytometry. At the same time, the evolution of the number of these phagocytic cells was realized in order to identify their function in the nanoparticle uptake. Finally, a histological study of the spleen (30 h after the nanoparticle administration) was investigated to highlight the splenic trapping of these stealth nanoparticles. This study has shown that the phagocytic circulating cells involved in the nanoparticle uptake were mainly neutrophilic granulocytes and some of them were found in the spleen.

Animals↗

PubMed2000

Biosynthesis and photodynamic efficacy of protoporphyrin IX (PpIX) generated by 5-aminolevulinic acid (ALA) or its hexylester (hALA) in rat bladder carcinoma cells.

Hexylester of 5-aminolevulinic acid (hALA) has been considered as an alternative to 5-aminolevulinic acid (ALA) for the treatment of malignancies of different origin. The present study addresses the ALA and hALA-induced PpIX pharmacokinetic profile using rat bladder carcinoma cells (AY27). The total PpIX content measured spectrofluorimetrically after extraction procedure at the end of 2 h incubation was at least 1.5-fold greater with hALA compared to ALA despite the difference in concentration of several orders between the two compounds (1 or 5 mM ALA and 5 or 10 x 10(-3) mM hALA). Considerable PpIX efflux was detected in the extracellular medium at the end of the incubation. With 5 mM ALA and 10 x 10(-3) mM hALA, PpIX build-up was continued beyond the incubation period pointing out to enzyme saturation in the biosynthetic pathway or/and the constitution of ALA reserve. Red laser light (lambda=630 nm) irradiation of AY27 cells after 2 h incubation with increasing ALA or hALA concentrations resulted in a nearly equal photocytotoxicity.

Aminolevulinic Acid↗

PubMed2000

Determination of the maximal carcinoma/normal skin ratio after HpD or m-THPC administration in Hairless mice (SKH-1) by fluorescence spectroscopy.

The two major steps in our study on the treatment of skin carcinomas by photochemotherapy (PCT) were the development of a skin tumor model in Hairless mice by a chemical carcinogenesis and the use of fluorescence spectroscopy, a semi-quantitative and non-invasive method, in order to determine the time after i.p. injection of photosensitizer when the tumor/normal skin ratio was the highest. A three-step carcinogenesis protocol provided mice bearing carcinomas and these were used to determine the tumor/normal skin ratios of two photosensitizers by fluorescence spectroscopy. Hematoporphyrin derivative (HpD) (5 mg/kg body weight) and m-tetra(hydroxyphenyl) chlorine (m-THPC) (0.3 mg/kg body weight) were injected i.p., and fluorescence was measured at 1, 4, 8, 12, 24, 48, 72 and 96 h after injection. The best carcinoma/normal skin ratio would be 3.2+/-1.4 for HpD and 2.7+/-2.1 for m-THPC, respectively. The delays required to reach these ratios were 72 h for HpD and 24 h for m-THPC. These results have to be considered with caution due to the high SEs and they must be confirmed by organic extraction. Photodynamic therapy with the same doses of HpD and m-THPC used in this pharmacokinetic study has to be carried out in order to compare the toxicities of the two photosensitizers and to determine which one is the best for this type of tumor.

Animals↗

PubMed2000

Systemic and renal hemodynamics after moderate hemodilution with HbOCs in anesthetized rabbits.

Hb-based O(2)-carrying solutions (HbOCs) have been developed as red blood cell substitutes for use in patients undergoing hemodilution. Variously modified Hb with diverse solution properties have been shown to produce variable hemodynamic responses. We examined, through pulsed-Doppler velocimetry, the systemic and renal hemodynamic effects of dextran-benzene-tetracarboxylate-conjugated (Hb-Dex-BTC), bis(3,5-dibromosalicyl)fumarate cross-linked (alphaalpha-Hb), and o-raffinose-polymerized (o-raffinose-Hb) Hb perfused in rabbits after moderate hemodilution (30% hematocrit), and we compared the effects of these Hb solutions with the effects elicited by plasma volume expanders. In addition, vascular hindrance (resistance/blood viscosity at 128.5 s(-1)) was calculated to determine whether a moderate decrease in the viscosity of blood mixed with HbOCs may impair vasoconstriction as a result of autoregulation after infusion of cell-free Hb. No changes were observed in renal hemodynamics after hemodilution with reference or Hb solutions. Increase in blood pressure and vascular resistance was found with Hb-Dex-BTC and alphaalpha-Hb (for 180 min) and, to a lesser extent, with o-raffinose-Hb (for 120 min). Furthermore, Hb-Dex-BTC (high viscosity) and o-raffinose-Hb (medium viscosity) induced comparable increases in vascular hindrance (from 0.091 to 0. 159 and from 0.092 to 0.162 cm(-1), respectively) but far less than that produced by alphaalpha-Hb (low viscosity, from 0.092 to 0.200 cm(-1)). These results suggest that maintaining the viscosity of blood by infusing solutions with high viscosity makes it possible to limit vasoconstriction due to autoregulation mechanisms and mainly caused by hemodilution per se.

Animals↗

PubMed1999

Preparation and characterization of protein C-loaded PLA nanoparticles.

This paper deals with the preparation and the characterization of poly(lactic acid) (PLA) nanoparticles containing protein C, a plasma inhibitor. Nanoparticles were prepared by the double emulsion method (w/o/w), using methylene chloride as an organic solvent and polyvinyl alcohol (PVA) or human serum albumin (HSA) as a surfactant. The influence of experimental constraints such as sonication and organic solvent on protein C activity was evaluated. It appears that a short time of sonication as well as the addition of acetone to methylene chloride (1/1) limited the lost of protein C activity. The study of protein C adsorption on blank PLA nanoparticles gave evidence to hydrophobic interactions between these two entities. The increase in PLA molecular weight on the characteristics of the protein C-loaded nanoparticles led to both a slightly decreased particle size and a lower polydispersity index, whereas the entrapment efficiency of protein C was not affected. The use of HSA as a surfactant allowed the increase in the entrapment efficiency of protein C but prevented its release. Finally, the evaluation of the activity of released protein C clearly illustrates that it was disturbed during the nanoparticle preparation. Thus, the obtained results emphasize the potential of protein C-loaded biodegradable nanoparticles for protein progressive delivery in plasma.

Anticoagulants↗

PubMed1999

MPEO-PLA nanoparticles: effect of MPEO content on some of their surface properties.

Nanoparticles of poly(lactic acid) (PLA) and of blends of PLA and monomethoxypoly(ethylene oxyde) (MPEO-PLA) were prepared by the double emulsion method. Sucrose was added to overcome nanoparticle aggregation with freeze-drying. Whereas PLA nanoparticles rapidly are phagocytosed by the mononuclear phagocyte system cells, the uptake of MPEO-PLA nanoparticles is delayed. Opsonization is one of the steps of phagocytosis, and serum complement is a major component of the opsonin system. The in vitro complement consumption of the prepared nanoparticles was evaluated as a function of time and of their surface area. To avoid the aggregation of MPEO-PLA nanoparticles due to MPEO crystallization, sucrose was necessary, and its concentration was dependent on MPEO proportion in the nanoparticle suspension. As expected, the complement consumption for PLA nanoparticles is faster and more important than for PLA/MPEO-PLA blends. The complement consumption decreases with the increase in MPEO surface density. Complement is less consumed with the lower surface density provided by a higher molecular weight than by the higher surface density provided by a lower molecular weight MPEO. The complement consumption seems to be dependent on the number of nanoparticles in contact with human serum. Finally, the model of steric repulsion of MPEO towards proteins and the importance of the surface density of MPEO were emphasized.

Biocompatible Materials↗

PubMed1999

Rheological behaviour of red blood cells suspended in hemoglobin solutions. In vitro study comparing dextran-benzene-tetra-carboxylate hemoglobin, stroma free hemoglobin and plasma expanders.

Circulating volume expansion for intentional hemodilution and/or resuscitation of hemorrhagic shock can be performed with hemoglobin-based oxygen carriers (HBOC) which, in addition to oxygen transport, have vasoactive effects through poorly documented mechanisms. Among these, the effects of HBOC on red blood cell (RBC) rheology are relatively unknown. The aim of the present in vitro study was to measure the rheological effects of human hemoglobin bound to benzene-tetracarboxylate substituted dextran (Dex-BTC-Hb) as an example of chemically modified hemoglobin. The viscosity was assessed with a capillary and a rotational viscometer for shear rates of 0.5-128 s-1. Erythrocyte aggregation was determined by analysis of the red light backscattered in a RBC suspension and with a rheoscope. The deformability was determined by the pressure-flow relationship of the RBC suspensions passed through polycarbonate filters. At hematocrit of 0.35 l/l and at low shear rates, the viscosity of RBC was higher in the presence of Dex-BTC-Hb as compared to free Hb, Dex-BTC, Dextran 40 (Plasmacair), modified fluid gelatin (MFG-Plasmion) or hydroxyethyl starch (HEA-Elohes). The effect on erythrocyte aggregation of Dex-BTC-Hb was greater than that of standard solutions, but close to that of MFG or HEA. There was no apparent change in RBC deformability. Dex-BTC-Hb, unlike free Hb, has a hyperaggregating effect on RBC, similar to that of some clinically used volume expanders. This hyperaggregating effect could influence the in vivo rheological behavior of substituted Hb by increasing shear stress.

Blood Substitutes↗

PubMed1999

Identification of protein C epitopes altered during its nanoencapsulation.

Protein C is a plasmatic inhibitor which regulates the blood coagulation mechanism by modulating the anticoagulant response. The improvement of its bioavailability would be beneficial for the treatment of the disorders caused by its homozygous deficiency or by an other plasmatic inhibitor deficiency. In this context, the protein C encapsulation into biodegradable nanoparticles could be used to approach the problem. However, the method used to prepare the nanoparticles requires the use of ultrasonication and of an organic solvent such as methylene chloride which interferes with protein activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that neither ultrasonication nor methylene chloride, singly or in combination, led to protein C aggregation or cleavage. Thus, a binding study using an ELISA assay with four characterized monoclonal antibodies was carried out to identify the epitopes damaged by these experimental constraints. The correlation between the immunological assay and a functional one i.e. by the means of the assay of its anticoagulant activity (activated partial thromboplastin time) made it possible to show that protein C amino acids 166-169 of the activation peptide were probably altered after ultrasonication and methylene chloride treatment. Indeed, it is likely that these residues were no longer surface-exposed but had moved inside the protein core.

Anticoagulants↗

PubMed1999

Presence of hemoglobin inside aortic endothelial cells after cell-free hemoglobin administration in guinea pigs.

The endothelium is the production site of several potent vasoactive factors that contribute to the modulation of the vascular tone. Because hemoglobin-based oxygen carriers (HBOC) have been demonstrated to cause vasoconstriction and thereby increase arterial pressure by interacting with endothelium-derived factors such as nitric oxide and endothelin-1, we hypothesized that hemoglobin could penetrate into the endothelial cells. Therefore, we investigated the presence of hemoglobin into guinea pig aortic endothelial cells by immunohistochemical staining after exchange transfusion with a hemoglobin-based oxygen carrier. Despite the large molecular size of HBOC due to chemical modifications designed to prevent hemoglobin subunit dissociation and extravascular leakage, hemoglobin was detectable by immunohistochemical staining into the endothelial cells. These findings suggest that the vascular endothelial cells could uptake hemoglobin by endocytosis mechanisms or could help hemoglobin to cross the endothelial barrier toward media by transcytosis mechanisms. These findings are very important to lead future investigations to the mechanisms by which HBOC cause vasoconstriction.

Animals↗

PubMed1999

Cardiovascular and hemorheological effects of three modified human hemoglobin solutions in hemodiluted rabbits.

The cardiovascular effects of human albumin (Alb) and three human hemoglobin (Hb) solutions, dextran-benzene-tetracarboxylate Hb, alphaalpha-crosslinked Hb, and o-raffinose-polymerized Hb were compared in anesthetized rabbits undergoing acute isovolemic hemodilution with Hct reduction from 41.4 +/- 2.7 to 28.8 +/- 1.6%. The impact of the vasoconstricting properties of Hb was examined by measuring heart rate (HR), mean arterial pressure (MAP), abdominal aortic, and femoral arterial blood flow, vascular resistance (VR), and aortic distension during the first 3 h after hemodilution. The impact of the hemorheological parameters was assessed by measurements of hemodiluted blood viscosity. In contrast to Alb, the Hb solutions elicited an immediate increase in MAP (20-38%). The effects of Alb and Hb solutions on HR, as well as on aortic and femoral arterial blood flow, were similar. VR decreased with Alb (20-28%) and increased with all three Hb solutions (30-90%), but the MAP and VR rising trends were different with each Hb solution. Aortic distension decreased in Hb groups compared with the Alb group for the first 60 min. The viscosity of hemodiluted blood was similar for all groups at high shear rates but was dependent on the viscosity of the solutions at low shear rates. We conclude that the vasoconstriction elicited by the Hb solutions overrides the vasodilation associated with viscosity changes due to hemodilution and would be the major factor responsible to the cardiovascular changes.

Albumins↗

PubMed1999

The effects of stroma-free and dextran-conjugated hemoglobin on hemodynamics and carotid blood flow in hemorrhaged guinea pigs.

Hemoglobin solutions are potential resuscitative fluids with volume expanding and oxygen delivery abilities developed to reduce the use of blood transfusion. Most hemoglobin solutions in clinical trials increase transiently arterial pressure by inhibiting nitric oxide-dependent vasodilation. Our objective was to compare the effects on central hemodynamics and carotid blood flow of two hemoglobin solutions after resuscitation from hemorrhage in anesthetized guinea pigs. After anesthesia and instrumentation, severe hemorrhage was induced by withdrawing 50% of the blood volume. Resuscitation was performed after 15 min of hypovolemia with 5% albumin, stroma-free hemoglobin, or hemoglobin conjugated to dextran-benzenetetracarboxylate (Dex-BTC-Hb). The mean arterial pressure (MAP), carotid blood flow (CBF), vascular resistance index and heart rate (HR) were monitored for 3 hours after resuscitation. After hemorrhage, MAP and CBF dropped to 57.6 +/- 4.4% and 58.9 +/- 3.7% of control values respectively. Albumin failed to maintain hemodynamics in the decompensatory phase of shock. Both hemoglobin solutions gave rise to a transient increase in MAP (35%); stroma-free hemoglobin increased the CBF (150%) and resistance index (24%) whereas Dex-BTC-Hb had no effect on CBF and vascular resistances. None of the solutions affected the HR. Modified hemoglobin has attenuated effects on CBF and resistance index compared to stroma-free hemoglobin. This may be due to a balance between the stimulation of nitric oxide synthesis by shear-stress and the inhibition of vasodilation by nitric oxide trapping.

Animals↗

PubMed1999

Induction of superficial bladder tumors in the female Fischer 344 rats with AY-27 tumor cells for the study of diffusion and localization of hemoglobin derived components (hematoporphyrin derivative) in view of photochemotherapy.

Photochemotherapy (PCT) consists in administration of a photosensitizer and subsequent irradiation of the tumor with visible light. Routinely, the photosensitizer is given intravenously (i.v.), but the major drawback of this procedure is the resulting skin photosensitivity. The goal of our study is to examine whether intravesical (i.b.) instillation of the photosensitizer for PDT of bladder cancer might be feasible in order to target the tumors and to avoid the photosensitization phenomenon. After first studying the biodistribution of hematoporphyrin derivative (HpD) in vivo in the rat bladder, two and four hours after intravesical administration, by fluorescence microscopy, we compared two different methods for the induction of superficial bladder tumors in rats with AY-27 tumor cell line in order to perform the same study on bladder tumors. The best results for the penetration depth of HpD in the normal bladder wall were obtained two hours after the bladder instillation where the photosensitizer was detected only in the bladder surface (urothelium and small part of the chorion). That's why we must choose the most appropriate bladder tumor model in order to obtain superficial bladder tumors that mimic the clinical behavior of superficial bladder cancer in man. Both techniques used in this study gave a high tumor take rate in a short time (> 90%). But we really obtained superficial bladder tumors directly attached to the bladder surface with one of the two methods of tumor induction consisting in the abrasion of the bladder surface prior to the administration of the tumoral cells in the bladder cavity.

Animals↗

PubMed1999

Determination of endogenous porphyrins and the maximal HpD tumor/normal skin ratio in SKH-1 hairless mice by light induced fluorescence spectroscopy.

The treatment of skin tumors is an application of photochemotherapy (PCT) which involves an initial administration of a photosensitizer (PS) followed by irradiation with a light beam that causes the PS to produce cytotoxic oxygen species within the tumors. As the PS is also present in normal skin, it is necessary to know how it is distributed between the two tissues. In this study, we have used SKH-1 hairless mice bearing papillomas or carcinomas chemically induced. The biodistribution of hematoporphyrin derivative (HpD) and the tissue autofluorescence measurements were studied by light induced fluorescence spectroscopy. The tumor and normal autofluorescence spectra measured on control mice with papillomas or carcinomas had a very similar shape. However, the principal endogenous porphyrin peak at about 630 nm showed a fluorescence signal amplitude 2 (for papilloma) and 1.5 (for carcinoma)-fold higher than the one found for the normal skin. Moreover, the fluorescence intensity of carcinoma spectrum is 1.4-fold lower than the one of papilloma spectrum at 630 nm. The tissue autofluorescence can be used to distinguish tumor from normal skin and benign from malignant tumor. This difference in fluorescence intensity at 630 nm was directly related to the concentration of endogenous porphyrins in the tumor. Fluorescence intensity ratios between tumor and normal skin were measured 4, 8, 24, 48, 72 and 96 hours after intraperitoneal injection of HpD (5 mg/kg body weight). The best tumor/normal skin ratio was 6.2 for HpD and the time required to reach this ratio was 48 h. HpD showed a moderate selectivity since the ratio was higher than 1 during the four first days. Photodynamic therapy with the same dose of HpD used in this biodistribution study must also be carried out to verify that the maximal tumor/skin ratio corresponds to the maximal efficiency of HpD.

Animals↗

PubMed1998

Microporous microparticles designed as stable immunoadsorbents.

We have developed a solid-phase immunoadsorbent based on encapsulated goat anti-apolipoprotein B polyclonal antibodies previously crosslinked with a 0.25% glutaraldehyde solution, and designed to remove by immunoaffinity the excess of apolipoproteins B from the plasma of patients affected by familial hypercholesterolemia. Compared to a classical immunoadsorbent prepared by activation of Sepharose CL-4B with cyanogen bromide, the resulting immunoadsorbent exhibits both optimal adsorption capacity and stability over the entire range of chemical and biochemical conditions during its practical handling. This approach will serve as a model system to demonstrate the applicability of microparticles as immunoadsorbents, which can be achieved for other encapsulated crosslinked proteins.

Adsorption↗

PubMed1998

Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method.

Nanoparticles were prepared by the double emulsion method (w/o/w), using methylene chloride as an organic solvent and polyvinyl alcohol (PVA) or human serum albumin (HSA) as a surfactant. Experimental parameters such as the preparation temperature, the solvent evaporation methods, the internal aqueous phase volume, the surfactant concentration and the polymer molecular weight were investigated for particle size, the zeta potential, the residual surfactant percentage and the polydispersity index. Preparation parameters leading to particles with well-defined characteristics such as an average size around 200 nm and a polydispersity index lower than 0.1 were identified. The conditions were optimized to ensure protein encapsulation: a cool temperature, a short processing time, a sufficient internal aqueous phase and careful washing. It appeared that the higher the surfactant concentration in the external aqueous phase was, the smaller the particles, the lower the polydispersity index and the higher the residual amount of surfactant were. For PVA or HSA, the agreement between the convenient surfactant concentration and its critical aggregation concentration could be emphasized. Otherwise, an increased polymer molecular weight led both to a slightly decreased particle size and to a lower polydispersity index. Moreover, multilayer absorption of PVA which does not depend on Poly(lactic-acid) molecular weight was exhibited. Finally, the zeta potential resulted from the polymer molecular weight and the residual PVA.

Drug Delivery Systems↗

PubMed1998

Hb Nancy and Hb Osler: two distinct genetic variants with identical clinical and hemoglobin phenotype.

Three hemoglobin variants (Hb Nancy, Osler and Fort Gordon), carrying the same Tyr-->Asp substitution at position beta 145 (HC2), have been independently described in 1975 in patients with marked polycythemia. The first one was found in a French caucasian family from Lorraine, and the two others in African Americans. Two unrelated individuals with Hb Osler have been recently reinvestigated at the DNA level and surprisingly, in their beta gene, codon 145 was found to be AAT which encodes for asparagine and not for aspartic acid, the aspartate at the protein level resulting, thus, from a very efficient posttranslational event. We reinvestigated a patient from the family of Hb Nancy and found that codon 145 was GAT, encoding for aspartate. This demonstrates that Hb Nancy is genetically distinct from Hb Osler despite an almost identical phenotype.

Asparagine↗