WO2001059455A2 - Materiau et procede pour l'immobilisation d'especes bioactives - Google Patents

Materiau et procede pour l'immobilisation d'especes bioactives Download PDF

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Publication number
WO2001059455A2
WO2001059455A2 PCT/NL2001/000103 NL0100103W WO0159455A2 WO 2001059455 A2 WO2001059455 A2 WO 2001059455A2 NL 0100103 W NL0100103 W NL 0100103W WO 0159455 A2 WO0159455 A2 WO 0159455A2
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WIPO (PCT)
Prior art keywords
blood
ligand
wire
microparticles
anx
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PCT/NL2001/000103
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English (en)
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WO2001059455A3 (fr
Inventor
Chris Peter Maria Reutelingsperger
Waander Laurens Van Heerde
Levinus Hendrik Koole
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Acspurt B.V.
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Priority to AU2001237805A priority Critical patent/AU2001237805A1/en
Publication of WO2001059455A2 publication Critical patent/WO2001059455A2/fr
Publication of WO2001059455A3 publication Critical patent/WO2001059455A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/70—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the isocyanates or isothiocyanates used
    • C08G18/81—Unsaturated isocyanates or isothiocyanates
    • C08G18/8108—Unsaturated isocyanates or isothiocyanates having only one isocyanate or isothiocyanate group
    • C08G18/8116—Unsaturated isocyanates or isothiocyanates having only one isocyanate or isothiocyanate group esters of acrylic or alkylacrylic acid having only one isocyanate or isothiocyanate group
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the present invention relates to a device and use thereof for the removal of microparticles, which exhibit procoagulant and pro-inflammatory activities, from the blood of a patient, so as to decrease the pathogenic consequences of the circulating microparticles.
  • the device is based on a polymer coated onto a carrier, which polymer has functional groups capable of binding to a ligand.
  • the carrier coated with the polymer is used to bind bioactive ligands, which recognize selectively procoagulant and pro-inflammatory structures of the microparticles.
  • US 5,897,955 describes how to modify a carrier to make it suitable for immobilizing bioactive substances.
  • the material according to this document consists of a carrier provided with a first layer of a cross-linked surfactant and a second layer of hydrophilic polymer having functional groups.
  • US 5,622,826 describes a method for immobilizing molecules on a surface of platinum. A platinum surface is covered by isocyanate or isothiocyanate monomers by physical absorbance. These immobilized reactive groups are used to subsequently immobilize compounds for the purpose of chemical analysis.
  • EP 0 965 647 discloses a silicon based surface, which is chemically modified with isothiocyanate groups to immobilise oligo- or polynucleotides.
  • US 3,981,775 describes the grafting of a monomer onto a polymer carrier and subsequently converting the grafted polymer into an isothiocyanate group to immobilise an enzyme on the polymeric carrier.
  • EP 0 353 460 relates to a flexible, homopolymeric, porous membrane having functional groups like isothiocyanate to immobilise peptides and proteins to allow for their sequence determination by chemical or enzymatic degradation.
  • Cavadore and Vallet, Anal. Biochem., 84:402 (1978) reported hydrophilic activated polyacrylamide, the free arylaminogroups of which were converted to isothiocyanate to immobilise proteins for chemical sequencing.
  • the fulminant progression of a bacterial scepsis is characterised by a high level of circulating microparticles, which, because of their procoagulant and pro-inflammatory nature, contribute to the pathogenesis (Nieuwland et al. Blood 95:930 (2000)).
  • HIV-infection Alignment et al. J Clin Invest 99, 1546 (1997)
  • paroxysmal nocturnal hemoglobinuria nocturnal Hugel et al. Blood 93:3451 (1999)
  • circulating microparticles which may cause thrombosis.
  • microparticles are quickly removed by the reticulo-endothelial system (RES) of the patient.
  • RES reticulo-endothelial system
  • the huge amount of generated microparticles in combination with the trauma of the surgery or the disease likely prevents the RES from performing this efficiently. Consequently, microparticles keep circulating in these patients during a longer time, and, consequently, are prone to cause thrombosis and form microemboli, which get trapped in the microcirculation of organs like the brain. There they may cause harm by inducing locally an inflammatory response because of their surface characteristics.
  • the surface of the microparticles contains amongst other stmctures the phospholipid phosphatidylserine (PS) to which the protein annexin A5 can bind (Nieuwland et al. Circulation 96:3543 (1997)).
  • PS phospholipid phosphatidylserine
  • Annexin A5 is well-known fcr its PS binding property (VanHeerde et al. Thromb. Haemost 73:172 (1995)).
  • tissue factor, CD14, and CD16 can be present on the surface depending on the disease of the patient.
  • the present invention relates to a novel medical device to remove microparticles from the blood of patients.
  • This device can be regarded as an artificial reticulo-endothelial system.
  • the device operates by specific interactions between an immobilized ligand and a surface antigen of the microparticle.
  • the medical device contains a mesh, which is composed of a flexible carrier, for example a metallic wire, with a polymeric coating.
  • the flexible carrier is folded and pressed together in such a way that four important conditions are met: a) the resulting mesh guarantees that the fluid can pass easily (i.e. the pressure drop is low). b) there is a large fluid-solid contact surface. c) the surface must be hemo/biocompatible. d) the passage per se does not induce damage to cells or other vulnerable constituents of the fluid.
  • a novel family of macromolecules was developed specifically to coat the flexible carrier in order to meet the above mentioned conditions while creating the possibility to immobilize a ligand for binding to the microparticles.
  • a device comprising: a) a carrier b) a polymeric coating physically adhered to the carrier and c) a ligand, which is covalently attached to the surface of the polymeric coating and which is capable of binding to microparticles in blood.
  • the flexible carrier is a metallic wire.
  • the carrier can also be a long metallic strip, but carriers based on different materials such as polymeric carriers can also be used.
  • the macromolecules of this invention consist of polymers, which are synthesised by copolymerizing a backbone monomer and a reactive monomer, which contains a functional group for capturing the ligand.
  • the functional groups are selected such that they can form a covalent bond with preferably a primary amino group of the ligand.
  • the choice of the functional group is not restricted as long as it is able to bind to the ligand, in particular by means of a covalent link.
  • Functional groups are preferably selected from the group consisting of isocyanate, isothiocyanate, arylisocyanate and arylisothiocyanate. It is advantageous to use arylisothiocyanate groups.
  • the reactive monomer contains an (aryl)isothiocyanate group.
  • the monomer containing the (aryl)isothiocyanate group is selected from compounds comprising an acrylic or methacrylic group and 4-isothiocyanato-phenylene group, wherein the acrylic or methacrylic acid group and the 4-isothiocyanato-phenylene group are optionally separated by a spacer and wherein the phenyl group is optionally substituted, for example by C ⁇ -C 4 alkyl, C ⁇ -C 4 alkoxy or a halogen atom.
  • the spacer may be hydrophobic or hydrophilic in nature and can be any spacer known to a skilled person.
  • the spacer may belong to the series of polymethylene [— (CH 2 )— ] n or polyethylene oxide [— CH 2 — CH 2 — O— ] m where n and m are maximally 30, but preferably in the range of 1 to 8.
  • the compound having isothiocyanate groups according to the present invention in particular has the formula (I):
  • R is H or CH 3 , preferably CH 3 ;
  • A is -O-, -O-(CH 2 ) deliberately- -N-(CH 2 ) n - -O-(CH 2 ) n -O-, -N-(CH 2 ) n -N-
  • n is 1 to 30, preferably 1 to 8.
  • the phenyl group can be substituted by Cj-C alkyl, C ⁇ -C 4 alkoxy or a halogen atom.
  • the compound having isothiocyanate groups is selected from compounds with formula (I), wherein R is CH 3 and A is selected from — O— CH 2 — CH 2 — O— C(O)— , — O— CH 2 — CH 2 — , — O— CH 2 — and — O— , i.e.
  • the compounds having isothiocyanate groups as described above can be obtained by means of processes which are known to a person skilled in the art of organic chemistry. An example of such a process is described in the experimental section, hereafter.
  • the backbone monomer can be selected preferably from the group of monomers, which polymerize into polyurethanes, polyesters, polyalcohols, polyacrylates or polymethacrylate.
  • these groups are bound to a backbone of an acrylate type polymer or copolymer, such as a polymer or copolymer which is prepared from the group of reactive monomers comprising alkylacrylates and alkylmethacrylates, in particulare acrylate, methacrylate, methylacrylate, methylmethacrylate or 2-hydroxyethylmethacrylate.
  • the co-polymer for coating of the metallic wire can be obtained by reaction of the backbone monomer with the reactive monomer in a molar ratio preferably ranging from 999 : 1 (backbone monomer : reactive monomer) to 190 : 1 (backbone monomer : reactive monomer) (based on weight).
  • the monomers described for instance the following copolymer can be obtained:
  • the (co)polymerization of the monomers can be carried out by conventional processes, by employing a suitable initiator and solvent.
  • Figure 1 shows a column of filter house in which a wire mesh is incorporated
  • Figure 2 shows schematically the attachment of a structure having primary amino groups to the material according to the present invention
  • Figure 3 shows the time temperature profile for polymerization according to the examples
  • Figure 4 shows prothrombinase activity of different wires incubated with PRP containing activated platelets
  • Figure 5 shows flow cytometric analysis of whole blood before perfusion through the filter house containing Anx V wire
  • Figure 6 shows flow cytometric analysis of whole blood that was perfused through the filter house containing Anx V wire
  • Figure 7 shows flow cytometric analysis of the EDTA wash of the Anx V-wire after its perfusion with patient's blood.
  • the copolymers, te ⁇ olymers, etc. can be dissolved in a variety of organic solvents, including dimethylformamide, N-methylpyrrolidone, and dimethylsulfoxide.
  • organic solvents including dimethylformamide, N-methylpyrrolidone, and dimethylsulfoxide.
  • a polymeric solution car be applied as a uniform layer onto the surface of the carrier, preferably a thin wire, which is usually a metallic wire but can also be a polymeric material. Controlled evaporation of the solvent in an oven then leads to the formation of a uniform thin coating of the isothiocyanate-containing polymer on the surface of the wire. The isothiocyanate groups are exposed on the surface of the coating and available for chemical reaction.
  • the coated carrier When the coated carrier is a wire, it is subsequently folded or treated otherwise in such a manner that a mesh is formed.
  • the dimensions of the mesh can be controlled.
  • the mesh is formed such that it fits in a column or in a filter house, as is depicted schematically in figure 1.
  • (1) is the filter house, (2) a mesh of polymer coated metallic wire with exposing NCS groups at the surface or exposing NCS coupled specific structures at the surface and (3) the flow of biological fluid, e.g. blood.
  • the present invention also relates to a flow-through medical device, preferably a column or a filter, wherein the stationary phase is a carrier coated with a polymer according to the present invention.
  • a solution of the ligand that is engaged in the specific recognition/association process as described above is passed through the filter or column.
  • the pH will be elevated to the level of, approximately, 10. Consequently, primary amino groups present in the structure of the ligand, will react with the isothiocyanate groups exposed on the surface of the coated wires in the mesh, according to scheme in Figure 2. This implies that the ligand is immobilised via an array of covalent chemical bonds to the stationary phase, i.e. to the poly- meric coating of the mesh.
  • the solution of the ligand is then removed from the column or filter.
  • the column or filter is then washed and stored, preferably in dry form.
  • the ligand is a biologically active substance or molecule, having a function in biochemical and/or biological recognition.
  • the ligand is covalently linked to the functional groups that are exposed at the material's surface.
  • the ligands are selected from the group consisting compounds capable of binding to phospholipids, in particular phosphatidylserine binding proteins, members of the Annexin protein family, immunoglobulins, biotin, avidin or streptavidin. Most preferred are members of the Annexin protein family and immunoglobulins.
  • a fluid in particular blood
  • a fluid is pumped through the column or filter house in such a manner that the fluid contacts the surface of the carrier.
  • the specific recognition/association occurs, leading to retention of cells on the carrier in the column or filter. Consequently, the effluent will be poorer in, or possibly even free of, those cells that are involved in the specific recognition.
  • the column must be flushed with a non-cellular medium and the condition must be chosen such as to minimise the affinity between cells and the non-cellular component that resides on the stationary phase.
  • the device of the invention is in particular suitable for the removal of microparticles from blood.
  • microparticles in blood which include platelet microparticles and cellular fragments or remnants, occur during trauma or surgery. It would be beneficial to the patient if these microparticles could be removed from the blood. By contacting the blood of the patient with the device of the invention, these microparticles can be removed.
  • Preferred ligands for this use are phosphatidylserine binding ligands, members of the Annexin protein family and immunoglobulins.
  • examples which illustrate the present invention are given.
  • the contents were thoroughly mixed and transferred into the teflon tubes.
  • the teflon tubes were placed in a thermostatic oil bath, equipped with a time-temperature control system (PM LAUD A, K ⁇ ningshofen, Germany).
  • the time-temperature profile as depicted in Figure 3 was then run. The polymerization proceeded successivefully, no residual monomer was seen.
  • This example describes a procedure to remove cells and/or cellular fragments with exposed phosphatidylserine (PS) from human blood. Such procedure would be of benefit to patients having elevated levels of PS-exposmg cells and/or cellular fragments in the circulating blood.
  • Reasons of this kind include, but are not limited to: auto-immune diseases, diabetes, cardiovascular disease, and neoplastic disease. Reasons of this kind also include, but are not limited to: clinical treatments such as cardiopulmonary bypass, whole-blood dialysis, radiotherapy, chemotherapy, and blood transfusion.
  • the elevation of PS-exposing cells and/or cellular fragments in the circulating blood means the elevation of sites which catalyse pro-inflammatory and procoagulant reactions.
  • Cells and structures with surface-exposed PS catalyze the formation of thrombin.
  • This pleiotropic enzyme acts not only in the haemostatic system by activating platelets and cleaving fibrinogen (soluble) into fibrin (insoluble), but also in the inflammatory system by acting on leukocytes and vascular endothelial cells.
  • Reduction of elevated levels hence, would reduce the inflammatory and procogulant potential of the blood.
  • a procedure accomplishing such reduction offers the patient the benefit of reducing the risk of complications, which might arise from elevated levels of PS- exposing cells and/or cellular fragment in their circulating blood for above-mentioned reasons.
  • the non-cellular component to be immobilised on the polymer-coated wire is selected from the Annexin family, which comprises proteins that bind to phospholipids.
  • This example descibes the immobilisation of Annexin V, which has a high affinity for PS.
  • the example also describes the power of the resulting combination to remove PS-exposing cells and/or cellular fragments from blood.
  • recombinant Annexin V (Anx V), recombinant des- Annexin V (des-Anx V), bovine serum albumin (BSA), recombinant Annexin V-FITC (Anx V-FITC), metallic wire, coated with the polymer as described above, polymer-coated wire, meshed in a filter house as described above and schematically depicted in Figure 1, precision infusion pump, buffer solution with pH > 8, bovine coagulation factors Xa, Va, and II, prothrombinase buffer, platelet-rich human blood plasma (PRP), whole blood, taken with informed consent from a patient during cardiopulmonary bypass surgery.
  • PRP platelet-rich human blood plasma
  • the recombinant Anx V, des-Anx V, and BSA are dissolved in the buffer with pH > 8.
  • the composition of the buffer has the only restriction that it should not contain compounds with primary amino groups.
  • the pH > 8.0 is chosen because in this pH range the coupling reaction between the isothiocyanate groups of the polymer and primary amino groups of amino acids of the proteins is facilitated.
  • the polymer-coated wire is immersed into the protein solution, and the coupling reaction is allowed to proceed for a certain time period. Then, the wire is removed from the protein solution, and washed. Unreacted isothiocyanate groups which are possibly still exposed at the polymer surface, are capped (inactivated) by incubation with compounds having one or more primary amino groups, such as, for example, glycine or Tris.
  • the platelets of the PRP were activated to ensure exposition of PS at their surface.
  • the PRP was anticoagulated with heparin and contained sufficient Ca2+ ions to mediate Anx V binding to the PS-exposing platelets.
  • the wires were processed for evaluation with scanning electron microscopy. This analysis showed that the Anx wire is capable of catching cells and cellular structures, whereas the des-Anx wire and the BSA wire lack this property (data not shown). Visualisation is not conclusive about the nature of the structures attached to the wire. Therefore, a biological assay was performed that measures the amount of PS-exposing cells attached to the surface.
  • the PRP-treated Anx V- wire, des Anx V-wire and the BSA-wire were incubated with a mixture of coagulation factors Xa, Va, II, and Ca2+ ions. This mixture generates factor Ila if PS-exposing cells are present. At various time points of the incubation period samples were withdrawn from the mixture to measure the amount of factor Ila, by means of a chromogenic assay.
  • Anx V is responsible for the observed capturing potential. It also demonstrates that the conju- gation procedure does not affect the capability of Anx V to bind PS-exposing cells and/or cellular fragments.
  • a filter house as depicted in Figure 1 , was filled with the Anx V-wire. Fifty mL of patient heparinized blood was perfused through the filter at a rate of 4 mL/min using the perfusion pump. When the blood was passed through the filter house, the house was flushed with isotonic buffer containing Ca2+-ions to remove unbound blood cells as good as possible.
  • Figure 5 depicts the dot plot analysis of the blood sample before perfusion through the filter.
  • the x-axis shows the forward scatter of the blood cells, which is a measure for the size of the cells.
  • the y-axis gives the FITC fluorescence of the blood cells indicating the amount of Anx V - FITC bound per cell in other words indicating whether the cells have surface-exposed PS.
  • the flow cytometric analysis of the patients blood before perfusion of the filter tells us that most blood cells have basal levels of FITC fluorescence and hence do not expose PS at their surface. Few percentage of the particles do have Anx V - FITC bound and, thus, expose PS at their surface. The forward scatter of these particles indicates that they are intact blood cells and fragments thereof.
  • the mesh was washed firstly with Ca2+-containing buffer to remove unbound cells as good as possible. Then, the mesh was flushed with EDTA, which will chelate the Ca2+ ions and by doing so liberate the cells and/or fragments from the wire, which were specifically bound through the Anx V-surface exposed PS interaction.
  • the EDTA effluent contained a high amount of PS-exposing particles ( Figure 7) demonstrating that the Anx V- wire is able to capture PS-exposing particles from whole blood in a Ca2+-dependent mode.
  • Anx V can be immobilized on the polymer coated wire without destruction of its PS-binding capacity.
  • the combination of Anx V and polymer- coated wire offers the possibility to remove PS-exposing cells and/or cellular fragments from whole blood by flowing the blood through a mesh of Anx V-wire.
  • a medical device can be constructed to reduce the number of pro-inflammatory and procoagulant structures in the blood of patients.

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Abstract

L'invention concerne un dispositif comprenant un support et un revêtement polymère adhérant physiquement au support, capable d'exposer les groupes fonctionnels à la surface du matériau. Les groupes fonctionnels sont capables de se lier à un ligand, lequel est de préférence une substance ou une molécule biologiquement active ayant une fonction dans le domaine de la reconnaissance biochimique et/ou biologique, avec liaison covalente aux groupes fonctionnels exposés à la surface du matériau. On peut utiliser le dispositif pour extraire des microparticules du sang.
PCT/NL2001/000103 2000-02-08 2001-02-08 Materiau et procede pour l'immobilisation d'especes bioactives WO2001059455A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001237805A AU2001237805A1 (en) 2000-02-08 2001-02-08 Material and method for immobilization of bioactive species

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EP00200415.8 2000-02-08
EP00200415 2000-02-08

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WO2001059455A2 true WO2001059455A2 (fr) 2001-08-16
WO2001059455A3 WO2001059455A3 (fr) 2002-01-31

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2255834A1 (fr) 2004-11-08 2010-12-01 K.U. Leuven Research and Development Échafaudage pour ingénierie tissulaire avec des facteurs d'homing
JP2013253068A (ja) * 2012-05-08 2013-12-19 Jsr Corp 新規（メタ）アクリル酸エステルおよびその重合体
WO2015113699A1 (fr) * 2014-01-30 2015-08-06 Fresenius Hemocare Italia Srl Dispositif de filtration
EP3216457B1 (fr) * 2008-02-22 2019-04-10 Annexin Pharmaceuticals AB Composés et procédés de prévention ou de traitement de la resténose

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2255834A1 (fr) 2004-11-08 2010-12-01 K.U. Leuven Research and Development Échafaudage pour ingénierie tissulaire avec des facteurs d'homing
EP3216457B1 (fr) * 2008-02-22 2019-04-10 Annexin Pharmaceuticals AB Composés et procédés de prévention ou de traitement de la resténose
JP2013253068A (ja) * 2012-05-08 2013-12-19 Jsr Corp 新規（メタ）アクリル酸エステルおよびその重合体
WO2015113699A1 (fr) * 2014-01-30 2015-08-06 Fresenius Hemocare Italia Srl Dispositif de filtration
CN105939782A (zh) * 2014-01-30 2016-09-14 意大利费森尤斯血液技术有限公司 过滤装置
JP2017509360A (ja) * 2014-01-30 2017-04-06 フレゼニウス・ヘモケア・イタリア・ソシエタ・ア・レスポンサビリタ・リミタータＦＲＥＳＥＮＩＵＳ ＨＥＭＯＣＡＲＥ ＩＴＡＬＩＡ Ｓ．ｒ．ｌ． 濾過装置
US10322216B2 (en) 2014-01-30 2019-06-18 Fresenius Hemocare Italia S.R.L. Filtering device

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