WO2018086556A1 - 捕捉或分离白血球的聚合物、装置、其制造方法及其应用 - Google Patents

捕捉或分离白血球的聚合物、装置、其制造方法及其应用 Download PDF

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WO2018086556A1
WO2018086556A1 PCT/CN2017/110177 CN2017110177W WO2018086556A1 WO 2018086556 A1 WO2018086556 A1 WO 2018086556A1 CN 2017110177 W CN2017110177 W CN 2017110177W WO 2018086556 A1 WO2018086556 A1 WO 2018086556A1
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polymer
white blood
blood cells
group
monomer
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PCT/CN2017/110177
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English (en)
French (fr)
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WO2018086556A8 (zh
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张雍
叶至诚
杨承臻
鍾政峯
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普瑞博生技股份有限公司
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Priority to EP17868975.8A priority Critical patent/EP3539581A4/en
Priority to US16/348,325 priority patent/US11833289B2/en
Priority to CN201780069531.8A priority patent/CN109963600B/zh
Publication of WO2018086556A1 publication Critical patent/WO2018086556A1/zh
Publication of WO2018086556A8 publication Critical patent/WO2018086556A8/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/36—Amides or imides
    • C08F222/38—Amides
    • C08F222/385—Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3496—Plasmapheresis; Leucopheresis; Lymphopheresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D39/00—Filtering material for liquid or gaseous fluids
    • B01D39/02—Loose filtering material, e.g. loose fibres
    • B01D39/06—Inorganic material, e.g. asbestos fibres, glass beads or fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D39/00—Filtering material for liquid or gaseous fluids
    • B01D39/14—Other self-supporting filtering material ; Other filtering material
    • B01D39/16—Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C08J7/16—Chemical modification with polymerisable compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A61M1/0281—Apparatus for treatment of blood or blood constituents prior to transfusion, e.g. washing, filtering or thawing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3621—Extra-corporeal blood circuits
    • A61M1/3627—Degassing devices; Buffer reservoirs; Drip chambers; Blood filters
    • A61M1/3633—Blood component filters, e.g. leukocyte filters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0439—White blood cells; Leucocytes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2239/00—Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04—Additives and treatments of the filtering material
    • B01D2239/0407—Additives and treatments of the filtering material comprising particulate additives, e.g. adsorbents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2239/00—Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04—Additives and treatments of the filtering material
    • B01D2239/0471—Surface coating material
    • B01D2239/0478—Surface coating material on a layer of the filter
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
    • C08J2333/24—Homopolymers or copolymers of amides or imides
    • C08J2333/26—Homopolymers or copolymers of acrylamide or methacrylamide

Definitions

  • Embodiments of the present invention relate to a material for capturing or separating cells, and more particularly to a polymer for capturing or separating white blood cells.
  • Blood transfusion is mainly done by infusion of blood from the donor to the patient to supplement the blood component of the patient.
  • the blood (or blood component) type mainly includes whole blood, plasma, washed erythrocyte, erythrocyte concentrate, leukocyte concentrate and platelet concentrate. .
  • the blood component of the input can be selected according to the patient's needs during blood transfusion.
  • cytokines cellular mediators released by white blood cells or white blood cells.
  • Adverse reactions include, for example, non-hemolytic febrile transfusion reactions (NHFTR), alloimmunization (alloimmunization), viral infection, and transfusion-associated graft versus host disease (TA-). GVHD) and so on. Therefore, many countries have already listed white blood cell depletion as a necessary procedure for blood transfusion. Whether it is red blood cell or platelet transfusion, the concentration of white blood cells in the blood must be reduced to a certain extent to avoid adverse reactions.
  • the separation of conventional white blood cells is the use of a filter material with a surface charge.
  • the separation of white blood cells by a charged surface is likely to cause an increase in the concentration of bradykinin, which is liable to cause a hypopotensive transfusion reaction during transfusion.
  • an anticoagulant is generally added to the blood sample, or a surface treatment is further performed on the surface of the filter material to prevent coagulation or activate platelets.
  • a surface treatment is further performed on the surface of the filter material to prevent coagulation or activate platelets.
  • One aspect of the invention is a polymer that is a material for capturing or separating white blood cells.
  • the polymer is prepared by a polymerization reaction of an amide group and a hydroxyl group-containing monomer, wherein the amide group and the hydroxyl group-containing monomer have the structure of the formula (1):
  • R 1 is independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxy, C1 to C12 long carbon chain and benzene ring
  • R 2 is independently selected from hydrogen
  • A a group consisting of a base, an ethyl group, a C1 to C6 long carbon chain, an amino group and a benzene ring
  • n is an integer of 1 to 5.
  • R 1 in formula (1) is hydrogen
  • R 2 is hydrogen
  • n is 1.
  • the amide group- and hydroxyl group-containing monomer is N-Hydroxyethyl acrylamide or N-(2-hydroxyethyl)acrylamide (N-(2) -Hyroxyethyl)acrylamide).
  • the polymer is a copolymer formed by copolymerization of the amide- and hydroxyl-containing monomer and at least one other monomer.
  • the other monomer is Butyl methacrylate (BMA) or Glycidyl methacrylate (GMA).
  • the polymer is a segment polymer.
  • the polymer is a crosslinked polymer.
  • the polymerization comprises the use of a crosslinking agent and the crosslinking agent has a functional group of a diacrylate.
  • the crosslinking agent is methylene bis acrylamide and ethylene glycol dimethacrylate, polylactic acid-polyethylene glycol-polylactic acid polyglycolic acid copolymer or polyethylene glycol diacrylic acid. ester.
  • One aspect of the invention is a device for capturing or separating white blood cells, comprising a housing and a body.
  • the body comprises a polymer and the polymer comprises the structure of formula (2):
  • n is an integer of 10 to 50.
  • the polymer has the structure of formula (3):
  • m is an integer of 50 to 90
  • p is an integer from 2 to 6
  • q is an integer from 1 to 6
  • r is an integer from 1 to 6
  • s is an integer from 1 to 6.
  • the polymer has the structure of formula (4):
  • t is an integer of 50 to 90,
  • the body comprises a substrate, wherein the polymer is disposed on the substrate in a coating, spraying or dipping manner.
  • the body comprises a substrate, wherein the polymer is anchored to the substrate in an anchoring manner.
  • the surface element of the filter material comprises carbon, oxygen and nitrogen, and the total mole percentage of carbon, oxygen and nitrogen is defined as 100 mol%, and the mole percentage of carbon is from about 76.22% to about 79.84%, oxygen The mole percentage is from about 18.1% to about 21.04%, and the mole percent of nitrogen is about 2.05% to about 2.75%.
  • One aspect of the invention is the use of a polymer comprising an amide group and a hydroxyl group for capturing or separating white blood cells.
  • the polymer containing an amide group and a hydroxyl group comprises the structure of the formula (2): Wherein n is an integer from 10 to 50.
  • the amide group-containing and hydroxyl group-containing polymer has a white blood cell capture rate of not less than 70%.
  • the amide group-containing and hydroxyl group-containing polymer has a platelet retention rate of not less than 85%.
  • One aspect of the invention is the use of a monomer comprising an amide group and a hydroxyl group for use in the manufacture of a filter material for capturing or separating leukocytes.
  • the amide group-containing and hydroxyl group-containing monomer has the structure of the formula (1):
  • R1 is independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxyl, C1 to C12 long carbon chain and benzene ring
  • R2 is independently selected from hydrogen, methyl, a group consisting of an ethyl group, a C1 to C6 long carbon chain, an amino group and a benzene ring
  • n is an integer of 1 to 5.
  • the step of making a filter material that captures or separates white blood cells comprises providing a substrate, and modifying the amide- and hydroxyl-containing monomers on the substrate.
  • the modification of the amide group-containing and hydroxyl group-containing monomer on the substrate comprises: providing an amide group-containing group and a hydroxyl group-containing monomer; providing at least one anchoring unit; polymerizing the amide group and the hydroxyl group The monomer and the anchoring monomer form a copolymer; and anchor the copolymer to the substrate by anchoring the monomer.
  • the amide group and the hydroxyl group-containing monomer account for the weight percent of the copolymer.
  • the ratio is about 20% to about 40%.
  • the anchoring monomer comprises from about 60 to about 80 percent by weight of the copolymer.
  • the anchoring monomer is selected from the group consisting of butyl methacrylate, glycidyl methacrylate, and combinations thereof.
  • the step of modifying the amide group-containing and hydroxyl group-containing monomer on the substrate comprises coating the amide group-containing and hydroxyl group-containing monomer on the substrate, and treating the amide group on the substrate with ultraviolet rays. And a monomer of a hydroxyl group.
  • One aspect of the present invention provides a method for preparing a white blood cell thick liquid comprising: providing a device for capturing or separating white blood cells as described above; providing a blood sample, wherein the blood sample comprises white blood cells and platelets; and passing the blood sample through the device.
  • the white blood cells are captured in the device, or are separated from the blood sample into the device; and the white blood cells captured or separated are desorbed.
  • One aspect of the present invention provides a method for preparing a red blood cell thick liquid, comprising: providing a device for capturing or separating white blood cells as described above; providing a blood sample, wherein the blood sample comprises red blood cells, white blood cells, and platelets; and the blood sample is passed through the device. A filtrate is obtained in which white blood cells are captured or separated from the blood sample; and the filtrate is treated as a red blood cell thick liquid.
  • One aspect of the present invention is a method of treating a plasma product prior to storage comprising: providing a device for capturing or separating white blood cells as described above; providing a blood sample, wherein the blood sample comprises plasma and white blood cells; and passing the blood sample through the device A filtrate is obtained in which white blood cells are captured in the device or separated from the blood sample.
  • One aspect of the present invention is a method for removing white blood cells in whole blood, comprising: providing a device for capturing or separating white blood cells as described above; providing a whole blood sample, wherein the whole blood sample comprises red blood cells, white blood cells, and platelets; The whole blood sample is passed through a device to obtain a filtrate in which the white blood cells are captured in the device or separated from the whole blood sample.
  • a method for preparing a platelet thick liquid comprising: providing a device for capturing or separating white blood cells as described above; providing a blood sample, wherein the blood sample comprises white blood cells and platelets; and passing the blood sample through the device to obtain a filtrate, wherein the white blood cells Captured in or isolated from the blood sample; and the treated filtrate is a platelet concentrate.
  • the blood sample may be a whole blood sample or a blood sample obtained by centrifugation to remove red blood cells.
  • the present invention provides a polymer, device, method of making the same, and use thereof for capturing or separating white blood cells.
  • a compound formed by a compound containing an amide group and a hydroxyl group or a monomer thereof provides a function of capturing or separating white blood cells. Since such compounds and polymers have high affinity for white blood cells, they can be separated from whole blood by, for example, capturing, adsorbing, attaching or adhering white blood cells, and it is important that almost It does not adsorb plasma proteins, and it hardly causes platelet adhesion and increases platelet retention. Referring to FIG. 1, two paths of white blood cell subtraction, path A and path B, are illustrated.
  • the mechanism of leukocyte depletion is mainly caused by the positively charged filter material F, which adsorbs plasma protein P 1 and causes platelet P 2 to attach.
  • the positively charged filter material F which adsorbs plasma protein P 1 and causes platelet P 2 to attach.
  • secretion of the cell medium and growth factor as a signal further causes white blood cell L to attach.
  • the surface of filter material F is generally modified to be positively charged.
  • the use of the positively charged filter material F tends to cause activation of platelet P 2 , and it is difficult to efficiently recover platelet P 2 while removing white blood cells L.
  • the leukocyte depletion mechanism of the present invention (path B of FIG. 1) is different from the conventional mechanism (path A of FIG.
  • the filter material F' prepared by using the embodiment of the present invention can solve the problem of white blood cell depletion in the past.
  • the problem. Accordingly, the present invention can effectively separate white blood cells, avoid activation of platelets or blood coagulation, and can also avoid increasing the concentration of bradykinin, resulting in an acute hypotensive reaction at the time of blood transfusion.
  • FIG. 1 is a schematic diagram showing the mechanism of leukocyte depletion in accordance with some embodiments of the present invention.
  • FIG. 2 is a filtration device for separating or capturing white blood cells, in accordance with some embodiments of the present invention.
  • 3 is a graph showing the results of equilibrium water content and oil contact angle for various water gel materials, in accordance with some embodiments of the present invention.
  • FIG. 4 is a graph showing the results of equilibrium water content, non-frozen water ratio, and dimensionless group of various water-gel materials, in accordance with some embodiments of the present invention.
  • FIG. 5 is a graph showing the results of relative protein adsorption in various enzyme-linked immunosorbent assays (ELISAs) according to some embodiments of the present invention.
  • FIG. 6 is a diagram showing protein adsorption on the surface of various water gel materials according to some embodiments of the present invention. A graph of the results of the amount and cell density.
  • Figure 7 is a graph showing the results of white blood cell attachment on various water gels, in accordance with some embodiments of the present invention.
  • Figure 8 is a chemical shift of the structural formula of each monomer, polymer compound, and its nuclear magnetic resonance spectrum signal, in accordance with some embodiments of the present invention.
  • NMR 9 is a Nuclear Magnetic Resonance spectroscopy (NMR) chemical structure map of each monomer and polymer compound, in accordance with some embodiments of the present invention.
  • Figure 10 is a graft graft density of a material on a polypropylene (PP) substrate and a polyethylene terephthalate (PET) substrate, in accordance with some embodiments of the present invention.
  • PP polypropylene
  • PET polyethylene terephthalate
  • FIG. 11 is a photoelectron spectrometer analysis map of nitrogen elements on the surface of a PP substrate and a PET substrate, in accordance with some embodiments of the present invention.
  • Figure 12 is a qualitative result plot of images attached to white blood cells, platelets, and red blood cells on various PP substrates, in accordance with some embodiments of the present invention.
  • Figure 13 is a graph showing the quantitative results of the amount of white blood cells, platelets, and red blood cells attached to various PP substrates, in accordance with some embodiments of the present invention.
  • Figure 14 is a qualitative result plot of images attached to white blood cells, platelets, and red blood cells on various PET substrates, in accordance with some embodiments of the present invention.
  • Figure 15 is a graph showing quantitative results for the amount of white blood cells, platelets, and red blood cells attached to various modified PET substrates, in accordance with some embodiments of the present invention.
  • a first component is formed in a second
  • the element "above” or “above” may include direct contact between the first element and the second element in the embodiment, or may include other additional elements between the first element and the second element such that the first element and the second element No direct contact.
  • component symbols and/or letters will be used repeatedly. This repetition is for the purpose of simplification and clarity and does not determine the relationship between various embodiments and/or structural configurations.
  • various features may be drawn in different proportions for simplicity and clarity.
  • relative spatial relationships may be used here to describe a component in the drawing or The relationship between a feature and another component or feature.
  • the terms of such relative spatial relationships are intended to encompass various orientations of the device in use or operation in addition to the orientation described. For example, elements that are “under” or “beneath” other elements or features are “above” other elements or features. Therefore, the example term “below” can encompass both the above and below.
  • the above device can be otherwise guided (rotated 90 degrees or in other directions), and the spatial relative relationship at this time can also be interpreted in the above manner.
  • capture refers to the blood cell in a blood sample that is exposed to the surface of the material and is attracted by the hydrophobic, hydrogen bonding, or electrostatic molecular forces between the material and the blood cell, causing various types of blood cells to be directly attached. On the surface of the material, or by first adsorbing smaller plasma proteins and platelets, larger blood cells are attached, and these processes are defined as “capture” of blood cells.
  • separation means that a white blood cell-containing sample can be separated from the sample by separating the white blood cell material, which also means that the white blood cell content in the sample can be reduced, and even the white blood cell content in the sample can be greatly reduced, thereby separating.
  • the filtrate of the subsequent filtrate has a white blood cell concentration smaller than that of the sample originally containing white blood cells.
  • samples containing white blood cells and platelets can be separated to achieve high recovery of platelets; or samples of white blood cells and red blood cells can be separated to achieve high recovery of red blood cells.
  • leukocyte depletion does not mean that all or substantially all of the white blood cells are completely removed. This term is used to broadly indicate that the number of white blood cells is reduced during separation or filtration.
  • platelet concentrate does not refer to a platelet concentrate limited to blood, but broadly comprises a cell suspension or blood sample containing platelets, which has been treated with a separation material or a filter material. filtrate. Among them, the volume of the filtrate may be more or less than the volume of the cell suspension or the blood sample.
  • leukocyte concentrate is a buffer or solvent that broadly contains a cell suspension or blood sample containing white blood cells, which has been treated with a separating material or a filter material and left on a separating material or a filter material. Or rinse solution after washing with other solutions.
  • the volume of the leukocyte-rich rinse may be more or less than the volume of the cell suspension or blood sample.
  • erythrocyte concentrate does not refer to a red blood cell thick liquid restricted to blood, but broadly contains a cell suspension or blood sample containing red blood cells, which is treated with a separation material or a filter material. filtrate. Among them, the volume of the filtrate may be more or less than the volume of the cell suspension or the blood sample.
  • One embodiment of the invention is a polymer for capturing or separating white blood cells.
  • the polymer can be used as a material for capturing or separating white blood cells, or as a material for capturing or separating white blood cells by being disposed on other substances or substrates alone or together.
  • the polymer is prepared by a polymerization reaction comprising a monomer containing an amide group and a hydroxyl group, for example, a polymerization reaction of a monomer containing an amide group and a hydroxyl group, or a monomer having an amide group and a hydroxyl group, and other compounds.
  • the copolymerization reaction is carried out. It should be noted that any suitable material can be used for this polymerization. Suitable materials generally refer to any compound that has not been subjected to polymerization, which is used to make a polymer that captures or separates white blood cells.
  • the polymerization reaction may include a monomer having an amide group and a hydroxyl group, and the monomer having an amide group and a hydroxyl group has a structure of the formula (1):
  • R 1 is independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxy, a C1 to C12 long carbon chain, and a benzene ring
  • R 2 is independently selected from hydrogen, a group consisting of a methyl group, an ethyl group, a C1 to C6 long carbon chain, an amino group and a benzene ring
  • n is an integer from 1 to 5; and at least one crosslinking agent.
  • the polymer is a copolymer formed by copolymerization of the amide- and hydroxyl-containing monomer and at least one other monomer.
  • the polymer is a segment polymer.
  • the polymer is a crosslinked copolymerized polymer.
  • the amide group- and hydroxyl group-containing monomer is N-Hydroxyethyl acrylamide or N-(2-hydroxyethyl)acrylamide (N-(2-hydroxyethyl) acrylamide).
  • Acrylamide, HEAA for example, the amide- and hydroxy-containing monomer has the structure of formula (1) wherein R 1 is hydrogen, R 2 is hydrogen, and n is 1, and the chemical structural formula is as follows:
  • N-hydroxyethyl acrylamide is a compound having both a hydroxy functional group (-OH) and an amide functional group (-R n C(O) x NR' 2 , wherein R and R' refer to a hydrogen atom or an organic group).
  • the polymer may be simply polymerized from an N-hydroxyethyl acrylamide monomer or a copolymer formed by copolymerization of N-hydroxyethyl acrylamide with other compounds.
  • the isolated white blood cell material contains N-hydroxyethyl acrylamide monomer, has high affinity for white blood cells, can specifically capture, adsorb, attach or adhere white blood cells, and hardly adsorbs plasma protein and attaches. Platelets (specific experimental data will be detailed later). Thereby, the isolated leukocyte material of the present invention can effectively separate white blood cells. In addition, the isolated leukocyte material of the present invention avoids activation of platelets or coagulation, avoids increasing the concentration of bradykinin, and results in an acute hypotensive response at the time of transfusion.
  • the foregoing polymerization reaction further comprises the use of a crosslinking agent such as, but not limited to, less than 10% by weight of a crosslinking agent.
  • a crosslinking agent such as, but not limited to, less than 10% by weight of a crosslinking agent.
  • the cross-linking agent is used to strengthen the mechanical properties of the hydrocolloid material, regardless of the blood compatibility of the material.
  • the crosslinking agent can be polymerized by mixing with an amide group-containing and hydroxyl group-containing monomer (for example, an N-hydroxyethyl acrylamide monomer).
  • This crosslinking agent can be a monomeric compound or a polymer.
  • the crosslinking agent when the crosslinking agent is a monomeric compound, it is selected from the group consisting of N, N'-Methylenebisacrylamide (NMBA) and Ethylene glycol dimethacrylate (EGDMA). ) the group consisting of.
  • NMBA N, N'-Methylenebisacrylamide
  • Ethylene glycol dimethacrylate Ethylene glycol dimethacrylate
  • the crosslinking agent when the crosslinking agent is a polymer, it is a polylactic acid-polyethylene glycol-polylactic acid polyglycolic acid copolymer (PLA-PEG-PLGA, PLA: Polylactic Acid, PEG: Polyethylene glycol, PLGA: Poly(lactic acid-co-glycolic acid) or poly(ethylene glycol) diacylate (PEGDA).
  • the crosslinking agent is preferably selected from NMBA and is at room temperature 25 ° C. The polymerization was carried out.
  • the chemical structure of the crosslinker is as follows:
  • the polymer used to capture or separate white blood cells can be a hydrocolloid material, such as a crosslinked polymer that undergoes a crosslinking reaction.
  • the polymer may be a powder material, such as a segmented polymeric copolymer formed by copolymerization with other monomers. The powder material can be dissolved in an alcohol solution to form a liquid, and then applied, sprayed or impregnated.
  • crosslinked polymers and copolymers please refer to the following description.
  • the foregoing polymerization reaction further comprises using an initiator or a catalyst.
  • the aforementioned N-hydroxyethyl acrylamide monomer can be mixed with an initiator or a catalyst to accelerate the polymerization.
  • the initiator is ammonium peroxodisulfate (APS).
  • the catalyst is N, N, N, N tetramethylethylenediamine (TEMED).
  • the white blood cell material used for capturing or separating after the completion of the polymerization reaction can be cut or shaped according to a desired size to have different shapes, such as a film shape, a plate shape, a block shape, and a fiber shape. , tubular, beaded, granulated, or powdered for subsequent separation or filtration processes.
  • the separation or filtration process can be continuous or batch, for example, the separation of white blood cell material can be a filter membrane, a filter plate; or a fiber, a particle or a powder filled in a column.
  • the device includes a housing and a body.
  • the body may be in the form of a film, a plate, a block, a fiber, a tube, a bead, a granule, or a powder, and the body includes a substrate and the above polymer.
  • the substrate is a major part of the shape of the body, and the polymer can be applied to the substrate by coating, spraying or dipping in whole or in part, for example, coating one or both sides of the film-shaped substrate, or all of the substrate. Immersion in the polymer.
  • the body has the function of capturing or separating white blood cells, so that in some embodiments or embodiments, the body may be referred to as a filter material.
  • One aspect of the present invention is an apparatus for capturing or separating white blood cells, comprising a housing and a body.
  • the body comprises a substrate and a polymer for capturing or separating white blood cells disposed on the substrate, the polymer having the structure of formula (2): Wherein n is an integer from 10 to 50.
  • the polymer can be a water gel material.
  • the means for capturing or separating white blood cells can be a filtration device.
  • a filter for capturing or separating white blood cells As shown in Figure 2, in accordance with some embodiments of the present invention, a filter for capturing or separating white blood cells.
  • the outer casing includes an upper casing 1 and a lower casing 3, and the filter material 2 is located between the upper casing 1 and the lower casing 3.
  • the filter can be a white blood cell filter, a platelet depletion white blood cell filter, or a red blood cell depletion white blood cell filter.
  • a filter for separating white blood cells is used to separate white blood cells from a blood sample.
  • the platelet depletion white blood cell filter passes a sample of a suspension containing white blood cells and platelets through a filter to separate white blood cells and platelets, thereby achieving high recovery of platelets.
  • the red blood cell depletion white blood cell filter passes a sample of a suspension containing white blood cells and red blood cells through a filter to separate white blood cells and red blood cells, thereby achieving high recovery of red blood cells.
  • the blood sample comprises a whole blood sample, a sample containing white blood cells and platelets, a sample containing plasma proteins, platelets and white blood cells, a sample containing white blood cells and red blood cells, or other sample containing a cell suspension.
  • the filter material of the present invention can be used for pre-transfusion or pre-storage storage of plasma products to reduce the concentration of white blood cells in whole blood, platelet thick liquid or red blood cell thick liquid to a certain extent ( Leukocyte depletion) to avoid or reduce adverse reactions and comply with national regulations.
  • AABB American Association of Blood Banks
  • the European standard is that the white blood cell content per unit of blood should be less than 1 ⁇ 10 6 .
  • the polymer in the filter material 2 can be obtained by a polymerization reaction in which an amide group-containing and hydroxyl group-containing monomer is mixed with a crosslinking agent.
  • the polymer of the filter material 2 has the structure of formula (3):
  • p is an integer from 2 to 6
  • q is an integer from 1 to 6
  • r is an integer from 1 to 6
  • s is an integer from 1 to 6.
  • Structure symbol used here Is used to indicate the state that has not been bonded. In other words, if a substituent is attached to this symbol, it is meant that the substituent may be further joined to any other substituent.
  • the structural symbol "*" used herein is used to indicate the position of attachment of a substituent in a chemical structure.
  • the polymer in the filter material 2 can be obtained by a polymerization reaction in which an amide group-containing and hydroxyl group-containing monomer is mixed with an adsorption monomer.
  • the polymer of the filter material 2 has the structure of formula (4):
  • t is a 50 to 90 integer.
  • the filter material 2 can be a membrane modified substrate.
  • the polymer having the formula (2), the formula (3) or the formula (4) is applied to a substrate to modify the surface of the substrate.
  • the surface element of the modified substrate comprises carbon, oxygen, and nitrogen, and the total mole percent of carbon, oxygen, and nitrogen It is defined as 100%, wherein the mole percent of carbon is from about 76.22% to about 79.84%, the mole percent of oxygen is from about 18.1% to about 21.04%, and the mole percent of nitrogen is from about 2.05% to about 2.75%.
  • One embodiment of the present invention is the use of a polymer containing an amide group and a hydroxyl group for capturing or separating white blood cells, and the polymer containing an amide group and a hydroxyl group comprises the structure of the formula (2):
  • n is a 10 to 50 integer.
  • the aforementioned polymer containing the structure represented by formula (2) is polymerized from N-hydroxyethyl acrylamide.
  • the sample with white blood cells is passed through a material containing N-hydroxyethyl acrylamide so that N-hydroxyethyl acrylamide can specifically capture, adsorb, attach or adhere white blood cells.
  • the use of separating white blood cells is to contact a sample with white blood cells with a material for capturing or separating white blood cells, and the contact may be by (flowing through), filtering or batch contact, contacting.
  • the liquid can then be collected or the material used to capture or separate the white blood cells.
  • the use of the aforementioned polymer of formula (2) allows the capture rate of white blood cells to be at least 87%.
  • One embodiment of the present invention is a use of an amide group-containing and hydroxyl group-containing monomer having the formula (1) as described above, which is a filter material for producing a white blood cell for capturing or separating.
  • the step of making a filter material that captures or separates white blood cells comprises providing a substrate and modifying the amide- and hydroxyl-containing monomer onto the substrate.
  • the substrate may be in the form of a film, a plate, a block, a fiber, a tube, a bead, a granule, or a powder.
  • the amide- and hydroxyl-containing monomers are modified to the surface of the substrate by the use and requirements of the visual separation or filtration process.
  • the N-hydroxyethyl acrylamide monomer or N-hydroxyethyl acrylamide monomer can be a coating material after polymerization with other compounds, and then applied to the surface of the substrate.
  • the subsequent separation or filtration process can be continuous or batch, for example, the filter material for capturing or separating white blood cells can be a filter, a filter plate or a fiber, a particle or a powder filled in the column. body.
  • the substrate is polypropylene or polyethylene terephthalate.
  • the method of modifying the surface of the substrate may be physical modification or chemical modification.
  • the physical modification method includes coating, which is a modification of the surface properties of the material by physical force, and the coating adsorption force includes van der Waals force, hydrogen bonding force, hydrophobic force, electrostatic force, and the like.
  • the chemical modification method includes grafting or etching.
  • the grafting method is to modify the surface properties of the material by chemical bonding, and the chemical grafting method comprises ozone, ultraviolet light, or plasma-initiated radical polymerization.
  • the surface property of the material may be modified by a coating method and a grafting method, and the material monomer or the copolymer polymer may be adsorbed on the surface of the substrate by coating, and then chemically connected by ozone, ultraviolet light or plasma treatment. branch.
  • the surface modification method is preferably coating, grafting or a combination thereof.
  • the method of surface modification of the substrate is by a physical modification method.
  • modifying an amide- and hydroxy-containing monomer onto a substrate comprises the steps described below. First, from about 20 to about 40 parts by weight of the amide group-containing and hydroxyl group-containing monomer are provided, and from about 60 to about 80 parts by weight of the adsorbent monomer is provided. Next, a monomer containing an amide group and a hydroxyl group and a monomer are adsorbed to form a copolymer. Finally, the copolymer is applied to the surface of the substrate.
  • the adsorbent monomer is selected from the group consisting of butyl methacrylate, glycidyl methacrylate, and combinations thereof.
  • the method of surface modification of the substrate is by chemical modification.
  • modifying an amide- and hydroxy-containing monomer onto a substrate comprises the steps described below. The monomer containing the amide group and the hydroxyl group is coated on the substrate. Next, the graft reaction is carried out by treating the substrate containing the amide group and the hydroxyl group on the substrate and the substrate with ultraviolet rays. It should be understood that additional operations may be used in this embodiment, and that the operations may be replaced, deleted, or alternated.
  • modifying the amide- and hydroxyl-containing monomer on the substrate comprises: treating the substrate with ultraviolet light; and coating the monomer containing the amide group and the hydroxyl group onto the substrate treated with ultraviolet rays for grafting reaction.
  • the first type of structure is 2-hydroxyethyl acrylate (HEA) and 2-Hdroxyethyl methacrylate (HEMA) having only hydroxyl group monomers, and the chemical structures are as follows:
  • the second type of structure is acrylamide (AAm) and methacrylamide (MAA) having only amide group monomers, and the chemical structures are as follows:
  • the third type of structure is N-(2-hydroxypropyl)methacrylamide (HPMA) and N-hydroxyethyl acrylamide (N-(2-hydroxypropyl)methacrylamide (HPMA)) having both a hydroxyl functional group and an amide functional group.
  • HPMA N-(2-hydroxypropyl)methacrylamide
  • HPMA N-(2-hydroxypropyl)methacrylamide
  • HEA N-(2-hydroxyethyl)acrylamide
  • the fourth type of structure is a compound having a double ionicity, which is [3-(methacrylamido)propyl]dimethyl(3-thiopropyl)ammonium hydroxide ([3-(Methacryloylamino)propyl]dimethyl) (3-sulfopropyl)ammonium hydroxide, SBAA), and [2-(methacryloyl)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide ([2-(methacryloyloxy)ethyl]dimethyl-) (3-sulfopropyl)ammonium hydroxide, SBMA), the chemical structures are as follows:
  • Ammonium peroxodisulfate (APS) is used as a starter in this test and has the following chemical structure:
  • N, N, N, N tetramethylethylenediamine (TEMED) is used as a catalyst in this test and has the following chemical structure:
  • crosslinking agent used in this test is methylene bisacrylamide (N, N'-Methylenebisacrylamide, NMBA), and the chemical structure is as shown in the foregoing discussion, and is not mentioned here.
  • Two amide functional group-containing water gels namely acrylamide (AAm) water gel and methacrylamide (MAA) water gel, are provided in this step.
  • AAm acrylamide
  • MAA methacrylamide
  • a crosslinking agent NMBA was added.
  • the AAm solution was stirred in an ice bath for 10 minutes while the MAA solution was stirred at room temperature for 10 minutes.
  • the initiator APS was added for 10 minutes, and then the catalyst TEMED was added.
  • the mixture was sucked into a glass mold by a syringe, and subjected to radical crosslinking polymerization at room temperature for 1 hour to form a hydrophilic water gel.
  • amide functional group-containing water gels namely hydroxyethyl acrylate (HEA) water gel and hydroxyethyl methacrylate (HEMA) water gel
  • HEA hydroxyethyl acrylate
  • HEMA hydroxyethyl methacrylate
  • a crosslinking agent NMBA was added and stirred for 10 minutes.
  • the initiator APS was added for 10 minutes, and then the catalyst TEMED was added.
  • the syringe was sucked into the glass mold, and subjected to radical crosslinking polymerization at room temperature for 1 hour to form a hydrophilic water gel.
  • two kinds of water gels having both hydroxy functional groups and amide functional groups namely N-(2-hydroxypropyl)methacrylamide (HPMA) water gel and N-hydroxyethyl acrylamide (HEAA) water, are provided. gum.
  • HPMA N-(2-hydroxypropyl)methacrylamide
  • HEAA N-hydroxyethyl acrylamide
  • gum First, after dissolving HPMA and HEAA in deionized water, respectively, a crosslinking agent NMBA was added and stirred for 10 minutes. After HPMA and HEAA were uniformly mixed with the crosslinking agent NMBA, respectively, the initiator APS was added for 10 minutes, and then the catalyst TEMED was added. Next, the syringe was sucked into the glass mold, and subjected to radical crosslinking polymerization at room temperature for 1 hour to form a hydrophilic water gel.
  • HPMA 2-hydroxypropyl)methacrylamide
  • HEAA N-hydroxyethyl acrylamide
  • Two kinds of diionic hydrocolloids are provided in this step, namely [3-(methacrylamido)propyl]dimethyl(3-thiopropyl)ammonium hydroxide (SBAA) water gel and [2-( Methacryloyl)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide (SBMA) water gel.
  • SBAA methacrylamido)propyl]dimethyl(3-thiopropyl)ammonium hydroxide
  • SBMA Methacryloylethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide
  • the hydrophilic polymer material can capture the water molecules in the water and the water to form a hydrate layer by hydrogen bonding or ionic bonding (diionic polymer) in an aqueous solution environment, thereby not only reducing the free energy of the contact interface between the material and the biomolecule. It also creates physical barriers that prevent proteins from being inaccessible and attached, and prevents biomolecules such as proteins from sticking (References Morisaku, T., J. Watanabe, T. Konno, M. Takai and K. Ishihara,: Hydration Of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis. Polymer, 2008.49(21): p.4652-4657. Chen, S., L.
  • Equilibrium water content can be calculated by the following formula (1):
  • the strength of hydration ability affects the degree to which the hydrophilic polymer material forms a hydration layer, and indirectly affects the effect of the material against biomolecular adhesion or protein adsorption.
  • the equilibrium water content can obtain the relationship between polymer materials and water molecules at the macroscopic scale, it cannot explain the affinity relationship between polymer materials and water molecules at the microscopic scale. The following experiments are further supported.
  • non-freezing water is the main reason for resisting the adhesion of biomolecules;
  • Freezable bound water frozen water is located outside the non-freezing water, frozen Water and polymer or non-freezing water are intermittent, so that the temperature of ice crystals is lower than 0 °C;
  • free water flowing water is outside the frozen water, flowing water is polymer or not The effect of frozen water is very slight, even unaffected, so that ordinary water will form ice crystals at 0 °C (Reference Higuchi, A.
  • the differential scanning calorimetry can be used to determine the amount of enthalpy change ( ⁇ Hf) in the water gel to determine the type of water molecules, so the content of different water molecules can be further identified (Ref. Literature Tanaka, M. and A. Mochizuki,: Effect of water structure on blood compatibility-thermal analysis of water in poly(meth)acrylate. Journal of Biomedical Materials Research Part A, 2004.68(4): p. 684-695. .
  • the higher the proportion of non-frozen water formed around the polymer the stronger the hydration ability at the microscopic scale, the denser the hydrated layer formed, and the more resistant to biomolecular adhesion or protein adsorption (Reference Morisaku, T., J.
  • the water gel was separately placed in deionized water, and the soaking solution was changed every 30 minutes and repeated 3 times to ensure that the methanol in the water gel was replaced. Subsequently, immersed in deionized water for 1 day, and remove excess water on the surface, take 3-4 mg of water gel in a special aluminum plate for differential scanning calorimetry (DSC), and set the DSC temperature range and conditions to After cooling from 25 ° C to -40 ° C, the temperature was raised to 40 ° C at 5 ° C per minute.
  • DSC differential scanning calorimetry
  • the conventional equilibrium moisture content is composed of non-frozen water and frozen water, so it can correspond to the following formula (3) and formula (4), and obtain the weight percentage of unfrozen water (w nonfreezable ):
  • the unit weight of unfrozen water and frozen water per unit weight of the water-gel polymer can be obtained by the following formula (5) and formula (5), wherein w polymer is the weight percentage of the polymer in the water gel:
  • W nonfreezable is the ratio of the polymer chain that forms unfrozen water (gH 2 O/g polymer). It can then be converted into the number of moles of unfrozen water bonded per mole of polymer repeating unit by the following formula, expressed as a dimensionless group N W .
  • M p is the molecular weight of each polymer repeat unit
  • M w is the molecular weight of the water molecule:
  • the various water gels prepared as described above were separately placed in deionized water and PBS, and the PBS was changed every 30 minutes and repeated three times to ensure that the methanol in the water gel was replaced. It was then transferred to a 24-well-tissue culture polystyrene plate (24-well TCPS disk) and washed 3 times with PBS. After immersing in 1 ml of PBS in each of the water gel samples and the empty TCPS wells for 2 hours in an oven at 37 ° C, the PBS was removed.
  • the target protein to be tested HSA, ⁇ -globulin and fibrinogen
  • PPP poor platelet plasma
  • BSA bovine serum albumin
  • a first antibody (1st antibody) specific for the target protein was added to each hydrocolloid sample, placed in an oven at 37 ° C for 30 minutes, and then washed three times with PBS to remove the solution.
  • the hydrocolloid sample was transferred to a new 24-well TCPS dish, and 0.5 ml of the developer 3,3',5,5'-tetramethylbenzidine was added to each hydrocolloid sample (3, 3',5,5'-Tetramethylbenzidine, TMB), after waiting for 6 minutes for color development, 0.5 ml of 1 M sulfuric acid was added to each water gel sample to terminate the reaction.
  • Each of the prepared water gels was separately immersed in PBS, and the PBS was changed every 30 minutes and repeated 3 times to ensure that the methanol in the water gel was replaced.
  • the water gel was then soaked in PBS and placed in an oven at 37 ° C for 1 day.
  • the PBS was removed and 1 ml of platelet concentrate and white blood cell thick solution were added to the water gel, and placed in an oven at 37 ° C for 30 minutes.
  • the water gel was then rinsed with PBS to remove unattached blood cells.
  • a 2.5 wt% glutaraldehyde solution was placed in a 24-well dish and placed in a refrigerator at 4 °C for 1 day to fix the blood cells.
  • the Nikon model A1R conjugated laser scanning microscope (CLSM) was used to observe the blood cell attachment, and the number of blood cell attachments per unit area was calculated.
  • the abscissa is a variety of different water gels, and the ordinate is the equilibrium moisture content (%).
  • the equilibrium water content of hydrophilic water gel (AAm, MAA, HEA, HEMA, HPMA and HEAA) in deionized water environment is higher than the equilibrium moisture content in PBS environment, because the amide of hydrophilic water gel Both the functional group and the hydroxy functional group can fully generate hydrogen bonds with water molecules, and the ionic interaction between the salt and water molecules in the PBS competes with the hydrogen bonding force of the hydrophilic water gel, resulting in a hydrophilic water gel.
  • the equilibrium moisture content in PBS is generally lower than the equilibrium moisture content in deionized water solutions.
  • the equilibrium water content of the double ionic water gel (SBAA and SMBA) in the deionized water environment is lower than the equilibrium water content in the PBS environment, because the positive and negative charges in the double ionic water gel structure are based on The ion-bonding method captures the water molecules and thus resists the salt ion interaction of the PBS, so that the double-ion water gel has a higher equilibrium moisture content in the PBS.
  • the equilibrium water content of the hydrophilic water gel is from low to high, hydroxy functional water gel (HEA, HEMA), amide functional water gel (AAm, MAA), and both hydroxy functional and amide functional water gel. (HPMA, HEAA).
  • the amide functional water gel is more hydrophilic than the hydroxy functional water gel, so if the water gel contains a hydroxyl functional group, further inclusion of the amide functional group increases the position at which hydrogen bonds are generated. Therefore, HPMA monomer water gel and HEAA monomer water gel have the highest equilibrium moisture content.
  • the equilibrium moisture content in deionized water, the proportion of non-frozen water and the dimensionless group in various water gels are shown.
  • the horizontal coordinate is a variety of different water gels, the left vertical coordinate is the equilibrium moisture content (%), and the right vertical coordinate is corresponding to the proportion of non-freezing water (gH 2 O/g polymer) and no cause Subgroup N w .
  • the non-freezing water content is represented by Wnonfrezzable.
  • the non-freezing water content of the amide functional water gel (AAm, MAA) in the hydrophilic water gel is significantly higher than that of the hydroxy functional water gel (HEA, HEMA) due to the amide.
  • the functional group is a hydrogen bond acceptor and a hydrogen bond donor, and the binding ability and binding probability for water molecules is higher than that of a hydrogen bond accepting only a hydrogen bond acceptor. Accordingly, the water gel having the HPMA monomer and the HEAA monomer can provide more hydrogen molecules at the same time because it has both a hydroxyl functional group and an amide functional group, so that more water molecules can be bonded around the polymer chain. This results in a more dense hydration layer.
  • NW the dimensionless group NW
  • the physical meaning is the number of moles of unfrozen water bonded per mole of polymer repeating unit
  • NW value Higher means that the amount of non-frozen water formed per mole of monomer is increased.
  • NW values from high to low are double ionic water gel (SBAA, SBMA), with both hydroxyl and amide based water gel (HPMA, HEAA), acyl Amine based water gel (AAm, MAA), hydroxy water gel (HEA, HEMA).
  • the chemical structure of the double-ionic water gel has a positive and negative charge, and the generated ion force is very strong, so the double-ionic water gel has good hydrophilicity and hydration ability.
  • the water-gel with HPMA monomer and HEAA monomer has hydroxy functional group and amide functional group in chemical structure, which can increase the position and probability of hydrogen bonding of water molecules, so the water hydration ability of water gel is no less than that of double ion. Sexual water gel.
  • Common plasma proteins are human serum albumin (HSA), immunoglobulin (Ig) and fibrinogen.
  • immunoglobulins can be divided into IgA, IgD, IgE, IgG and IgM, and most of IgG is ⁇ -globulin.
  • ⁇ -globulin has a structure in which saccharide residues and platelet enzyme glycosyltransferase cause platelet aggregation. Therefore, in this experiment, HSA, ⁇ -globulin and fibrinogen were used as target proteins to observe the adsorption effect of water gel on target proteins in plasma proteins.
  • the protein adsorption was tested by ELISA, because the relative adsorbed protein solution contained only one protein per ELISA test, and the environment lacked competition of other proteins in adsorption. Therefore, it is possible to show the maximum adsorption amount of different water gel materials for various proteins. As shown in Figure 5, the abscissa is a variety of different water gels, and the ordinate is the relative amount of protein adsorbed.
  • HSA Hydroxyl functional water gel
  • AAm amide functional water gel
  • HPMA both hydroxy functional and amide functional water gel
  • SBMA diionic water gel
  • the abscissa is a variety of different water gels.
  • the relative adsorption amount of fibrinogen corresponds to the left ordinate, and the cell density of platelets. (cells/mm2) corresponds to the ordinate of the right side.
  • the small figure in the figure is an enlarged schematic view of a part indicated by a broken line.
  • N,N-dimethylaminoethyl methacrylate, DMAEMA) and Trimellitic anhydride (TMA) are two kinds of positively charged water gels.
  • the adsorption amount of fibrinogen and the amount of platelets attached are quite high. Because the platelet surface is negatively charged, it is exposed to positively charged DMAEMA water gel and TMA water gel will cause irreversible adsorption due to electrostatic force and adsorb a large amount of platelets.
  • the hydroxyl functional water gel, the amide functional water gel of the invention, the hydroxy functional group and the amide functional water gel and the diionic water gel have relatively low adsorption amount and platelet adhesion amount in the fibrinogen, and are much smaller than Adsorption of fibrinogen and platelets by DMAEMA water gel and TMA water gel. It is noteworthy that the amount of HEAA monomer hydrogel adsorbed to fibrinogen is less than 3% (preferably less than 2%, more preferably less than 1%), while in Figure 6, the amount of fibrinogen and platelet adhesion is even Both tend to be close to zero.
  • the HEAA monomer water gel hardly adsorbs fibrinogen, and thus hardly adheres to platelets, thereby reducing platelet activation.
  • the reduction in platelet activation in turn reduces the clotting reaction in contact with blood, so that it is not necessary to add an anticoagulant when used to recover the platelet process.
  • conventionally known filter materials with charged surfaces tend to increase the concentration of bradykinin.
  • the use of HEAA monomer water gel with no surface charge can also avoid acute hypotensive reactions during transfusion due to an increase in the concentration of bradykinin.
  • the white blood cells are attached in a large amount by the electrostatic force between the white blood cells and the white blood cells. It is noteworthy that the white blood cell attachment amount of HPMA monomer water gel and HEAA monomer water gel having both hydroxy functional group and amide functional water gel is significantly different. Specifically, the HPMA monomer water gel has a very low white blood cell adhesion, and the HEAA monomer water gel has a very high white blood cell adhesion, even higher than the positively charged water gel DMAEMA, indicating that the HEAA monomer water gel has white blood cells. Extremely high affinity.
  • HPMA monomer water gel does not attach white blood cells, but HEAA monomer water gel has a large number. White blood cells are attached and hardly adsorb plasma proteins, and platelets are hardly attached.
  • the use of HEAA monomers or/and polymers polymerized with other compounds allows a large number of white blood cells to adhere to the polymer without the need to assist in the adsorption of white blood cells via plasma proteins and platelets.
  • the HEAA-containing polymer may directly adsorb to the surface of white blood cells, or it may be because a small amount of intercellular adhesion molecules are adsorbed on the HEAA-containing polymer to assist the affinity of white blood cells. Adsorption.
  • the surface of the substrate was modified to further verify the ability of the present embodiment to capture or separate white blood cells.
  • test materials of the examples are the aforementioned N-Hydroxyethyl acrylamide (HEAA) having both a hydroxyl functional group and an amide functional group, and have the following chemical structures:
  • test material of the comparative example was a positively charged N,N-dimethylaminoethyl methacrylate (DMAEMA) having the following chemical structure:
  • BMA and GMA are used for the occlusal end of the polymer to be tested, so that the polymer to be tested can be physically adsorbed.
  • the surface of the substrate. BMA and GMA have the following chemical structures:
  • the material of the substrate is polypropylene (PP) and polyethylene terephthalate (PET).
  • PP and PET have the following chemical structures:
  • BMA and GMA were used as the substrate occlusion end, HEAA and DMAEMA were used as functional end, and the ratio (about 70% of the occlusal end and about 30% of the active end) was mixed, and the initiator ACVA was added, and the solvent was ethanol.
  • polymerization was carried out for 24 hours in an environment of 70 ° C to prepare BMA-r-HEAA, BMA-r-DMAEMA, BMA-r-GMA-r-HEAA, BMA-r-GMA-r-DMAEMA polymer. After the reaction was completed, the product was precipitated with deionized water as a precipitating agent and dried.
  • PP and PET were selected as the substrates to be modified.
  • BMA-r-HEAA, BMA-r-DMAEMA, BMA-r-GMA-r-HEAA, BMA-r-GMA-r-DMAEMA polymer were respectively prepared, and ethanol was used as a solvent to prepare a polymer solution.
  • the PP substrate was taken, cut to a suitable size, immersed in the polymer solution for 1 minute, and then the surface residual solution was washed with deionized water and dried. According to this, BMA-r-HEAA, BMA-r-DMAEMA, respectively, can be obtained.
  • the HEAA and DMAEMA monomers were taken and used as a solvent to prepare a monomer solution.
  • the PET substrate was taken, cut to a suitable size, immersed in a monomer solution, and placed under 7200 W of UV light for 2 minutes, and then the surface residual solution was washed with deionized water and dried. Accordingly, a surface-modified PET substrate grafted with HEAA and DMAEMA monomers can be obtained separately.
  • the PP and PET substrates were weighed by a microbalance. After the surface has been modified, the substrate is dried and the weight of the surface is weighed via a microbalance. The weight of the polymer modified on the surface of the substrate can be obtained by calculating the difference in weight of the substrate before and after the experiment. Finally, the weight of the polymer per unit area can be obtained by conversion, that is, the surface coating density.
  • the surface composition elements were analyzed by X-ray photoelectron spectroscopy (XPS) on the surface of PP and PET modified by HEAA. The characteristic peaks of the map are then used to compare the nitrogen content of each modified surface.
  • XPS X-ray photoelectron spectroscopy
  • the substrate was first immersed in PBS buffer for half an hour. Next, after the liquid was blotted dry, 1 ml of red blood cells, white blood cells, and platelet thick liquid were respectively applied to the surface of the substrate, and placed in an oven at 37 ° C for 2 hours. Then, the unattached blood cells on the surface of the substrate were washed with PBS buffer, immersed in glutaraldehyde for one day, and the blood cells were observed on the surface of the polypropylene disk by a conjugated laser scanning electron microscope (LSCM). Attachment situation.
  • LSCM conjugated laser scanning electron microscope
  • the modified substrate was cut into a circle having a diameter of 2.6 cm and stacked in 5 layers, and locked in an acrylic filter (similar to the exemplary embodiment shown in Fig. 2). 5 ml of platelet thick solution was taken for filtration. Next, the blood samples before and after the filtration were examined using a blood cell counter to calculate the white blood cell removal rate and the platelet retention rate.
  • Fig. 8 shows the structural formula and chemical shift of each monomer and polymer compound.
  • polymer compounds such as BMA-r-HEAA, BMA-r-DMAEMA, BMA-r-GMA-r-HEAA, BMA-r-GMA-r- are herein.
  • DMAEMA is abbreviated as BrH, BrD, BrGrH, BrGrD.
  • Fig. 9 is an NMR chart of each monomer and polymer structure. After nuclear magnetic resonance analysis, the characteristic peak of HEAA is mainly reflected in a, and the characteristic peak of DMAEMA is mainly reflected in b. It can be seen from the nuclear magnetic resonance spectrum that the polymer compound used in this test has been successfully synthesized.
  • the PP film is a PP substrate coated with the polymer compounds BrH, BrD, BrGrH, and BrGrD.
  • the PET film is a PET substrate with monomer grafted HEAA and DMAEMA.
  • the surface of the modified substrate can be analyzed by surface X-ray photoelectron spectroscopy. From the analysis of nitrogen, the difference before and after the modification can be seen. As shown in Fig. 11, it is an analytical map of nitrogen element on the upper surface of the substrate. Both the PP control group and the PET control group are unmodified PP substrates and PET substrates. The proportions of surface elements before and after HEAA modification are detailed in Table 2 below. Referring to Figure 11 and Table 2 below, there is no nitrogen signal on the surface of the unmodified PP substrate. On the contrary, the ratio of nitrogen elements on the surface of PET and PP substrates modified by HEAA monomer or polymer increased significantly, and the characteristic peaks of nitrogen appeared at positions of 399-402eV.
  • Figures 12 to 15 show blood cell attachment images and their counts before and after surface modification of PP and PET substrates.
  • FIG. 12 and FIG. 13 the result of attaching blood cells to the PP substrate after physical coating modification is shown.
  • the platelet plasma and red blood cell images on the HEAA-containing PP substrate PP B-r-H and PP B-r-G-r-H
  • the white blood cell images on the PP substrate containing B-r-H are obviously deeper, meaning that white blood cells are easier to attach.
  • the upper graph shows the results of the white blood cell attachment amount
  • the lower graph shows the results of the red blood cell attachment amount.
  • the surface of the PP substrate containing HEAA containing PP substrate containing HEAA can effectively capture white blood cells, and the adhesion amount of platelets and red blood cells is low.
  • the surface of PP substrate containing DMAEMA not only captures white blood cells, but also the amount of platelets and red blood cells attached. Higher.
  • FIG. 14 and FIG. 15 Please continue to refer to FIG. 14 and FIG. 15 for the result of attaching blood cells to the PET substrate after chemical graft modification.
  • the platelet plasma and red blood cell images on the PET substrate (PET HEAA (UV)) grafted with HEAA are shallow, meaning that platelet plasma and red blood cells are less likely to be attached.
  • the white blood cell images on the PET substrate grafted with HEAA are significantly deeper, meaning that white blood cells are easier to attach.
  • the upper graph shows the results of the white blood cell attachment amount
  • the lower graph shows the results of the red blood cell attachment amount.
  • the surface of the PET substrate grafted with HEAA has the ability to capture white blood cells, and at the same time, the amount of platelets and red blood cells can be reduced. Conversely, all three blood cells are attached to the surface of the PET substrate grafted with DMAEMA.
  • the red blood cell thick liquid actually contains trace white blood cells
  • the HEAA-containing substrate can still be filled with the red blood cell thick liquid. Capture traces of white blood cells.
  • the surface of the HEAA modified substrate is shown to be specific for white blood cell capture.
  • the blood filtration test as proposed in the above test method uses a modified substrate to filter the platelet thick liquid. After the blood cell counter is detected, the blood cell contents in the blood before and after the filtration can be known, and the white blood cell removal rate and the platelet retention rate are calculated accordingly.
  • WBC is a white blood cell
  • PLT is a platelet
  • the substrate modified by HEAA has a white blood cell capture rate of at least 87%, and can retain more than 86% of platelets. That is, when a platelet thick liquid (a platelet thick liquid containing white blood cells) flows through the modified substrate, most of the white blood cells adhere to the substrate, and most of the remaining platelets are retained in the remaining filtrate. Accordingly, the embodiment of the present invention has a very high specificity for white blood cell capture (does not cause red blood cells and platelets to be captured or attached), and is a good material for reducing white blood cells as a platelet thick liquid. Of course, in other embodiments, it is also possible to directly prepare a platelet thick solution by, for example, passing a blood sample of whole blood through the above substrate and then removing the red blood cells.

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Abstract

一种聚合物，用于捕捉或分离白血球。聚合物是以含酰胺基及羟基的单体进行聚合反应制备而得。此含酰胺基及羟基的单体具有式(1)的结构。在式(1)中，R 1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环所组成的群组，R 2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数。

Description

捕捉或分离白血球的聚合物、装置、其制造方法及其应用 技术领域
本发明实施例是关于一种捕捉或分离细胞的材料，特别是一种用于捕捉或分离白血球的聚合物。
背景技术
输血主要是藉由输注来自捐赠者的血品至病人体内，以补充病人缺乏的血液成份。血品(或称血液成份)种类主要包含全血(whole blood)、血浆、洗涤红血球(washed erythrocyte)、红血球浓厚液(erythrocyte concentrate)、白血球浓厚液(leukocyte concentrate)与血小板浓厚液(platelet concentrate)。输血时可视病人需求选择输入的血液成份。
目前已知输血后的许多不良反应，与白血球或白血球所释放出的细胞介质(cytokines)有关。不良反应例如有：非溶血性发热反应(non-hemolytic febrile transfusion reactions，NHFTR)、异体免疫反应(alloimmunization)、病毒感染及与输血相关移植体抗宿主疾病(transfusion-associated graft versus host disease，TA-GVHD)等。因此，目前已有许多国家将白血球减除列为输血的必要程序，不论是红血球或血小板的输血皆须将血液中的白血球浓度减至一定程度，以避免病患产生不良反应。
习知白血球的分离是利用表面带有电荷的过滤材料。然而，以带电荷的表面来分离白血球，容易造成缓激肽(bradykinin)的浓度增加，易引发输血时的急性低血压反应(hypotensive transfusion reaction)。
此外，为避免于输血或是血液处理的过程中引发凝血或活化血小板，一般在血液样品中需要添加抗凝血剂，或于过滤材料表面进一步进行表面处理，以防止凝血或活化血小板。目前，在白血球减除技术领域中，尚未有一种适合的过滤材料，可有效解决上述习知技术的问题。
发明内容
本发明的目的为提供具有高度白血球捕捉或分离能力的材料，较佳地是该材料具有可避免纤维蛋白原(fibrinogen)吸附及/或血小板贴附，从而在白血球减 除过程中具有高度的血小板保留率。
本发明的一态样为一种聚合物，为用于捕捉或分离白血球的材料。此聚合物是以一含酰胺基及羟基的单体进行一聚合反应制备而得，其中该含酰胺基及羟基的单体具有式(1)的结构：
Figure PCTCN2017110177-appb-000001
在式(1)中，R1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环所组成的群组，R2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数。
根据本发明的一些实施方式，其中式(1)中的R1为氢，R2为氢，以及n为1。
根据本发明的一些实施方式，该含酰胺基及羟基的单体为N-羟乙基丙烯酰胺((N-Hydroxyethyl acrylamide)，或N-(2-羟乙基)丙烯酰胺(N-(2-Hyroxyethyl)acrylamide)。
根据本发明的一些实施方式，聚合物为共聚物(copolymer)，其是由该含酰胺基及羟基的单体及至少另一单体共聚合而成。
根据本发明的一些实施方式，另一单体是甲基丙烯酸甲酯(Butyl methacrylate，BMA)或甲基丙烯酸缩水甘油酯(Glycidyl methacrylate，GMA)。
根据本发明的一些实施方式，聚合物为链段高分子。
根据本发明的一些实施方式，聚合物为交联聚合物。
根据本发明的一些实施方式，聚合反应包含使用一交联剂，且该交联剂具有双丙烯酸酯的官能基。
根据本发明的一些实施方式，交联剂是亚甲基双丙烯酰胺及二甲基丙烯酸乙二醇酯、聚乳酸-聚乙二醇-聚乳酸聚乙醇酸共聚物或聚乙二醇二丙烯酸酯。
本发明的一态样为一种用于捕捉或分离白血球的装置，包含外壳以及本 体。本体包含聚合物，且此聚合物包含式(2)的结构：
Figure PCTCN2017110177-appb-000002
在式(2)中，n为10至50的整数。
根据本发明的一些实施方式，聚合物具有式(3)的结构：
Figure PCTCN2017110177-appb-000003
在式(3)中，m为50至90的整数，且
Figure PCTCN2017110177-appb-000004
Figure PCTCN2017110177-appb-000005
Figure PCTCN2017110177-appb-000006
其中p为2至6的整数，q为1至6的整数，r为1至6的整数，s为1至6的整数。
根据本发明的一些实施方式，聚合物具有式(4)的结构：
Figure PCTCN2017110177-appb-000007
在式(4)中，t为50至90的整数，
Figure PCTCN2017110177-appb-000008
Figure PCTCN2017110177-appb-000009
根据本发明的一些实施方式，本体包括基材，其中聚合物是以涂布(coating)、喷洒或浸渍的方式设置于基材上。
根据本发明的一些实施方式，本体包括基材，其中聚合物是以锚定(anchoring)的方式固定于基材上。
根据本发明的一些实施方式，过滤材料的表面元素包含碳、氧及氮，且碳、氧及氮的总摩尔百分比定义为100mol％，碳的摩尔百分比为约76.22％至约79.84％，氧的摩尔百分比为约18.1％至约21.04％，以及氮的摩尔百分比为约 2.05％至约2.75％。
本发明的一态样为一种含酰胺基及羟基的聚合物的用途，其是用于捕捉或分离白血球。含酰胺基及羟基的聚合物包含式(2)的结构：
Figure PCTCN2017110177-appb-000010
其中，n为10至50的整数。
根据本发明的一些实施方式，含酰胺基及羟基的聚合物其白血球的捕捉率不低于70％。
根据本发明的一些实施方式，含酰胺基及羟基的聚合物其血小板的保留率不低于85％。
本发明的一态样为一种含酰胺基及羟基的单体的用途，其是用于制造捕捉或分离白血球的过滤材料。此含酰胺基及羟基的单体具有式(1)的结构：
Figure PCTCN2017110177-appb-000011
在式(1)中，R1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环所组成的群组，R2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数。
根据本发明的一些实施方式，其中制造捕捉或分离白血球的过滤材料的步骤包含提供基材，以及将含酰胺基及羟基的单体修饰于此基材上。
根据本发明的一些实施方式，含酰胺基及羟基的单体修饰于基材上包含：提供含酰胺基及羟基的单体；提供至少一锚定单体(anchoring unit)；聚合含酰胺基及羟基的单体及锚定单体形成共聚物；以及通过锚定单体锚定共聚物于基材上。
根据本发明的一些实施方式，含酰胺基及羟基的单体占共聚物的重量百分 比为约百分之20至约百分之40。
根据本发明的一些实施方式，锚定单体占共聚物的重量百分比为约百分之60至约百分之80。
根据本发明的一些实施方式，锚定单体是选自由甲基丙烯酸丁酯、甲基丙烯酸缩水甘油酯及其组合所组成的群组。
根据本发明的一些实施方式，其中将含酰胺基及羟基的单体修饰于基材上的步骤包含涂布含酰胺基及羟基的单体于基板上，以及以紫外线处理基板上的含酰胺基及羟基的单体。
本发明的一态样为一种白血球浓厚液的制备方法，包含：提供如前述用于捕捉或分离白血球的装置；提供血液样品，其中血液样品包含白血球及血小板；将血液样品通过此装置，使得白血球被捕捉于此装置中，或自血液样品中被分离至此装置中；以及脱附被捕捉或分离的白血球。
本发明的一态样为一种红血球浓厚液的制备方法，包含：提供如前述用于捕捉或分离白血球的装置；提供血液样品，其中血液样品包含红血球、白血球及血小板；将血液样品通过装置而获得滤液，其中白血球被捕捉于此装置中或自该血液样品中被分离出来；以及处理滤液为红血球浓厚液。
本发明的一态样为一种血浆制品储存前的处理方法，包含：提供如前述用于捕捉或分离白血球的装置；提供血液样品，其中血液样品包含血浆及白血球；以及将血液样品通过装置而获得滤液，其中白血球于装置中被捕捉或自血液样品中分离出来。
本发明的一态样为一种去除全血血液中白血球方法，包含：提供如前述用于捕捉或分离白血球的装置；提供全血血液样品，其中全血血液样品包含红血球、白血球及血小板；以及将全血血液样品通过装置而获得滤液，其中白血球于装置中被捕捉或自全血血液样品中被分离出来。
一种血小板浓厚液的制备方法，包含：提供如前述用于捕捉或分离白血球的装置；提供一血液样品，其中该血液样品包含白血球及血小板；将血液样品通过此装置而获得一滤液，其中白血球被捕捉于此装置中或自此血液样品中分离出来；以及处理滤液为血小板浓厚液。其中，血液样品可以为全血血液样品，或者为经过离心处理去除红血球的血液样品。
综上所述，本发明是提供一种用于捕捉或分离白血球的聚合物、装置、其 制造方法及其用途。以含酰胺基及羟基的化合物或其作为单体形成的聚合物，提供捕捉或分离白血球的功能。由于此类化合物及聚合物对白血球具有高的亲和性，可通过捕抓、吸附、贴附或粘附白血球而使其例如自全血血品中分离出来，且重要的是，过程中几乎不会吸附血浆蛋白质，亦几乎不会造成血小板贴附，提高血小板的保留率。请参照图1，绘示白血球减除的两种路径，路径A及路径B。一般白血球减除的作用机制主要是藉由带正电的过滤材料F，吸附血浆蛋白质P1后，引起血小板P2贴附。被贴附的血小板P2活化后，分泌作为信号传递的细胞介质和生长因子(growth factor)会进一步引起白血球L贴附。由于血小板P2及白血球L带负电，故过滤材料F表面一般修饰成带正电荷。然而，使用带正电的过滤材料F，易导致血小板P2活化，不易在除去白血球L的同时高效率回收血小板P2。本发明的白血球减除作用机制(如图1的路径B)与习知机制(如图1的路径A)不同，利用本发明实施方式所制备的过滤材料F’可解决过去白血球减除所面临的问题。据此，藉由本发明可有效分离白血球、避免活化血小板或凝血，亦可避免增加缓激肽的浓度，而导致输血时的急性低血压反应。
附图说明
当结合附图阅读以下详细描述时将更好地理解本揭露内容的态样。但须注意依照本产业的标准做法，各种特征未按照比例绘制。事实上，各种特征的尺寸为了清楚的讨论而可被任意放大或缩小。
图1是根据本发明一些实施方式，绘示白血球减除作用机制的示意图。
图2是根据本发明一些实施方式，绘示一种用于分离或捕捉白血球的过滤装置。
图3是根据本发明一些实施方式，绘示各种水胶材料的平衡含水量及油接触角的结果图。
图4是根据本发明一些实施方式，绘示各种水胶材料的平衡含水量、不冻水比例及无因次群的结果图。
图5是根据本发明一些实施方式，绘示各种水胶于酶结合免疫吸附(enzyme-linked immunosorbent assay，ELISA)试验中，相对蛋白质吸附的结果图。
图6是根据本发明一些实施方式，绘示各种水胶材料表而上的蛋白质吸附 量及细胞密度的结果图。
图7是根据本发明一些实施方式，绘示各种水胶上的白血球贴附量的结果图。
图8是根据本发明一些实施方式，为各单体、高分子化合物的结构式及其核磁共振图谱信号的化学位移。
图9是根据本发明一些实施方式，为各单体、高分子化合物的核磁共振(Nuclear Magnetic Resonance spectroscopy，NMR)化学结构图谱。
图10是根据本发明一些实施方式，为聚丙烯(polypropylene，PP)基材及聚对苯二甲酸乙二酯(polyethylene terephthalate，PET)基材上的材料接枝披覆密度。
图11是根据本发明一些实施方式，为PP基材及PET基材表面的氮元素的光电子能谱仪分析图谱。
图12是根据本发明一些实施方式，为各种PP基材上的白血球、血小板、与红血球贴附影像的定性结果图。
图13是根据本发明一些实施方式，为各种PP基材上其白血球、血小板、与红血球贴附量的定量结果图。
图14是根据本发明一些实施方式，为各种PET基材上的白血球、血小板、与红血球贴附影像的定性结果图。
图15是根据本发明一些实施方式，为各种改质的PET基材上其白血球、血小板、与红血球贴附量的定量结果图。
其中，符号说明：
A路径                B路径
F过滤材料            F’过滤材料
L白血球              P1血浆蛋白质
P2血小板             1上壳体
2过滤材料            3下壳体
具体实施方式
本揭露接下来将会提供许多不同的实施方式或实施例以实施本揭露中不同的特征。各特定实施例中的组成及配置将会在以下作描述以简化本揭露。这些为实施例仅作为式范并非用于限定本揭露。例如，一第一元件形成于一第二 元件「上方」或「之上」可包含实施例中的第一元件与第二元件直接接触，亦可包含第一元件与第二元件之间更有其他额外元件使第一元件与第二元件无直接接触。此外，在本揭露各种不同的范例中，将重复地使用元件符号及/或字母。此重复乃为了简化与清晰的目的，而其本身并不决定各种实施例及/或结构配置之间的关系。此外，各种特征乃为了简化与清晰可能会依不同比例做绘制。
更进一步，像是「之下」、「下面」、「较低」、「上面」、「较高」、以及其他类似的相对空间关系的用语，可用于此处以便描述图式中一元件或特征与另一元件或特征之间的关系。该等相对空间关系的用语乃为了涵盖除了图式所描述的方向以外，装置于使用或操作中的各种不同的方向。举例来说，若于图中的装置被翻转过来，原先被描述为在其他元件或特征「之下」或「下面」的元件则变成在其他元件或特征「上面」。因此，范例用语「之下」皆能包含上面及之下的方位。上述装置可另有其他导向方式(旋转90度或朝其他方向)，此时的空间相对关系也可依上述方式解读。
用语「捕捉」是指血液样品中的血球细胞，接触到材料表面上，受到材料与血球细胞之间的疏水、氢键、或静电分子作用力的吸引，造成各式血球细胞可能直接吸贴附于材料表面上，或先吸附体积较小的血浆蛋白质与血小板，才导致较大型的血球细胞贴附，这些过程被定义成血球「捕捉」。
用语「分离」是指将含有白血球的样品通过分离白血球的材料后，可将白血球由样品中分离出来，亦指可减少样品中的白血球含量，甚至可大量减少样品中的白血球含量，而使得分离后的滤液的白血球浓度小于原来含有白血球的样品。例如，可将含有白血球和血小板的样品分离，以达到血小板的高回收率；或将白血球和红血球的样品分离，以达到红血球的高回收率。
用语「减除白血球(leukocyte depletion)」并非是指所有的或实质上所有的白血球被完全去除。该用语是用以广义地指出白血球的数目在分离或过滤的过程中减少。
用语「血小板浓厚液(platelet concentrate)」并非是指限制在于血品中的血小板浓厚液，而是广泛地包含将含有血小板的细胞悬浮液或血液样品，经过分离材料或过滤材料处理过所得到的滤液。其中，滤液的体积与细胞悬浮液或血液样品的体积相较，可能变多或变少。
用语「白血球浓厚液(leukocytes concentrate)」是广泛地包含将含有白血球的细胞悬浮液或血液样品，经过分离材料或过滤材料处理过，留在分离材料或是过滤材料上的物质经缓冲液、溶剂或其他溶液冲洗后的冲洗液。富有白血球的冲洗液的体积与细胞悬浮液或血液样品的体积相较，可能变多或变少。
用语「红血球浓厚液(erythrocyte concentrate)」并非是指限制在于血品中的红血球浓厚液，而是广泛地包含将含有红血球的细胞悬浮液或血液样品，经过分离材料或过滤材料处理过所得到的滤液。其中，滤液的体积与细胞悬浮液或血液样品的体积相较，可能变多或变少。
本发明的一实施态样是一种聚合物，用于捕捉或分离白血球。聚合物可以作为制造捕捉或分离白血球的材料，或者藉由设置于其他物质或基材上单独或共同成为捕捉或分离白血球的材料。
聚合物是利用包含含酰胺基及羟基的单体进行聚合反应后制备而得，例如完全以含酰胺基及羟基的单体进行聚合反应，或是以含酰胺基及羟基的单体及其他化合物进行共聚合反应。应说明的是，此聚合反应可使用任何适合的材料。适合的材料泛指任何尚未进行聚合反应的化合物，其是用于制造捕捉或分离白血球的聚合物。在一实施例中，聚合反应中可包括含酰胺基及羟基的单体，此含酰胺基及羟基的单体具有式(1)的结构：
Figure PCTCN2017110177-appb-000012
在该式(1)中，R1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环所组成的群组，R2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数；以及至少一交联剂。
在一实施例中，聚合物为共聚物(copolymer)，其是由该含酰胺基及羟基的单体及至少另一单体共聚合而成。在一实施例中，聚合物为链段高分子。在一实施例中，聚合物为交联共聚高分子。在一实施例中，含酰胺基及羟基的单体为N-羟乙基丙烯酰胺(N-Hydroxyethyl acrylamide)或称为N-(2-羟乙基)丙烯酰胺(N-(2-Hyroxyethyl)acrylamide，HEAA)。举例来说，含酰胺基及羟基的单 体具有式(1)的结构，其中R1为氢，R2为氢，以及n为1，化学结构式如下：
Figure PCTCN2017110177-appb-000013
N-羟乙基丙烯酰胺是同时具有羟基官能基(-OH)和酰胺官能基(-RnC(O)xNR′2，其中R和R′指氢原子或有机基团)的化合物。在一实施例中，聚合物可单纯只由N-羟乙基丙烯酰胺单体聚合而成，或是由N-羟乙基丙烯酰胺与其他化合物的经共聚合反应而形成的共聚物。
分离白血球材料中包含N-羟乙基丙烯酰胺单体，对白血球具有高的亲和性，可专一性捕抓、吸附、贴附或粘附白血球，且几乎不会吸附血浆蛋白质及贴附血小板(具体的实验数据将详述于后)。藉此，本发明的分离白血球材料即可有效分离白血球。另外，本发明的分离白血球材料可避免活化血小板或凝血，避免增加缓激肽的浓度，而导致输血时的急性低血压反应。
在一些实施例中，前述聚合反应中更包含使用交联剂，例如但不限于使用小于10wt％的交联剂。交联剂是用于强化水胶材料的机械性质，与材料的血液相容性无关。交联剂可与含酰胺基及羟基的单体(例如：N-羟乙基丙烯酰胺单体)混合进行聚合反应。此交联剂可为单体化合物或聚合物。在一些实施例中，交联剂为单体化合物时，其是选自由亚甲基双丙烯酰胺(N，N’-Methylenebisacrylamide，NMBA)及二甲基丙烯酸乙二醇酯(Ethylene glycol dimethacrylate，EGDMA)所组成的群组。在一些实施例中，交联剂为聚合物时，其是聚乳酸-聚乙二醇-聚乳酸聚乙醇酸共聚物(PLA-PEG-PLGA，PLA：Polylactic Acid，PEG：Polyethylene glycol，PLGA：poly(lactic acid-co-glycolic acid)或聚乙二醇二丙烯酸酯(Poly(ethylene glycol)diacylate，PEGDA)。在一些实施例中，交联剂较佳选自NMBA，并于室温25℃下进行聚合反应。
交联剂的化学结构式如下：
Figure PCTCN2017110177-appb-000014
Figure PCTCN2017110177-appb-000015
Figure PCTCN2017110177-appb-000016
Figure PCTCN2017110177-appb-000017
在一些实施例中，用于捕捉或分离白血球的聚合物可为水胶材料，例如经过交联反应的交联聚合物。在本发明其他一些实施例中，聚合物可为粉体材料，例如经过与其他单体进行共聚反应所形成的链段高分子共聚物。粉体材料可以溶于醇类溶液中形成液态，再进行涂布、喷洒或浸渍等应用。有关交联聚合物以及共聚物的具体实例请参考以下说明。
在一些实施例中，前述聚合反应中进一步包含使用起始剂(initiator)或催化剂(catalyst)。举例来说，前述N-羟乙基丙烯酰胺单体可与起始剂(initiator)或催化剂(catalyst)混合，以加速聚合反应。在一些实施例中，起始剂为过硫酸铵(ammonium peroxodisulfate，APS)。在一些实施例中，催化剂为四甲基乙二胺(N，N，N，N tetramethylethylenediamine，TEMED)。
在一实施例中，聚合反应完成后用于捕捉或分离的白血球材料，可依所需要的尺寸进行切割或塑型，以成为不同的形状，例如为膜状、板状、块状、纤维状、管状、珠状、颗粒状、或粉状，以供后续分离或过滤流程的进行。要补 充的是，分离或过滤流程可为连续式或是批次式，例如分离白血球材料可为滤膜、滤板；或是充填于管柱中的纤维、颗粒或粉体。
本发明的一实施态样是一种用于捕捉或分离白血球的装置。该装置包括一外壳以及一本体。该本体可以为膜状、板状、块状、纤维状、管状、珠状、颗粒状、或粉状，且该本体包括一基材以及上述的聚合物。其中基材为构成本体形状的主要部分，而聚合物是可以通过全部或部分涂布、喷洒或浸渍的方式设置于基材，例如涂布于膜形基材的一面或两面，或基材全部浸渍于聚合物中。通过聚合物，本体具有捕捉或分离白血球的功能，是以，在一些实施例或实施态样中，本体可以称之为过滤材料。
本发明的一实施态样是一种用于捕捉或分离白血球的装置，包含外壳以及本体。在一些实施例中，本体包括基材以及设置于基材上的捕捉或分离白血球的聚合物，聚合物具有式(2)的结构：
Figure PCTCN2017110177-appb-000018
其中，n为10至50的整数。在一些实施例中，聚合物可为水胶材料。在一些实施例中，用于捕捉或分离白血球的装置可为过滤装置。
如图2所示，是根据本发明的一些实施例，为用于捕捉或分离白血球的过滤器。外壳包含上壳体1及下壳体3，而过滤材料2则位于上壳体1及下壳体3之间。过滤器可为分离白血球的过滤器、血小板减除白血球过滤器、或是红血球减除白血球过滤器。具体而言，分离白血球的过滤器是用以从一血液样品中分离白血球。血小板减除白血球过滤器是将含有白血球和血小板的悬浮液样品通过过滤器，使得白血球和血小板分离，进而达到血小板的高回收率。红血球减除白血球过滤器则是将含有白血球和红血球的悬浮液样品通过过滤器，使得白血球和红血球分离，进而达到红血球的高回收率。在一实施例中，血液样品包含全血血液样品、含有白血球及血小板的样品、含有血浆蛋白质、血小板及白血球的样品、含有白血球及红血球的样品、或其他含有细胞悬浮液的样品。
输血前或是血浆制品储存前，可使用本发明的过滤材料以进行输血前或是血浆制品储存前的处理，以将全血、血小板浓厚液或红血球浓厚液中的白血球浓度减至一定程度(白血球减除)，以避免或减少产生的不良反应，且符合各国法规规定。例如美国血库协会(American Association of Blood Banks，AABB)规定，每单位血品的白血球含量必须小于5×106，欧洲普遍标准为每单位血品的白血球含量需低于1×106。
过滤材料2中的聚合物可藉由含酰胺基及羟基的单体与交联剂混合进行聚合反应而得。在一些实施例中，前述用于捕捉或分离白血球的过滤器中，过滤材料2的聚合物具有式(3)的结构：
Figure PCTCN2017110177-appb-000019
其中，m为50至90的整数，且R1＝
Figure PCTCN2017110177-appb-000020
Figure PCTCN2017110177-appb-000021
Figure PCTCN2017110177-appb-000022
Figure PCTCN2017110177-appb-000023
其中p为2至6的整数，q为1至6的整数，r为1至6的整数，s为1至6的整数。
此处所用的结构符号
Figure PCTCN2017110177-appb-000024
是用于表示尚未键结的状态。换言之，若一取代基连接此符号泛指此取代基可再行连接其他任何取代基。此处所用的结构符号「*」是用于表示取代基在化学结构中的连接位置。
过滤材料2中聚合物可藉由含酰胺基及羟基的单体与吸附单体混合进行聚合反应而得。在一些实施例中，前述用于捕捉或分离白血球的过滤器中，过滤材料2的聚合物具有式(4)的结构：
Figure PCTCN2017110177-appb-000025
其中，t为50至90整数。
Figure PCTCN2017110177-appb-000026
在一些实施例中，过滤材料2可为膜改质基板。举例来说，将前述具有式(2)、式(3)或式(4)的聚合物涂布至基材上对基材表面进行改质。在一些实施例中，经改质后的基材表面元素包含碳、氧及氮，且碳、氧及氮的总摩尔百分比 定义为100％，其中碳的摩尔百分比为约76.22％至约79.84％，氧的摩尔百分比为约18.1％至约21.04％，以及氮的摩尔百分比为约2.05％至约2.75％。
本发明的一实施态样是一种含酰胺基及羟基的聚合物的用途，其是用于捕捉或分离白血球，含酰胺基及羟基的聚合物包含式(2)的结构：
Figure PCTCN2017110177-appb-000027
其中，n为10至50整数。
举例来说，在一些实施例中，前述含式(2)所示结构的聚合物是由N-羟乙基丙烯酰胺聚合而成。将带有白血球的样品通过含N-羟乙基丙烯酰胺的材料，以使得N-羟乙基丙烯酰胺能专一性捕抓、吸附、贴附或粘附白血球。在另一实施例中，分离白血球的用途则是将带有白血球的样品与用于捕捉或分离白血球的材料行接触反应，而接触方式可为通过(流过)、过滤或批次接触，接触后可收集液体，或取出用于捕捉或分离白血球的材料。利用前述含式(2)的聚合物，可使得白血球的捕捉率至少为87％。
本发明的一实施态样是一种如前述具有式(1)的含酰胺基及羟基的单体的用途，其是用于制造捕捉或分离白血球的过滤材料。
在一些实施例中，制造捕捉或分离白血球的过滤材料的步骤包含提供基材，以及将含酰胺基及羟基的单体修饰于基材上。基材可为膜状、板状、块状、纤维状、管状、珠状、颗粒状、或粉状基材。可视分离或过滤流程的用途及需求，而将含酰胺基及羟基的单体修饰至基材表面。举例而言，N-羟乙基丙烯酰胺单体或N-羟乙基丙烯酰胺单体与其他化合物行聚合反应后可为一涂布材料，随后涂布于基材的表面。要补充的是，后续分离或过滤流程可为连续式或是批次式，例如用于捕捉或分离白血球的过滤材料可为滤膜、滤板或是充填于管柱中的纤维、颗粒或粉体。在一些实施例中，基材为聚丙烯或聚乙烯对苯二甲酸酯。
此外，基材表面修饰的方法可为物理性修饰或化学性修饰。物理性修饰方法包含涂布(coating)，是藉由物理作用力进行材料表面性质修饰，而涂布吸附作用力包含凡德瓦力、氢键作用力、疏水作用力、静电作用力等。化学性修饰方法则包含接枝(branching)或蚀刻(etching)，接枝方法为藉由化学键结进行材料表面性质修饰，化学接枝方法包含臭氧、紫外线、或等离子体起始自由基聚合法。此外，亦可结合涂布方法与接枝方法进行材料表面性质修饰，可先以涂布将材料单体或共聚高分子吸附于基材表面，再以臭氧、紫外线、或等离子体处理进行化学接枝。表面修饰方法较佳为涂布、接枝或其组合。
在一些实施例中，基材表面修饰的方法是采用物理性修饰方法。举例来说，将含酰胺基及羟基的单体修饰于基材上包含下述步骤。首先，提供约20至约40重量份的含酰胺基及羟基的单体，及提供约60至约80重量份的吸附单体。接着，聚合含酰胺基及羟基的单体及吸附单体而形成共聚物。最后，涂布此共聚物于基材表面上。在一些实施例中，吸附单体是选自由甲基丙烯酸丁酯、甲基丙烯酸缩水甘油酯及其组合所组成的群组。
在一些实施例中，基材表面修饰的方法是采用化学性修饰方法。举例来说，将含酰胺基及羟基的单体修饰于基材上包含下述步骤。涂布含酰胺基及羟基的单体于基板上。接着以紫外线处理基板及基板上的含酰胺基及羟基的单体进行接枝反应。应理解的是，额外的操作可用于本实施方式中，且操作可被取代、删除或其顺序可交替使用。在另一实施例中，将含酰胺基及羟基的单体修饰于基材上包含：以紫外线处理基板；以及涂布含酰胺基及羟基的单体至以紫外线处理过后的基板上进行接枝反应。
为证实本发明实施方式捕捉或分离白血球的效果，遂进行以下试验。应注意的是，下述实施例仅提供作为示范目的，而非限制本发明。
实验方法及材料
一、水胶材料的实验
1.1水胶制备的实验材料
第一类型结构为仅具有羟基官能基(hydroxyl group)单体的丙烯酸羟乙酯(2-Hydroxyethyl acrylate，HEA)及甲基丙烯酸羟乙酯(2-Hdroxyethyl methacrylate，HEMA)，化学结构分别如下：
Figure PCTCN2017110177-appb-000028
第二类型结构为仅具有酰胺官能基(amide group)单体的丙烯酰胺(Acrylamide，AAm)及甲基丙烯酰胺(Methacrylamide，MAA)，化学结构分别如下：
Figure PCTCN2017110177-appb-000029
第三类型结构为同时具有羟基官能基和酰胺官能基的N-(2-羟丙基)甲基丙烯酰胺(N-(2-Hydroxypropyl)methacrylamide，HPMA)、N-羟乙基丙烯酰胺(N-Hydroxyethyl acrylamide，或称N-(2-羟乙基)丙烯酰胺，N-(2-Hyroxyethyl)acrylamide，HEAA)，化学结构分别如下：
Figure PCTCN2017110177-appb-000030
Figure PCTCN2017110177-appb-000031
第四类型结构为具有双离子性的化合物，为[3-(甲基丙烯酰氨基)丙基]二甲基(3-硫代丙基)氢氧化铵([3-(Methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide，SBAA)，以及[2-(甲基丙烯酰基)乙基]二甲基-(3-磺酸丙基)氢氧化铵([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide，SBMA)，化学结构分别如下：
Figure PCTCN2017110177-appb-000032
过硫酸铵(Ammonium peroxodisulfate，APS)在本试验中是作为起始剂，具有下列化学结构：
(NH4)2S2O8
四甲基乙二胺(N，N，N，N tetramethylethylenediamine，TEMED)在本试验中是作为催化剂，具有下列化学结构：
Figure PCTCN2017110177-appb-000033
在本试验中所使用的交联剂为亚甲基双丙烯酰胺(N，N’-Methylenebisacrylamide，NMBA)，化学结构如前述讨论所示，在此不多做赞述。
1.2水胶材料的制备
1.2.1酰胺官能基水胶
此步骤中提供两种含酰胺官能基的水胶，即丙烯酰胺(AAm)水胶及甲基丙烯酰胺(MAA)水胶。首先，将AAm及MAA分别溶于去离子水后，分别加入交联剂NMBA。AAm溶液于冰浴中搅拌10分钟，而MAA溶液则于室温下搅拌10分钟。待AAm及MAA分别于冰浴中及室温下与交联剂NMBA均匀混合后，加入起始剂APS反应10分钟，再加入催化剂TEMED。接着，以针筒吸取上述混合液分别注入玻璃模具，于室温进行自由基交联聚合反应1小时形成亲水性水胶。
1.2.2酰胺官能基水胶
此步骤中提供两种含酰胺官能基的水胶，即丙烯酸羟乙酯(HEA)水胶及甲基丙烯酸羟乙酯(HEMA)水胶。首先，将HEA及HEMA分别溶解于甲醇(methanol)后，分别加入交联剂NMBA搅拌10分钟。待HEA及HEMA分别与交联剂NMBA均匀混合后，加入起始剂APS反应10分钟，再加入催化剂TEMED。接着，以针筒吸取溶液注入玻璃模具，于室温进行自由基交联聚合反应1小时形成亲水性水胶。
1.2.3同时具有羟基官能基和酰胺官能基水胶
此步骤中提供两种同时具有羟基官能基和酰胺官能基的水胶，即N-(2-羟丙基)甲基丙烯酰胺(HPMA)水胶及N-羟乙基丙烯酰胺(HEAA)水胶。首先，将HPMA及HEAA分别溶解于去离子水后，分别加入交联剂NMBA搅拌10分钟。待HPMA及HEAA分别与交联剂NMBA均匀混合后，加入起始剂APS反应10分钟，再加入催化剂TEMED。接着，以针筒吸取溶液注入玻璃模具，于室温进行自由基交联聚合反应1小时形成亲水性水胶。
1.2.4双离子性水胶
此步骤中提供两种双离子性水胶，即[3-(甲基丙烯酰氨基)丙基]二甲基(3-硫代丙基)氢氧化铵(SBAA)水胶及[2-(甲基丙烯酰基)乙基]二甲基-(3-磺酸丙基)氢氧化铵(SBMA)水胶。首先，将SBAA及SBMA分别溶于去离子水后，分别加入交联剂NMBA搅拌10分钟。待SBAA及SBMA分别与交联剂NMBA均匀 混合后，加入起始剂APS反应10分钟，再加入催化剂TEMED，以针筒吸取溶液注入玻璃模具，于室温进行自由基交联聚合反应1小时形成双离子性水胶。
1.3水合能力(hydration ability)量测
亲水性高分子材料于水溶液环境中能够藉由氢键或离子键(双离子性高分子)抓取水中的水分子与水产生水合层，不仅能够降低材料与生物分子接触介面上的自由能，亦产生物理上的障碍，使蛋白质无法接近及贴附，可避免蛋白质等生物分子沾粘(参考文献Morisaku，T.，J.Watanabe，T.Konno，M.Takai and K.Ishihara，：Hydration of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis.Polymer，2008.49(21)：p.4652-4657.Chen，S.，L.Li，C.Zhao and J.Zheng，：Surface hydration：Principles and applications toward low-fouling/nonfouling biomaterials.Polymer.2010.51(23)：p.5283-5293.)。藉由平衡含水率可判断材料整体的亲水程度，平衡含水率愈高表示材料整体愈亲水。将水胶分别浸泡于去离子水和磷酸盐缓冲液(phosphate buffered saline，PBS)中，每30分钟更换浸泡溶液并重复3次，以确保水胶中的甲醇被置换。随后，将水胶分别浸泡于去离子水和PBS放置于37℃烘箱24小时，并移除表面多余水分后秤得湿重(WW)。接着，再将水胶移入新的24孔盘中，放置真空烘箱中，于40℃下抽干24小时后，秤得干重(WD)。平衡含水率(Equilibrium water content，EWC)可由下列公式(1)算得：
Figure PCTCN2017110177-appb-000034
水合能力的强弱影响亲水性高分子材料形成水合层的程度，更间接影响材料抵抗生物分子沾粘或蛋白质吸附的效果。平衡含水率虽可得到宏观尺度下高分子材料与水分子的关系，但却无法在微观尺度下解释高分子材料与水分子的亲和力作用关系，遂进行下述试验进一步佐证。
1.4水分子型态的分析
在微观尺度下水分子与亲水高分子结构接触时会产生三种不同的水分子型态：(1)不冻水(nonfreezable bound water)，不冻水与高分子有强烈的作用力， 即使在-100℃仍不会形成冰晶，根据文献不冻水是主要抵抗生物分子沾粘的主因；(2)结冻水(freezable bound water)，结冻水位于不冻水的外侧，结冻水与高分子或不冻水为间歇性作用，使其形成冰晶的温度低于0℃；(3)流动水(free water)，流动水在结冻水的外侧，流动水受到高分子或不冻水的影响非常轻微，甚至不受影响，故如同一般的水会在0℃形成冰晶(参考文献Higuchi，A.and T.Iijima，：D.s.c.investigation of the states of water in poly(vinyl alcohol-co-itaconic acid)membranes.Polymer，1985.26(12)：p.1833-1837.Higuchi，A.and T.Iijima，：D.s.c.investigation of the states of water in poly(vinyl alcohol-co-itaconic acid)membranes.Polymer，1985.26(8)：p.1207-1211.Tanaka，M.and A.Mochizuki，：Effect of water structure on blood compatibility-thermal analysis of water in poly(meth)acrylate.Journal of Biomedical Materials Research Part A，2004.68(4)：p.684-695.)。
藉由微分扫描热量计(differential scanning calorimetry，DSC)可测量水胶中冰晶融化的焓变化量(ΔHf)以判别各型态水分子，故可进一步鉴定不同水分子型态之间的含量(参考文献Tanaka，M.and A.Mochizuki，：Effect of water structure on blood compatibility-thermal analysis of water in poly(meth)acrylate.Journal of Biomedical Materials Research Part A，2004.68(4)：p.684-695.)。高分子周围形成不冻水的比例越高，表示在微观尺度下有较强的水合能力，形成的水合层较为致密，应较能抵抗生物分子沾粘或蛋白质吸附(参考文献Morisaku，T.，J.Watanabe，T.Konno，M.Takai and K.Ishihara，：Hydration of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis.Polymer，2008.49(21)：p.4652-4657.)。
将水胶分别置于去离子水中，每30分钟更换浸泡溶液并重复3次，以确保水胶中的甲醇被置换。随后，浸泡于去离子水中1天，并移除表面多余水分，取3～4毫克水胶于微分扫描热量计(differential scanning calorimetry，DSC)专用铝盘中，将DSC温度范围及条件设定为由25℃降温至-40℃后，以每分钟5℃升温至40℃。
计算水胶吸热峰(endothermic peak)曲线下的积分面积为冰晶融化时相变化的吸热焓变化量(ΔHf)，一般冰溶化吸热焓(ΔHw)为333.5J/g。以下列公式(2)计算结冻水(freezable bound water)的重量百分率(wfreezable)：
Figure PCTCN2017110177-appb-000035
另外，习知平衡含水率是由不冻水和结冻水组成，因此可对应下列公式(3)及公式(4)，而得不冻水的重量百分率(wnonfreezable)：
EWC＝wnonfreezable+wfreezable  (3)
wnonfreezable＝EWC-wfreezable  (4)
每单位重量的水胶高分子所含单位重量的不冻水及结冻水，可由下列公式(5)及公式(5)获得，其中wpolymer为水胶中高分子的重量百分率：
Figure PCTCN2017110177-appb-000036
Figure PCTCN2017110177-appb-000037
Wnonfreezable是高分子链所能形成不冻水的比例(gH2O/g polymer)。接着可由下列公式转换成每摩尔高分子重复单元所键结不冻水的摩尔数，以无因次群NW表示。Mp是每个高分子重复单元的分子量，Mw是水分子的分子量：
Figure PCTCN2017110177-appb-000038
1.5酶结合免疫吸附(ELISA)
将前述所制备而得的各种水胶分别置于去离子水及PBS中，每30分钟更换PBS并重复3次，以确保水胶中的甲醇被置换。接着移入24孔盘中(24well-tissue culture polystyrene plate，即24孔的TCPS盘)再用PBS清洗3次。在每个水胶样品以及空的TCPS孔位加入1毫升PBS于37℃烘箱浸泡2小时后，移除PBS。
在每个水胶样品中加入欲测试的目标蛋白质(HSA、γ-globulin及fibrinogen)或血小板稀少血浆(poor platelet plasma，PPP)，其中单一蛋白质浓度为1毫克/毫升(mg/ml)。接着静置于37℃烘箱30分钟后，移除欲测试的目标蛋 白质(或PPP)，并用PBS清洗3次后移除溶液。
于每个水胶样品加入1毫升浓度为1mg/ml的牛血清白蛋白(BSA)于37℃烘箱静置30分钟，以填补水胶样品上未吸附目标蛋白质的部分，再用PBS清洗3次，以去除多余的BSA。
于每个水胶样品中加入对目标蛋白质具有专一性的第一抗体(1st antibody)，放置于37℃烘箱30分钟，再以PBS清洗3次后移除溶液。
于每个水胶样品再加入1毫升浓度为1mg/ml的BSA，于37℃烘箱静置30分钟，再以PBS清洗3次后移除溶液。
于每个水胶样品加入1毫升的第二抗体(2sec antidoby)，其对第一抗体具有专一性只会与第一抗体产生键结，放入37℃烘箱30分钟，再以PBS清洗5次后移除溶液(测试γ-globulin吸附无须此步骤)。
移除PBS之后，将水胶样品移置新的24孔TCPS盘中，于每个水胶样品中加入0.5毫升显色剂3，3′，5，5′-四甲基联苯胺(3，3′，5，5′-Tetramethylbenzidine，TMB)，等待6分钟使其显色后，再于每个水胶样品中加入0.5毫升1M的硫酸以终止反应。由每个水胶样品中(包括TCPS空孔位)吸取200微升溶液于96孔盘中，藉由Bio-tek型号PowerWare XS的微量盘式分析仪(microplate reader)在UV波长450nm下检视读值，经回推得知水胶对于蛋白质的吸附程度。
1.6血细胞贴附测试
将制得的各种水胶分别浸泡于PBS，每30分钟更换PBS并重复3次，以确保水胶中的甲醇被置换。接着把水胶浸泡在PBS，放置于37℃烘箱1天。移除PBS并于水胶中分别加入1毫升血小板浓厚液与白血球浓厚液，放入37℃烘箱30分钟。然后以PBS冲洗水胶以去除未贴附的血球。将2.5wt％戊二醛(glutaraldehyde)溶液加入24孔盘中，置于4℃冰箱1天，以固定血球。隔日以Nikon型号A1R的共轭焦激光扫描显微镜(confocal laser scanning microscope，CLSM)观察血球贴附情况，并计算单位面积下的血球贴附数量。
二、水胶材料的检测结果
2.1水胶的平衡含水率分析
如图3所示，横座标为各种不同的水胶，纵座标为平衡含水率(％)。首先， 亲水性水胶(AAm、MAA、HEA、HEMA、HPMA及HEAA)在去离子水环境下的平衡含水率皆高于在PBS环境下的平衡含水率，这是因为亲水性水胶的酰胺官能基或羟基官能基皆能充分与水分子产生氢键，而PBS中的盐类与水分子的离子作用力会和亲水性水胶的氢键作用力互相竞争，导致亲水性水胶在PBS中的平衡含水率普遍低于在去离子水溶液的平衡含水率。其次，双离子性水胶(SBAA及SMBA)在去离子水环境下的平衡含水率皆低于在PBS环境下的平衡含水率，这是因为双离子性水胶结构中的正负电荷是依据离子键方式抓取水分子，因而能够抵抗PBS的盐类离子作用力，而使双离子水胶在PBS中有较高的平衡含水率。此外，亲水性水胶的平衡含水率由低至高依序是羟基官能基水胶(HEA、HEMA)、酰胺官能基水胶(AAm、MAA)以及同时具有羟基官能基和酰胺官能基水胶(HPMA、HEAA)。据此可知，酰胺官能基水胶比羟基官能基水胶更为亲水，故若在水胶包含羟基官能基的情形下，进一步包含酰胺官能基可增加产生氢键的位置。因此，HPMA单体水胶和HEAA单体水胶的平衡含水率最高。
2.2水胶的水分子型态分析
如图4所示，绘示各种不同水胶中在去离子水中的平衡含水率、不冻水所占的比例及其无因次群。横座标为各种不同的水胶，左边的纵座标为平衡含水率(％)，右边的纵座标则分别对应至不冻水所占的比例(gH2O/g polymer)及无因次群Nw。
不冻水含量以Wnonfrezzable表示，在亲水性水胶中具有酰胺官能基水胶(AAm、MAA)所键结的不冻水含量明显高于羟基官能基水胶(HEA、HEMA)，由于酰胺官能基是氢键受体也是氢键予体，对于水分子的结合能力和结合机率高于仅是氢键受体的羟基官能基。据此，具有HPMA单体及HEAA单体的水胶由于同时具有羟基官能基和酰胺官能基可提供更多产生氢键作用的位置，故能键结更多的水分子于高分子链周围，进而形成更致密的水合层。
须说明的是，由于在水胶系统中难以计算其分子量和聚合程度，所以使用无因次群NW，其物理意义为每摩尔高分子重复单元所键结不冻水的摩尔数，故NW值越高表示每摩尔单体所形成的不冻水量越多。NW值由高到低是双离子性水胶(SBAA、SBMA)、同时具有羟基和酰胺基水胶(HPMA、HEAA)、酰 胺基水胶(AAm、MAA)、羟基水胶(HEA、HEMA)。双离子性水胶的化学结构因具有正负电荷，产生的离子作用力非常强，故双离子性水胶有良好的亲水性及水合能力。然而，具有HPMA单体及具有HEAA单体的水胶于化学结构上具有羟基官能基和酰胺官能基能增加水分子发生氢键的位置和机率，故其水胶的水合能力不亚于双离子性水胶。
2.3水胶与单一血浆蛋白质的吸附
常见的血浆蛋白质为人类血清白蛋白(human serum albumin，HSA)、免疫球蛋白(immunoglobulin，Ig)及纤维蛋白原(fibrinogen)，其中，免疫球蛋白又可分为IgA、IgD、IgE、IgG和IgM，而IgG中大多数为γ-globulin。γ-globulin除了与免疫系统息息相关之外，其结构中具有醣基(saccharide residues)会与血小板的酶醣基转化酶作引起血小板聚集。因此，本实验是以HSA、γ-globulin和fibrinogen为目标蛋白质，来观察水胶对于血浆蛋白质中的目标蛋白质的吸附效果。
本实验分别配置1mg/ml HSA、γ-globulin和fibrinogen后，以ELISA测试蛋白质吸附，因每次经由ELISA测试相对吸附的蛋白质溶液仅含有一种蛋白质，环境中缺少其他蛋白质在吸附作用的竞争，因此，可显示出不同水胶材料对于各种蛋白质的最大吸附量。如图5所示，横座标为各种不同的水胶，纵座标为蛋白质的相对吸附量。羟基官能基水胶(HEA、HEMA)、酰胺官能基水胶(AAm、MAA)、同时具有羟基官能基和酰胺官能基水胶(HPMA、HEAA)及双离子性水胶(SBAA、SBMA)的血浆蛋白质相对吸附值皆在10％以下。值得注意的是，HEAA单体水胶不论是对HSA、γ-globulin或fibrinogen的吸附量皆相当低，远小于3％，甚至吸附率趋近于0。
2.4水胶与纤维蛋白原(fibrinogen)及血小板的吸附或贴附实验
本实验是使用1mg/ml fibrinogen及1毫升血小板进行吸附实验，如图6所示，横座标为各种不同的水胶，fibrinogen的相对吸附量对应至左方的纵座标，血小板的细胞密度(cells/mm2)则对应至右方的纵座标。另外，为使数值之间的差异更为清楚地呈现，图中的小图是虚线所示意的部分的放大示意图。
聚甲基丙烯酸N，N-二甲基氨基乙酯(N，N-dimethylaminoethyl methacrylate， DMAEMA)和偏苯三酸酐(Trimellitic anhydride，TMA)是二种带正电荷水胶，对于fibrinogen的吸附量和血小板的贴附量皆相当高，因血小板表面带负电，故接触带正电的DMAEMA水胶和TMA水胶时会因静电作用力造成不可逆吸附，吸附大量的血小板。本发明的羟基官能基水胶、酰胺官能基水胶、同时具有羟基官能基和酰胺官能基水胶及双离子性水胶在fibrinogen的吸附量和血小板的贴附量皆相当低，且远小于DMAEMA水胶和TMA水胶对fibrinogen和血小板的吸附量。令人值得注意的是，HEAA单体水胶对于fibrinogen的吸附量是小于3％(较佳是小于2％，更佳是小于1％)，而在图6中fibrinogen和血小板的贴附量甚至皆趋近于0。由此可知，HEAA单体水胶几乎不会吸附fibrinogen，进而也几乎不会贴附血小板，故可减少血小板的活化。血小板活化的减少进而可减少与血液接触时的凝血反应，故用于回收血小板制程时，甚至可不需添加抗凝血剂。另，习知带电荷表面的过滤材料容易使得缓激肽浓度增加。利用表面不带电荷的HEAA单体水胶亦可避免因缓激肽浓度的增加，而导致输血时的急性低血压反应。
2.5水胶与白血球的吸附或贴附实验
本实验是以白血球浓厚液进行白血球吸附或贴附实验，且由共轭焦激光扫描式显微镜(CLSM)观测白血球贴附于水胶表面的荧光信号并计算白血球的荧光信号数量。结果如图7所示，酰胺官能基水胶(AAm、MAA)和双离子性水胶(SBAA、SBMA)的白血球贴附量低于其他水胶，羟基官能基水胶(HEA、HEMA)、同时具有羟基官能基和酰胺官能基水胶中的HEAA单体水胶及带正电荷水胶(DMAEMA、TMA)则有较多白血球贴附，其中带正电荷水胶(DMAEMA、TMA)是藉由与白血球之间的静电作用力，而使白血球大量贴附。令人值得注意的是，同时具有羟基官能基和酰胺官能基水胶的HPMA单体水胶和HEAA单体水胶的白血球贴附量却具有显著差异。具体来说，HPMA单体水胶的白血球贴附量非常低，HEAA单体水胶的白血球贴附量却非常高，甚至高于带正电荷水胶DMAEMA，显示HEAA单体水胶对白血球具有极高的亲和性。
另外，由前述提及的水合层理论、血浆蛋白质吸附实验及血小板贴附实验皆显示HPMA单体水胶和HEAA单体水胶具有良好的抗生物分子沾粘效果。值得注意的是，HPMA单体水胶并不会贴附白血球，但HEAA单体水胶却有大量 白血球贴附且几乎不会吸附血浆蛋白质，亦几乎不会贴附血小板。换言之，利用HEAA单体或/及与其他化合物聚合而成的聚合物，可使得白血球大量贴附于此聚合物上，且不需经由血浆蛋白质及血小板来协助吸附白血球。推测可能是含HEAA的聚合物可直接与白血球的表面有亲和性吸附，或者可能是因为有微量的ICAMs分子(intercellular adhesion molecules)吸附于含HEAA的聚合物上，进而协助白血球的亲和性吸附。
三、基材改质的实验方法
3.1基材改质的实验材料
本试验对基材表面进行改质，进一步验证本实施方式的捕捉或分离白血球的能力。
实施例的试验材料为前述同时具有羟基官能基和酰胺官能基N-羟乙基丙烯酰胺(N-Hydroxyethyl acrylamide，HEAA)，具有化学结构如下：
Figure PCTCN2017110177-appb-000039
比较例的试验材料为带正电荷的聚甲基丙烯酸N，N-二甲基氨基乙酯(N，N-dimethylaminoethyl methacrylate，DMAEMA)，具有化学结构如下：
Figure PCTCN2017110177-appb-000040
另外，甲基丙烯酸丁酯(Butyl methacrylate，BMA)及甲基丙烯酸缩水甘油酯(Glycidyl methacrylate，GMA)则是用于待测聚合物中的咬合端，使得待测聚合物能以物理方式吸附于基材表面。BMA及GMA分别具有化学结构如下：
Figure PCTCN2017110177-appb-000041
Figure PCTCN2017110177-appb-000042
4，4′-偶氮双(4-氰戊酸)(4，4′-Azobis(4-cyanovaleric acid)，ACVA)做为起始剂，具有化学结构如下：
Figure PCTCN2017110177-appb-000043
基材的材料则为聚丙烯(polypropylene，PP)及聚对苯二甲酸乙二酯(polyethylene terephthalate，PET)。PP及PET分别具有化学结构如下：
Figure PCTCN2017110177-appb-000044
3.2基材改质的高分子制备
以BMA与GMA作为基材咬合端，以HEAA与DMAEMA作为功能作用端，依比例(咬合端约70％，作用端约30％)混合，并加入起始剂ACVA，而溶剂为乙醇。接着，在70℃的环境下进行聚合反应24小时，制备出BMA-r-HEAA、BMA-r-DMAEMA、BMA-r-GMA-r-HEAA、BMA-r-GMA-r-DMAEMA高分子。待反应完成后，以去离子水作为析出剂将产物析出并干燥。
3.3基材表面改质
选用PP与PET作为欲改质的基材。
3.3.1物理吸附作用力改质PP基材
分别取BMA-r-HEAA、BMA-r-DMAEMA、BMA-r-GMA-r-HEAA、BMA-r-GMA-r-DMAEMA高分子，以乙醇为溶剂，配制成高分子溶液。取PP基材，裁剪为合适大小，浸泡于高分子溶液中1分钟，再以去离子水洗去表面残余溶液并干燥。据此，可分别获得经BMA-r-HEAA、BMA-r-DMAEMA、 BMA-r-GMA-r-HEAA、BMA-r-GMA-r-DMAEMA涂布(coating)的表面改质的PP基材。
3.3.2UV处理方式改质PET基材
取HEAA、DMAEMA单体，以乙醇为溶剂，分别配制成单体溶液。取PET基材，裁剪为合适大小，浸泡于单体溶液中，置于7200W的UV光照下反应2分钟，再以去离子水洗去表面残余溶液并干燥。据此，可分别获得经HEAA及DMAEMA单体接枝(grafting)的表面改质的PET基材。
3.4核磁共振(NMR)的鉴定
将上述高分子各秤取10mg，分别溶于1mL的甲醇(d-MeOH)中，配置为浓度10mg/mL的溶液并装入NMR试管，送交中国台湾国立中央大学贵重仪器中心委测。再由图谱的特征峰，分析并计算高分子的化学结构与单体比例。
3.5表面改质密度量测
进行改质前，先将PP、PET基材以微量天平秤量其重量。当表面完成改质后，将基材干燥后并经由微量天平秤量该表面的重量。经由计算实验前后的基材的重量差，可得到改质于基材表面的高分子重量。最后经由换算可得每单位面积的高分子重量，即为表面披覆密度。
3.6X射线光电子能谱分析仪(XPS)的量测
将经由HEAA改质的PP、PET表面利用X射线光电子能谱分析仪(XPS)进行表面组成元素分析。再由图谱的特征峰比较各改质表面的氮元素含量。
3.7血细胞贴附测试
首先将基材浸泡于PBS buffer中半小时。接着，将液体吸干后，分别取1ml红血球、白血球及血小板浓厚液覆盖于基材表面，并置于37℃烘箱中贴附2小时。接着以PBS buffer冲洗基材表面未贴附的血球细胞，浸泡于戊二醛(glutaraldehyde)中一天进行固定，以共轭焦激光扫瞄式电子显微镜(LSCM)观察血球细胞于聚丙烯盘表面的贴附情形。
3.8血液过滤测试
将改质后的基材，裁剪成直径2.6cm的圆形并堆叠5层，置于压克力过滤器中锁紧(类似于图2中所例示性的实施例)。取5ml血小板浓厚液进行过滤。接着使用血球计数仪对过滤前、过滤后的血品进行检验，计算出白血球移除率以及血小板保留率。
四、基材改质的检测结果
4.1核磁共振(NMR)的分析结果
图8为各单体、高分子化合物的结构式及其化学位移。另外，如下表一所示，为方便后续说明，在此将高分子化合物如BMA-r-HEAA、BMA-r-DMAEMA、BMA-r-GMA-r-HEAA、BMA-r-GMA-r-DMAEMA各缩写为B-r-H、B-r-D、B-r-G-r-H、B-r-G-r-D。图9为各单体及高分子结构的NMR图谱。经核磁共振分析后，HEAA的特征峰主要体现于a处，DMAEMA的特征峰主要体现于b处。藉由核磁共振的图谱可得知本试验所用的高分子化合物已成功进行合成。
表一
Figure PCTCN2017110177-appb-000045
4.2PP、PET基材披覆密度量测结果
如图10所示，为各种高分子化合物的披覆密度结果图。PP膜是涂布高分子化合物B-r-H、B-r-D、B-r-G-r-H及B-r-G-r-D的PP基材。PET膜是单体接枝HEAA及DMAEMA的PET基材。藉由对改质前后的基材重量进行秤量，并量测基材的表面积，可算得基材表面的各高分子披覆密度，介于约0.1至约0.25 mg/cm2。
4.3X射线光电子能谱分析仪(XPS)的量测结果
经改质后的基材表面可利用X射线光电子能谱分析仪进行表面元素分析，从氮元素的分析可看出改质前后的差异。如图11所示，为基材上表面的氮元素的分析图谱。PP控制组及PET控制组皆为未经改质的PP基材及PET基材。HEAA改质前后的表面元素的比例详细汇整于下表二中。参照图11及下表二可知，未经改质的PP基材表面并无氮的信号产生。反之，经HEAA单体或高分子改质的PET、PP基材表面氮元素比例均明显升高，且在399～402eV位置均出现氮元素的特征峰。
表二
Figure PCTCN2017110177-appb-000046
4.4血细胞贴附测试结果
图12至图15为PP及PET基材表面改质前后的血液细胞贴附影像及其计数。
首先请参照图12及图13，为PP基材经物理性涂布改质后其血液细胞贴附的结果图。图12中可明显看出含HEAA的PP基材(PP B-r-H及PP B-r-G-r-H)上的血小板血浆及红血球细胞影像较浅，意即较不易贴附血小板血浆及红血球细胞。除此之外，含B-r-H的PP基材上的白血球细胞影像明显较深，意即较易贴附白血球细胞。图13中，上图为白血球贴附量的结果图，下图为红血球贴附量的结果图。由此可知，含有含HEAA的PP基材含HEAA的PP基材表面可以有效捕捉白血球，并且血小板与红血球的贴附量较低。而含DMAEMA的PP基材(PPB-r-D及PP B-r-G-r-D)表面不仅会捕捉白血球，其血小板与红血球的贴附量亦相 对较高。
请继续参照图14及图15，为PET基材经化学性接枝改质后其血液细胞贴附的结果图。图14中可明显看出接枝HEAA的PET基材(PET HEAA(UV))上的血小板血浆及红血球细胞影像较浅，意即较不易贴附血小板血浆及红血球细胞。除此之外，接枝HEAA的PET基材上的白血球细胞影像明显较深，意即较易贴附白血球细胞。图14中，上图为白血球贴附量的结果图，下图为红血球贴附量的结果图。由此可知，经UV处理过后，接枝HEAA的PET基材表面具有捕捉白血球的能力，同时可以降低血小板与红血球的贴附量。反之，3种血液细胞均会贴附于接枝DMAEMA的PET基材表面。
除此之外，在血细胞贴附试验中，红血球浓厚液中实际上仍含有微量白血球，而在前述图12及图14的贴附影像中，含HEAA的基材仍能在充满红血球浓厚液体中捕捉其中的微量白血球。显示经HEAA改质的基材表面对于白血球捕捉具有专一性。
4.5血液过滤测试结果
如前述试验方法中所提的血液过滤测试，利用改质后的基材对血小板浓厚液进行过滤。经由血球计数仪检测后可得知过滤前与过滤后的血品中各种血液细胞含量，依此计算出白血球移除率与血小板保留率。
如下表三所示，WBC是白血球(White blood cell)，PLT是指血小板(Platelet)。根据表三可知经HEAA改质后的基材对白血球的捕捉率至少达到87％，并且能保留86％以上的血小板。意即，血小板浓厚液(含有白血球的血小板浓厚液)流经改质后的基材时，大部分白血球会贴附于基材上，剩下的滤液中能保留大部分的血小板。据此，本发明的实施例对于白血球捕捉有极高的专一性(不会造成红血球与血小板的捕捉或贴附)，是作为血小板浓厚液减除白血球的良好材料。当然，在其他实施态样中，亦可以使用例如全血血液样品通过上述基材，再通过去除红血球的技术，直接制备血小板浓厚液。
相对于使用B-r-D、B-r-G-r-D以及DMAEMA等聚合物进行改质的习知捕捉、分离或过滤白血球的方式而言，不仅是白血球捕捉率提高，更重要的是同时维持极高的血小板保留率，是故明显可知，通过B-r-H、B-r-G-r-H以及HEAA是通过不同于过去的作用机转来达成功效。
表三
Figure PCTCN2017110177-appb-000047
前文概述数个实施例的特征以使得熟习该项技术者可更好地理解本揭露的态样。熟习该项技术者应了解，可容易地将本揭露内容用作设计或修改用于实现相同目的及/或达成本文引入的实施例的相同优点的其他制程及结构的基础。熟习该项技术者亦应认识到，此类等效物构造不违背本揭露内容的精神及范畴，且可在不违背本揭露内容的精神及范畴的情况下于此作出各种变化、替代以及变更。

Claims (30)

  1. 一种聚合物，用于捕捉或分离白血球，该聚合物是以包含一含酰胺基及羟基的单体进行聚合反应制备而得，其特征在于，其中该含酰胺基及羟基的单体具有式(1)的结构：
    在式(1)中，R1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环组成的群组，R2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数。
  2. 如权利要求1所述的聚合物，其特征在于，其中R1为氢，R2为氢，以及n为1。
  3. 如权利要求1所述的聚合物，其特征在于，其中该含酰胺基及羟基的单体为N-羟乙基丙烯酰胺((N-Hydroxyethyl acrylamide)，或N-(2-羟乙基)丙烯酰胺(N-(2-Hyroxyethyl)acrylamide)。
  4. 如权利要求1所述的聚合物，其特征在于，其为共聚物(copolymer)，且是由该含酰胺基及羟基的单体及至少另一单体共聚合而成。
  5. 如权利要求4所述的聚合物，其特征在于，其中该另一单体是甲基丙烯酸甲酯(Butyl methacrylate，BMA)或甲基丙烯酸缩水甘油酯(Glycidyl methacrylate，GMA)。
  6. 如权利要求1所述的聚合物，其特征在于，其为链段高分子。
  7. 如权利要求1所述的聚合物，其特征在于，其为交联共聚高分子。
  8. 如权利要求7所述的聚合物，其特征在于，其中聚合反应包含使用一交联剂，且该交联剂具有双丙烯酸酯的官能基。
  9. 如权利要求8所述的聚合物，其特征在于，其中该交联剂是亚甲基双丙烯酰胺、二甲基丙烯酸乙二醇酯、聚乳酸-聚乙二醇-聚乳酸聚乙醇酸共聚物或聚乙二醇二丙烯酸酯。
  10. 一种用于捕捉或分离白血球的装置，其特征在于，包含：
    一外壳；以及
    一本体，其中该本体包含一聚合物，该聚合物包含式(2)的结构：
    Figure PCTCN2017110177-appb-100002
    其中，n为10至50的整数。
  11. 如权利要求10所述的用于捕捉或分离白血球的装置，其特征在于，其中该聚合物具有式(3)的结构：
    其中，m为50至90的整数，且
    Figure PCTCN2017110177-appb-100004
    Figure PCTCN2017110177-appb-100005
    Figure PCTCN2017110177-appb-100006
    其中p为2至6的整数，q为1至6的整数，r为1至6的整数，s为1至6的整数。
  12. 如权利要求10所述的用于捕捉或分离白血球的装置，其特征在于，其中该聚合物具有式(4)的结构：
    Figure PCTCN2017110177-appb-100007
    其中，t为50至90的整数，
    Figure PCTCN2017110177-appb-100008
  13. 如权利要求10所述的装置，其特征在于，其中该本体包括一基材，该聚合物是涂布(coating)、喷洒或浸渍的方式设置于该基材。
  14. 如权利要求10所述的装置，其特征在于，其中该本体包括一基材，该聚合物是以锚定(anchoring)的方式固定于该基材。
  15. 如权利要求10所述的装置，其特征在于，其中该本体的表面元素包含碳、氧及氮，且碳、氧及氮的总摩尔百分比定义为100mol％，碳的摩尔百分比为约76.22％至约79.84％，氧的摩尔百分比为约18.1％至约21.04％，以及氮的摩尔百分比为约2.05％至约2.75％。
  16. 一种含酰胺基及羟基的聚合物的用途，其特征在于，其是用于捕捉或分离白血球，该含酰胺基及羟基的聚合物包含式(2)的结构：
    Figure PCTCN2017110177-appb-100009
    其中，n为10至50的整数。
  17. 如权利要求16所述的含酰胺基及羟基的聚合物的用途，其特征在于，其中白血球的捕捉率不低于70％。
  18. 如权利要求16所述的含酰胺基及羟基的聚合物的用途，其特征在于，其中血小板的保留率不低于85％。
  19. 一种含酰胺基及羟基的单体的用途，其特征在于，其是用于形成捕捉或分离白血球的材料，该含酰胺基及羟基的单体具有式(1)的结构：
    Figure PCTCN2017110177-appb-100010
    在该式(1)中，R1是独立选自由氢、甲基、乙基、羟基、C1至C12长碳链其中任一及苯环组成的群组，R2是独立选自由氢、甲基、乙基、C1至C6长碳链其中任一、氨基及苯环所组成的群组，以及n为1至5的整数。
  20. 如权利要求19所述的用途，其特征在于，其中形成捕捉或分离白血球的材料的步骤包含：
    提供一基材；以及
    将该含酰胺基及羟基的单体修饰于该基材上。
  21. 如权利要求20所述的用途，其特征在于，其中将该含酰胺基及羟基的单体修饰于该基材上包含：
    提供该含酰胺基及羟基的单体；
    提供至少一锚定单体(anchoring unit)；
    聚合该含酰胺基及羟基的单体及该锚定单体形成一共聚物；以及
    通过该锚定单体锚定该共聚物于该基材上。
  22. 如权利要求21所述的用途，其特征在于，其中该含酰胺基及羟基的 单体占该共聚物的重量百分比为约百分之20至约百分之40。
  23. 如权利要求21所述的用途，其特征在于，其中该锚定单体占该共聚物的重量百分比为约百分之60至约百分之80。
  24. 如权利要求21所述的用途，其特征在于，其中该锚定单体是选自由甲基丙烯酸丁酯、甲基丙烯酸缩水甘油酯及其组合所组成的群组。
  25. 如权利要求20所述的用途，其特征在于，其中将该含酰胺基及羟基的单体修饰于该基材上包含：
    涂布该含酰胺基及羟基的单体于该基板上；以及
    以紫外线处理该基板及该基板上的该含酰胺基及羟基的单体。
  26. 一种白血球浓厚液的制备方法，其特征在于，包含：
    提供如权利要求10所述的装置；
    提供一血液样品，其中该血液样品包含白血球及血小板；
    将该血液样品通过该装置，使得白血球被捕捉于该装置中或自该血液样品中被分离至该装置中；以及
    脱附被捕捉或分离出来的白血球。
  27. 一种红血球浓厚液的制备方法，其特征在于，包含：
    提供如权利要求10所述的装置；
    提供一血液样品，其中该血液样品包含红血球、白血球及血小板；
    将该血液样品通过该装置而获得一滤液，其中白血球被捕捉于该装置中或自该血液样品中被分离出来；以及
    处理该滤液为该红血球浓厚液。
  28. 一种血浆制品储存前的处理方法，其特征在于，包含：
    提供如权利要求10所述的装置；
    提供一血液样品，其中该血液样品包含血浆及白血球；以及
    将该血液样品通过该装置而获得一滤液，其中白血球于该装置中被捕捉或自该血液样品中分离出来。
  29. 一种去除全血血液中白血球的方法，其特征在于，包含：
    提供如权利要求10所述的装置；
    提供一全血血液样品，其中该全血血液样品包含红血球、白血球及血小板；
    将该全血血液样品通过该装置而获得一滤液，其中白血球被专一性地捕捉于该装置中。
  30. 一种血小板浓厚液的制备方法，其特征在于，包含：
    提供如权利要求10所述的装置；
    提供一血液样品，其中该血液样品包含白血球及血小板；
    将该血液样品通过该装置而获得一滤液，其中白血球被捕捉于该装置中或自该血液样品中分离出来；以及
    处理该滤液为该血小板浓厚液。
PCT/CN2017/110177 2016-11-09 2017-11-09 捕捉或分离白血球的聚合物、装置、其制造方法及其应用 WO2018086556A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI790567B (zh) * 2020-03-27 2023-01-21 康博醫創股份有限公司 一種用於富集和檢測生物樣品中微生物的方法和裝置

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113769120A (zh) * 2021-09-18 2021-12-10 北京脑陆科技有限公司 双层导电水凝胶的制备方法
TWI808510B (zh) * 2021-10-15 2023-07-11 中原大學 抗生物惰性效能衰變之雙離子型材料、薄膜及其應用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6168454A (ja) * 1984-09-11 1986-04-08 Mitsubishi Rayon Co Ltd Ｎ−２−ヒドロキシエチルアクリルアミドもしくはｎ−２−ヒドロキシエチルメタクリルアミドの製造方法
CN101358015A (zh) * 2007-07-31 2009-02-04 赛拉尼斯国际公司 利用具有c2－c10羟烷基的丙烯酰胺/n－羟烷基丙烯酰胺交联混合物的自交联分散体
CN102292115A (zh) * 2009-03-30 2011-12-21 泰尔茂株式会社 表面处理剂及过滤器用过滤材料以及血液处理过滤器
CN105813663A (zh) * 2013-12-13 2016-07-27 旭化成医疗株式会社 去除白血球的过滤材料、以及去除白血球的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60262805A (ja) * 1984-06-08 1985-12-26 Mitsubishi Rayon Co Ltd ポリｎ−（２−ヒドロキシエチル）メタクリルアミド又はアクリルアミド
DE69319471T2 (de) 1992-03-17 1999-04-15 Asahi Medical Co Filtermedium mit begrenzter negativer Oberflächenladung für die Behandlung von Blutmaterial
ATE425995T1 (de) * 2001-07-31 2009-04-15 Asahi Kasei Medical Co Ltd Leukozytenfilter mit polymerbeschichtung
TWI410269B (zh) * 2010-09-16 2013-10-01 私立中原大學 白血球過濾材料及其過濾方法
TWI481442B (zh) * 2013-11-14 2015-04-21 中原大學 雙離子電荷偏差型血球篩選用材料
CN203763551U (zh) * 2013-12-26 2014-08-13 中原大学 血液过滤装置
US9522971B2 (en) 2014-12-31 2016-12-20 Chung Yuan Christian University Amine modifying material and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6168454A (ja) * 1984-09-11 1986-04-08 Mitsubishi Rayon Co Ltd Ｎ−２−ヒドロキシエチルアクリルアミドもしくはｎ−２−ヒドロキシエチルメタクリルアミドの製造方法
CN101358015A (zh) * 2007-07-31 2009-02-04 赛拉尼斯国际公司 利用具有c2－c10羟烷基的丙烯酰胺/n－羟烷基丙烯酰胺交联混合物的自交联分散体
CN102292115A (zh) * 2009-03-30 2011-12-21 泰尔茂株式会社 表面处理剂及过滤器用过滤材料以及血液处理过滤器
CN105813663A (zh) * 2013-12-13 2016-07-27 旭化成医疗株式会社 去除白血球的过滤材料、以及去除白血球的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN, S.L. LIC. ZHAOJ. ZHENG: "Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials", POLYMER, vol. 51, no. 23, 2010, pages 5283 - 5293, XP027437364
HIGUCHI, A.T. LIJIMA: "D.s.c. investigation of the states of water in poly(vinyl alcohol-co-itaconic acid) membranes", POLYMER, vol. 26, no. 12, 1985, pages 1833 - 1837, XP024113910, DOI: doi:10.1016/0032-3861(85)90011-4
HIGUCHI, A.T. LIJIMA: "D.s.c. investigation of the states of water in polyvinyl alcohol-co-itaconic acid) membranes", POLYMER, vol. 26, no. 8, 1985, pages 1207 - 1211
MORISAKU, T.J. WATANABET. KONNOM. TAKAIK. ISHIHARA: "Hydration of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis", POLYMER, vol. 49, no. 21, 2008, pages 4652 - 4657, XP025471139, DOI: doi:10.1016/j.polymer.2008.08.025
TANAKA, M.A. MOCHIZUKI: "Effect of water structure on blood compatibility-thermal analysis of water in poly (meth) acrylate", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, vol. 68, no. 4, 2004, pages 684 - 695

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI790567B (zh) * 2020-03-27 2023-01-21 康博醫創股份有限公司 一種用於富集和檢測生物樣品中微生物的方法和裝置
AU2021240657B2 (en) * 2020-03-27 2024-01-25 Micronbrane Medical Co., Ltd. Method and device for enriching and detecting microorganisms in biological samples

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