Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

• the present invention relates to a method of preparation of a substrate surface containing carboxybetaine functional groups with bound bioactive substances, which significantly increases surface resistance against undesirable biological deposition upon contact with biological media.
• biological deposition on their surface takes place, which begins with the adsorption of biological molecules, especially proteins.
• biological molecules especially proteins.
• cells and microorganisms can adhere on them, followed by other biological processes such as blood coagulation, inflammatory and immune reactions, or formation of bacterial biofilms.
• Resulting biological deposits may impair the function of materials and equipment that work in biological media such as body fluids, cell- containing media, food and media from biological production, and from biological environment in general. The problem is particularly critical for materials used in contact with blood serum, plasma, or blood.
• surfaces that prevent non-specific formation of biological deposits in biological media and simultaneously facilitate binding of bioactive elements enabling the specific interaction of the surface with target components of the biological environment are very important for many biotechnological and medical applications.
• Such applications include biosensors, membranes and particles for separation and accumulation of biological agents and cells, drug carriers and diagnostic particles applied to blood stream, blood-contacting materials and cell carriers or scaffolds for tissue engineering.
• hydrophilic electroneutral polymers such as non-ionogenic (poly(oligo(hydroxy ethylene glycol) methacrylate) (polyHOEGMA), poly(2-hydroxyethyl methacrylate) polyHEMA, poly(3- hydroxypropyl methacrylate) (polyHPMA), poly(N-(2-hydroxypropyl methacrylamide) (polyHPMAA), and zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), and poly(carboxybetaine acrylamide) (polyCBAA) from the substrate surface (the "grafting from” method) using the surface -initiated atom transfer radical polymerization (SI-ATRP).
• SI-ATRP surface -initiated atom transfer radical polymerization
• the resulting so-called polymer brush is a layer of densely arranged polymer chains bonded with one end to the surface.
• Brushes made of polyCBAA, polyCBMA, and polyHPMAA are the only ones effectively suppressing even deposition from undiluted blood plasma and serum.
• An alternative method is to graft polymer chains prepared by polymerization in solution to a surface, or the so-called "grafting to” method. Smaller density of polymer chains achieved in the brushes prepared by this method versus brushes prepared by the "grafting from” method gives the surface a weaker resistance to biological deposition.
• PCT/CZ2016/050011 which relates to PV 2015-313, also includes the preparation of brushes from poly(HPMAA-co-CBMAA) prepared by polymerization in a solution and covalently grafted onto the surface of substrate.
• the state of the art also include hydrogels of poly(HEMA-co-CBMAA) copolymers resistant to biological deposition (Kostina et al., Biomacromolecules 2012,13,4164- 4170) or sorbents whose surface is modified by grafting of carboxybetaine zwitterions (WO2014165421 Al).
• Bioactive substances containing one or more amino groups are bound to the carboxybetaine groups virtually exclusively by reaction with the carboxybetaine zwitterion carboxylate which is first activated into an intermediate product easily reacting with the nucleophilic amino group of the bound bioactive substance.
• This activation is mainly accomplished by reacting with l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS) or its derivatives, resulting in the formation of an active NHS ester (EDC/NHS activation).
• EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
• NHS N-hydroxysuccinimide
• the bioactive substance is subsequently bonded by reaction of its amino group with an active NHS ester formed by activating the carboxyl in the carboxybetaine group.
• the current state of the art of technology provides a method of deactivating the aforementioned active esters using amino acids, especially glycine.
• the present invention describes a significantly more efficient method of deactivating active esters on the substrate surface.
• the use of this more efficient method of deactivation leads, in most cases, to complete or almost complete restoration of the original (prior to activation and binding of bioactive substances) ability of the surface to withstand non-specific deposition from biological media. Disclosure of the Invention
• the present invention relates to a method for preparing surfaces which by reacting with an acid of general formula (I) regenerate carboxyl groups from active NHS esters remaining after binding of bioactive substances.
• Parts of a product or device, the surface of which in a biological medium has an active function to perform that could be affected by biological deposition, and whose surface contains carboxybetaine groups, may be from organic and inorganic materials. They may have any morphology such as particles, membranes, pipes, hoses, wafers, porous material, and fibre webs and may have various uses in contact with biological media, such as biosensors, affinity particles and membranes for separation and accumulation of biological agents, targeted drug delivery carriers, biomaterials for tissue engineering, and antithrombogenic materials for contact with blood.
• the present invention relates on the fact that immediately after the activation of carboxybetaine groups and binding of bioactive substances, the substrate surface is incubated with a solution of an acid of general formula (I).
• the subject of the present invention is a method of preparation of a substrate surface containing carboxybetaine groups, with increased resistance to undesirable deposition of biological media components on the substrate surface, comprising the following steps:
• step c) is carried out in which the product of step b) reacts with an acid of general formula (I)
• n 1 to 4, wherein the acid of general formula (I) reacts with those active esters, which have not undergone covalent bonding of the bioactive substance in step b).
• Carboxybetaine groups can either be directly a part of the substrate material or may be attached to the substrate surface, e.g., as a part of molecules grafted onto the substrate surface, or the substrate can be coated with a layer containing these groups, e.g. a polymer brush prepared by grafting from the substrate surface using surface initiated polymerization ("grafting from”) or prepared by grafting of the polymer chains prepared by polymerization in solution to surface (“grafting to").
• grafting from surface initiated polymerization
• grafting to prepared by grafting of the polymer chains prepared by polymerization in solution to surface
• Carboxybetaine groups are well known to a person skilled in the art, and are generally defined as neutral chemical groups containing a quaternary ammonium cation that carries no hydrogen atom and a negatively charged carboxyl group not directly adjacent to quaternary ammonium cation (IUPAC, Compendium of Chemical Terminology).
• the acid of general formula (I) is (2-aminoethoxy)acetic acid (AEAA).
• concentration of the acid of general formula (I) in step c) of the method according to the present invention is in the range of from 0.5 M to 2 M, more preferably in the concentration range of from 0.7 M to 1.5 M, most preferably at the concentration of 1 M.
• the pH of step c) is preferably neutral or basic in the range of from pH 7 to 9, more preferably from pH 7 to 8.5, most preferably at pH 8.
• the substrate is an object which is resistant to the deposition of biological media components or which is to be coated with a polymeric layer giving it resistance to the deposition of biological media components.
• a polymer brush can be grafted to it or it can be coated with a polymer.
• a substrate thus can be:
• the polymeric layers formed on objects (1), (2), (3), and (4) always contain at least one homopolymer or copolymer containing carboxybetaine zwitterions in side chains.
• the layers on objects (2) and (4) may contain polymers containing carboxybetaine zwitterions in a mixture with hydrophilic polymers, preferably selected from polyHPMAA, polyHOEGMA, polyHEMA, and polyHPMA.
• the layers on objects (1), (2), (3), and (4) are formed from a homopolymer selected from the group comprising polyCBMAA, polyCBMA, and polyCBAA or from a copolymer poly(A-co- B), where A is a monomelic unit selected from the group comprising HPMAA, HOEGMA, HEMA, HPMA, and B is a monomelic unit at a concentration from 1 to 99 mol%, selected from the group comprising CBMAA, CBMA, CBAA.
• the shape, dimensions, morphology, and chemical nature of the substrate are not critical. They may be planar or differently shaped objects, tubes, fibres, particles, membranes, microparticles, nanoparticles, porous materials, metals, silicon, silicate- or aluminosilicate-based materials (such as glass), polymers, inorganic materials, and the like.
• the active carboxybetaine ester is a product of a reaction of carboxyl group of carboxybetaine with N-substituted carbodiimides, i.e. O-acylurea, and/or a product of a reaction of carboxyl group of carboxybetaine with N-substituted carbodiimides and N-hydroxysuccinimide (NHS) or derivatives thereof.
• active esters are NHS ester or sulfo-NHS ester.
• step c) is carried out by first rinsing the product of step b) with a solution or buffer, which was used as the solvent in step b), preferably water, an aqueous NaCl or PBS, followed by rinsing with a buffer, which will be used in the next step for incubation, preferably PBS buffer. Then the product is incubated with a solution of aminoethoxyacetic acid of general formula (I) in a buffer (preferably PBS buffer). Then it is rinsed with the buffer previously used for incubation, and then rinsed with a solution into which it is then deposited for storage or with water and dried.
• a solution or buffer which was used as the solvent in step b
• a buffer which will be used in the next step for incubation
• a buffer which will be used in the next step for incubation
• PBS buffer preferably PBS buffer
• the bioactive substance of the present invention is a substance containing at least one NH 2 group selectively interacting with the target component of the biological medium.
• the bioactive substance may have affinity for the target component.
• the bioactive substance is a natural antibody, antigen, lectin and the cellular receptor and their synthetic analogues and parts prepared by recombinant techniques as well as synthetic oligopeptide sequences, nucleic acids and portions thereof, and synthetic oligonucleotide sequences and aptamers.
• the bioactive substance can catalyse chemical conversion of the target substance, such as enzyme, coenzyme, and their synthetic analogues.
• the bioactive substance can induce a biological response, such as an anticoagulant including heparin, proteins and oligopeptide sequences responding to cellular integrins, growth factors, hormones, and derivatives of drugs and vitamins.
• Bioactive substances may also include nanoparticles functionalized by the NH 2 group, in particular metallic, polymeric, and silicon nanoparticles, as well as nanoparticles based on metal oxides, or polymeric nanoparticles with magnetic core.
• the bioactive substance is a substance with affinity for the target component, selected from the group consisting of antibody, antigen, lectin, cellular receptor and analogues thereof, as well as parts prepared by recombinant techniques, synthetic oligopeptide sequences, nucleic acids and parts thereof, synthetic oligonucleotide sequences, and aptamers; a substance catalysing chemical transformation of the target substance, selected from the group comprising enzymes, coenzymes, and synthetic analogues thereof; a substance that induces biological response, selected from the group comprising anticoagulants, proteins and oligopeptide sequences reacting with cellular integrins, growth factors, hormones, and drugs.
• a substance with affinity for the target component selected from the group consisting of antibody, antigen, lectin, cellular receptor and analogues thereof, as well as parts prepared by recombinant techniques, synthetic oligopeptide sequences, nucleic acids and parts thereof, synthetic oligonucleotide sequences, and
• the biological medium for the purposes of the present invention is a fluid containing biological agents, that is biomolecules and their associates (proteins, saccharides, polysaccharides, lipids, nucleic acids, lipoproteins, glycoproteins, organelles etc.), viruses, cells, microorganisms, and fragments thereof.
• the biological medium is, for example, blood and other body fluids, blood plasma and serum, tissue extracts, cell lysates and suspensions, as well as food extracts.
• the carboxybetaine groups are contained in a polymeric layer on the substrate surface as a polymer brush, prepared by surface polymerization or grafting of polymers to the substrate surface, or a polymer coating, preferably having a thickness ranging between 5 nm and 5 um.
• the polymeric layer containing carboxybetaine groups is a polymer brush of poly(carboxybetaine methacrylamide) (polyCBMAA), poly(carboxybetaine methacrylate) (polyCBMA), poly(carboxybetaine acrylamide) (polyCBAA) or copolymer poly (A-co-B), where A is a monomeric unit from the group of N-(2-hydroxypropyl) methacrylamide (HPMAA), 2- hydroxyethyl methacrylate, 3-hydroxypropyl methacrylate and oligo(hydroxy ethylene glycol) methacrylate, and B is a monomeric unit from the group of carboxybetaine methacrylamide, carboxybetaine methacrylate and carboxybetaine acrylamide at a concentration of from 1 to 99 mol%, especially poly(HPMAA-co-CBMAA/xmol%), where x (molar concentration of CBMAA) ranges between 1 and 99 mol%.
• this layer has
• the substrate is selected from the group comprising particles, porous membranes, cell carriers (“scaffolds”), and biosensors.
• Particles are preferably from material selected from the group containing gold, silver, magnetic materials, silicon, S1O2, and polymers falling within the definitions of substrate (1) to (6) and preferably have their diameter of from 5 nm to 1 mm.
• Particles with bioactive substances bound on activated carboxybetaine groups on the surface of particles including the inner surfaces of porous particles are applicable as carriers for targeted therapy and in vivo diagnostics, separation and accumulation of biological agents, and for enzymatic catalysis in bioreactors.
• Porous membranes are membranes designed for affinity separation of biological substances from biological media.
• Cells carriers or scaffolds are used for example for tissue engineering such as:
• the substrate is a biosensor detection surface for direct detection or multistage detection of analytes in complex biological media, such as by optical or mass biosensors.
• the detection surface is coated with a polyCBMAA, polyCBMA, polyCBAA or poly (HPMAA-co-CBMAA) polymer brush with bound bioreceptors.
• Bioreceptors in this application are bioactive agents that have selective affinity for the target components of the analysed medium.
• the substrate is intended for contact with blood in vitro, ex vivo, and in vivo.
• the method of preparation of a substrate surface containing carboxybetaine groups according to the present invention provides a very rapid, cheap, and effective mitigation of problems with deposition of undesirable components of biological media.
• the claimed use of (2-aminoethoxy)acetic acid is more effective, as illustrated in the following comparative examples.
• FIG. 1 Diagram of activation of carboxybetaine groups (a), binding of bioactive substance (b), and incubation with an aqueous solution of (2-aminoethoxy)acetic acid (AEAA) (c).
• AEAA (2-aminoethoxy)acetic acid
• Fig. 2 Example of detection of carcinoembryonic antigen (CEA, 10 ug/mL) in undiluted blood plasma using an SPC biosensor coated with a polyCBAA brush functionalized by anti-CEA binding and subsequently deactivated by AEAA.
• CEA carcinoembryonic antigen
• Fig. 3 Example of detection of Salmonella typhimorium (1 ⁇ 10 7 CFU/mL) in a homogenized cucumber solution using a SPR biosensor coated with a polyCBAA brush functionalized by anti- Salm binding and subsequently deactivated by AEAA.
• the figure shows reference compensatory data obtained by subtracting the SPR response from the reference channel with anti-Salm incubated in a homogenized cucumber solution from the anti-Salm measuring channel incubated in Salmonella-added homogenized cucumber solution (1 ⁇ 10 7 CFU/mL). Examples
• Example 1 Method of treating a surface of the substrate containing activated carboxybetaine functional groups by reaction with (2-aminoethoxy)acetic acid
• Fig. 1 shows the method of treating a surface of the substrate containing carboxybetaine functional groups.
• the carboxybetaine groups are chemically activated by converting the carboxyl groups of carboxybetaine into an active NHS ester or sulfo-NHS ester by reaction with l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or sulfo NHS.
• EDC l-ethyl-3- (3-dimethylaminopropyl)carbodiimide
• NHS N-hydroxysuccinimide
• sulfo NHS N-hydroxysuccinimide
• step (c) a reaction of those active NHS esters or sulfo-NHS esters with (2-aminoethoxy)acetic acid (AEAA) takes place, which did not react with bioactive substance in step b).
• AEAA (2-aminoethoxy)acetic acid
• Comparative Example 1 Regeneration of resistance of EDC/NHS activated polyCBAA brushes against biological deposition by a reaction with deactivating agents
• SPR surface plasmon resonance
• the chip covered with the polyCBAA brush was rinsed with water and mounted into the SPR sensor chamber with four flow microfluidic channels.
• Carboxybetaine groups of brush in the 2 nd , 3 rd , and 4 th channels were activated by reaction with an aqueous solution of N-hydroxysuccinimide (NHS, 0.1 M) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 0.5 M) for 20 minutes with 10 mM NaCl at 20 °C (step (a) in Example 1).
• NHS N-hydroxysuccinimide
• EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
• the activated brush was exposed to deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) for 30 minutes or 40 minutes in the 2 nd channel, to the solution of (2- aminoethoxy)acetic acid (1 M, water pH 7, 30 minutes) in the 3 rd channel, and to the glycine solution (1 M, water pH 7, 30 minutes) in the 4 th channel at 20 °C (step (c) in Example 1).
• a SPR sensor was prepared in this way, in which the 1 st channel contained an inactivated brush; the 3 rd channel contained the brush after deactivation by (2-aminoethoxy)acetic acid; and the 4 th channel contained the brush after deactivation by glycine.
• the brush was always rinsed for 5 minutes with water and 10 minutes with PBS.
• the SPR resonance wavelength ( ⁇ ) was measured, the surface was incubated with undiluted blood plasma for 10 minutes, rinsed with PBS, measured ⁇ 2 , incubated with high ionic strength solution (PBS* 1 , PBS containing 0.75 M NaCl, pH 7.4) for 5 minutes, rinsed with PBS, and measured ⁇ 3 .
• Activation of the polyCBAA brush, binding of bioactive substances, and incubation with deactivating agents was carried out in four microfluidic channels.
• the brush was activated by EDC/NHS as described in Comparative example 1. After rinsing with water, the brush was incubated with an antibody against carcinoembryonic antigen solution (anti- CEA, 50 ⁇ g/ml, 10 mM borate buffer, pH 8) for 15 minutes, the achieved level of bound antibody was 200 ng/cm 2 according to SPR, or with solution of NH 2 -DNA oligonucleotide probes (NH 2 - DNA-ON, 22-mer, 2 ⁇ , 10 mM borate buffer, pH 8), the level of bound probes was 75 ng/cm 2 according to SPR, or with anti-Salmonella typhimorium (Anti-Salm, 50 ⁇ g / ml, 10 mM borate buffer, pH 8), the achieved levels of bound antibody were 180
• the brush was deactivated by (2-aminoethoxy)acetic acid or glycine or deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8, 40 minutes) and then incubated with undiluted blood plasma as described in Comparative example 1.
• Plasma deposits which remained on the surface after rinsing with PBS, and deposits after further rinsing with a high ionic strength solution (PBS* 1 , PBS containing 0.75 M NaCl, pH 7.4) were determined by SPR measurement as in Comparative example 1.
• Table 2 Deposition from undiluted human blood plasma and undiluted human cerebrospinal fluid on the polyCBAA brush functionalized by antibody against carcinoembryonic antigen (anti- CEA) or NH 2 -DNA oligonucleotide probes for detection of microRNA (NH 2 -DNA-ON) and subsequently deactivated by deactivation reagents
• deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) 40 minutes anti-CEA, 125 ng/cm 2
• Blood plasma deposit on polyCBAA with bound anti-CEA and deactivated by AEAA was under the SPR detection limit.
• glycine On the same brush deactivated by glycine, 7.5 ng/cm 2 of blood plasma was deposited. Only slightly more, namely 10.8 ng/cm 2 , was deposited on the brush deactivated by spontaneous hydrolysis of active esters in the deactivation buffer.
• Table 3 Deposition from homogenized hamburger, undiluted blood plasma, and cerebrospinal fluid on the polyCBAA brush functionalized by anti-Salmonella (anti-Salm) binding followed by deactivation by deactivating agents
• Fig. 3 shows reference- compensated data obtained by subtracting the SPR response from the reference channel with anti- Salm incubated in a homogenized cucumber solution from the measuring channel with anti-Salm incubated in Salmonella-added homogenized cucumber solution (1 ⁇ 10 7 CFU/mL).
• Comparative Example 3 Reaction of functionalized poly(HPMAA-co-CBMAA/ 15 mol%) brushes with (2-aminoethoxy)acetic acid increases their resistance to biological deposition
• the golden surface of the SPR chip was covered with a copolymer brush containing 15 mol% CBMAA (poly(HPMAA-co-CBMAA/15 mol%) with a thickness ranging from 19 nm to 31 nm according to the protocol described in PCTCZ2016050011, PV 2015-313.
• the brush was activated, functionalized by covalent bonding of an antibody to Staphylococcal enterotoxin B (anti-SEB) or NH 2 -DNA oligonucleotide probes, deactivated by AEAA or glycine by the procedure described in Comparative example 2.
• Table 4 Deposition from non-diluted blood plasma on poly(HPMAA-co-CBMAA/15 mol%) brushes functionalized by binding of an antibody to Staphylococcal enterotoxin B (anti-SEB) or NH 2 -DNA-oligonucleotide probes (NH 2 -DNA-ON) for detection of microRNA and subsequently deactivated by deactivating agents.
• anti-SEB Staphylococcal enterotoxin B
• NH 2 -DNA-oligonucleotide probes NH 2 -DNA-ON
• Comparative Example 4 Resistance of carboxybetaine polymer brushes to biological deposition from undiluted blood plasma and food samples after EDC/NHS activation and after reaction with deactivating agents at different concentrations
• Carboxybetaine groups of the brush were activated in the 2 nd to 6 th channel by reaction with an aqueous solution of N-hydroxysuccinimide (NHS, 0.1 M) and 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 M) for 20 minutes at 20 °C (step (a) in Example 1).
• NHS N-hydroxysuccinimide
• EDC 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide
• the SPR sensor was prepared in which the 1 st channel contained an inactivated brush, the 2 nd channel brush after activation, the 3 rd channel brush after deactivation by glycine, the 4 th channel brush after deactivation by (2-aminoethoxy)acetic acid, the 5 th channel brush after deactivation by ethanolamine, and the 6 th channel brush after deactivation by deactivation buffer.
• the brush was rinsed with water for 5 minutes and with PBS for 10 minutes.
• the SPR resonance wavelength ( ⁇ was measured, the surface was incubated with undiluted blood plasma for 10 minutes, rinsed with PBS and again ⁇ 2 was measured.
• the size of the biological deposit in ng/cm 2 was calculated from the difference ⁇ 2 -
• Tables 5 and 6 present deposition from all tested biological samples for both types of carboxybetaine polymer brushes after deactivation by incubation with a solution of (2- aminoethoxy)acetic acid. This is comparable or slightly lower than deposition after deactivation by incubation with 10 times more concentrated glycine solution and significantly lower compared with traditional deactivation by hydrolysis in deactivation buffer or by ethanolamine binding. This example demonstrates that even at much lower concentrations of (2-aminoethoxy)acetic acid compared to glycine, deactivation by AEAA is comparable or even more effective than deactivation by glycine.

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