WO2010063725A2 - Structuring particles into useful structures and their use in life science - Google Patents

Structuring particles into useful structures and their use in life science Download PDF

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WO2010063725A2
WO2010063725A2 PCT/EP2009/066161 EP2009066161W WO2010063725A2 WO 2010063725 A2 WO2010063725 A2 WO 2010063725A2 EP 2009066161 W EP2009066161 W EP 2009066161W WO 2010063725 A2 WO2010063725 A2 WO 2010063725A2
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particles
process according
cross
suspension
macroporous
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PCT/EP2009/066161
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French (fr)
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WO2010063725A3 (en
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Bo Mattiasson
Igor Galaev
Harald Kirsebom
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Bo Mattiasson
Igor Galaev
Harald Kirsebom
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Publication of WO2010063725A2 publication Critical patent/WO2010063725A2/en
Publication of WO2010063725A3 publication Critical patent/WO2010063725A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28047—Gels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group

Definitions

  • the present invention relates to cryostructuration of particulate material. More particularly, the present invention relates to a process for the production of a self- supporting macroporous material by fusing suspended particulate material (e.g. microgel particles, latex particles, microbial cells) using a combination of cryostructuration and chemical cross-linking, a macroporous material and use of said material. Even more specifically, the invention relates to a macroporous structure.
  • suspended particulate material e.g. microgel particles, latex particles, microbial cells
  • Cryostructuration is a technique relying on rejection of solutes or suspended particles from growing ice crystals when an aqueous solution or suspension is frozen at moderate sub-zero temperatures (see Lozinsky 2003, Lozinsky 2002, Zhang 2007).
  • Macroporous structures from hard particles like alumina, hydroxyapatite and silica- PVA have been produced earlier in a multi-step procedure by cryostructuration of the suspension followed by freeze drying to remove frozen solvent and fixation of the macroporous structure by sintering (see Zhang 2007, Deville 2006, Deville 2006).
  • Plieva 2008, Galaev 2007, Lozinsky 2001, Plieva 2007, Hedstr ⁇ m 2008 and WO-A-2007/108770 all discloses methods for preparation of cryogels in the form of macroporous gels prepared by freezing a solution of monomers or polymers, whereby the monomers or polymers crosslink during this state and during subsequent thawing forming the macroporous material.
  • water soluble polymers such as agarose, chitosan, PVA or monomers such as acrylamide, HEMA, DMAAm etc are used.
  • a polymeric matrix is also required to form macroporous gels as described by
  • Orrego 2009 discloses how solutions of chitosan may be used to create gels with or without chemical cross-linking at ambient temperature.
  • the gels are subsequently freezed and freeze dried in order to make them porous.
  • this is a complicated and costly process.
  • the present invention seeks to mitigate, alleviate, circumvent or eliminate at least one, such as one or more, of the above-identified deficiencies by combining the cryo-structuration of particles from a suspended state, into a self-supporting macroporous material, with the simultaneous fixation of the formed structure by cross- linking of particles in a semi- frozen state.
  • the cross-linking is achieved either between groups on the surface of the particles directly, thereby forming linkages between the particles, which in turn preserves the macroporous structure formed during the cryo-structuation, or the cross-linking is achieved by added cross-linking agent which reacts with groups present on the surface of particles forming linkages between them and thus preserving the macroporous structure formed during cryo-structuration also after melting the solvent crystals, or cross-linking may take place according to a combination of the above.
  • a process is provided for the production of a self-supporting macroporous material, comprising cross-linked particles, from a suspension of particles in a liquid.
  • Said particles comprise groups at the surface for cross-linking of said particles.
  • Said process comprise the steps of; a) freezing said suspension of particles, thereby forming crystals from the liquid of the suspension; and cross-linking said particles; and b) melting the crystals to release said self-supporting macroporous structure comprising cross-linked particles.
  • a macroporous material is provided, which is obtainable by the process according to the first aspect of the invention.
  • the macroporous material according to the second aspect is a structure obtained by freezing said suspension in a mould.
  • Fig. 1 is a microscopy photography of a cryogel according to an embodiment
  • Fig. 2 is a microscopy photography of a cryogel according to another embodiment
  • Fig. 3 is a microscopy photography of a cryogel according to yet another embodiment
  • Fig. 4 is a microscopy photography of a cryogel according to a further embodiment
  • Fig. 5 is a graph showing biological stability of a cryogel according to yet a further embodiment.
  • Fig. 6 is a graph showing that the macroporous material according to an embodiment comprises micropores.
  • cross-linking in a suspension may be carried out in combination with cryo-structuration to obtain a self-supporting macroporous material, comprising cross-linked particles.
  • An advantage with this is that it is possible to build a material where the particles have free access to the pore liquid without being hindered by polymer layers. Yet another advantage is that the material allows for potentially a very high density of particles, such as cells, in the structure, since the particles themselves may form the stable structure.
  • the surface area may also be large which makes it possible for the material to interact effectively with its surrounding.
  • a further advantage is that the surface may be rough, which makes the material a good scaffold, i.e. for growing cells.
  • said freezing causes a first group on a first particle to interact with a first group on a second particle to cross-link said particles. Furthermore, a second group on said first particle or a second group on a second particle interacts with a first group on a third particle to cross-link said particles. Accordingly, several particles may be cross- linked to each other in order to form a network being a self-supporting macroporous material.
  • a small amount of cross-linking agent is present to achieve cross-linking.
  • a small amount of cross-linking reagent may be between 0.1% to 10% of the total content used. Accordingly, a majority, such as 90% to 99.9% of the final material consists of the structured cross-linked particles.
  • a cross- linking reactant e.g. a chemical like glutaraldehyde is present, it is also concentrated in this area in so called non-frozen microphase, which volume may be defined by the concentration of the solutes, depression of freezing temperature and/or amount of polymer bound water (Kirsebom 2008).
  • the non-frozen phase will then act as a reaction medium for the cross-linking reaction which will proceed under semi-frozen conditions with high concentration of both particles and cross-linking agent promoting the binding of particles together into self-supporting materials.
  • the system is thawed with melted ice crystals leaving complementary pores.
  • said freezing may cause said first group said first particle and said first group said second particle to interact with said cross-linking agent to cross-link said particles.
  • Cryo-structuration of a suspension is different from cryo-polymerization according to prior art, since cryo-structuration results in polymeric systems of significantly different morphology than those from polymerization in homogeneous solution according to prior art.
  • the liquid of the suspension is substantially water, thus forming an aqueous suspension.
  • an aqueous suspension may comprise water-miscible solvent, such as DMSO, ethanol, methanol or acetone
  • liquid of the invention is water
  • other liquids such as DMSO, formamide or dioxane, may also be used.
  • Water is preferred as it is compatible with sensitive particles such as cells. Further, water has a suitable freezing point. In addition the formation of crystals when water freezes is pronounced.
  • the diameter, such as the Feret's diameter, of the particles is between between 40 nm and 100 ⁇ m.
  • Feret's diameter is the statistical mean distance apart of two parallel lines which are tangential to the particle.
  • the particles are selected from the group consisting of microgels made from natural or synthetic polymers, or mixtures thereof, latexes or microbial cells or their combination, as the particles may be poly(7V- isopropylacrylamide) (pNIPA) microgels with the size from 40 nm to 100 ⁇ m, rod shaped bacterial cells ⁇ Escherichia coli and Caldicellulosiruptor saccharolyticus) with the approximate size of 2 x 0.4 ⁇ m, spherical bacteria (Ralstonia eutropha and Lactococcus lactis) cells with the diameter of about 1 ⁇ m and baker's yeast cells which are 5-10 ⁇ m in diameter.
  • pNIPA poly(7V- isopropylacrylamide)
  • the main condition for the particles to be suitable for this procedure is the existence on their surface of groups which could be used for cross-linking.
  • the cross linking may be chemical, ionical, or hydrophobical cross-linking.
  • Prepared pNIPA microgels contain amino groups whereas the microorganisms contain inherent amino groups at the cell surface.
  • Such particles are suitable for cross-linking, such as cross-linking with amino reactive cross-linking agents, e.g. glutaraldehyde.
  • the particles may be both soft, i.e. particles which can be at least partly compressed or deformed by external stimuli e.g.; mechanical forces, temperature or pH, or hard, i.e. particles not deformed or compressed by external stimuli e.g.; mechanical forces, temperature or pH.
  • the particles may be metal, metal oxides, carbon, silicate, sol-gel particles, minerals and has or has been modified to contain reactive groups on the particle surface such that the particles can be cross-linked using a bi- or multifunctional reagent or through reactions between the surface groups.
  • the particles may be of different types, such as at least two types, either of the same size or with different size distributions.
  • examples on such mixtures of particles are different microorganisms which are capable of catalyzing a reaction sequence, one catalytically active unit and one adsorbent which can either bind the substrate or the product, or it could be a catalytically active unit combined with a material that captures toxic compounds that otherwise may destroy the biocatalyst.
  • it could be one type of particle but with a broad particle size distribution, or any combination thereof. Examples of this are seen in Example 5 and 10 below.
  • a macroporous material is defined by IUPAC as a material containing pores larger than 50 nm. However in this case macroporous material is considered with pores of larger than 1 ⁇ m.
  • the size of pores is larger than 1 ⁇ m.
  • the size of the pores are typically less than 200 ⁇ m.
  • the materials may also contain micropores, which may occur between the particles in the material, or in the particles themselves.
  • the morphology of produced cryogels is very similar to that formed after cryostructuration of the chemically cross-linked gels produced either by radical polymerization, as described by Plieva 2005, or via covalent cross-linking of preformed polymers, as described by Plieva 2006.
  • the present invention has the further advantages that it is possible to build a material where the particles have free access to the pore liquid without being hindered by polymer layers.
  • Yet another advantage with the present invention is that it allows for potentially a very high density of particles, such as cells, in the material, since the particles themselves may form the stable material.
  • porous polymer particles When porous polymer particles are used high binding capacities may be obtained. This is normally difficult using conventional cryo-structurization, and then usually traditional adsorbents have to be used, but such are either big with long diffusion distances or small particles with massive back pressure when used in chromatography.
  • the structure produced according to some embodiments has the advantage of good kinetics and high capacity when adsorbents are used.
  • cryogels formed according to the present invention may have a rough surface. This may be advantageous for cell cultivation, since the cryogels may then be a very suitable scaffold for the cell cultivation. Furthermore this invention will allow production of a material which has a lower polymer density in the pore walls than previous work on macroporous gels. Since cryo-concentration of suspended particles do not proceed in the same way as with soluble polymers, this change in concentration will result in porosity in the walls and thus a possible higher total porosity.
  • the present invention also allows for co -immobilization of particles with different functionalities in close proximity to each other. In prior art super-macroporous gels the particles are diluted with polymer, which do not allow the close proximity of the particles. Further, the particles may be immobilized in vicinity to each other. Thereby molecules may rapidly diffuse from one particle to another. This may be advantageous when using the material for catalytic applications.
  • the process to form macroporous material from particles comprise the following steps: First, particles are suspended, thus forming a suspension. Second, the suspension is frozen which allows forming of cross-links between constituents of the suspension. Third, the frozen suspension is thawed, resulting in a self-supporting macroporous material, held together by the cross-links formed between the particles.
  • the particles themselves carry groups on their surface which may be cross-linked even in the absence of a cross-linking agent.
  • the cross-links are formed during freezing between said groups and the resulting self-supporting macroporous material is held together by the cross-links formed between the particles.
  • the groups may be any reactive groups suitable to form cross-links during freezing, such as amino-, hydroxy-, carboxy-, sulfhydryl-, hydrophobic-, charged- or affinity groups, or their combination.
  • Hydrophobic groups may be e.g. linear alkyl residues preferably comprising at least 4 carbon atoms. Such residue may be obtained by amidation of amino groups by use of a carboxylic acid, such as a fatty acid.
  • the particles may be any kind of particle suitable to create self-supporting macroporous material, such as particles selected from metal, metal oxides, carbon, silicate, sol-gel particles, mineral particles that contain, or have been modified to contain, said groups for cross-linking.
  • Affinity groups may be any known entity for creating affinity bindings, such as biotin, nucleotides, amino acids, chelating groups, carbohydrates, DNA-probes or textile dyes, or their combination.
  • Example 11 is an example of affinity binding.
  • a chemical reactant i.e. a cross-linking agent
  • the cross-links are formed during freezing as a result of an interaction, such as reaction, of groups on the particles with the chemical reactant, which may interact with at least two groups .
  • the self-supporting macroporous material is then formed by the combination of chemical reagents and particles, since the particles are held together by the cross-links formed by the chemical reagents.
  • the chemical reactant, or cross-linking agent may be any chemical reactant suitable to form cross-links during freezing, such as Bis(Sulfosuccinimidyl) suberate, Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, (l,5-Difiuoro-2,4-dinitrobenzene), Dimethyl pimelimidate»2 HCl, Dimethyl Suberimidate»2 HCl, Disuccinimidyl glutarate, Dithiobis(succinimidyl) propionate, Disuccinimidyl suberate, Disuccinimidyl tartrate, Dimethyl 3,3'-dithiobispropionimidate»2 HCl, 3,3 '-
  • the cross-linking agent may have at least two reactive groups capable of interacting with any of the groups exposed on the surfaces of the particle.
  • the cross-linking agent may be of polymeric nature, , such as polymers with many aldehyde groups, a polymer with boronate groups which interacts with PVA or with carbohydrate particles etc.
  • the cross-linking agent may also comprise a polymer chain, such as oxidized dextran.
  • the cross-linking agent comprises a polymer chain with reactive groups.
  • reactive groups may be such that they cause covalent interactions, or non-covalent interactions with the particles that are to be cross- linked.
  • Examples of such structures are e.g. oxidized carbohydrates that contain aldehyde groups that will interact with amino groups of particles such as cells, polymers containing boronate groups that will complex with carbohydrate groups on the particle surface, polymers with weak hydrophobic groups. Examples of these are short alkyl chains that are too short to cause any interaction per se, when present individually. However, when present in a plurality on a polymer each may cause a weak interaction that taken together results in a strong interaction and a multipoint attachment.
  • Still another alternative is a polymer with charged groups that will form a multitude of interactions with charged structures on the particles, thereby causing a strong cross- linking of said particles.
  • Advantages with polymeric cross-linkers are obvious in cases of living organisms that are cross-linked. The polymeric structure will not penetrate into the cell and thereby it is less toxic to the cell.
  • the particles themselves carry reactive groups on their surface and a chemical reactant is added to the suspension before freezing.
  • the cross-links are formed both between said reactive groups of the particles and between the molecules in the chemical reactant.
  • the particles are packed together since they are expelled from the growing crystals.
  • the packed particles are linked in the semi- frozen state either through cross-linking with a cross-linker or due to selfcross-linking between particles.
  • An example of this is e.g. carbohydrate - lectin, hydrophobic groups packing together to form interactions, multiply weak affinity bonds, which create strong bonds through multiple point attachment.
  • the freezing temperature may be any temperature below crystallization temperature of the liquid, down to 40 K below the crystallization point. In the case the liquid is water, or a water based solution, the temperature is typically between -I 0 C and -3O 0 C.
  • the time between freezing and thawing may be any period long enough to allow forming of the cross-links, such as between 0.5h and 7 days, such as between 1Oh and 24h.
  • the particles are cells.
  • the cryogels formed from the resulting cell suspensions may be similar to cryogels formed from polymers.
  • An advantage with this is that the cryogels formed from cell suspensions are sponge-like and elastic, withstanding significant mechanical deformation and restoring completely their shape when the stress is released. This may be advantageous if the material constitutes a biologic reactor, and substrate is fed into the material and the product is transported away from the material.
  • the cryogels are produced from thermoresponsive particles.
  • the gels may then exhibit a very rapid response to changes in temperature.
  • the unique material with large pores and small particles enable a rapid transition in size. This material would allow the capture and release of substances, such as pharmaceuticals, controlled by temperature change.
  • hydrophobicity changes with temperature hydrophobic compounds may thus be trapped at one temperature and released at another.
  • the method according to some embodiments is relatively friendly to the living microbial cells, provided the cross-linking agent is not toxic.
  • biocompatible alternative cross-linkers like partially oxidized dextran or another polymeric crosslinker, allows for producing cryogels from bacteria e.g. Caldicellulosiruptor saccharolyticus cells maintaining their viability. This observation in combination with sufficient mechanical stability of macroporous gels formed via cryostructuration of cell suspensions opens an interesting perspective of carrier- free immobilization of cells and using them in flow-though bioreactors either for the synthesis of target product or for selective bioconversion of e.g. toxic compounds in effluents.
  • the macroporous material may have catalytic activity, such that bioconversions or combined chemical-biochemical conversions may be carried out.
  • the macroporous material may be loaded with biologically active compound or a drug. Said biologically active compound or drug, may be slowly release form said particles into surrounding media.
  • the method of cryo-structuration according to an aspect of the invention is very much separated from prior art methods for making composite materials. This is advantageous; because the particle in the resulting gel has free access to the pore liquid, since no hindering polymer layers are needed for stabilization of the material.
  • the macroporous material according an aspect of the invention may be formed into a desired structure by freezing said suspension in a mould. Any kind of mould may be used, as long as the cross-linking is possible. Thus the material may be shaped into structures with desired geometry.
  • the macroporous structure has catalytic activity.
  • the particles of the macroporous structure are particles with affinity ligands or molecular structures to which dedicated molecules bind.
  • Affinity ligands may be any known entity for creating affinity bindings, such as biotin, nucleotides, amino acids, chelating groups, carbohydrates, DNA-probes or textile dyes, or their combination.
  • use of a macroporous material with affinity ligands, in studies of affinity separation and affinity binding is provided. Such structure is seen in example 10.
  • the process according to an aspect may further comprise a step of loading said material with biologically active compound(s).
  • said biologically active compound may be released from the macroporous material by changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
  • the particles of the macroporous material may also themselves be released in response to external changes.
  • particles in said macroporous material may be released in response to changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
  • Example 1 Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus cells.
  • the structured bacterium in the gel was confirmed using Scanning electron microscopy (SEM).
  • SEM Scanning electron microscopy
  • the gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C.
  • the samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried.
  • the dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 1.
  • Fig. 1 is shown that the walls of the material comprise of closely packed bacteria and macropores above 40 micrometer. The walls are only composed of bacteria and not immobilized in a polymeric matrix. This material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of
  • Example 2 Preparation of a cryo-structured cryogel of 15% Lactobacillus casei cells.
  • Cell pellet of Lactobacillus casei (0.15 g) was suspended in 1 ml of sterile deionized water and the suspension was cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with sterile water.
  • a thermostat Liauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigshofen, Germany
  • Example 3 Preparation of a cryo-structured cryogel of 5% poly N- isopropylacrylamide (pNIPA)-co-allylamine particles.
  • Synthesized pNIPA particles (0.1 g) of a size between 200-500 nm was suspended in 2 ml H 2 O and cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h.
  • a thermostat Liauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-K ⁇ nigshofen, Germany
  • Example 4 Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles.
  • MMA-GMA particles of a size between 200-500 nm were reacted with ethylenediamine to introduce reactive amino groups.
  • the activated particles (0.130 g) were suspended in 1.3 ml of H 2 O and cooled. Thereafter 40 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
  • the particulate structure of the gel was confirmed using Scanning electron microscopy (SEM).
  • SEM Scanning electron microscopy
  • the gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C.
  • the samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried.
  • the dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 3. It is clearly shown in Fig. 3 that the walls of the structure are composed of densely packed polymer particles, which make up the macroporous structure of the material.
  • the material is composed of hydrophobic particles; therefore it is possible to prepare hydrophobic materials with this method.
  • the reactive epoxy groups are possible to use for coupling of different ligands.
  • Example 5 Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus and Saccharomvces cerevisiae Cell pellet of Caldicellulosiruptor saccharolyticus (0.136 g) and Saccharomyces cerevisiae (0.014 g) was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co.
  • the dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 4. It is shown in Fig. 4 that the material consists of two different types of micro organisms; bacteria and yeast. The material is only composed of crosslinked micro organisms and no inert matrix used to immobilize the cells. This structure will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products. The use of two organisms allows using sequential processes.
  • Example 6 Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus and MMA-GMA particles
  • Example 7 Preparation of a cryo-structured cryogel of 6% bovine serume albumin (BSA) particles
  • Example 8 Preparation of a cryo-structured cryogel of 20% silica particles
  • Silica particles (0.2 g) activated with amino groups with a size less than 1 ⁇ m were suspended in ImI of deionized water containing 1% (weight of silica particles) of anionic dispersant and the suspension was cooled. Thereafter 20 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. This material is suitable for further modifications and can then be used for analytical applications.
  • Example 9 Preparation of a cryo-structured cryogel of 3% chitosan particles containing insulin
  • Chitosan nanoparticles (0.03 g) containing insulin as a model compound was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. Short diffusion distances in the particles will give an efficient release of the insulin. This exemplifies the possibility to use this material for the delivery of macromolecular compounds.
  • Example 10 Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles containing imminoacetic acid. Synthesised MMA-GMA particles of a size between 200-500 nm were reacted with immidoacetic (0.1 M in 1 M NaCO 3 ) for 12 h. After which the particles were reacted with ethylenediamine to introduce reactive amino groups. The activated particles (0.130 g) were suspended in 1.3 ml of H 2 O and cooled.
  • the prepared gel was then used for IMAC purification of the protein ⁇ - glucosidase.
  • First the gel was then loaded with 0.1 M copper sulfate and then non bound copper was washed out with running buffer (20 mM Tris-HCl, pH 7.4 containing 10 mM imidazole and 0.5 M NaCl) after equilibrating the system with running buffer. Then 5 ml of clarified protein sample (in running buffer) containing his-tagged ⁇ - glucosidase was applied at a flow rate of 1 ml/min.
  • the non bound protein was washed out with running buffer after which the ⁇ -glucosidase was eluted using elution buffer (20 mM Tris-HCl, pH 7.4 containing 200 mM imidazole and 0.5 M NaCl).
  • the target protein ( ⁇ -glucosidase) was eluted as could be confirmed with SDS-PAGE and of activity of the enzyme. This shows that the material has a potential for efficient affinity purification of proteins.
  • Example 11 Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles containing trypsin.
  • MMA-GMA particles of a size between 200-500 nm were coupled with 10 mg/ml trypsin solution in 0.1 M Na-phosphate buffer pH 8.0. After which the particles were reacted with ethylenediamine to introduce reactive amino groups and block free epoxy groups. The use of ethylenediamine was to introduce additional reactive sites for the following crosslinking reaction with glutaraldehyde.
  • the activated particles (0.130 g) were suspended in 1.3 ml of H 2 O and cooled. Thereafter 40 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co.
  • BAPNA N-benzoyl-DL- arginine-4-nitroanilide
  • the BAPNA containing buffer was then added to the gel and incubated 15 min at 37°C after which the solution was squeezed out of the gel and absorbance at 410 nm measured.
  • the resulting material is suitable material for use as an enzyme reactor which can be used for the degradation of proteins, the macroporous material will enable an efficient mass transfer in the system.
  • Example 12 Preparation of a cryo-structured cryogel of 15%
  • Cell pellet of Caldicellulosiruptor saccharolyticus (0.15 g) was suspended in 0.9 ml of deionized water and the suspension was cooled. Thereafter 0.1 ml of solution of partially oxidized dextran containing aldehyde groups (5% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-K ⁇ nigshofen, Germany) set at - 12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. This material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products.
  • Example 13 Preparation of a cryo-structured cryogel of 5% poly N- isopropylacrylamide(pNIPA)-co-allylamine particles-co-2-(dimethylamino) ethylmethacrylate (DMAEMA)
  • Synthesized pNIPA particles (0.1 g) containing groups with ion-exchange capability (DMAEMA) of a size between 200-500 nm was suspended in 2 ml H 2 O and cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-K ⁇ nigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The resulting material has a potential for use as a material for ion exchange separations.
  • DMAEMA groups with ion-exchange capability
  • Example 14 Preparation of a cryo-structured cryogel of 3% chitosan particles Chitosan nanoparticles (0.03 g) in ImI of deionized water and the suspension was cooled. Thereafter 10 ⁇ l of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-K ⁇ nigshofen, Germany) set at - 12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The produced material is both biocompatible and biodegradable.
  • Example 15 Preparation of a cryo-structured symbiotic cryogel of 15% Lactobacillus casei and Saccharomvces cerevisiae Using two different organisms which can act in a symbiotic pathway is modeled by using Lactobacillus casei and Saccharomyces cerevisiae, Saccharomyces cerevises can degrade surose to glucose which can then be used as a carbon source for Lactobacillus casei. This can be seen as a model for co-immobilizing two microorganisms which can act in symbiosis.
  • Example 16 Preparation of a cryo-structured cryogel of 15% Escherichia coli cells expressing poly-His peptides for affinity capture.
  • the capture of copper was checked by passing 0.1 M CuSO 4 and then washes with plenty of buffer (20 mM Tris-HCl, pH 7.4 containing 10 mM imidazole) to remove all non bound copper.
  • the bound copper was then eluted with 0.1 M EDTA and absorbance measured of the eluted fraction. This shows that the material has a potential for efficient affinity capture of target compounds.
  • Example 17 Use of structured recombinant Escherichia coli gel for the enzymatic conversion of a substrate
  • Escherichia coli expressing the protein beta-glucosidase was cultured. 0.15 g of the cell pellet was suspended in 1 ml deionized water and the suspension was cooled.
  • Example 18 Preparation of structured particles using particles with crosslinkable groups.
  • Synthesized pNIPA particles (0.1 g) of a size between 200-500 nm containing N-hydroxymethyl groups was suspended in 2 ml 0.1 M HCl after which the suspension was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- K ⁇ nigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
  • the structure shows fast response to temperature since the diffusion distances are short in the material. The material changes its hydrophobicity when changing the temperature. Higher temperature makes the material more hydrophobic and thus more suitable to capture hydrophobic substances.
  • Example 19 Incorporation of carbon particles as fillers in a particle structured gel
  • This example illustrates the mixture of synthetic particles with micro organisms.
  • the particles can in a system act as a reservoir or to capture substances which the cells might metabolise.
  • Example 20 Temperature response of gels. Gels made from structured pNIPA particles (5% w/v) according to example 18 were fully reswollen in water at ambient temperature. The gels were then placed in water at +45 0 C and the change in size was recorded. Shrinkage to 40% in volume was observed within 3 minutes. The shrunken gel was then placed in water at ambient temperature and reswell to original volume within 3 minutes. The material changes its hydrophobicity with change in temperature; this phenomena can be used to capture compounds. The change in volume will make it possible to elute the captured compounds in a smaller volume and thus concentrating the compounds.
  • Example 21 Micro porosity of particle structured gels.
  • Gels made from structured pNIPA particles (5% w/v) according to example 3 were prepared with the addition of porous carbon particles (1% w/v).
  • marcoporous cryogel according to prior art was prepared from a monomeric solution (5% w/v) of the same monomeric composition as the used particles, this monomeric solution was cryopolymerized with porous carbon particles (1% w/v) at -12°C.
  • Prepared gels were washed and thereafter freeze dried and the porosity was analyzed using mercury porosity to determine the porosity of the samples.
  • Fig. 6 shows that the particle structured material described in this invention contains micropores, as marked by (A). These micropores show that pores of the carbon particles are not blocked.
  • the gel from monomers according to prior art do not have any micropores showing that the pores of the carbon particles are blocked with polymer, as marked by ( ⁇ ).

Abstract

The present invention regards a process for the production of a self-supporting macroporous material, comprising cross-linked particles, from a suspension of particles in a liquid. Furthermore, a material produced by said process, specific structures and uses of said material are also provided.

Description

STRUCTURING PARTICLES INTO USEFUL STRUCTURES AND THEIR USE IN LIFE SCIENCE
FIELD OF THE INVENTION The present invention relates to cryostructuration of particulate material. More particularly, the present invention relates to a process for the production of a self- supporting macroporous material by fusing suspended particulate material (e.g. microgel particles, latex particles, microbial cells) using a combination of cryostructuration and chemical cross-linking, a macroporous material and use of said material. Even more specifically, the invention relates to a macroporous structure.
BACKGROUND OF THE INVENTION
Cryostructuration is a technique relying on rejection of solutes or suspended particles from growing ice crystals when an aqueous solution or suspension is frozen at moderate sub-zero temperatures (see Lozinsky 2003, Lozinsky 2002, Zhang 2007). Macroporous structures from hard particles like alumina, hydroxyapatite and silica- PVA have been produced earlier in a multi-step procedure by cryostructuration of the suspension followed by freeze drying to remove frozen solvent and fixation of the macroporous structure by sintering (see Zhang 2007, Deville 2006, Deville 2006). The procedures used are complicated, require large energy consumption for freeze-drying and sintering and most of all these procedures are not applicable for the production of macroporous structures from soft particles like microgels, latexes and microbial cells, as freeze drying will remove water present in the system not only as ice crystals but also as an integral part of soft particle hence destroying the integrity of soft particles. Moreover, the sintering at high temperatures used in the state of the art methods completely destroys organic material form which soft particles are composed.
Furthermore, a simple melting of the frozen cryostructurated particulate system, composed either from soft or hard particles, results in the complete destruction of the structure as the particles are not bound together in any way during the cryostructuration. In order to keep after melting the cryostructured particles integral, it has been suggested to cement them with the cryostructurated polymer formed either in situ as the result of polymerization of monomers (see Le Noir 2007) or as the result of physical or chemical cross-linking of pre-synthesized polymer during cryostructuration (Savina 2005). However, in this case the particles are surrounded by a dense polymer phase. Thus, the access of large molecules to the particles is practically prevented with the exception of only small area of particles surface which is exposed into the pore the surrounding polymer thus blocks or hinders access to the pores of the entrapped particle.
Plieva 2008, Galaev 2007, Lozinsky 2001, Plieva 2007, Hedstrόm 2008 and WO-A-2007/108770 all discloses methods for preparation of cryogels in the form of macroporous gels prepared by freezing a solution of monomers or polymers, whereby the monomers or polymers crosslink during this state and during subsequent thawing forming the macroporous material. However, only solutions of water soluble polymers, such as agarose, chitosan, PVA or monomers such as acrylamide, HEMA, DMAAm etc are used. A polymeric matrix is also required to form macroporous gels as described by
Savina in 2005. However, the particles are randomly distributed in the polymer matrix, and the polymer constitutes a majority of the material.
Cross-linking of soft particles such as cells in suspension by chemical cross- linkers like e.g. glutaraldehyde in ambient temperature, where the solvent is in a liquid state, has also been suggested (see Hua 2004, Cassidy 1996, Fukada 1995). However, since this process is carried out at ambient temperatures in liquid suspensions, cross- linking results in non-structured aggregates of cells.
Orrego 2009 discloses how solutions of chitosan may be used to create gels with or without chemical cross-linking at ambient temperature. The gels are subsequently freezed and freeze dried in order to make them porous. However, this is a complicated and costly process.
SUMMARY
The present invention seeks to mitigate, alleviate, circumvent or eliminate at least one, such as one or more, of the above-identified deficiencies by combining the cryo-structuration of particles from a suspended state, into a self-supporting macroporous material, with the simultaneous fixation of the formed structure by cross- linking of particles in a semi- frozen state.
More specifically, the cross-linking is achieved either between groups on the surface of the particles directly, thereby forming linkages between the particles, which in turn preserves the macroporous structure formed during the cryo-structuation, or the cross-linking is achieved by added cross-linking agent which reacts with groups present on the surface of particles forming linkages between them and thus preserving the macroporous structure formed during cryo-structuration also after melting the solvent crystals, or cross-linking may take place according to a combination of the above. According to a first aspect of the invention, a process is provided for the production of a self-supporting macroporous material, comprising cross-linked particles, from a suspension of particles in a liquid. Said particles comprise groups at the surface for cross-linking of said particles. Said process comprise the steps of; a) freezing said suspension of particles, thereby forming crystals from the liquid of the suspension; and cross-linking said particles; and b) melting the crystals to release said self-supporting macroporous structure comprising cross-linked particles.
According to a second aspect of the invention, a macroporous material is provided, which is obtainable by the process according to the first aspect of the invention. According to a third aspect of the invention, the macroporous material according to the second aspect is a structure obtained by freezing said suspension in a mould.
According to a fourth aspect of the invention, a use of the macroporous structure according to the third aspect, in studies of affinity separation and affinity binding is provided. Further, advantageous features of various embodiments of the invention are defined in the dependent claims and within the detailed description below.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which
Fig. 1 is a microscopy photography of a cryogel according to an embodiment;
Fig. 2 is a microscopy photography of a cryogel according to another embodiment;
Fig. 3 is a microscopy photography of a cryogel according to yet another embodiment;
Fig. 4 is a microscopy photography of a cryogel according to a further embodiment; and Fig. 5 is a graph showing biological stability of a cryogel according to yet a further embodiment.
Fig. 6 is a graph showing that the macroporous material according to an embodiment comprises micropores.
DETAILED DESCRIPTION
Several embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in order for those skilled in the art to be able to carry out the invention. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The embodiments do not limit the invention, but the invention is only limited by the appended patent claims. Furthermore, the terminology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.
The inventors surprisingly found that cross-linking in a suspension may be carried out in combination with cryo-structuration to obtain a self-supporting macroporous material, comprising cross-linked particles.
An advantage with this is that it is possible to build a material where the particles have free access to the pore liquid without being hindered by polymer layers. Yet another advantage is that the material allows for potentially a very high density of particles, such as cells, in the structure, since the particles themselves may form the stable structure.
The surface area may also be large which makes it possible for the material to interact effectively with its surrounding.
A further advantage is that the surface may be rough, which makes the material a good scaffold, i.e. for growing cells.
In general, it was found that freezing of liquid suspension of particles results in the rejection of the particles by the growing front of crystal- liquid interface. The particles are essentially pressed together into the interstitial space between growing crystals. The non-frozen phase will then act as a reaction medium for the cross-linking reaction which will proceed under semi-frozen conditions with high concentration of particles promoting the binding of particles together into self-supporting materials. After completion of the reaction, the system is thawed and melted crystals leave complementary pores, forming a self-supporting macroporous material from said cross- linked particles.
Thus, said freezing causes a first group on a first particle to interact with a first group on a second particle to cross-link said particles. Furthermore, a second group on said first particle or a second group on a second particle interacts with a first group on a third particle to cross-link said particles. Accordingly, several particles may be cross- linked to each other in order to form a network being a self-supporting macroporous material.
According to an embodiment of the invention, a small amount of cross-linking agent is present to achieve cross-linking. A small amount of cross-linking reagent, may be between 0.1% to 10% of the total content used. Accordingly, a majority, such as 90% to 99.9% of the final material consists of the structured cross-linked particles. If a cross- linking reactant, e.g. a chemical like glutaraldehyde is present, it is also concentrated in this area in so called non-frozen microphase, which volume may be defined by the concentration of the solutes, depression of freezing temperature and/or amount of polymer bound water (Kirsebom 2008). The non-frozen phase will then act as a reaction medium for the cross-linking reaction which will proceed under semi-frozen conditions with high concentration of both particles and cross-linking agent promoting the binding of particles together into self-supporting materials. After completion of the reaction, the system is thawed with melted ice crystals leaving complementary pores.
Thus, wherein the suspension further comprises a cross-linking agent, said freezing may cause said first group said first particle and said first group said second particle to interact with said cross-linking agent to cross-link said particles.
Cryo-structuration of a suspension is different from cryo-polymerization according to prior art, since cryo-structuration results in polymeric systems of significantly different morphology than those from polymerization in homogeneous solution according to prior art.
In an embodiment, the liquid of the suspension is substantially water, thus forming an aqueous suspension. Further, such an aqueous suspension may comprise water-miscible solvent, such as DMSO, ethanol, methanol or acetone
Although, it is preferred if the liquid of the invention is water, other liquids, such as DMSO, formamide or dioxane, may also be used.
Water is preferred as it is compatible with sensitive particles such as cells. Further, water has a suitable freezing point. In addition the formation of crystals when water freezes is pronounced.
Herewith is described a generic method as for production of macroporous materials of cross-linked small particles, of different chemical or structural nature and of broad range of particle sizes.
In an embodiment, the diameter, such as the Feret's diameter, of the particles is between between 40 nm and 100 μm. As well known to the skilled person, Feret's diameter is the statistical mean distance apart of two parallel lines which are tangential to the particle.
In an embodiment, the particles are selected from the group consisting of microgels made from natural or synthetic polymers, or mixtures thereof, latexes or microbial cells or their combination, as the particles may be poly(7V- isopropylacrylamide) (pNIPA) microgels with the size from 40 nm to 100 μm, rod shaped bacterial cells {Escherichia coli and Caldicellulosiruptor saccharolyticus) with the approximate size of 2 x 0.4 μm, spherical bacteria (Ralstonia eutropha and Lactococcus lactis) cells with the diameter of about 1 μm and baker's yeast cells which are 5-10 μm in diameter.
The main condition for the particles to be suitable for this procedure is the existence on their surface of groups which could be used for cross-linking. The cross linking may be chemical, ionical, or hydrophobical cross-linking.
Prepared pNIPA microgels contain amino groups whereas the microorganisms contain inherent amino groups at the cell surface. Thus such particles are suitable for cross-linking, such as cross-linking with amino reactive cross-linking agents, e.g. glutaraldehyde. The particles may be both soft, i.e. particles which can be at least partly compressed or deformed by external stimuli e.g.; mechanical forces, temperature or pH, or hard, i.e. particles not deformed or compressed by external stimuli e.g.; mechanical forces, temperature or pH. The particles may be metal, metal oxides, carbon, silicate, sol-gel particles, minerals and has or has been modified to contain reactive groups on the particle surface such that the particles can be cross-linked using a bi- or multifunctional reagent or through reactions between the surface groups.
The particles may be of different types, such as at least two types, either of the same size or with different size distributions. Examples on such mixtures of particles are different microorganisms which are capable of catalyzing a reaction sequence, one catalytically active unit and one adsorbent which can either bind the substrate or the product, or it could be a catalytically active unit combined with a material that captures toxic compounds that otherwise may destroy the biocatalyst. Alternatively, it could be one type of particle but with a broad particle size distribution, or any combination thereof. Examples of this are seen in Example 5 and 10 below. Cryo-structuration of the suspension accompanied with structured aggregation and thereafter cross-linking of particle results in the formation of macroporous materials with highly interconnected pores with diameters in the range from 1 to 200 micrometers. Furthermore, interstitial pores appear between the particles that are fused to build the macroporous material. Besides these, there are pores in many of the particles used for creation of the material. A macroporous material is defined by IUPAC as a material containing pores larger than 50 nm. However in this case macroporous material is considered with pores of larger than 1 μm.
Thus, in an embodiment, the size of pores is larger than 1 μm. Evidently, there is also an upper limit for the size of pores. Accordingly, the size of the pores are typically less than 200 μm.
In addition to the macropores, the materials may also contain micropores, which may occur between the particles in the material, or in the particles themselves.
The morphology of produced cryogels is very similar to that formed after cryostructuration of the chemically cross-linked gels produced either by radical polymerization, as described by Plieva 2005, or via covalent cross-linking of preformed polymers, as described by Plieva 2006. However, the present invention has the further advantages that it is possible to build a material where the particles have free access to the pore liquid without being hindered by polymer layers. Yet another advantage with the present invention is that it allows for potentially a very high density of particles, such as cells, in the material, since the particles themselves may form the stable material.
When porous polymer particles are used high binding capacities may be obtained. This is normally difficult using conventional cryo-structurization, and then usually traditional adsorbents have to be used, but such are either big with long diffusion distances or small particles with massive back pressure when used in chromatography. The structure produced according to some embodiments has the advantage of good kinetics and high capacity when adsorbents are used.
Furthermore, cryogels formed according to the present invention may have a rough surface. This may be advantageous for cell cultivation, since the cryogels may then be a very suitable scaffold for the cell cultivation. Furthermore this invention will allow production of a material which has a lower polymer density in the pore walls than previous work on macroporous gels. Since cryo-concentration of suspended particles do not proceed in the same way as with soluble polymers, this change in concentration will result in porosity in the walls and thus a possible higher total porosity. The present invention also allows for co -immobilization of particles with different functionalities in close proximity to each other. In prior art super-macroporous gels the particles are diluted with polymer, which do not allow the close proximity of the particles. Further, the particles may be immobilized in vicinity to each other. Thereby molecules may rapidly diffuse from one particle to another. This may be advantageous when using the material for catalytic applications.
The process to form macroporous material from particles comprise the following steps: First, particles are suspended, thus forming a suspension. Second, the suspension is frozen which allows forming of cross-links between constituents of the suspension. Third, the frozen suspension is thawed, resulting in a self-supporting macroporous material, held together by the cross-links formed between the particles.
In an embodiment, the particles themselves carry groups on their surface which may be cross-linked even in the absence of a cross-linking agent. According to this embodiment, the cross-links are formed during freezing between said groups and the resulting self-supporting macroporous material is held together by the cross-links formed between the particles.
The groups may be any reactive groups suitable to form cross-links during freezing, such as amino-, hydroxy-, carboxy-, sulfhydryl-, hydrophobic-, charged- or affinity groups, or their combination. Hydrophobic groups may be e.g. linear alkyl residues preferably comprising at least 4 carbon atoms. Such residue may be obtained by amidation of amino groups by use of a carboxylic acid, such as a fatty acid.
The particles may be any kind of particle suitable to create self-supporting macroporous material, such as particles selected from metal, metal oxides, carbon, silicate, sol-gel particles, mineral particles that contain, or have been modified to contain, said groups for cross-linking.
Affinity groups may be any known entity for creating affinity bindings, such as biotin, nucleotides, amino acids, chelating groups, carbohydrates, DNA-probes or textile dyes, or their combination. Example 11 is an example of affinity binding.
In another embodiment, a chemical reactant, i.e. a cross-linking agent, is added to the suspension before freezing. According to this embodiment, the cross-links are formed during freezing as a result of an interaction, such as reaction, of groups on the particles with the chemical reactant, which may interact with at least two groups . When the frozen suspension is thawed, the self-supporting macroporous material is then formed by the combination of chemical reagents and particles, since the particles are held together by the cross-links formed by the chemical reagents. The chemical reactant, or cross-linking agent, may be any chemical reactant suitable to form cross-links during freezing, such as Bis(Sulfosuccinimidyl) suberate, Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, (l,5-Difiuoro-2,4-dinitrobenzene), Dimethyl pimelimidate»2 HCl, Dimethyl Suberimidate»2 HCl, Disuccinimidyl glutarate, Dithiobis(succinimidyl) propionate, Disuccinimidyl suberate, Disuccinimidyl tartrate, Dimethyl 3,3'-dithiobispropionimidate»2 HCl, 3,3 '-
Dithiobis[sulfosuccinimidylpropionate], Ethylene glycol bis[succinimidylsuccinate], Ethylene glycol bis[sulfosuccinimidylsuccinate], β-[Tris(hydroxymethyl) phosphino] propionic acid, Tris-succinimidyl aminotriacetate, 1 ,4-bismaleimidobutane, 1 ,4 bismaleimidyl-2,3-dihydroxybutane, bismaleimidohexane, Bis-Maleimidoethane, 1,4- Di-[3 '-(2'-pyridyldithio)-propionamido]butane, Dithio-bismaleimidoethane, 1,6- Hexane-bis-vinylsulfone, Tris[2-maleimidoethyl]amine, epichlorohydrin, divinyl sulfone, glutaric aldehyde, azobenzoyl hydrazide, 4-(N-maleimidomethyl)cyclohexane- 1-carboxyl hydrazide hydrochloride, N-hydroxysuccinimidyl-4-azidosalicylic acid, 3- (2-pyridyldithio)propionyl hydrazide, dimethyladipimidate*2HCl, N-succinimidyl-6(4'- azido-2'-nitrophenylamino)hexanoate, sulfosuccinimidyl-(4'- azidosalicylamido)hexanoate, di- and triglycidyl compounds, or their combination. As elaborated above, the cross-linking agent may have at least two reactive groups capable of interacting with any of the groups exposed on the surfaces of the particle. Further, the cross-linking agent may be of polymeric nature, , such as polymers with many aldehyde groups, a polymer with boronate groups which interacts with PVA or with carbohydrate particles etc. The cross-linking agent may also comprise a polymer chain, such as oxidized dextran.
Thus, in an embodiment the cross-linking agent comprises a polymer chain with reactive groups. These reactive groups may be such that they cause covalent interactions, or non-covalent interactions with the particles that are to be cross- linked. Examples of such structures are e.g. oxidized carbohydrates that contain aldehyde groups that will interact with amino groups of particles such as cells, polymers containing boronate groups that will complex with carbohydrate groups on the particle surface, polymers with weak hydrophobic groups. Examples of these are short alkyl chains that are too short to cause any interaction per se, when present individually. However, when present in a plurality on a polymer each may cause a weak interaction that taken together results in a strong interaction and a multipoint attachment. Still another alternative is a polymer with charged groups that will form a multitude of interactions with charged structures on the particles, thereby causing a strong cross- linking of said particles. Advantages with polymeric cross-linkers are obvious in cases of living organisms that are cross-linked. The polymeric structure will not penetrate into the cell and thereby it is less toxic to the cell.
In yet another embodiment, the particles themselves carry reactive groups on their surface and a chemical reactant is added to the suspension before freezing. According to this embodiment, the cross-links are formed both between said reactive groups of the particles and between the molecules in the chemical reactant. When the frozen suspension is thawed, the self-supporting macroporous material is then formed by the combination of chemical reagents and particles, since the particles are held together by the cross-links formed between the particles and by the cross-links between the chemical reagents.
During the freezing, the particles are packed together since they are expelled from the growing crystals. The packed particles are linked in the semi- frozen state either through cross-linking with a cross-linker or due to selfcross-linking between particles. An example of this is e.g. carbohydrate - lectin, hydrophobic groups packing together to form interactions, multiply weak affinity bonds, which create strong bonds through multiple point attachment.
The freezing temperature may be any temperature below crystallization temperature of the liquid, down to 40 K below the crystallization point. In the case the liquid is water, or a water based solution, the temperature is typically between -I0C and -3O0C. The time between freezing and thawing may be any period long enough to allow forming of the cross-links, such as between 0.5h and 7 days, such as between 1Oh and 24h.
In an embodiment, the particles are cells. The cryogels formed from the resulting cell suspensions may be similar to cryogels formed from polymers. An advantage with this is that the cryogels formed from cell suspensions are sponge-like and elastic, withstanding significant mechanical deformation and restoring completely their shape when the stress is released. This may be advantageous if the material constitutes a biologic reactor, and substrate is fed into the material and the product is transported away from the material. In an embodiment, the cryogels are produced from thermoresponsive particles.
This is advantageous, since the gels may then exhibit a very rapid response to changes in temperature. The unique material with large pores and small particles enable a rapid transition in size. This material would allow the capture and release of substances, such as pharmaceuticals, controlled by temperature change. Furthermore, since the hydrophobicity changes with temperature, hydrophobic compounds may thus be trapped at one temperature and released at another.
The method according to some embodiments is relatively friendly to the living microbial cells, provided the cross-linking agent is not toxic. The use of biocompatible alternative cross-linkers like partially oxidized dextran or another polymeric crosslinker, allows for producing cryogels from bacteria e.g. Caldicellulosiruptor saccharolyticus cells maintaining their viability. This observation in combination with sufficient mechanical stability of macroporous gels formed via cryostructuration of cell suspensions opens an interesting perspective of carrier- free immobilization of cells and using them in flow-though bioreactors either for the synthesis of target product or for selective bioconversion of e.g. toxic compounds in effluents.
The macroporous material may have catalytic activity, such that bioconversions or combined chemical-biochemical conversions may be carried out.
The macroporous material may be loaded with biologically active compound or a drug. Said biologically active compound or drug, may be slowly release form said particles into surrounding media. The method of cryo-structuration according to an aspect of the invention is very much separated from prior art methods for making composite materials. This is advantageous; because the particle in the resulting gel has free access to the pore liquid, since no hindering polymer layers are needed for stabilization of the material. The macroporous material according an aspect of the invention may be formed into a desired structure by freezing said suspension in a mould. Any kind of mould may be used, as long as the cross-linking is possible. Thus the material may be shaped into structures with desired geometry.
In an embodiment, the macroporous structure has catalytic activity. In a further embodiment, the particles of the macroporous structure are particles with affinity ligands or molecular structures to which dedicated molecules bind. Affinity ligands may be any known entity for creating affinity bindings, such as biotin, nucleotides, amino acids, chelating groups, carbohydrates, DNA-probes or textile dyes, or their combination. In an aspect, use of a macroporous material with affinity ligands, in studies of affinity separation and affinity binding is provided. Such structure is seen in example 10.
The process according to an aspect may further comprise a step of loading said material with biologically active compound(s). Specifically, said biologically active compound may be released from the macroporous material by changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
The particles of the macroporous material may also themselves be released in response to external changes. Thus, in an embodiment, particles in said macroporous material may be released in response to changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
The following documents have been referred to above:
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[2] V. I. Lozinsky, Russian Chemical Reviews 2002, 71, 489 - 511 [3] H. Zhang, A. I. Cooper, Adv. Mater. 2007, 19, 1529-1533
[5] S. Deville, E. Saiz, R. K. Nalla, A. P. Tomsia, Science 2006, 311, 515-518 [6] S. Deville, E. Saiz, A. P. Tomsia, Biomaterials 2006, 27, 5480-5489
[7] H. Kirsebom, G. Rata, D. Topgaard, B. Mattiasson, I. Yu. Galaev, Polymer, 2008, in press
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[9] F. Plieva, A. Oknianska, E. Degerman, I. Yu. Galaev, B. Mattiasson, J. Biomater. Sci.-Polym. Ed. 2006, 17, 1075-1092.
[10] L. Hua, Z.-H. Sun, P. Zheng, Y. Xu, Enzyme Microb. Technol. 2004, 35, 161-166. [11] M. B. Cassidy, H. Lee, J. T. Trevors, J. Ind. Microbiol. Biotechnol. 1996,
16, 79-101.
[12] H. Fukuda, Immobilized Microorganism Bioreactors in Bioreactor System Design A. A. Asenjo, J. C. Merchuk (eds) CRC Press, 1995.
[13] S. Basha, Z. P. V. Murty, Seaweeds for engineering metal biosorption: A Review in Focus on Hazardous Materials Research A. G. Mason (ed), Nova Science Publishers Inc., 2007.
[14] N. Savina, V. I. Lozinsky, A. Hanora, F. M. Plieva, I. Yu. Galaev, B. Mattiasson, J. Appl. Polym. Sci. 2005, 95, 529-538.
[15] M. Le Noir, F. Plieva, T. Hey, B. Guieysse and B. Mattiasson, Journal of Chromatography A, 1154 (2007) 158-164
[16] Plieva, F. M. et al. (2008) Industrial and Engineering Chemistry Research 47 (12) 4132-4141
[17] Orrego, CE. et al. (2009) Bioprocess and Biosystems Engineering 32 (2) 197-206 [18] Galaev, LYu. et al. (2007) Langmuir 23 (1) 35-40
[19] Lozinsky, V.I et al. (2001) Bioseparation 10 (4-5) 163-188
[20] Plieva, F.M. et al. (2007) Journal of Separation Science 30 (11) 1657-1671
[21] Hedstrόm, M. et al. (2008) Analytical and Bioanalytical Chemistry 390 (3) 907-912 [22] Kirsebom, H. et al. (2008) Macromolecules 2009, 42, 5208-5214 Examples are shown below to further describe the present invention in detail. These examples should not be construed as limiting, as the invention is limited only by the appended claims
Example 1 : Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus cells.
Cell pellet of Caldicellulosiruptor saccharolyticus (0.15 g) was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
The structured bacterium in the gel was confirmed using Scanning electron microscopy (SEM). The gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C. The samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried. The dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 1. In Fig. 1 is shown that the walls of the material comprise of closely packed bacteria and macropores above 40 micrometer. The walls are only composed of bacteria and not immobilized in a polymeric matrix. This material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products.
Example 2: Preparation of a cryo-structured cryogel of 15% Lactobacillus casei cells.
Cell pellet of Lactobacillus casei (0.15 g) was suspended in 1 ml of sterile deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with sterile water.
The viability and productivity of the bacteria was confirmed by recirculation of
20 ml media (Per liter of media: 10 g yeast extract, 0.5 g K2HPO4, 0.5 g KH2PO4, 1.0 g sodium citrate, 0.005 g MgSO4.7H2O, 0.0031 g MnSO4.H2O, 0.002 g FeSO4.H2O, and 0.005 g ascorbic acid; and 100 g sugar using definite amounts of both glucose and lactose) at +42°C. HPLC analysis indicated the production of lactic acid by the cryo- structured cells. This structured material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products.
Example 3: Preparation of a cryo-structured cryogel of 5% poly N- isopropylacrylamide (pNIPA)-co-allylamine particles. Synthesized pNIPA particles (0.1 g) of a size between 200-500 nm was suspended in 2 ml H2O and cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The particulate structure of the gel was confirmed using Scanning electron microscopy (SEM). The gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C. The samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried. The dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 2. It is clearly shown in Fig. 2 that the walls of the structure are composed of densely packed polymer particles, which make up the macroporous structure of the material. The structure shows fast response to temperature since the diffusion distances are short in the material. The material changes its hydrophobicity when changing the temperature. Higher temperature makes the material more hydrophobic and thus more suitable to capture hydrophobic substances. Example 4: Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles.
Synthesised MMA-GMA particles of a size between 200-500 nm were reacted with ethylenediamine to introduce reactive amino groups. The activated particles (0.130 g) were suspended in 1.3 ml of H2O and cooled. Thereafter 40 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
The particulate structure of the gel was confirmed using Scanning electron microscopy (SEM). The gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C. The samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried. The dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 3. It is clearly shown in Fig. 3 that the walls of the structure are composed of densely packed polymer particles, which make up the macroporous structure of the material. The material is composed of hydrophobic particles; therefore it is possible to prepare hydrophobic materials with this method. The reactive epoxy groups are possible to use for coupling of different ligands.
Example 5: Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus and Saccharomvces cerevisiae Cell pellet of Caldicellulosiruptor saccharolyticus (0.136 g) and Saccharomyces cerevisiae (0.014 g) was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The particulate structure of the gel with both microorganisms was confirmed using Scanning electron microscopy (SEM). The gels used for SEM were cut into thin discs and fixed in 2.5% (w/v) glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4) over night at 4°C. The samples were dehydrated in ethanol (0, 20, 75, 95 and 99.5%) and then critical point dried. The dried samples were sputter-coated with gold/palladium (40/60) and examined using a JEOL JSM-5000LV scanning microscope, see Fig. 4. It is shown in Fig. 4 that the material consists of two different types of micro organisms; bacteria and yeast. The material is only composed of crosslinked micro organisms and no inert matrix used to immobilize the cells. This structure will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products. The use of two organisms allows using sequential processes.
Example 6: Preparation of a cryo-structured cryogel of 15% Caldicellulosiruptor saccharolvticus and MMA-GMA particles
Cell pellet of Caldicellulosiruptor saccharolyticus (0.136 g) and activated MMA-GMA particles (0.014 g) was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. This example illustrates the mixture of synthetic particles with micro organisms. The particles can in a system act as a reservoir or to capture substances which the cells might metabolise.
Example 7: Preparation of a cryo-structured cryogel of 6% bovine serume albumin (BSA) particles
Nanoparticles of BSA (0.06 g), 50 nm in size, were suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 5 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The produced material is both biocompatible and biodegradable.
Example 8: Preparation of a cryo-structured cryogel of 20% silica particles
Silica particles (0.2 g) activated with amino groups with a size less than 1 μm were suspended in ImI of deionized water containing 1% (weight of silica particles) of anionic dispersant and the suspension was cooled. Thereafter 20 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. This material is suitable for further modifications and can then be used for analytical applications.
Example 9: Preparation of a cryo-structured cryogel of 3% chitosan particles containing insulin
Chitosan nanoparticles (0.03 g) containing insulin as a model compound was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. Short diffusion distances in the particles will give an efficient release of the insulin. This exemplifies the possibility to use this material for the delivery of macromolecular compounds.
Example 10: Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles containing imminoacetic acid. Synthesised MMA-GMA particles of a size between 200-500 nm were reacted with immidoacetic (0.1 M in 1 M NaCO3) for 12 h. After which the particles were reacted with ethylenediamine to introduce reactive amino groups. The activated particles (0.130 g) were suspended in 1.3 ml of H2O and cooled. Thereafter 40 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
The prepared gel was then used for IMAC purification of the protein β- glucosidase. First the gel was then loaded with 0.1 M copper sulfate and then non bound copper was washed out with running buffer (20 mM Tris-HCl, pH 7.4 containing 10 mM imidazole and 0.5 M NaCl) after equilibrating the system with running buffer. Then 5 ml of clarified protein sample (in running buffer) containing his-tagged β- glucosidase was applied at a flow rate of 1 ml/min. The non bound protein was washed out with running buffer after which the β-glucosidase was eluted using elution buffer (20 mM Tris-HCl, pH 7.4 containing 200 mM imidazole and 0.5 M NaCl). The target protein (β-glucosidase) was eluted as could be confirmed with SDS-PAGE and of activity of the enzyme. This shows that the material has a potential for efficient affinity purification of proteins.
Example 11 : Preparation of a cryo-structured cryogel of 10% methylmethacrylate(MMA)-co-glycidylmethacrylate(GMA) particles containing trypsin.
Synthesised MMA-GMA particles of a size between 200-500 nm were coupled with 10 mg/ml trypsin solution in 0.1 M Na-phosphate buffer pH 8.0. After which the particles were reacted with ethylenediamine to introduce reactive amino groups and block free epoxy groups. The use of ethylenediamine was to introduce additional reactive sites for the following crosslinking reaction with glutaraldehyde. The activated particles (0.130 g) were suspended in 1.3 ml of H2O and cooled. Thereafter 40 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The activity of trypsin in the prepared gel was check using N-benzoyl-DL- arginine-4-nitroanilide (BAPNA) as a substrate. BAPNA (10 mg) was dissolved in 10 ml of 50% ethanol and the mixted with 0.3 M Tris-HCl containing O.lmM CaCl2 pH 7.6 with a ratio 1 :2.5 BAPNA:Buffer. The BAPNA containing buffer was then added to the gel and incubated 15 min at 37°C after which the solution was squeezed out of the gel and absorbance at 410 nm measured. The resulting material is suitable material for use as an enzyme reactor which can be used for the degradation of proteins, the macroporous material will enable an efficient mass transfer in the system.
Example 12: Preparation of a cryo-structured cryogel of 15%
Caldicellulosiruptor saccharolvticus cells.
Cell pellet of Caldicellulosiruptor saccharolyticus (0.15 g) was suspended in 0.9 ml of deionized water and the suspension was cooled. Thereafter 0.1 ml of solution of partially oxidized dextran containing aldehyde groups (5% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at - 12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. This material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resistance, therefore the material will facilitate the transportation of reactants and products.
Example 13: Preparation of a cryo-structured cryogel of 5% poly N- isopropylacrylamide(pNIPA)-co-allylamine particles-co-2-(dimethylamino) ethylmethacrylate (DMAEMA)
Synthesized pNIPA particles (0.1 g) containing groups with ion-exchange capability (DMAEMA) of a size between 200-500 nm was suspended in 2 ml H2O and cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The resulting material has a potential for use as a material for ion exchange separations.
Example 14: Preparation of a cryo-structured cryogel of 3% chitosan particles Chitosan nanoparticles (0.03 g) in ImI of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at - 12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The produced material is both biocompatible and biodegradable.
Example 15: Preparation of a cryo-structured symbiotic cryogel of 15% Lactobacillus casei and Saccharomvces cerevisiae Using two different organisms which can act in a symbiotic pathway is modeled by using Lactobacillus casei and Saccharomyces cerevisiae, Saccharomyces cerevises can degrade surose to glucose which can then be used as a carbon source for Lactobacillus casei. This can be seen as a model for co-immobilizing two microorganisms which can act in symbiosis. Cell pellet of Lactobacillus casei (0.140 g) and Saccharomyces cerevisiae (0.010 g) was suspended in 1 ml of sterile deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with sterile water.
The viability and productivity of the bacteria was confirmed by recirculation of 20 ml media (Per liter of media: 10 g yeast extract, 0.5 g K2HPO4, 0.5 g KH2PO4, 1.0 g sodium citrate, 0.005 g MgSO4.7H2O, 0.0031 g MnSO4.H2O, 0.002 g FeSO4.H2O, and 0.005 g ascorbic acid; and 100 g sucrose) at +42°C. Samples were taken of the media after certain times to check the production of lactic acid using HPLC. This material will yield a very high density of cells since there is no additional matrix. Since the cells are not encapsulated in a polymer there will be no additional diffusion resitance, therefore the material will facilitate the transportation of reactants and products. The use of two organisms allows using sequential processes.
Example 16: Preparation of a cryo-structured cryogel of 15% Escherichia coli cells expressing poly-His peptides for affinity capture.
Escherichia coli, K12 strain pop 6510 {thr, leu, tonB, thi, lacYl, recA, dex5, metA, supE and dex5) with plasmid pLH2 encoding the hybrid LamBHis (two 6 x His) monomers (His-tagged E. coli), displaying poly-His peptides on the surface was used for affinity capture of copper. Cell pellet of Escherichia coli (0.15 g) was suspended in 1 ml of deionized water and the suspension was cooled. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water.
The capture of copper was checked by passing 0.1 M CuSO4 and then washes with plenty of buffer (20 mM Tris-HCl, pH 7.4 containing 10 mM imidazole) to remove all non bound copper. The bound copper was then eluted with 0.1 M EDTA and absorbance measured of the eluted fraction. This shows that the material has a potential for efficient affinity capture of target compounds.
Example 17: Use of structured recombinant Escherichia coli gel for the enzymatic conversion of a substrate,
Escherichia coli expressing the protein beta-glucosidase was cultured. 0.15 g of the cell pellet was suspended in 1 ml deionized water and the suspension was cooled.
Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R.
WOBSER GmbH & Co. KG, Lauda-Kόnigshofen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The produced gel was placed in a continuous flow through system at +450C and a 20 rnM Tris-HCl buffer containing 20 rnM para-nitrophenyl-β-D-glucoside was pumped through the system and the absorbance at 455 nm was measured to confirm the activity of enzyme and stability of the gel since the product has a maximum absorbance at 455 nm. Fig. 5 shows that the enzymatic activity of the prepared material remains stable over 24 hours. A suitable material to use as an enzyme reactor which can be used for the degradation of proteins, the macroporous material will enable an efficient mass transfer in the system.
Example 18: Preparation of structured particles using particles with crosslinkable groups.
Synthesized pNIPA particles (0.1 g) of a size between 200-500 nm containing N-hydroxymethyl groups was suspended in 2 ml 0.1 M HCl after which the suspension was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH & Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. The structure shows fast response to temperature since the diffusion distances are short in the material. The material changes its hydrophobicity when changing the temperature. Higher temperature makes the material more hydrophobic and thus more suitable to capture hydrophobic substances.
Example 19: Incorporation of carbon particles as fillers in a particle structured gel
Cell pellet of Escherichia coli (0.15 g) was suspended in 1 ml of deionized water and the suspension was cooled and 1% of carbon particles were added. Thereafter 10 μl of glutaraldehyde solution (50% w/v) was added to the mixture after which the sample was placed in a thermostat (Lauda RK20KP from LAUDA Dr.R. WOBSER GmbH &
Co. KG, Lauda- Kόnigsho fen, Germany) set at -12°C and kept frozen for 14 h. After which the produced gel was thawed at room temperature and washed with water. . This example illustrates the mixture of synthetic particles with micro organisms. The particles can in a system act as a reservoir or to capture substances which the cells might metabolise.
Example 20: Temperature response of gels. Gels made from structured pNIPA particles (5% w/v) according to example 18 were fully reswollen in water at ambient temperature. The gels were then placed in water at +450C and the change in size was recorded. Shrinkage to 40% in volume was observed within 3 minutes. The shrunken gel was then placed in water at ambient temperature and reswell to original volume within 3 minutes. The material changes its hydrophobicity with change in temperature; this phenomena can be used to capture compounds. The change in volume will make it possible to elute the captured compounds in a smaller volume and thus concentrating the compounds.
Example 21 : Micro porosity of particle structured gels. Gels made from structured pNIPA particles (5% w/v) according to example 3 were prepared with the addition of porous carbon particles (1% w/v). For comparison marcoporous cryogel according to prior art was prepared from a monomeric solution (5% w/v) of the same monomeric composition as the used particles, this monomeric solution was cryopolymerized with porous carbon particles (1% w/v) at -12°C. Prepared gels were washed and thereafter freeze dried and the porosity was analyzed using mercury porosity to determine the porosity of the samples. Fig. 6 shows that the particle structured material described in this invention contains micropores, as marked by (A). These micropores show that pores of the carbon particles are not blocked. As a contrast, the gel from monomers according to prior art do not have any micropores showing that the pores of the carbon particles are blocked with polymer, as marked by (■).
Although the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims.

Claims

1. Process for the production of a self-supporting macroporous material, comprising cross-linked particles, from a suspension of particles in a liquid, said particles comprising groups at the surface for cross-linking of said particles, which comprise the steps of: a) freezing said suspension of particles, thereby the liquid solidifies into crystals; and cross-linking said particles; and b) melting the crystals to release said self-supporting macroporous structure comprising cross-linked particles.
2. The process according to claim 1, wherein said freezing causes a first group on a first particle to interact with a first group on a second particle to cross-link said particles.
3. The process according to claim 2, wherein a second group on said first particle or a second group on a second particle interacts with a first group on a third particle to cross-link said particles.
4. The process according to any of the preceding claims, wherein the suspension further comprises a cross-linking agent, and wherein said freezing causes said first group on said first particle and said first group on said second particle to interact with said cross-linking agent to cross-link said particles.
5. The process according to claim 4, wherein said cross-linking agent is glutaraldehyde.
6. The process according to any of the preceding claims, wherein said suspension is an aqueous suspension, optionally said suspension comprises a water- miscible solvent.
7. The process according to any of the preceding claims, wherein said particles are selected from the group consisting of microgels made from natural or synthetic polymers, or mixtures thereof, latexes or microbial cells or their combination.
8. The process according to claim 7, wherein said particles are microgels.
9. The process according to claim 8, wherein said microgel comprises pNIPA.
10. The process according to claim 7, wherein said particles are microbial cells.
11. The process according to any of the preceding claims, wherein the Feret's diameter of said particles is between 40 nm and 100 μm.
12. The process according to any of the preceding claims, wherein the size of pores is larger than 1 μm.
13. The process according to any of the claims 1 to 12, wherein said particles can be at least partly compressed or deformed by external stimuli.
14. The process according to any of the claims 1 to 12, wherein said particles are not deformed or compressed by external stimuli.
15. The process according to any of the preceding claims, wherein said groups at the surface are selected from the group consisting of amino-, hydroxy-, carboxy-, sulfhydryl-, hydrophobic-, charged- or affinity groups, or their combination.
16. The process according to claim 15, wherein said groups at the surface are amino groups.
17. The process according to any of the above claims, wherein said particles are selected from metal, metal oxides, carbon, silicate, sol-gel particles, mineral particles and have or have been modified to contain said groups for cross-linking.
18. The process according to claim 17, wherein said particles are carbon particles.
19. The process according to claim 17, wherein said particles are silica particles.
20. The process according to claim 3 or any claim dependent on claim 3, wherein said cross-linking agent is selected from the group consisting Bis(Sulfosuccinimidyl) suberate, Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, (l,5-Difluoro-2,4-dinitrobenzene), Dimethyl pimelimidate»2 HCl, Dimethyl Suberimidate»2 HCl, Disuccinimidyl glutarate, Dithiobis(succinimidyl) propionate, Disuccinimidyl suberate, Disuccinimidyl tartrate, Dimethyl 3,3'- dithiobispropionimidate»2 HCl, 3,3 '-Dithiobis[sulfosuccinimidylpropionate], Ethylene glycol bis[succinimidylsuccinate], Ethylene glycol bis[sulfosuccinimidylsuccinate], β- [Tris(hydroxymethyl) phosphino] propionic acid, Tris-succinimidyl aminotriacetate, 1 ,4-bismaleimidobutane, 1,4 bismaleimidyl-2,3-dihydroxybutane, bismaleimidohexane, Bis-Maleimidoethane, l,4-Di-[3 '-(2'-pyridyldithio)-propionamido]butane, Dithio- bismaleimidoethane, 1 ,6-Hexane-bis-vinylsulfone, Tris[2-maleimidoethyl]amine, epichlorohydrin, divinyl sulfone, glutaric aldehyde, azobenzoyl hydrazide, 4-(N- maleimidomethyl)cyclohexane-l-carboxyl hydrazide hydrochloride, N- hydroxysuccinimidyl-4-azidosalicylic acid, 3-(2-pyridyldithio)propionyl hydrazide, dimethyladipimidate*2HCl, N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate, sulfosuccinimidyl-(4'- azidosalicylamido)hexanoate, di- and triglycidyl compounds, or their combination.
21. The process according to claim 3 or any claim dependent on claim 3, wherein said cross-linking agent comprises a polymer.
22. The process according to claim 21, wherein said cross-linking agent is oxidized dextran.
23. The process according to any of the preceding claims, wherein said particles are of at least two different types.
24. Macroporous material, which is obtainable by the process according to any of the preceding claims.
25. The macroporous material according to claim 24, wherein said material is a structure obtained by freezing said suspension in a mould.
26. The macroporous material according to claim 24 or 25, wherein said structure has catalytic activity.
27. The macroporous material according to any of the claims 24 to 26, wherein said particles are particles with affinity ligands or molecular structures to which dedicated molecules bind.
28. Use of the macroporous material according to claim 27 in studies of affinity separation and affinity binding.
29. Process according to any of the claims 1 to 23, wherein said process further comprising the step of loading said material with biologically active compound(s).
30. The macroporous material according to claim 24 or 25, wherein said particles are loaded with biologically active compound.
31. The macroporous material according to claim 30, wherein said biologically active compound may be released from the macroporous material by changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
32. The macroporous material according to any of the claims 24 to 25 and 30 to 31, wherein particles in said macroporous material may be released in response to changes in pH, temperature, electric or magnetic field or in the presence of particular chemicals.
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