Device and method for crosslinking macromonomers

Abstract

A device (100) for crosslinking macromonomers (1) is described, comprising a mold (11) with an at least partially open side surface, a membrane (33, 33a, 33b) which is passable for crosslinker molecules (5) and at least one crosslinker attachment (22, 22a, 22b) with a cavity (24), wherein the casting mold (11) and the at least one crosslinking attachment (22, 22a, 22b) can be placed on top of one another, so that the membrane (33, 33a, 33b) forms an interface between the casting mold (11) and the cavity (24) of the at least one crosslinking attachment (22, 22a, 22b).

Description

The present invention relates to an apparatus and to a method for crosslinking macromonomers according to the preamble of the independent claims.Prior ArtHydrogels are used in biology for various applications. These are polymeric networks which are not soluble in a specific medium, for example water, but are highly swellable. The polymers can be of natural (biopolymers) as well as synthetic origin. The high water content allowing a high diffusion rate makes these materials very interesting for biological questions, for example for encapsulation of living cells for three-dimensional cultivation or for drug delivery systems.Polymeric networks are usually produced physically, for example by charge interactions, also called chelating, or chemically, for example by covalent crosslinking. In both cases, for example, crosslinker molecules are used which function as bridging agents between the polymer chains, for example ions for ionic crosslinking or crosslinkers having at least two reactive groups for covalent linkage of the macromonomers.EP 261035 A1 relates to dried hydrogels and the production of vitrgel membranes from the hydrogels by vitrifying, a nylon membrane being introduced as carrier into a casting mold in which the hydrogel is situated.WO 02 / 36342 A1 describes a casting mold for the production of a hydrogel contact mask from a precursor. The mold comprises two halves, the first half being made of an elastomer and deforming to accommodate changes in shape in the precursor.Disclosure of the InventionMacromonomers are understood to mean large chain oligomers or polymers which frequently consist of repeating similar structural units having functional groups.To initiate crosslinking, the crosslinking agent must be introduced into the macromonomer solution. This can be done either by direct mixing or by overlaying / recoating the macromonomer solution with crosslinker solution. Depending on the speed of the crosslinking process, introduction of the crosslinking agent into the macromonomer solution is impractical, in particular if the polymer solution is still to be brought into a specific shape before curing, for example by means of a casting mold. Frequently, therefore, the macromonomer solution is introduced into a desired casting mold and then coated over or over with crosslinking agent solution. However, when the crosslinker solution is added, especially at its interface, uncontrolled mixing of the two solutions and thus dilution of the macromonomer solution can occur. This causes an undefined change in macromonomer concentration which may result in changes in the mechanical properties of the resulting hydrogel, for example in strength or degree of swelling. In addition, the macromonomer solution forms a meniscus when introduced into the casting mold, which meniscus is also maintained by the resulting hydrogel. This therefore does not have a planar surface and a uniform thickness, which is restrictive for various analyses sought, such as for optical or rheological analyses.In contrast, according to the invention, a device and a method for crosslinking macromonomers having the characterizing features of the independent patent claims are provided.The device according to the invention for crosslinking macromonomers comprises a casting mold having at least one partially open side face, a membrane which can be passed for crosslinker molecules, and at least one crosslinker attachment having a cavity. The casting mold and the at least one crosslinker attachment can be placed on top of one another, such that the membrane forms an interface between the at least one, at least partially open side surface of the casting mold and the cavity of the at least one crosslinker attachment.The casting mold is, for example, rectangular, square, cylindrical or other shape. The casting mold has at least one, at least partially open side surface, wherein this side surface can be, for example, the cover surface and / or the bottom surface. An at least partially open side surface can be understood to mean that it is completely or partially recessed.A membrane is understood to mean a defined-permeable mechanical barrier, regardless of the type and thickness of material.The crosslinker attachment forms a cavity or comprises at least one such cavity. The cross-linking agent attachment can be placed on the casting mold or placed under the casting mold. The casting mold comprises, for example, any desired capacity.It is advantageous in the device according to the invention that with the aid of these, no uncontrolled mixing of the macromonomer solution and the crosslinker solution occurs and dilution of the macromonomer solution by entering solvent from the crosslinker solution is prevented. This prevents undefined changes in the macromonomer concentration, so that the mechanical properties of the resulting hydrogel, such as strength and degree of swelling, are not influenced.Moreover, mixing of the macromonomer solution and the crosslinker solution is also no longer dependent on the respective experimenter and / or the experimental procedure, which leads to comparable results. The result is, even at the interface to the crosslinker solution, a homogeneous concentration of the macromonomers in the macromonomer solution and thus a homogeneous crosslinking of the macromonomers in the polymeric network or hydrogel.Because the membrane forms an interface between the at least one, at least partially open side face of the casting mold and the cavity of the crosslinker attachment, no meniscus can form. Instead, a planar surface of the macromonomer solution is forced at the interface and hydrogels of uniform thickness are thus produced, which is advantageous for various further, for example rheological or optical, analyses or only allows a valid statement from these. In addition, the choice of membrane material and the pore size of the crosslinker top makes it possible to control and control the rate of diffusion of the crosslinker molecules into the macromonomer solution and hence the crosslinking rate. Thus, for example, as long as this diffusion rate is below the diffusion rate of the macromonomer solution, the crosslinking conditions can be controlled independently of the macromonomer solution and made comparable for different samples.Further advantageous embodiments of the present invention are the subject matter of the dependent claims.In a particularly advantageous embodiment, at least one side surface, in particular the bottom surface, of the crosslinker attachment is configured as a membrane. In this embodiment, the cross-linker attachment is placed on the at least partially open side of the casting mold, which is in particular the cover surface. In this way, the membrane finally forms the interface between the casting mold and the cavity of the crosslinker attachment.It is advantageous here that the casting mold can be filled with macromonomer solution before the crosslinker attachment is placed thereon and the crosslinker attachment can also be filled in a simple manner again without separate fluidic connections to the casting mold and the crosslinker attachment being necessary.In an alternative or additional embodiment, the casting mold has a membrane on one or more at least partially open side surfaces, for example the bottom surface.In this embodiment, the casting mold is placed, for example with the membrane bottom surface, on the cavity of the crosslinker attachment. In this way, the membrane finally forms the interface between the casting mold and the cavity of the crosslinker attachment.It is advantageous here that the filling of the crosslinker top piece with crosslinker solution can take place before the casting mold is placed thereon. The casting mold in turn can also be easily filled without separate fluidic connections to the casting mold and the cross-linker attachment being necessary.In an advantageous embodiment, the casting mold and the cross-linker attachment have the same length and width or the same diameter. It is advantageous here that the casting mold and the cross-linker attachment can thus be placed on top of one another in a simple manner and a compact and handy device is provided. Furthermore, this also has advantages, for example, with regard to the mixing, since this can be effected uniformly on account of the same dimensions of the mutually adjoining surfaces of the casting mold and of the crosslinking agent attachment.Furthermore, in an advantageous embodiment, the membrane comprises a porous material, in particular a polymer having pore sizes in the nanometer range. Suitable polymer membranes are those produced by track etch methods, in particular from polyethylene terephthalate (PET, hydrophilic) and / or polycarbonate (PC, hydrophobic) and / or polyimide (PI).Alternatively, membranes made of metal, for example made of aluminum and / or silicon, can also be used. It is advantageous in these cases that the material of the membrane is comparatively rigid. The space or volume in which the macromonomer solution is located is defined. For example, the volume is increased by a certain expansion of the membrane. The more inflexible the material of the membrane, the better an increase in volume and an associated dilution of the macromonomer solution by migrating solvent of the crosslinker solution can be avoided.It is also advantageous if the pores of the membrane have a diameter of 5-50 nm and particularly advantageously of 5-15 nm. It is advantageous here that the crosslinker molecules of the crosslinker solution can pass through the pores of the membrane, but not the macromonomers from the macromonomer solution. This ensures that only the crosslinker molecules can diffuse into the macromonomer solution, but not the macromonomers into the crosslinker solution. In this way, a homogeneous distribution of the macromonomers in the macromonomer solution is ensured and uncontrolled mixing of the solutions and thus an undefined change in the macromonomer concentration are prevented. The concentration of the macromonomers in the final hydrogel is thus uniformly distributed over the entire gel.A further advantageous embodiment provides that the cross-linker attachment or the casting mold-depending on which of the two components is located on top of the device-has a lid, in particular an irreversibly closable lid. It is advantageous here that evaporation of the crosslinking agent solution during the crosslinking process is prevented. In addition, the crosslinker attachment can be removed quickly and comfortably without prior removal of the crosslinker solution without the risk of the crosslinker solution being wasted.In a further advantageous embodiment, the device comprises two cross-linking agent attachments which can be placed or placed onto the casting mold from different sides, in particular on the top and bottom surfaces, of the casting mold, in such a way that a membrane forms an interface between the casting mold and the cavity of the respective cross-linking agent attachment. At the bottom, for example, there is a first cross-linker attachment with a fixed cavity bottom. The cavity is open at the top. A casting mold with a membrane base is placed on the first cross-linker attachment, for example, which casting mold further has an open lid surface. A second cross-linker attachment with a membrane base is again placed on the casting mold. It is advantageous here that the macromonomer solution is supplied with crosslinking molecules from below and from above via the respective membrane.The present invention further provides a process for crosslinking macromonomers, comprising the steps: a) fully filling the casting mold with a macromonomer solution. Complete filling means that the casting mold is filled at least with the calculated volume, so that no air layer is present later, which separates the macromonomer solution and the crosslinker solution from one another and prevents diffusion. Optically, the casting mold may not be completely filled, since, for example, a meniscus forms. b) placing and / or placing the at least one crosslinker cap on or under an at least partially open side surface of the casting mold, such that the membrane, in particular the casting mold and / or the crosslinker cap, forms an interface between the casting mold and the cavity of the crosslinker cap. By placing the crosslinker top piece, the macromonomer solution is distributed in the mold in a mold-filling manner, such that no meniscus is formed any longer and the macromonomer solution is in physical contact over the entire surface with the membrane. c) Filling the cavity of the crosslinker top piece with a crosslinker solution comprising crosslinker molecules. In this case, process steps a) - c) can also proceed in interchanged order. d) Incubating, such that crosslinker molecules diffuse from the cavity of the at least one crosslinker top piece through the membrane into the macromonomer solution and bring about crosslinking of the macromonomers with one another.The pore size of the membrane is larger than the size of the cross-linking molecules. Due to the concentration gradient of cross-linking molecules between the macromonomer solution and the cross-linking solution, the cross-linking molecules diffuse into the macromonomer solution. In the casting mold, the covalent linkage thereof and thus the hydrogel formation is initiated again by the meeting of the crosslinker molecules and the macromonomers. The incubation takes place at least for the desired or necessary crosslinking time, in particular in a quiet manner. This is understood to mean resting, wherein the device is not moved.After the desired incubation period has elapsed, the crosslinker solution and the crosslinker attachment are removed again, for example, carefully. In this case, the crosslinker solution can also be removed separately at first. Finally, the crosslinked hydrogel can be dissolved out of the macromonomer casting mold and removed and used for further experiments.The method according to the invention has the same advantages as already described with respect to the device according to the invention.In a particularly advantageous embodiment, the device is tempered and / or gassed during the incubation in step d).A temperature control can take place in particular at 37° C. and offers the advantage that the crosslinking of the macromonomers is accelerated and the incubation time can thereby be reduced (reaction rate temperature (RGT) rule). Moreover, it is advantageous for living cells, for example cells to be embedded, to be incubated in the hydrogel at a physiological temperature of 37° C., since this does not adversely affect the viability of the cells.Furthermore, the entire structure, including possibly living embedded cells, can be gassed if necessary. In this case, gassing typical for a cell culture, in particular 5% carbon dioxide (CO 2) and a saturated air humidity, is particularly expedient. Thus, the polymerization takes place with regard to gassing and temperature under optimum culture conditions for the cells and a reduction in cell viability is avoided. The saturated humidity can also reduce or prevent possible evaporation of the macromonomer and / or crosslinker solution, in particular when operating at elevated temperature.BRIEF DESCRIPTION OF THE DRAWINGAdvantageous embodiments of the present invention are illustrated in the drawings and are explained in more detail in the following description. It shows: FIG. 1 a : shows a schematic illustration of ionic crosslinking to form a polymeric network according to the prior art, FIG. 1 b : shows a schematic illustration of covalent crosslinking to form a polymeric network according to the prior art, FIG. 2 : shows a schematic illustration of the sequence of hydrogel production in a sectional view according to the prior art, FIG. 3 a : shows a schematic illustration of a sectional view of a first step of the method according to the invention in a first embodiment, FIG. 3 b : shows a schematic illustration of a sectional view of a second step of the method according to the invention in a first embodiment, FIG. 3 c : shows a schematic illustration of a sectional view of a third step of the method according to the invention in a first embodiment, FIG. 3 d : shows a schematic illustration of a sectional view of a fourth step of the method according to the invention in a first embodiment, and FIG. 4 : shows a schematic illustration of an apparatus according to the invention in an embodiment with a three-part construction in a sectional view.FIG. 1 ashows ionic crosslinking to form a polymeric network 10 according to the prior art, the coupling of the macromonomers 1 to one another taking place by charge interactions (chelating).First, macromonomer molecules 1 are present in the form of polymer chains 2 each having a plurality of unbound ions 3. The addition of crosslinker molecules 5 causes the crosslinking of the polymer chains 2 with one another in that the crosslinker molecules 5 each bind to two or more ions 3 via charge interactions and thus link the polymer chains 2 to one another to form a polymeric network 10.A popular hydrogel system based on ionic cross-linking is alginate / alginic acid, derived from brown algae, for example. Here, divalent cations function as crosslinking molecules 5, for example calcium ions, which, by ionotropic incorporation, bring about reversible gel formation of the alginate macromonomers 1.FIG. 1 bshows a chemical crosslinking to form a polymeric network 10 according to the prior art, the linkage of the macromonomer molecules 1 taking place by covalent bonds. Macromonomer molecules 1 are initially present in the form of polymer chains 2 each having different reactive groups 3a, 3b. Crosslinking agent molecules 5 are added, which act as bridging agents between the reactive groups 3 a, 3 b.The crosslinking of the polymer chains 2 with one another takes place in that the crosslinking agent molecules 5 each bind to two different reactive groups 3 a, 3 band in this way link the polymer chains 2 with one another to form a polymeric network 10.Macromolecules which carry amino and aldehyde groups, for example proteins, can be crosslinked, for example, by dialdehyde crosslinkers such as glyoxal, glutaraldehyde or genipin by generating so-called Schiff bases. Also known are tyramine-modified (bio)polymers which can be crosslinked by means of horseradish peroxidase and hydrogen peroxide.FIG. 2 shows a procedure for hydrogel production according to the prior art. A macromonomer solution 1a is placed in a casting mold 11. A meniscus bulging centrally downward is naturally formed. The macromonomer solution 1a is overlaid with a crosslinker solution 5a having crosslinker molecules 5. Crosslinking agent molecules 5 diffuse from the crosslinking agent solution 5 ainto the macromonomer solution 1 aand crosslink the macromonomers 1 with one another there, so that a polymeric network 10 in the form of a hydrogel is formed. In this case, uncontrolled mixing of the two solutions 1 a, 5 aand thus dilution of the macromonomer solution 1 acan occur at the interface between the macromonomer solution 1 aand the crosslinker solution 5 a. The resulting hydrogel 10 also maintains the meniscus.FIG. 3 ashows a first step of the method according to the invention. A casting mold 11 is provided. This has an upwardly open side face via which a macromonomer solution 1a is introduced into the casting mold 11. The casting mold 11 is completely filled in this case. The macromonomer solution 1a forms a meniscus bulging centrally downwards. The macromonomers 1 of the macromonomer solution 1a are, for example, polysaccharides and carry, for example, carboxyl and hydroxyl groups.FIG. 3 bshows a second step of the method according to the invention. A cross-linker attachment 22 is placed on the open side surface of the filled casting mold 11. The crosslinker attachment 22 has an upwardly open cavity 24. The bottom surface of the cavity 24 is designed as a membrane 33 made of a porous material, in particular a polymer. In the mounted state, the membrane 33 forms an interface between the casting mold 11 and the cavity 24 of the crosslinker attachment 22. The pore size of the membrane 33 is, for example, 5-15 nm, so that crosslinker molecules 5 of a crosslinker solution 5 acan pass through the membrane 33, but not macromonomers 1 of the macromonomer solution 1 a. The casting mold 11 and the cross-linking agent attachment 22 have the same length and width.FIG. 3 cshows a third step of the method according to the invention. The cavity 24 of the crosslinker attachment 22 is filled with a crosslinker solution 5 acomprising crosslinker molecules 5. The cross-linking agent solution 5 ais, for example, a dialdehyde cross-linking agent solution, wherein the cross-linking agent molecules 5 are, for example, glyoxal, glutaraldehyde or genipin.Alternatively and in particular for the production of cell-free hydrogels, formaldehyde, for example, functions as the crosslinking molecule.For an alginate-based hydrogel system, divalent cations function as cross-linker molecules 5, for example calcium ions.FIG. 3 dillustrates a fourth step of the method according to the invention, with incubation taking place such that the crosslinker molecules 5 diffuse from the cavity 24 through the membrane 33 into the macromonomer solution 1 aand bring about crosslinking of the macromonomers 1 with one another via covalent bonds. A polymeric network in the form of a hydrogel is formed.If the macromolecules carry, for example, amino and / or aldehyde groups and the cross-linking agent solution is, for example, a dialdehyde cross-linking agent with cross-linking agent molecules 5 such as glyoxal, glutaraldehyde or genipin, Schiff bases are formed, via which the macromonomers are cross-linked with one another to form a hydrogel.In the case of an alginate-based hydrogel system, the bivalent cations, for example calcium ions, as crosslinker molecules 5, bring about reversible gel formation of the alginate macromonomers 1 by ionotropic incorporation.In FIG. 4, a device 100 according to an embodiment of the invention is shown with two cross-linker attachments 22 a, 22 b. On the left, the individual components, namely a first cross-linker attachment 22 a, a casting mold 11 and a second cross-linker attachment 22 bare shown. The first crosslinker attachment 22 aincludes a cavity 24 and a base which is configured as a first membrane 33 a. The first cross-linker attachment 22 ais placed on an open side surface of the casting mold 11, here the cover surface of the casting mold, so that the first membrane 33 aof the first cross-linker attachment 22 aconstitutes an interface between the casting mold 11 and the cavity 24 of the first cross-linker attachment 22 a.The casting mold 11 here has an open side surface, namely the cover surface. The bottom surface of the mold is formed by a second diaphragm 33b.The second crosslinker attachment 22 bincludes a cavity 24 having an open side surface, but no membrane.The second cross-linking attachment 22 bis placed under the second membrane 33 bof the casting mold 11, or the casting mold 11 is placed on the second cross-linking attachment 22 b, so that the second membrane 33 bof the casting mold 11 forms an interface between the casting mold 11 and the cavity 24 of the second cross-linking attachment 22 b.The membranes 33 a, 33 bare each permeable to crosslinker molecules 5 of a crosslinker solution 5 a, but not to macromonomers 1.On the right, the completely assembled device 100 is shown.A crosslinker solution 5 ais introduced into both crosslinker attachments 22 a, 22 b, such that crosslinker molecules 5 diffuse from above and from below into the macromonomer solution 1 alocated in the casting mold 11, and thus a rapid and homogeneous crosslinking of the macromonomers to form a polymeric network in the form of a hydrogel takes place.The filling of at least one of the cross-linking agent attachments 22 a, 22 band the casting mold 11 is effected, for example, via fluidic connections, not shown in FIG. 4, to the respective components.Alternatively, instead of being arranged above and below the casting mold 11, the cross-linking agent attachments 22 a, 22 bmay also be arranged on, for example, opposite side surfaces of the casting mold 11 which connect the cover and the base of the casting mold 11 to one another.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedEP 261035 A1

[0004] WO 02 / 36342 A1

[0005]

Claims

Device (100) for crosslinking macromonomers (1) comprising a casting mold (11) having at least one, at least partially open side face, further comprising a membrane (33, 33a, 33b) which can be passed for crosslinking agent molecules (5), and at least one crosslinking agent attachment (22, 22a, 22b) having a cavity (24), wherein the casting mold (11) and the at least one crosslinking agent attachment (22, 22a, 22b) can be placed on one another, such that the membrane (33, 33a, 33b) forms an interface between the casting mold (11) and the cavity (24) of the at least one crosslinking agent attachment (22, 22a, 22b).Device (100) according to claim 1, wherein at least one side surface, in particular the bottom surface, of the cross-linking agent attachment (22, 22a) is configured as a membrane (33, 33a).Device (100) according to one of the preceding claims, wherein the casting mould (11) has a membrane (33, 33b) on one or more at least partially open side faces.The device (100) according to any one of the preceding claims, wherein the casting mold (11) and the at least one cross-linker attachment (22, 22a, 22b) have the same length and widthDevice (100) according to any one of the preceding claims, wherein the membrane (33, 33a, 33b) comprises a porous material, in particular a polymer.Device (100) according to one of the preceding claims, wherein the pore size of the membrane (33, 33a, 33b) is 5-50 nm, preferably 5-15 nm, so that these cross-linking agent molecules (5) can pass through a cross-linking agent solution (5a), but no macromonomers (1).Device (100) according to one of the preceding claims, wherein the cross-linker attachment (22, 22a) or the casting mould (11) has a lid, in particular a lid which can be closed irreversibly.Device (100) according to one of the preceding claims, comprising two cross-linking agent attachments (22a, 22b) which can be placed or placed onto the casting mold (11) from different sides, in particular on the top and bottom surfaces, in such a way that a membrane (33a, 33b) forms an interface between the casting mold (11) and the cavity 24 of the respective cross-linking agent attachment (22a, 22b).Method for crosslinking macromonomers (1) by means of a device (100) according to one of Claims 1-8, having the following steps: a) complete filling of the casting mould (11) with a macromonomer solution (1a) b) placing and / or subjecting at least one crosslinking agent cap (22, 22a, 22b) on or under an open side face of the casting mould (11) or under a side face designed as a membrane (33b), such that the membrane (33, 33a, 33b), in particular of the casting mould (11) and / or of the crosslinking agent cap (22, 22a, 22b), forms an interface between the casting mould (11) and the cavity 24 of the crosslinking agent cap (22, 22a, 22b), and c) filling of the cavity (24) of the at least one crosslinking agent cap (22, 22a, 22b) incubating with a crosslinker solution (5a) comprising crosslinker molecules (5)d) so that crosslinker molecules (5) diffuse from the cavity (24) through the membrane (33, 33a, 33b) into the macromonomer solution (1a) and bring about crosslinking of the macromonomers (1) with one another, it also being possible for the steps to proceed in a different order.The method of claim 9, wherein the device (100) is tempered and / or gassed during the incubation in step d).

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