WO2018046129A1

WO2018046129A1 - Lectin for reversible cell immobilisation

Info

Publication number: WO2018046129A1
Application number: PCT/EP2017/001071
Authority: WO
Inventors: Frank Rosenau; Nicholas BODENBERGER
Original assignee: Baden-Württemberg Stiftung Ggmbh
Priority date: 2016-09-12
Filing date: 2017-09-11
Publication date: 2018-03-15
Also published as: DE102016011057A1; DE102016011057B4

Abstract

The invention relates to methods for producing hydrogels for reversibly immobilising glycoproteins and/or cells on the hydrogel, involving lectins being coupled to the hydrogel, corresponding lectin-coupled hydrogels, the use thereof in 3D cell culture, and corresponding methods for reversibly immobilising glycoproteins and/or cells on such hydrogels.

Description

Lectin for the reversible immobilization of cells

description

The present invention relates to processes for the preparation of hydrogels for the reversible immobilization of glycoproteins and / or cells on the hydrogel, comprising the coupling of lectins to the hydrogel, corresponding lectin-coupled hydrogels, their use in 3D cell culture, and the like Process for the reversible immobilization of glycoproteins and / or cells on such hydrogels.

The immobilization of cells plays an important role, for example in SD cell culture, in the 3D printing of cell-supporting systems, in the cultivation of artificial tissue, in the basic research of cell systems in a native environment (eg tumor model formation), as well as in the testing potential agents on immobilized cells. Likewise, the immobilization of proteins on surfaces is interesting for many applications. As a carrier material for such immobilization, hydrogels are becoming increasingly important. Hydrogels are generally insoluble polymers that can bind significant amounts of water, but the matrix itself is insoluble in water. The molecules are linked by covalent or ionic bonds into a three-dimensional network. Hydrogels have become an important branch of research in recent years due to their wide application potential. The application of hydrogel systems in medicine ranges, for example, from the treatment of open wounds via drug delivery to the cultivation of pharmaceutically and / or biotechnologically relevant organisms or stem cells for transplantation medicine, for example in cancer therapy. Due to the wide variety of possible uses for hydrogels, it is expected that the number of applications and the possible fields of application will increase considerably in the coming years. Basically, one differentiates between two types of hydrogels. With permanent hydrogels there is a covalent cross-linking of the matrix building blocks. In the case of reversible hydrogels, on the other hand, the molecules involved are linked only by noncovalent bonds, ie ionic interactions, hydrogen bonds, hydrophobic interactions, or mechanical interweaving. Different classes of molecules (polymers) can be used as basic building blocks for hydrogels. For example, hydrogels are used from sugars, their derivatives or polysaccharides such as alginates, chitosan or dextran for tissue engineering and drug delivery applications. Likewise, proteins such as fibrin, elastin, lysozyme and collagen are now common starting materials for hydrogeism, with some excellent properties being attributed to them. DNA as a potentially versatile material has also been used as a starting material for hydrogels, although the possibilities for effective and cost-effective production of tailor-made DNA in biotechnological, application-relevant or even industrial standards are, at best, to be regarded as very limited. Also chemically-synthetic, non-biodegradable polymers such as polyvinyl alcohol or polyhydroxy methacrylates are used as the basis for hydrogels.

Protein hydrogels (protein hydrogels) are emerging as a highly promising category of materials, as proteins have unique principal properties due to their biological synthesis principle by translating a matrix from the point of view of the polymer chemist. As a precision polymer with a defined chain length, they are monodisperse and inspired by their function as a universal building material of nature, they are provided with a multitude of almost freely determinable physicochemical and biological properties, which goes far beyond the obvious argument of biocompatibility / degradability. In protein hydrogels, the matrix consists of a backbone of protein, which is usually chemically linked by suitable binding molecules (linker molecules) as cross-linker. Such linker molecules carry, for example, reactive groups such as hydroxyl, carboxyl or amino groups. By covalently linking the reactive groups with residues on the surface of proteins, three-dimensional networks are generated. For example, a gel of human protein (serum albumin) was used for cell culture Turning covalently linked via maleimide functions to a spatial gel structure. Other gels are based, for example, on the connective tissue protein collagen, which is cross-linked via terminal amino groups and a corresponding linker molecule. For the administration of drugs to patients, serum albumin-based gels have also been described.

Conventional methods for the immobilization of cells or proteins on surfaces such as hydrogels include the (chemical) functionalization of materials with cell-adhesive peptides, whereby the cells can be enzymatically detached from the material, for example by means of trypsin or accutase. Further, a direct encapsulation of cells in materials and dissolution of the material by heat without damaging the cells or the use of cationized material to ensure sufficient cell adhesion and detachment of the cells by proteolytic digestion are described. Other possibilities include, for example, the use of UV-sensitive detachable peptides, an increase in the hydrophobicity of a material by means of UV light, the destruction of the cell-carrying structure (for example the destruction of a hydrogel by enzymatic degradation), the activation and inactivation of cell-adhesive sequences (US Pat. RGD) by chemically blocking the sequences and using cell-adhesive aptamers and degrading them by DNAses.

However, these processes generally require relatively harsh conditions to dissolve the cells out of the material, for example, by increasing the temperature, using UV radiation, or using proteases. Toxic products are often generated during the release of the cells, for example during enzymatic degradation and / or decomposition of the matrix. Such unwanted by-products can either damage the cells themselves or the material and interfere with subsequent applications. Finally, in such procedures often the long time to detachment of the cells of disadvantage.

The present invention is therefore based on the object of providing a process for the simple and reversible immobilization of cells and / or proteins on surfaces or in porous systems based on hydrogels. Continue to corresponding hydrogels and methods for their preparation are provided.

This object is achieved by the embodiments characterized in the claims. In particular, a method for producing a hydrogel for the reversible immobilization of glycoproteins and / or cells on the hydrogel, a corresponding hydrogel, its use in SD cell culture, and a corresponding method for the reversible immobilization of glycoproteins and / or cells on a Hydrogel provided.

Accordingly, an article of the present invention relates to a process for the preparation of a hydrogel for the reversible immobilization of glycoproteins and / or cells on the hydrogel, comprising the steps:

(a) providing a polymerizable, biocompatible starting material for the preparation of a hydrogel,

(b) glycosylating the provided starting material with a sugar molecule,

(c) at least partially reacting the glycosylated material obtained in step (b) with a linker molecule such that crosslinking of the glycosylated material takes place and a hydrogel is obtained,

(d) lyophilizing the hydrogel obtained in step (c), thereby producing a macroporous structure in the hydrogel,

(e) washing the lyophilized hydrogel, and

(f) incubating the washed hydrogel with a solution containing a lectin having two or more binding sites for the specific binding of the sugar molecule used for the glycosylation in step (b), whereby the lectin is bound to the hydrogel.

The term "reversible immobilization" as used herein refers to an immobilization of cells and / or proteins on a carrier material which can be reversed by suitable measures, ie the possibility of removing and / or removing the cells and / or proteins from and / or or from the carrier material. The term "glycoproteins" as used herein refers to proteins comprising one or more covalently linked carbohydrate moieties.

In step (a) of the process of the invention, a polymerizable, biocompatible starting material is provided for the preparation of a hydrogel. Corresponding materials are not particularly limited and are known in the art. Examples of such materials include collagen, elastin, fibrin, lysozyme, alginate, chitosan, dextran, polyethylene glycol diacrylates (PEGDA), polyvinyl alcohols (PVA), polyethylene glycols (PEG), bovine serum albumin (BSA), and mixtures of these components. In a preferred embodiment, the material is bovine serum albumin (BSA).

In step (b) of the process according to the invention, the starting material provided is glycosylated with a sugar molecule. Methods for the glycosylation of certain materials are not particularly limited and are known in the art. Likewise, suitable sugar molecules are not particularly limited and are known in the art. Possible sugar molecules in this context include sucrose, maltose, glucose, fucose, fructose, galactose and lactose. In a preferred embodiment, the sugar molecule is sucrose. In step (c) of the method according to the invention, the glycosylated material is at least partially reacted with a linker molecule, so that a crosslinking of the glycosylated material takes place and a hydrogel is obtained. In this case, the proportion of the reacted material is at least so high that a hydrogel is formed. Suitable linker molecules are not particularly limited and are known in the art. In this connection, preference is given to linker molecules which are present at least homo- or heterobifunctional and are directed against primary amines, carboxyl, sulfhydryl or carbonyl groups occurring in proteins. Examples of possible linker molecules include NHS esters, imidoesters, maleimides, haloacetyls, pyridyl disulfides, hydrazides, alkoxyamines, diazirines and arylazides. Another concrete example of a possible linker molecule is tetrakis (hydroxymethyl) phosphonium chloride. Methods for reacting materials with linker molecules to produce a hydrogel are not particularly limited and are known in the art. Preferably, the linker molecule is added in the form of a solution at a concentration of 10 to 50 mg / ml, for example 30 mg / ml, of the linker molecule to the glycosylated material. The mixing ratio can be 1: 1, based on the volume of the solutions.

In step (d) of the process of the invention, the resulting hydrogel is lyophilized to produce a macroporous structure in the hydrogel. Corresponding methods of lyophilizing hydrogels are not particularly limited and are known in the art.

In step (e) of the process of the invention, the lyophilized hydrogel is washed. This is preferably carried out in a phosphate-buffered saline solution at physiological pH (pH 7.4), preferably at room temperature.

In step (f) of the method according to the invention, lectins are coupled to the hydrogel. The term "lectin" as used herein refers to complex proteins or glycoproteins which bind specific carbohydrate structures and thereby are capable, for example, of binding specifically to cells and cell membranes and eliciting biochemical reactions therefrom, but lectins do not exert any enzymatic activity The coupling of the lectins to the hydrogel is accomplished by incubating the washed hydrogel with a solution containing a lectin having at least one binding site for the specific binding of the sugar molecule used for the glycosylation in step (b), thereby binding the lectin to the hydrogel Appropriate lectins for binding to the sugar molecule used in step (b) of the process of the invention are known in the art Possible lectins include lectins such as concavalin A and j common multimeric plant or bacterial lectins. A specific example of a lectin in this context is the lectin PA-IIL (lecB) derived from Pseudomonas aeruginosa. Preferably, the lectin is a multimeric lectin. Further, said solution preferably contains the lectin in an amount of 50 to 400 μΜ, more preferably 200 to 300 μΜ. In addition, the lectin used is preferably matched to the cells and / or proteins to be immobilized, that is, chosen such that the cells and / or proteins to be immobilized have sugar residues which can bind to the selected lectin. Sugar residues present on cells and / or glycoproteins, as well as corresponding lectins which can bind them, are known in the art.

Another object of the present invention relates to a hydrogel comprising a glycosylated, biocompatible starting material for the preparation of a hydrogel, wherein

(i) the starting material is crosslinked by means of a linker molecule, and

(ii) a lectin is bound to the sugar molecules used for the glycosylation which has two or more binding sites for the specific binding of said sugar molecule.

In this connection, all relevant definitions and restrictions mentioned above for the method according to the invention for the production of a hydrogel are also applicable to the hydrogel according to the invention. In particular, the biocompatible starting material, the sugar molecule used for the glycosylation, the linker molecule and the lectin are preferably as defined above.

In a preferred embodiment, the hydrogel according to the invention is obtainable by the preparation process according to the invention.

Another object of the present invention relates to the use of a hydrogel according to the invention for the 3D cell culture. Methods for 3D cell culture are not particularly limited, are known in the art, and can be readily adapted for use with the hydrogels of the present invention.

A further subject matter of the present invention relates to a process for the reversible immobilization of glycoproteins and / or cells on a hydrogel, comprising the steps:

(a) providing a hydrogel according to the present invention, and (b) incubating the provided hydrogel with the glycoproteins and / or cells whereby the glycoproteins and / or cells are immobilized by binding of sugar residues present on the glycoproteins and / or cells to the lectins present on the hydrogel.

In this connection, all the relevant definitions and restrictions mentioned above for the process according to the invention for the preparation of a hydrogel also apply to the process according to the invention for the reversible immobilization of glycoproteins and / or cells on a hydrogel. In particular, the biocompatible starting material, the sugar molecule used for the glycosylation, the linker molecule and the lectin are preferably as defined above.

In a preferred embodiment, the method defined above also includes the detachment of the glycoproteins and / or cells from the hydrogel. In particular, the method preferably comprises the step:

(c) incubating the hydrogel carrying the immobilized glycoproteins and / or cells with a solution comprising the sugar molecule used for the glycosylation and / or a sugar molecule which has a higher affinity for the lectins than this.

As already described above, the lectin used is preferably matched to the cells and / or proteins to be immobilized, that is selected such that the cells and / or proteins to be immobilized have sugar residues which can bind to the selected lectin. Sugar residues present on cells and / or glycoproteins, as well as corresponding lectins which can bind them, are known in the art. Further, the affinities of various sugar molecules for a given lectin are known in the art. Thus, a sugar molecule which has a higher affinity for the lectins than the sugar molecule used for the glycosylation can be readily selected.

With the aid of the present invention, (synthetically) glycosylated materials can be modified by binding lectin as an adapter molecule and thereby cells or proteins can be reversibly immobilized on corresponding surfaces or in porous 3D matrices and subsequently gently removed from / from these. The detachment takes place within a short time by the addition of the corresponding binder sugar in low concentration and is therefore exceptionally gentle.

The method developed here ensures a gentle, reversible integration of cells and / or proteins on surfaces or macroporous 3D atrices which can be detached from the material within minutes by the simple addition of suitable sugars in low concentration. Possible applications are, for example, in 3D cell culture as well as in research on artificial tissue, in the basic research of cell systems in a native environment, as well as in the testing of potential active ingredients.

The sugar and sugar binding molecules play a major role in cell adhesion has long been known, but lectins are here for the first time for the functionalization of a synthetic material used to introduce cells and / or proteins in this reversible to immobilize there and a To cause adhesion. Furthermore, the cells can be dissolved out of the material by the simple addition of sugars specific for lectins in a gentle process

The figure shows:

Figure 1: Replacement of cells from a hydrogel according to the present invention over time.

The present invention will be further illustrated by the following non-limiting example.

Example:

The recombinantly produced lectin is a sugar-binding protein which forms a homotetramer. Each of these subunits is able to bind a sugar molecule, mainly through cooperative hydrogen bridges is realized. The recognition of sugars is specific to the different types of lectin. For the protein used here, the following affinity was found: L-fucose>L-galactose>D-mannose> D-fructose. On the basis of these findings, a special affinity chromatography was used in which mannose is immobilized on a solid phase to which lectin can bind. After binding, the lectin PA-IIL (lecB) was released from the column with an excess of mannose. The same principle was used in cellular adhesion; The tetrameric lectin acts as a link between the components of the hydrogel and the cells that carry various sugars on the surface. The separation of the cells from the matrix was then carried out either by adding a sugar with higher affinity or by adding excess sugars.

In order to facilitate the adhesion of the lectin to BSA protein (from which the resulting hydrogels are made), BSA was glycosylated via a dry process and polymerized to gels with the aid of a linker molecule (tetrakis (hydroxymethyl) phosphonium chloride, four arms, reacted with lysine groups in proteins). These gels were lyophilized (FreezeDryer Epsilon 1-6D, Christ, Osterode am Harz, Germany) to produce superior, macroporous structures by sublimation of the ice crystals formed in the frozen gel. After extensive washing in phosphate buffered saline at physiological pH (7.4) and room temperature, the gels were incubated with lectin (50-400 μΜ) so that lectin could bind to the network in sufficient concentration. In the next step, lung and breast carcinoma cells were added to the porous hydrogels to ensure attachment. At the same time, cells and hydrogel-forming BSA molecules could be bound via the tetrameric structure of the lectins. Subsequently, the gels were solubilized to provide optimal growth conditions for the cells. The dissolution of the cells from the gels was carried out by adding a sugar with a very high affinity to lectin. Cells were washed out of the gel and were subsequently in the medium from which they could be collected by centrifugation for further use. This method, described for the first time for the immobilization of cells with the aid of a microbial protein, is very gentle on the cell and reversible due to the simple release of the cells. Figure 1 shows the detachment of cells from a hydrogel according to the present invention over time.

A detailed description of the above experimental data can be found in Bodenberger et al. (Bodenberger, N. et al., Lectin-mediated reversible immobilization of human cells into a glycosylated macroporous protein hydrogel as a cell culture matrix; Scientific Reports, 7; 2017; Article number 6151).

Claims

claims

1. A process for the preparation of a hydrogel for the reversible immobilization of glycoproteins and / or cells on the hydrogel, comprising the steps:

(b) glycosylating the provided starting material with a sugar molecule,

(d) lyophilizing the hydrogel obtained in step (c) to produce a macroporous structure in the hydrogel,

(e) washing the lyophilized hydrogel, and

2. The method of claim 1, wherein the starting material provided in step (a) for the preparation of a hydrogel is selected from the group consisting of collagen, elastin, fibrin, lysozymes, alginates, chitosan, dextran, polyethylene glycol diacrylates (PEGDA), Polyvinyl alcohols (PVA), polyethylene glycols (PEG), bovine serum albumin (BSA) and mixtures of these components.

3. The method of claim 1 or claim 2, wherein the sugar molecule used for the glycosylation is selected from the group consisting of sucrose, maltose, glucose, fucose, fructose, galactose and lactose.

4. The method according to any one of claims 1 to 3, wherein the linker molecule is selected from the group consisting of NHS esters, imido esters, maleimides, haloacetylene, pyridyl disulfides, hydrazides, alkoxyamines, diazirines and antacids.

5. The method according to any one of claims 1 to 4, wherein the washing in step (e) is carried out in a phosphate buffered saline solution at physiological pH (pH 7.4) and room temperature.

6. The method according to any one of claims 1 to 5, wherein the lectin used in step (f) is a multimeric lectin.

7. The method according to any one of claims 1 to 6, wherein the solution from step (f) contains the lectin in an amount of 50 to 400 μΜ.

A hydrogel comprising a glycosylated biocompatible starting material for the preparation of a hydrogel, wherein

(i) the starting material is crosslinked by means of a linker molecule, and

9. Hydrogel according to claim 8, which is obtainable by a method according to any one of claims 1 to 7.

10. Use of a hydrogel according to claim 8 or claim 9 for SD cell culture.

11. A method for the reversible immobilization of glycoproteins and / or cells on a hydrogel, comprising the steps:

(a) providing a hydrogel according to claim 8 or claim 9, and

(b) incubating the provided hydrogel with the glycoproteins and / or cells whereby the glycoproteins and / or cells bind to one another by binding sugar residues present on the glycoproteins and / or cells immobilized to the hydrogel lectins are immobilized.

2. The method of claim 11, wherein the detachment of the glycoproteins and / or cells from the hydrogel comprises the step of: