WO2017121428A1

WO2017121428A1 - Porous glycopolymer-functionalized cryogels and use thereof

Info

Publication number: WO2017121428A1
Application number: PCT/DE2017/100014
Authority: WO
Inventors: Michael Gottschaldt; Ulrich Sigmar SCHUBERT
Original assignee: Friedrich-Schiller-Universität Jena
Priority date: 2016-01-12
Filing date: 2017-01-11
Publication date: 2017-07-20
Also published as: DE102016000458A1

Abstract

The invention relates to novel cryogels, the surface of which is functionalized with glycopolymers, whereby the cryogels have the property of interacting with specific bacteria or cells in a specified manner. The aim of the invention is to provide porous cryogel structures which can be flushed with aqueous suspensions of cells or bacteria and which are capable of binding specific cells or bacteria to the solid phase and becoming enriched by means of a specific interaction between the cryogel structure and said cells or bacteria. According to the invention, this is achieved by cryogel structures which consist of a base structure of the general formula (I), comprising a cryogel: a hydrogel which is polymerized in a frozen state; a linker: any alkyl or aryl group, preferably connected to the cryogel and glycopolymer via an ether, ester, and/or amid bond; and a glycopolymer: a polymer which has monosaccharides or disaccharides at all or at individual repeating units.

Description

Description of the invention

Porous Glycopolymer-Functionalized Cryogels and Their Use

The invention relates to novel cryogels which are functionalized on their surface with glycopolymers, thereby possessing the property of undergoing specific interactions with particular bacteria or cells.

Cryogels are hydrogels which are frozen in preparation in aqueous solution and are therefore hydrophilic and swellable in water, and in particular have a very large pore size (VI; Galaev, IY; Plieva, FM; Savina, IN; Jungvid, H. Mattiasson. B., Polymeric cryogels as promising materials of biotechnological interest Vladimir Lozinsky, Trends Biotechnol., 2003, 21 (10), 445-451).

Cryogels can be treated with an active substance, which can then be released slowly and in a controlled manner into the solution that can flow through the gel. Cryogels of various monomer types have been used for the controlled release of the hydrophilic active ingredient verapamil hydrochloride (Kostova, Bistra, Momekova, Denitsa, Petrov, Petar, Momekov, Georgi, Toncheva-Moncheva, Natalia, Tsvetanov, Christo B., Lambov, Nikolai, Poly. ethoxytriethylene glycol acrylate) cryogels as novel sustained drug release system for oral application ", Polymer (2011), 52 (5), 1217-1222 DOI 10.1016 / j.polymer.2011.01.049).

Cryogels were used as a porous carrier material for catalytically active substances. Thus, enzymes can be immobilized on a cryogel surface and then perform their function there. This gives a surface-immobilized catalyst that can be handled very easily (heterogeneous catalysis). Immobilization may be non-covalently by entrapping enzymes and then using the cryoprotectant with active surface (Petrov, Petar, Pavlova, Severina, Tsvetanov, Christo B, Topalova, Yana, Dimkov, Raycho, "In situ entrapment of urease in cryogels of poly (N-isopropylacrylamide): An effective strategy for noncovalent immobilization of enzymes ", Journal of Applied Polymer Science (2011), 122 (3), 1742-1748, DOI 10.1002 / app.34063.) Laccase can also be applied to cryogel surfaces immobilized while retaining enzyme activity (Nieto, Marina, Nardecchia, Stefania, Peinado, Carmen; Catalina, Fernando; Abrusci, Concepcion; Gutierrez, Maria C; Ferrer, M. Luisa; del Monte, Francisco Soft, "Enzyme-induced graft polymerization for preparation of hydrogels: synergetic effect of laccase-immobilized-cryogels for pollutants adsorption", Soft Matter (2010), 6 (15), 3533-3540, DOI 10.1039 / c0sm00079e) can be used to immobilize bacteria so that they can be used as a bioreactor, keeping the bacteria alive and functioning, so that bacteria that are able to break down phenol are encased in polyethylene oxide cryograms can then be used to degrade phenolic contaminants in industrial water wastes (G. Satchanska, Y. Topalova, R. Dimkov, V. Groudeva, P. Petrov, C. Tsvetanov, S. Selenska- Pobell, E. Golovinsky, "Phenol Degradation by environmental bacteria entrapped in cryogels ", Biotechnology & Biotechnological Equipment 2015, 29, 514-521.) Photobacteria were included in poly (vinyl alcohol) based cryogels and used as biosensors (EN Efremenko, OV Senko, LE Aleskerova, KA Alenina, MM Mazhul, AD Ismailov, "Biosensors Based on the Luminous Bacteria Photobaterium Phosphoreum Immobilized in Polyvinyl Alcohol Cryogel for the Monitoring of Ecotoxicants", Applied Biochemistry and Microbiology 2014, 50, 477 -482).

When it comes to extraction resulting in the removal of an analyte, specially functionalized surfaces can be used as the extraction matrix. Functionalized cryogels are used for this (O.G. Potter, E.F. Hilder, "Porous polymer monoliths for extraction: Diverse applications and platforms", J. Sep. Sci. 2008, 31, 1881-1906, DOI: 10.1002 / jssc.200800116).

Cryogels are used as a stationary material for affinity chromatography with a biological background. The reason for this is the large pore size, through which the gel has no general filter properties and thus does not retain non-specific relatively large objects such as bacteria or cells. Retention of certain analytes is by specific interaction with surface-fixed ligands (KKR Tetala, TA van Beek, J. Sep. Sci. 2010, 33 (3), 422-438, DOI 10.1002 / jssc.200900635.). Following such a principle, surface-fixed cryogels were PDMAEMA (cationic polymer) for DNA enrichment Hanora, I. Savina, FM Plieva, VA Izumrudov, B. Mattiasson, IY Galaev, J. Biotechnol., 2006, 123, 343-355).

A big advantage of Cryogels is their ability to elute whole cells due to their large pore size and thus to make cells and cell mixtures of biological samples accessible for analysis by chromatography (KKR Tetala, TA van Beek, J. Sep. Sei. 2010, 33 (3), 422-438, DOI 10.1002 / jssc.200900635.).

A concept for more selective retention of E. coli bacteria is the use of 2- (dimethylamino) ethyl groups as anion exchangers together with an immobilized metal affinity (IMA) ligand (Cu ²⁺ complexed by iminodiacetic acid, IDA). As a cryogel material Poly (acrylamide) used. (P. Arvidsson, FM Plieva, IN Savina, VI Lozinsky, S. Fexby, L. Bulow, IY Galaev, B. Mattiasson, J. Chromatogr. A 2002, 977, 27-38, DOI 10.1016 / S0021-9673 (02 ) 01114-7). Cu ²⁺ / IDA ligands immobilized on a poly (acrylamide) cryogel were used to separate E. coli and Bacillus halodurans bacteria (MB Dainiak, FM Plieva, IY Galaev, R. Hatti-Kaul, B. Mattiasson, Biotechnol., Prog , 21, 644-649.). The enrichment or separation of the E. coli is based in the cases described on nonspecific electrostatic interactions (anion exchange gels) or also known to be non-specific interactions with metal chelates (Cu ²⁺ / IDA gels) and it is assumed that a variety of cells and bacteria are nonspecifically bound to the gels and released again.

Concanavalin A was also used as the immobilized affinity ligand for the separation of Saccharomyces cerevisiae and Escherichia coli bacteria (M.B. Dainiak, I.Y. Galaev, B. Mattiasson, J. Chromatogr. A 2006, 1123, 145-150.). Concanavalin A has also been used on a poly (vinyl alcohol) cryogel for affinity chromatography of glycoproteins (Hajizadeh, H. Kirsebom, A. Leistner, B. Mattiasson, J. Sep. Sei. 2012, 35, 2978-2985).

A cryogel based on methacrylic acid as a backbone and poly (ethylene glycol) diacrylate as a crosslinker was used as a stationary material for liquid chromatography for separation / purification of chicken egg-type lysosome. Here, the carboxyl groups forming the cryogel polymer backbone served as a ligand for the necessary chemical interaction, similar to a cation exchange resin (polyethylenes) glycol diacrylate-based supermacroporous monolithic cryogel as high-performance liquid chromatography Separation Chen, Zhiyong; Xu, Li; Liang, Yuan; Wang, Jianbin; Zhao, Meiping; Li, Yuanzong Journal of Chromatography A (2008), 1182 (1), 128-131, DOI 10.1016 / j.chroma.2007.12.084). By using cryogel-immobilized antibodies specific antigens could be bound (GC Ingavle, LWJ Baillie, Y. Zheng, EK Lis, IN Savina, C.A. Howell, SV Mikhalovsky, SR Sandeman, "Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen ", Biomaterials 2015, 50, 140-153, DOI: 10.1016 / j.biomaterials.2015.01.039).

Glycopolymers are known to be capable of being amplified by the "cluster glycoside effect" (JJ Lundquist, EJ Toone, Chem. Rev. 2002, 102, 555-578.), With receptors called lectins, stable and Chen, G., Stenzel, MH Polym. Chem., 2010, 1, 1392-1412.) It is described that glycopolymers bind to bacteria, resulting in agglomeration of the bacteria (Yang, Q. Strathmann, M .; Rumpf, A., Schaule, G .; Ulbricht, M. ACS Appl., Mat., Interf., 2010, 2, 3555-3562.) Such interactions have also been described for binding to E. coli bacteria (Song, W .; Xiao, C; Cui, L .; Tang, Z .; Zhuang, X .; Chen, X. Coli., Surf., B: Bioint., 2012, 93, 188-194, Pasparakis, G .; Cockayne, A .; Alexander, C. Am. Chem. Soc., 2007, 129, 11014-11015; Ting, SRS; Min, EH; Zetterlund, PB; Stenzel, MH Macromolecules 2010, 43, 5211-5221; Toyoshima, M., Miura, Y. Polym., Sci., Part A: Polym. Chem., 2009, 47, 1412-1421) B should not be used for easy separation, concentration, etc. The invention is therefore based on the object to provide porous cryogel structures, which can be flushed with aqueous suspensions of cells or bacteria and which are capable of specific interaction of the cryogel structure with certain cells or bacteria, these on the solid phase to bind and enrich.

According to the invention cryogel structures are proposed to solve this problem, which consist of a basic structure of general formula I:

With

Cryogel: a hydrogel polymerized in the frozen state,

Linker: any alkyl or aryl radical, preferably via ether, ester and / or

Amide bond associated with cryogel and glycopolymer, glycopolymer: a polymer which is attached to all or to individual repeating units

Monosachharide or disaccharides contributes.

Cryogel structures, i. Cryogels, which are functionalized by polymerization, additionally have sugar residues in the grafted polymer, and are therefore able to interact with biological systems containing lectins, specific interactions.

A cryogel made from N, N-dimethylacrylamide with N-hydroxyethylacrylamide as a functional comonomer has proved to be advantageous. On the surface of this cryogel, a polymerization initiator can be bound by esterification. Subsequently, the cryogel can be functionalized by means of known atom transfer radical polymerization (ATRP) with various acrylamide-based glycopolymers. Thus, functionalized cryogels can be obtained according to formula II below:

CG-ManOH CG-GICOH

Thus, e.g. in particular E. coli bacteria from a gel which carries mannose residues bound, since Escherichia coli (E. coli) bacteria have corresponding receptors that can bind to mannose. In this way, these E. coli can be enriched from dilute solutions on the gel and thus isolated on a solid phase. By adding small amounts of free mannose, the bacteria can then be released from the cryogel structure again, e.g. further analyzes are supplied. The invention will be explained in more detail below with reference to the said cryogel structure in formula II.

Synthesis of the Cryogel: To 190 ml of an aqueous solution of the monomers (151 mg of N-hydroxyethylacrylamide, 6.11 g of N, N-dimethylacrylamide and 404 mg of Ν, Ν'-methylenebisacrylamide) is added 49.9 mg of ammonium peroxodisulfate and one hour with argon degassed. Argon-purged stainless steel columns (30 cm x 8 mm 0) are each filled with 14.5 mL of the monomer solution and shortly before the closing of the columns with 3 μΐ _^ Tetraethylmethylenediamin (Me ₆ TREN), and then frozen at -13 ° C and stored at -20 ° C for 40 h to ensure complete polymerization. Functionalization of the Cryogel with Linker or Initiator Group: After thawing and washing the gels with water, the gels become dry Washed N, N-dimethylformamide. Thereafter, each gel is treated with a solution of 595.8 μΐ _^ Bromoisobutyrylbromid and added 672 .mu.l · dry triethylamine in dry N, N-dimethylformamide (DMF) and then washed extensively with dry DMF and then with water. The gels thus obtained are transferred into glass tubes and pre-dried by means of a continuous stream of air before they can be completely dried in vacuo. The gels obtained in this way have a bromine content of 1.28% in the elemental analysis (0.16 mmol / g).

Functionalization of the Cryogel with Glycopolymer: In order to synthesize various glycopolymers on the surface of the cryogels thus obtained, appropriate amounts of N-isopropylacrylamide and of the glycomonomer (10 mol% relative to N-isopropylacrylamide) and CuBr ₂ and Me ₆ TREN in a mixture Methanol and water (1: 2) dissolved (resulting monomer concentration 0.42 mol / L). The theoretical concentration of the initiator is set to 1.25 mmol / L. The ratio of monomer: initiator: CuBr ₂ : ligand: reducing reagent is set to 300: 1: 1: 2: 0.9. Prior to the addition of ascorbic acid, the solution is pumped continuously through the column and degassed with argon in a surge tank to give the solution oxygen free (60 min). Thereafter, the ascorbic acid can be added in the form of an aqueous solution and the polymerization can be started with it. The reaction solution is then pumped continuously through the cryogel for 21 h (flow rate 6 mL / min). Finally, the functionalized cryogels are rinsed extensively with water and dried in vacuo. In this principled manner, it is possible to obtain, for example, functionalized cryogels corresponding to formula II using the specified amounts:

Cryogel without saccharide residues (CG-PNiPAM): 421 mg (3.7 mmol) of n-isopropylacrylamide (MPAm) dissolved in 143 DMF and 9.327 mL of methanol / water (1: 2). 274 (5.7 mg, 0.025 mmol) of a Me ₆ TREN solution (41.58 mg in 2.00 mL of solvent) are added, followed by 618.6 μΐ _^ (2.76 mg, 0.012 mmol) of a CuBr ₂ solution ( 4.47 mg in 1.00 mL solvent mixture). The solution is pumped through the cryoprotectant functionalized with the ATRP initiator (77.37 mg cryogel, 0.16 mmol Br per gram) and then 159.4 μL x (1.96 mg, 0.011 mmol) ascorbic acid solution (61.55 mg in 5.0 mL Solvent mixture) was added to start the polymerization. After reaction and work-up, 139.0 mg of functionalized cryogel are obtained.

Mannose-functionalized cryogel (CG-PMan): 417 mg (3.68 mmol) of MPAm together with 125 mg (0.41 mmol) of D- (mercaptoethylmethacryloyl) -mannoside (ManMAmOH) are dissolved in 157 DMF and 9.236 mL of methanocewater (1: 1). 2) solved. 240 (6.28 mg, 0.027 mmol) of a Me ₆ TREN solution (52.30 mg in 2.00 mL solvent mixture) are added followed by 480.7 μL x (3.04 mg, 0.014 mmol) of a CuBr ₂ solution ( 6.33 mg in 1.00 mL solvent mixture). The solution is pumped through the Cryogel functionalized with the ATRP initiator (85.15 mg cryogel, 0.16 mmol Br per gram) and then 175.4 μL x (2.16 mg, 0.012 mmol) ascorbic acid solution (61.55 mg in 5.0 mL solvent mixture) was added to start the polymerization. After reaction and work-up, 188.0 mg of functionalized cryogel are obtained.

Glucose Functionalized (CG-PGlc): 413 mg (3.64 mmol) of MPAm together with 125 mg (0.40 mmol) of β-D- (mercaptoethylmethacryloyl) glucoside (GlcMAmOH) are dissolved in 156 DMF and 9.094 mL of methanocewater (1: 1). 2) solved. 255 μΐ, (6.22 mg, 0.027 mmol) of a Me ₆ TREN solution (48.77 mg in 2.00 mL solvent mixture) are added, followed by 327.1 μL x (3.01 mg, 0.013 mmol mmol) of a CuBr ₂ solution (18.43 mg in 2.00 mL solvent mixture). The solution is pumped through the functionalized with the ATRP initiator Cryogel (84.36 mg Cryogel, 0.16 mmol Br per gram) and then 213.0 μΐ _^ (2.14 mg, 0.012 mmol) (a solution of ascorbic acid 40, 17 mg in 4.0 mL solvent mixture) was added to start the polymerization. After reaction and workup, 122.0 mg of functionalized cryogel are obtained.

Interaction with E. coli bacteria as an example analyte: W3110 was investigated as the Escherichia coli strain (E. coli obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, DSM No. 6302)). The E. coli bacteria were first cultured (overnight, at 37 ° C. in LB medium (LB Broth, Luria / Miller, Carl Roth)). The bacterial suspension was then transferred to 15 mL vials, centrifuged (10,000 g for 15 min) and washed three times with PBS buffer. The bacterial solution was then diluted with PBS buffer to an optical density of 1.0 (at 600 nm). The prepared cryogel structures were swollen in PBS buffer and incubated with the E. coli solutions (1 mL, room temperature). Thereafter, they were washed extensively with cold PBS buffer (each 30 min) and the gels were subjected to electron microscopic examination (SEM, samples fixed with 2% glutaraldehyde, dehydrated with alcohol, dried (Leica EM CPD300), then steamed with gold (BAL-TEC SCD 005 Sputter Coater) and examined with a Sigma VP Field Emission SEM (Zeiss, Germany) with SE detector at 10 kV).

It was found that no bacteria on the cryogel without glycopolymer (CG-PNiPAM) settled, only a few bacteria were detected on the glucose-functionalized gel (CG-PGlc), whereas the mannose-functionalized gel (CG-PMan) strongly with E. coli bacteria was colonized. By rinsing the CG-PMan gel with a solution of methyl a-D-mannoside in water, the bound E. coli bacteria were again completely detached from the gel and reconverted into solution.

Claims

claims

1. Cryogel structure with immobilized on the surface glycopolymer according to the general formula I:

with the ingredients:

a) cryogel: a hydrogel polymerized in the frozen state;

b) linker: any alkyl or aryl radical, preferably via ether, ester and / or amide bond bonded to cryogel and glycopolymer; c) Glycopolymer: Polymers which are present at all or at individual repeat units

Monosaccharides or disaccharides wear.

2. cryogel structures according to claim 1, characterized in that as monomers in the polymerization of the cryogel (formula III):

1) water-soluble acrylamide (R ^ H, X = NH), methacrylamide (R ^ CHs, X = NH), acrylate (R ^ H, X = 0) or methacrylate (R ^ CHs, X = 0) with any aliphatic or aromatic radicals R ³ and R ⁴ , which in turn can carry chemical functionalities, and

2) cross-linking agent based on a di (acrylamide) s (R ^ H) or di (methacrylamide) s (R ^ CHs) with any alipathischer chain R ⁵ as a link, and

3) functional comonomers based on an acrylate (R 1 H, X = O), methacrylates (R 1 CH 3, X = O), acrylamides (R 1 H, X = NH) or methacrylamide (R 1 CH 4, X = NH) which carry a functional group R which allows the subsequent functionalization of the cryogel, in particular OH, NH ₂ , COOH. radical

Polymerization in water

in frozen

Status

2) 3)

3. cryogel structure according to claim 1 or 2, characterized in that this glycol copolymer is preferably present as a random copolymer of formula IV or block copolymer of formula V or as a block copolymer of formula VI,

wherein R is a group which carries a monosaccharide or disaccharide, preferably glucose, mannose, fructose, galactose, lactose, and R ⁶ = H or CH ₃ and X = 0 or NH, and m and n are integers, and A is a repeating unit, which is preferably formed from one of the water-soluble comonomers in formula VII.

p = integer between 0 and 20

(VII)

4. cryogel structures according to claim 1, 2 or 3, characterized in that are selected for coupling the cryogel with the glycopolymer by means of known chemical coupling techniques, linkers which may contain aliphatic or aromatic groups and exemplified but not exclusively via amide, ester -, ether, thioether bond to the cryogel and the glycopolymer are bound.

5. cryogel structures according to claim 1, 2 or 3, characterized in that they are formed by modification of the surface with initiators for the polymerization of the glycopolymer s, for which purpose a corresponding linker with the functional groups of the cryogel (functionality R in formula III) is reacted, preferably halide-containing linker of formula VIII

where "Alk" denotes an arbitrarily long, branched or unbranched alkyl chain and "Hal" denotes a halogen atom (chlorine or bromine) and R = 0 or NH, or linkers with azo initiator structures according to Formula IX,

wherein "azo initiator" means any diazo structure which decomposes into free radicals under thermal action and R ⁹ = 0 or NH or COO.

6. cryogel structures according to claim 1, 2 or 3, characterized in that they are formed by modifying the surface for participation of the cryogel in a polymerization as comonomer, for which purpose a corresponding linker with the functional groups of the cryogel (functionality R in formula III), preferably double bond-containing linker according to formula X,

7. Use of the cryogel structures according to one of claims 1 to 6 for the enrichment, separation, isolation or targeted release of bacteria or cells.