NZ625119B2 - Methods for the preparation of hydrogels using lipase enzymes - Google Patents

Abstract

Provided is a process for preparing alginate hydrogels comprising the use of a lipase, a triacylglycerol ester, a divalent cation releasing carbonate, and an alginate. The hydrogels may be useful for the immobilisation and preservation of biological material, such as spermatozoa.

Description

/073434 METHODS FOR THE PREPARATION OF HYDROGELS USING LIPASE ENZYMES Field of the invention The present invention s to an ed method for preparing hydrogels, in particular alginate hydrogels. The els of the present invention have a wide area of application and are in particular useful for immobilisation of biological material, more particularly for the immobilisation and preservation of cellular material, such as e.g. spermatozoa.

Background of the invention Hydrogels consist of r chains forming a hydrophilic, water ning network having a wide area of application within various rial fields, in particular within the biotechnological and pharmaceutical industry. For example, hydrogels are used to immobilize biological material such as cells for transplantation or as delivery system for e. g. pharmaceutical active ingredients or nutrients. Hydrogels may also be useful as wound ngs. Furthermore, hydrogels, in particular alginate hydrogels, is widely used as thickening agents.

A widely used polymer for the formation of hydrogels is alginate. Alginate are naturally ing, anionic polysaccharides consisting of 1,4-1inked-B-D- mannuronic acid (M) and 0L-L-g1ucur0nic acid (G). (Smidsrod and Skjak-Braek, 1990, Trends in biotechnology, v01. 8, no. 3, pp 71-78). Commercial alginates are extracted from seaweed, such as Ascophyllum nodosum, Macrocystz's pyrz'fera, and Laminarz’a hyperborea, and also to some extent Laminarz’a digitata, Laminarz’a japonica, 'a maxima, Lesonia negrescens and Sargassum Sp. tes may also be prepared from some alginate producing bacteria, e. g. from some Pseudomonas species and from Azotobacter vinelandz'z' (Smidsrod and Skjak- Braek, 1990, supra).

Alginates are ly used inter a1ia in the food industry, e. g. as stabilizers for viscosity l, or as thickening agents. Alginates are also widely used within the pharmaceutical industry and cosmetic industry, also as stabilizers, thickening agent or disintegrant. For the various purposes, alginates being rich in either guluronic acid or mannuronic acid, respectively, are available ni et a1. (1999), Journal of Food ering 39, 369-378, WO8603781, US 4,990,601, US 5,639,467).

Due to alginates patibility and ability to gel in presence of divalent cations 40 such as e.g. calcium ions, alginate is also commonly used for encapsulation of cells (Nebe1,R.L.,Ba1me,J., Saacke, R.G. and Lim. F. (1985), J. Anim. Sci. 60:1631- 1639, Lim, F and Sun, A.M., (1980) Science 210: 908-9100, , EP0922451, 310, Torre et al. (1998), S.T.P. Pharma Sciences, 8 (4), pp. 233-236, Torre et al., (2000), Biomaterials, 21, pp. 1493-1498, Torre et al. (2002), Journal of Controlled Release, 85, pp. 83-89, Faustini et al, (2004),Theriogenology, 61, 173-184, Weber et al. (2006), Journal ofBiotechnology, 123, pp. 155-163.

Alginate gels are also useful for immobilising various materials. For example, WO2008/004890 describes biopolymer particles useful for vation of spermatozoa, and wherein the biological material is embedded in a polymer particle being solid throughout the whole diameter of the particle. By embedding the spermatozoa in the alginate hydrogels instead of ulating the spermatozoa, leaving the spermatozoa in the fluid core of the capsules, the cells are immobilized within the alginate gel network, so restricting the motility of the cells during storage.

Alginate hydrogels, e.g. alginate gels used for encapsulation or entrapment of various materials, may be prepared by mixing a solution of the material to be entrapped with a sodium alginate solution, and adding this on into a solution containing multivalent cations, usually nt cations such as calcium ions (e. g. a solution of CaClZ) rod and Skjak-Braek, 1990, supra).

US 6,497,902 disclose another method for preparing biocompatible hydrogels, such as alginate hydrogels, comprising mixing the cells to be embedded, alginate salt and a calcium releasing agent, and thereafter adding a calcium releasing compound to said mixture to form a cross-linked gel. According to US patent 6,497,902, the m releasing agent may be ono-S-lactone (GDL).

The method disclosed in US patent 6,497,902 may be used to immobilize spermatozoa useful for artificial insemination (e. g. for the preparation of alginate hydrogels disclosed in ). For example, diluted and cooled (4 °C) spermatozoa may be added to a solution comprising dissolved sodium alginate and ded calcium carbonate, and optionally a otectant such as glycerol, and thereafter initiating gelling and obtaining the d alginate hydrogel by adding a solution comprising GDL. The addition ofthe GDL results in the formation of gluconic acid which in turn will react with water and form H30+. The increase in H301 and the presence of calcium ate, results in the e of C02 and Ca2+.

The providing of Ca2+ by the adding the GDL results in formation of the alginate hydrogel with ed spermatozoa.

After gelling has occurred, the containers filled with spermatozoa embedded in te may be eserved in liquid nitrogen, thus providing cryopreservation of spermatozoa having exceptionally long shelf life. 40 r, the present inventors have experience that the use of GDL in preparing alginate hydrogels according to the method disclosed in US patent 6,497,902 W0 2013/076232 involves several drawbacks. When preparing alginate gel comprising immobilized spermatozoa according to the method bed above, it is vital that GDL is added to the solution immediately after the GDL solution is prepared to avoid spontaneous gelling. GDL must be added in dissolved form rather than as powder to avoid local zones with high concentrations of gluconic acid initially, which would be detrimental to the spermatozoa. Furthermore, after the addition of GDL, a period of increasing viscosity will follow as a result of the initiation of the gelling reaction.

Due to the increasing viscosity, the container used to form the hydrogel must be filled rather quickly. During the accrued time for transforming the dissolved GDL to glucuronic acid, the solution is therefore transferred to suitable containers (such as e.g. mini straw ed from IMV, L’Aigle, France) for further gelling and lizing of the desired biological material. The method disclosed in the prior art do therefore result in a rather short and inflexible time le for preparing the hydrogels comprising the desired biological material and are a substantial disadvantage from an industrial point of view.

Due to the drawbacks of the method bed above, there is a need for an improved method for preparing hydrogels, in particular a method being suitable for preparing hydrogels on an industrial scale.

Various other methods for preparing hydrogels have been described in the prior art.

Various reports disclose the utilization of enzymes which upon being subjected to a specific substrate provides for various reactions that in the end results in crossbinding of s types of polymers.

For example, CN 101439206 discloses inter alia the use of polymers comprising a phenolic hydroxyl unit and dioxygenase in an enzyme catalyzed process for the ation of r gels.

Johnsen et al. (2010), ACS Applied als & Interfaces, 2(7), pp. 972, report the preparation of PEG based hydrogel being polymerised using glucose oxidase. The glucose oxidase catalyses the oxidation ofB-D-glucose, and the subsequent use of oxygen to te the flavin e dinucleotid enzyme cofactor, results in formation of H202. By combining ferrous ions with this enzymatic H202 production, primary hydroxyl radical species are produced that further reacts with the ted monomers.

The use of H202 and horse radish peroxidase in preparation of hydrogels have also been reported, of e.g. Kurisawa et al., (2010), J. of Materials try, 20(26), pp. 5371-5375, Sakai et al, , Biomaterials, , pp. 3371-3377, Sakai and Kawakami (2006), Acta Biomaterialia, 20017, 3, pp. 495-501, Lee et al. (2009), J. of Controlled Release, 134, pp. 186-193, Wang et al.(2010), Biomaterials, 31, pp. 1148-1157.

Hydrogels prepared by the use of oxidases and H202 are inappropriate for some 40 application since H202 is a strong oxidant. 2012/073434 There is therefore still a need for an improved and simplified process for the preparation of alginate hydrogels, in particular hydrogels suitable for immobilizing biological material.

Summary of the invention The object of the present invention is to e an improved process for ing alginate hydrogels that is not attended with the drawbacks of the processes of the prior art.

Another object of the present invention is to e simplified process for the preparation of alginate hydrogels suitable for lizing biological material.

Thus, the invention relates to an alginate hydrogel wherein the gelling thereof is initiated by utilising a hydrolase and a substrate being hydrolysable by the hydrolase ing in the formation of H30+ and subsequent the release of a divalent cation due to the presence of a divalent cation releasing agent.

According to one aspect of the present invention, a process for the preparation of an alginate hydrogel is provided, said process comprising the mixing of a solution comprising a hydrolase with a solution sing a ate being hydrolysable by the hydrolase, and wherein the solution comprising the hydrolase or the solution comprising the substrate being hydrolysable by the hydrolase in addition comprises alginate, and a divalent cation releasing compound. Upon mixing of the two solutions, the binding of the substrate to the ase s in hydrolysis of said substrate and the uent ion of H30+. The tion of H30+ furthermore result in the release of divalent cation which thus initiate the formation of the hydrogel.

According to one embodiment, the divalent cation releasing compound and the alginate is present in the solution comprising the hydrolase. According to another embodiment, the divalent cation releasing compound and the alginate is present in the solution comprising the said substrate.

Said hydrolase may be an esterase, such as a lipase. According to one embodiment of the invention, the hydrolase used in the process according to the invention is a triacylglycerol lipase.

According to yet another ment of the invention, the divalent cation releasing compounds releases divalent cations selected from the group consisting of Ca2+, Ba2+, and Sr2+.

According to another embodiment of the present invention, the divalent cation releasing compound is a calcium releasing compound, such as e.g. calcium carbonate.

According to yet another embodiment of the present ion, the substrate is an ester of an organic acid.

According to yet r ment of the present invention, a process is provided comprising the steps of mixing a solution comprising a triacylglycerol lipase with a solution comprising a substrate being hydrolysable by said hydrolase, wherein the substrate is a compound of formula I: R OWOR wherein R1, R2, and R3 independently are the same or ent and represents a straight or branched, substituted or bstituted C1-C12-alkyl carbonyl chain, and n the solution comprising the said lipase or the solution sing the compound of formula I in addition comprises alginate and a divalent cation releasing compound. According to one embodiment, the R1, R2, and R3 are independently the same or different and represents a straight, non-substituted C1- C12-alkyl carbonyl chain, such as a straight, non-substituted C1-C3-alkyl carbonyl chain.

According to yet an embodiment of the present invention, R1, R2, and R3 of formula I are the same of different and represents a straight or branched, substituted or non- substituted C1-C3-alkyl carbonyl chain According to one embodiment of the present invention, R1, R2, and R3 of formula I is selected from the group consisting of methanone, ne, acetone, ne, pentanone, hexanone, heptanone, octanone, ne, decanone, or dodecanone..

In particular, e. g. when tozoa are to be embedded in the alginate hydrogel, the substrate may be selected from the group consisting of triacetin, pionin and tributyrin, preferably tripropionin and tributyrin.

According to another embodiment of the present invention, the solution comprising the hydrolase or the on comprising substrate being hydrolysable by the hydrolase may in addition comprise an object to be ed in the alginate hydrogel.

The object to be embedded in the alginate gel may ing to one aspect of the invention represent biological material, e.g. cellular material such as spermatozoa. ing to one embodiment of the present invention, a process for preparing alginate els comprising spermatozoa is provided, wherein the process comprises the steps of i) forming a first solution by ng spermatozoa with a solution comprising a triacylglycerol lipase; ii) adding to the first solution obtained in step i), a second solution comprising alginate, a divalent cation releasing compound, and a compound of formula I: /0R3 wherein R1, R2, and R3 independently are the same or different and represents a straight or branched, substituted or bstituted C1- C12-alkyl carbonyl chain, initiating gelling; iii) erring the solution ed in step ii) to a container for gelling of the alginate hydrogel; iv) optionally subjecting the alginate hydrogel of iii) to cryopreservation.

According to yet another embodiment of the present invention, a process for preparing te hydrogels sing spermatozoa is provided, wherein the process comprises the steps of i) forming a first solution by diluting spermatozoa with a solution comprising a compound of formula I: R1O\)\/0R3 wherein R1, R2, and R3 independently are the same or different and represents a straight or ed, substituted or non-substituted C1- C12-alkyl carbonyl chain; ii) adding to the solution obtained in step i) a second solution comprising alginate, a divalent cation releasing compound, and a triacylglycerol lipase initiating the gelling; 2012/073434 iii) transferring the solution obtained in ii) to container for gelling of the alginate hydrogel; iV) optionally subjecting the alginate hydrogel of iii) to cryopreservation.

According to one embodiment, the first and second solution of i) and ii) comprises in addition a cryoprotectant, and the solution obtained in iii) is further subjected to cryoconservation. Furthermore, when preparing alginate els comprising spermatozoa according to the present invention as outline above, the containers obtained in iii) may furthermore be subjected to cryopreservation, and n solution of step i) and/or step ii) comprises a cryoprotectant.

The cryoprotectant present in the on of step i) and/or step ii) according to this embodiment of the present ion may be selected from the group consisting of glycerol, ethylene glycol, methanol, DMA, DMSO, propylene glycol, trehalose, glucose.

When preparing alginate hydrogels sing spermatozoa according to the present invention, the divalent cation releasing compound may preferably be calcium carbonate, and the substrate being hydrolysable by the ase may ably be selected from the group consisting of triacetin, tripropionin and tributyrin.

According to one embodiment of the present invention, the te is alginate being rich in guluronic acid.

According to another aspect of the present invention, an te hydrogel prepared according to the present invention is provided.

The alginate hydrogel according to the present invention may in one embodiment of the invention comprise: a. alginate; b. optionally an object to be embedded in the alginate hydrogel; c. a hydrolase used in the preparation of the alginate hydrogel.

The alginate hydrogel according to the present invention is formed by gelling of the alginate, and wherein the gelling is achieved by the release of divalent cations from a nt cation releasing compound as a result of hydrolyse of a ase substrate. The formed hydrogel will thus comprise the hydrolase used in the formation of the gel. The presence of hydrolase in the hydrogel according to the t invention thus indicate that the process of the invention have been d.

According to another embodiment, the alginate hydrogel according to the invention comprises an embedded object. According to another embodiment, said object to be embedded is a ical material, such as cellular material, e.g. spermatozoa.

According to yet another aspect of the present invention, an alginate hydrogel kit is provided comprising one container comprising a hydrolase and a second container sing a substrate being ysable by the hydrolase.

One embodiment according to this aspect of the present invention s an alginate hydrogel kit comprising one container comprising a solution comprising a hydrolase, and a second container comprising a on comprising alginate, a divalent cation ing compound and a ate being hydrolysable by the hydrolase.

Yet r embodiment of this aspect regards an alginate hydrogel kit comprising one container sing a solution comprising a substrate being hydrolysable by a hydrolase, and a second container comprising a solution comprising alginate, a divalent cation releasing compound and a hydrolase.

Finally, the present invention provides the use of a hydrolase and a substrate being hydrolysable by the hydrolase in the ation of an alginate hydrogel. According to one embodiment, the enzyme used according to this aspect is an se, such as a lipase, e.g. a triacylglycerol lipase.

According to one embodiment of this aspect, the invention provides the use of a hydrolase and a ate in the preparation of an alginate hydrogel embedding biological material, preferably spermatozoa.

Detailed description of the invention.

The present invention provides a novel method for preparing alginate hydrogels useful for various areas of applications. In particular, the t invention provides a novel method for preparing hydrogels that in particular is useful for embedding and immobilising biological materials.

Various types of alginate may be used to prepare hydrogels according to the present invention. The ratio of guluronic acid: mannuronic acid is not critical, i.e. the type of alginate and the content of guluronic acid vs. mannuronic acid may be selected ing on the desired strength, stability, swellability, erosion characteristics etc.

The use of G-rich alginates will e.g. provide for stronger, more stable hydrogels.

According to one embodiment, the alginate used to form the hydrogels is an alginate being rich in guluronic acid. The term “alginate being rich in nic acid” or “G- rich alginate” as used herein means alginate comprising higher amounts of guluronic acid compared to mannuronic acid in the polysaccharide polymer chain of the alginate used. Opposite, the term h alginate” or ate being rich in mannuronic acid” as used herein means alginate comprising higher amounts of 40 mannuronic acid compared with guluronic acid. The term “rich” used in connection with alginates comprising either higher amounts of onic acid or guluronic acid, respectively, is well known and commonly used by the skilled person, cf. e.g.

Britt Iren Glaerum Svanem et al Journal of Biological Chemistry Vol 276, No 34, Aug 24 2001 pp3l542-3l550, Sumita Jain et al lar Microbiology (2003) 47(4), ppl 123-1 133, Marco Mancini et al Journal of Food Engineering 39 (1999) pp369-378, Applied and Environmental Microbiology, Sept 1982, Vol. 44 No.3 pp754-756, US patent 5,639,467, US application 2006/0159823, Ji Minghou (M. H.

Chi) et al iologia 116/1 17 (1984), pp554-556.

Non-limiting es of suitable alginate types to be used according to the present invention are FMC LF 10/40, FMC LF 10/60 and FMC LF 20/60 available from FMC Biopolymer AS, Drammen, Norway, A2033 from Sigma, Oslo, Norway, or Pronova UP MYG, Pronova UP LVG alginates available from e. g. NovaMatrix, Sandvika, Norway.

The function of the alginate hydrogel according to the present invention is independent of the three dimensional shape of the hydrogel formed, i.e. the alginate el may have different shapes such as e.g. a cal or cylindrical shape.

Various shapes of the alginate gel may be obtained dependent on the container used for gelling.

Also other polymers that upon being subjected to an enzyme and a substrate resulting in the release of nt cation form els may be used according to the present invention.

According to the present invention, an enzyme, i.e. a hydrolase, together with a suitable substrate thereto, is used to initiate gelling of the alginate. The term “hydrolase” as used herein is meant to encompass a hydrolase enabling the tion of H30+ when mixing a solution comprising substrate(s) with another solution sing the hydrolase. According to one embodiment of the invention, the hydrolase is a lipase. According to yet another embodiment of the present invention, the lipase is an acyl hydrolase, more preferably a triacylglycerol lipase, such as for example the triacylglycerol lipase isolated from the yeast Candida rugosa. A le lipase is available from Sigma-Aldrich Co. LLC (Ll754 - Type VII or L3001 Type 1, CAS number 90011).

It is to be understood that any hydrolase resulting in net production of H30+ upon to the hydrolysis of its substrate may be used according to the present invention. A hydrolase that may be used may thus be selected from the group ting of 40 carboxylic ester hydrolases, idases, and enzymes acting on carbon-carbon bonds in ketonic substances.

Non-limiting examples of ylic ester hydrolases are carboxylesterase, triglycerol lipases, acetyl se, sterol esterase, L-arabinonolactonase, gluconolactonase, ycerol lipase, g-acetylglucose deacetylase, otein lipase, fatty acyl ethyl ester synthase, and diacylglycerol acylhydrolase.

Non-limiting examples of hydrolases acting on carbon-carbon bonds in ketonic nces are acylpyruvate hydrolase, and acetyl pyruvate hydrolase.

A non-limiting example of a glycosidase is a-galacturonidase.

When g the alginate gel according to the present invention, the hydrolase and the substrate thereof are t in different solutions, which upon mixing result in initiating of the gelling process, and wherein one of the said two solutions in addition comprises alginate and a divalent cation releasing compound The sequence of the mixing, i.e. whether the solution comprising the enzyme is added to a solution comprising the substrate, or vice versa, or whether it is the solution comprising the enzyme which in addition comprises alginate and the divalent cation ing compound, or vice versa, is not critical.

The substrate used in the forming of an alginate hydrogel according to the present invention is a substrate which upon binding to the enzyme results in the production of H30+. The substrate may thus vary depending on the type of hydrolase used according to the present invention. le substrates according to the present invention are esters of organic acids, such as carboxylic acids.

According to one embodiment of the present invention, the substrate is a compound of formula 1: R10 0R3 wherein R1, R2, and R3 independently are the same or different and represents a straight or ed, substituted or non-substituted C1-C12 alkyl carbonyl chain, such as e.g. methanone, ethanone, acetone, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, none etc. ing to one embodiment, R1, R2, and R3 are each methanone. According to another embodiment, R1, R2, and R3 are each ethanone. According to yet another embodiment, R1, R2, and R3 is e. ates of the formula I is in particular useful when forming 40 alginate hydrogels using a triacylglycerol lipase as the hydrolase according to the present invention.

Upon binding to the enzyme present in the first t, said ester of formula I is split into glycerol and a carboxylic acid, i.e. thus providing H30+.

The alkyl carbonyl chain may be branched or unbranced. The alkyl carbonyl chain may rmore be substituted or non-substituted. The skilled person will acknowledge, based on the ng herein, that various substrate covered by the formula I may be used and may based on the teaching herein select the proper substrate to be used ing to the present invention. The skilled person will thus acknowledge that the alkyl chain length may vary without affecting the ability of the enzyme to produce glycerol and a carboxylic acid of the substrate, thus resulting in the release of H30+ ions.

According to a preferred embodiment of the present invention, the substrate is selected from the group triacetin, tripropionin and tributyrin, of the formulas: O o o o o koyok0‘ \Aofiflok/ AAOMOM Wo W0 W0 o o o tin Tripropionin Tributyrin Thus, according to one embodiment, R1, R2, and R3 represent Cl-C4 alkyl carbonyl.

According to yet another embodiment of the present invention, the substrate present is selected from the group consisting of tripropionin and tributyrin.

According to the present invention, the mixing of the hydrolase and the substrate defined above results in the production of H30+. Said H30+ furthermore result in the release of divalent cations from the divalent cation releasing agent. The term divalent cation ing agent is meant to encompass compounds resulting in the e of e.g. Ca2+, Br2+or Sr2+.

According to one ment of the t invention, the divalent cation releasing agent is a carbonate salt, such as C3C03, BrC03 or SI'COg. According to a preferred embodiment, the divalent cation releasing agent is a calcium releasing agent, such as e.g. calcium carbonate. ing to one embodiment of the present invention, the method for the t invention provides for embedding various materials in the polymer matrix of the hydrogel. The term “embed” or “embedding” as used herein should be understood as lising a material in the alginate hydrogels of the present invention, and wherein the alginate network is present throughout the whole diameter of the hydrogel (in contrast to ulation wherein the hydrogel forms a wall around a liquid core not sing a polymer network).

The term biological material is to be understood to encompass any type of biological material suitable for being lised or encapsulated in biocompatible hydrogels. A miting list of biological material is e.g. cells for transplantation, such as e.g. insulin producing cells, hybridoma cells producing monoclonal dies, or spermatozoa for utilisation in cial insemination and other reproduction technologies.

In case the material to be embedded is spermatozoa, the embedding results in that the spermatozoa are prevented from haVing their natural possibility of movement.

The degree of immobilisation may vary dependent on the teristics of alginate hydrogel, such as mechanical strength and ability to disintegrate e. g. after insemination in a recipient animal.

According to one preferred embodiment, the biological material to be immobilised according to the present process is tozoa. tozoa immobilised in an alginate hydrogel prepared according to the present invention may optionally be cryopreserved for storage in liquid nitrogen. The alginate hydrogel comprising immobilized tozoa prepared according to the present invention may be prepared in ready-to-use insemination doses by immobilising a suitable amount of spermatozoa in the alginate hydrogel, and ming the gelling in a container, such as a straw commonly used in artificial insemination procedures haVing the desired size, shape and volume.

For embedding (immobilising) spermatozoa in a hydrogel according to one embodiment of the present ion, spermatozoa are diluted after harvesting in a diluent comprising either the enzyme or the substrate. Said diluted on of spermatozoa is optionally cooled to approx. 4 °C. The solution used to dilute the spermatozoa is also herein named an extender solution (cf. e.g. example 1).

According to a preferred embodiment of the invention, the spermatozoa are first diluted in an extender solution not comprising the substrate or the enzyme. The substrate or the enzyme is then added in a second dilution step wherein the extender solution then used in addition comprises either the enzyme or the substrate.

According to one embodiment, the spermatozoa is diluted a second time in an extender solution comprising the enzyme hydrolase. The so obtained on is 40 then mixed with a second solution comprising alginate, calcium carbonate and ally a cryoprotectant, such as glycerol, and a ate for the enzyme t in first solution comprising the diluted spermatozoa and the enzyme. Upon adding and mixing the two solutions, the enzyme will bind to the substrate resulting in the formation of an acid and thus an increase in the level of H30+. The calcium carbonate acting as a buffer will prevent an increase in pH resulting in the release of Ca2+ and the production of C02. The release of Ca2+ results in crossbinding of the polysaccharide chains of the alginate and thus formation of the alginate hydrogel.

It is to be understood that the spermatozoa may also be diluted in an extender on comprising the substrate being hydrolysable by the enzyme to be used in ance with the present invention which are then mixed with a second solution comprising the enzyme, calcium carbonate and optionally a otectant.

Thus, the spermatozoa may first be diluted in an extender solution, and thereafter subjected to a second dilution by adding more of the extender solution furthermore comprising the substrate. The so obtained on may then be mixed with a solution comprising the enzyme, alginate, calcium ate and optionally a cryoprotectant, such as ol. Upon mixing of the two solutions, the gelling is initiated.

The ature of the solvent used to dilute the spermatozoa, and the temperature of the further steps of forming of the gel may vary ent e. g. on the type and source of tozoa. The process of the present invention may thus be carried out in refrigerated conditions or at room temperature (e. g. 20- 24 °C) or even at higher temperature, such as e.g. 30 - 35 °C.

The process of the present invention renders it possible to l the rate of the gelling s by varying the amount of enzyme used. In addition, the concentration of the alginate used influences the mechanical characteristics of the hydrogel formed, and therefore also the dissolution teristics of the hydrogel.

The alginate concentration may thus vary dependent on the desired characteristics of the hydrogel, dependent on the area of application of the hydrogel. The skilled person will based on the teaching herein be able to select the le amount of enzyme, substrate and te to be used in order to obtain the desired gel strength and the desired gelling time.

For example, the use of 3 g triacylglycerol lipase per litre and 0.3 — l g substrate per 100 ml in the spermatozoa/alginate solution obtained after mixing the two solutions according to the present invention results in a gelling time of about 2-3 hr when the solution is kept at about 4°C. The amount of substrate may in addition vary according to the type of enzyme used. 40 In addition, the amount of acid and thus the amount of H30+ to be produced when mixing the two solutions may be controlled by the amount of substrate present in the second diluent. It is thus possible to control both the gelling rate and the final pH ofthe process, which is an advantage from an industrial point of view. It provides for an improved and easier production process as one may perform the gelling whenever it is suitable from a production point of view as the gelling starts when mixing the spermatozoa diluted in an extender solution sing either the enzyme or the substrate.

For embedding biological material, it is to be understood that solution to be mixed may in addition comprise compounds useful for example for preservative purposes, such as e.g. antibiotics, extenders, antioxidants, buffers, etc., see .

The tration of biological material, e.g. living cells, ed in the alginate hydrogels according to the present invention may vary ing on the type/source of the material. In case of embedding tozoa, the concentration may further vary depending on the breed, recipient animal, insemination techniques, the ce of additional compounds (e.g. for preservative purposes) etc., see e.g. WO 2008/004890. gh the present invention is specifically useful for embedding biological al in form of cells or s in an alginate gel, it is to be understood that alginate gels prepared according to the present invention also have a range of other applications, including ing or immobilising of other s than biological al, and also applications ing the hydrogels per se. The present invention is thus not to be construed as limited to the applications specifically disclosed in the examples in the specification.

Thus, other objects of interest may also be embedded in an alginate hydrogel prepared according to the present invention. The term “object to be embedded in the alginate hydrogel” used herein is meant to encompass any material which is suitable for being immobilised in a hydrogel. For example, in case the alginate hydrogel according to the present invention is to be used as a controlled release system, the object to be embedded may be a ceutical active ingredient. For example, alginate gels prepared according to the present invention may be used as a controlled release or sustained release delivery system for a range of compounds, such as pharmaceutically active compounds, see e.g. WO98/46211, US 4,695,463, US 6,656,508, which are incorporated herein by reference. A non-limiting group of ceutically active ingredients to be embedded in an alginate hydrogel according to the present invention are e.g. recombinant or naturally occurring proteins or polypeptides, natural or tic or semi-synthetic compounds, such as growth factors, hormones, antibodies, interferones, eukins, etc. The alginate hydrogels according to the present invention may also have utilities within the food industry, e. g. for the embedding of nutritive.

The hydrogels prepared according to the present invention may also be used as such for other industrial purposes, both in pharmaceutical industry or cosmetic industry (e.g. as a thickening agent, in wound dressings, in dental health products and methods), or in the human and animal food industry (e. g. in preparation of restructured food, as thickening agent, etc).

The present invention provides advantages compared with the prior art. For example, when using the technique described in US 6,497,902, the reaction rate as well as the amount of acid produced can be influenced by changing the concentration of GDL. Changes in the GDL-concentration will however simultaneously also affect the amount of acid produced and thus the final pH in the gel. The preparation of an alginate hydrogel using an enzyme and substrate ysable by said enzyme to initiate gelling allows a significantly better l of reaction rates and the amount of acid produced (the pH in the gel) as the on rate and the final pH can be dually controlled by changing the concentration of enzyme and substrate, respectively. ing to yet another aspect of the present invention, the present invention provides an alginate hydrogel kit comprising a container comprising a hydrolase, and another container comprising a substrate being hydrolysable by the hydrolase.

The kit according to the present invention may also contain other nds necessary for casting an alginate hydrogel according to the present invention. Thus, the kit may also comprise te and a divalent cation releasing compound. Said compounds may be contained in the ner comprising the hydrolase, in the container comprising the substrate being ysable by the said hydrolase, or they may be contained in separate container(s).

The containers may also comprise other compounds of particular interest depending on the use of the te hydrogel to be casted using the kit of the present invention.

For example, in case the kit is to be used for casting gels comprising biological material, such as cellular al, e.g. tozoa, and wherein the resulting alginate hydrogel is to be r subjected to cryopreservation, the kit may furthermore contain a cryoprotectant such as e. g. glycerol, ethylene glycol, methanol, DMA, DMSO, propylene glycol, trehalose, glucose. Said cryoprotectant(s) may be contained in the container comprising the hydrolase, in the ner comprising the substrate being hydrolysable by the said hydrolase, or they may be contained in separate container(s).

WO 76232 The kit may also comprise other compounds useful for preservative purposes, such as e.g. antibiotics, extenders, idants, buffers, etc. known to the skilled person in the art, see e.g. .

For illustration purposes, the following examples are given. It is to be understood that the examples below are not to be construed as limiting the scope of the present invention. The foregoing description of various embodiments of the present invention s the general nature of the invention and the skilled person will by applying the general knowledge within the area of hydrogels, readily modify and/or adapt the method of the present invention, without undue mentation, without departing from the general t of the present invention and the scope of the enclosed claims. Such adaptations or modifications are thus intended to be within the meaning of a range of equivalents of the sed embodiments, based on the teaching and guidance presented herein. It is to be understood that the terminology used herein is for the purpose of description and not of limitation. Thus, the terminology of the present specification is to be interpreted by the skilled person in light of the teachings and guidance presented herein, in combination with the dge of the d person.

Example 1: Immobilization of bovine spermatozoa MATERIALS AND METHODS Materials The following chemicals were used: trizma hydrochloride and EDTA from Sigma (St. Luis, USA), , NaCl, glycerol (>99 %), sodium citrate and sodium pyruvate from Riedel de Haen (Seelze, Germany), fructose and glucose monohydrate from Norsk Medisinaldepot (Oslo, Norway), calcium carbonate from KSL staubtechnik gmbh (Lauingen, Germany) and sodium alginate NAL LF 10/60) from FMC Biopolymer A/S (Drammen, Norway).

Source of spermatozoa Bovine spermatozoa were collected at the Geno facilities at Hallsteingard in Trondheim and Store Ree in Stange, Norway.

Buffer solutions The following extender solutions were used: er for first dilution of spermatozoa: 1.45 g l'1 Trizma hydrochloride glucose, 0.4 g 1'1 sodium citrate, l g l'1 fructose, 0.22 g 1'1 sodium pyruvate and 200 ml 1'1 egg yolk. The pH ofthe solution was ed to 6.4 by addition ofNaOH.

Extender solution for secondary dilution of spermatozoa: 4 g 1'1 m ate (unless otherwise stated), 54 g l"1 fructose, 170 g 1'1 glycerol and 24 g l"1 LFlO/6O sodium alginate. Both ers contain rd antibiotic cocktail giving at least the final concentration required in EU dir 88/407. Modified IVT: 3 g 1'1 glucose, 20 g 1'1 sodium citrate, 2.1 g l'1 NaHCOg, 1.16 g 1'1 NaCl, 3 g 1'1 EDTA, pH 7.35. The extender solutions were added either enzyme (Sigma Ll754) or substrate as specified below.

Dilution, immobilization and cryoconservation of bull spermatozoa Bovine spermatozoa were harvested at the Geno facilities and diluted as described below: Immediately after harvesting, the spermatozoa were diluted to a concentration of 219 x 106 cells per ml in the extender solution for a first dilution.

This solution was then immediately mixed with an equal volume of extender for r dilution where said er now comprised either enzyme or substrate. The resulting solution containing diluted spermatozoa was then cooled to 4 0C.

After cooling to 4 CC, the on was mixed with an equal volume of the extender solution for a third dilution where said extender now comprised either substrate (triacetin, pionin or of yrin) if the diluted spermatozoa from the previous step comprised the enzyme or enzyme if the diluted spermatozoa from the previous step comprised the substrate. In addition, the extender now comprised calcium carbonate as a divalent cation releasing compound and alginate. The resulting solutions was then transferred to insemination straws and equilibrated at 4 CC for approximately 3 hours. The insemination straws was then transferred to a Nz-freezer and frozen according to standard procedures for bull semen.

Assessment of motility The motility of the spermatozoa was assessed through a microscopic evaluation.

Frozen semen straws were thawed by holding the straws in water bath at 37 °C for 30 seconds. Prior to ement the alginate gel was liquefied in modified IVT solution. The content of an insemination straw was added to 0.9 ml of IVT solution and shaken lly on a tube-tumbler for approximately 10 minutes. Prior to ment of motility, the tubes were ted for minimum 15 minutes in a heat- block at 37 °C. Approximately 3 ul of the solution was added to a preheated microscope slide and immediately inspected using a light microscope. The number of motile spermatozoa in each sample was estimated to the nearest 5% interval. If practically possible, the operator was unaware of the sample identity during the assessment.

RESULTS a) Immobilization of Bovine spermatozoa using triacetin as a ate Spermatozoa was collected and diluted according to the procedures described above. The extender solution for second dilution was added enzyme (Sigma Ll754) to a concentration of 6 g l'1 in the final solution containing spermatozoa. The extender solution for third on contained 8 g 1'1 calcium carbonate and was added tin to a concentration of 0.5 g/ 100 ml. Approximately 4 hours after the secondary dilution a gel was formed immobilizing the spermatozoa. After approximately 24 hours of storage at 4 °C the gel was liquefied and the motility of the immobilized spermatozoa was assessed. Approximately 60 % of the tozoa were motile at that time. b) Immobilization of Bovine spermatozoa using tripropionin as a ate Spermatozoa were ted, diluted, immobilized and cryoconserved according to the ures described above. The extender solution for the second dilution was added enzyme (Sigma Ll754) to a concentration of 6 g l'1 in the final on containing diluted spermatozoa. The extender solution for third dilution contained 4 g 1'1 calcium carbonate and was added tripropionin to a concentration of 0.30 g/ 100 ml. Approximately 3 hours after the secondary dilution a gel was formed and the straws containing the immobilized spermatozoa were frozen. The semen straws were stored in liquid N2 for several days until thawing and assessment of motility.

Approximately 60 % of the spermatozoa were motile after thawing and liquefying of the gel. c) Immobilization of Bovine spermatozoa using tripropionin as a substrate tozoa were ted, diluted, immobilized and cryoconserved according to the procedures described above. The extender solution for the second dilution was added tripropionin to a concentration of 0.30 g/ 100 ml in the final solution containing diluted spermatozoa. The extender solution for the third dilution contained 4 g 1'1 m carbonate and was added enzyme Sigma Ll754 to a tration of 6 g l'l. Approximately 2 hours after the secondary dilution a gel was formed. After approximately 24 hours of storage at 4 °C the gel was liquefied and the motility of the immobilized spermatozoa was assessed. Approximately 60 % of the spermatozoa were motile at that time. d) Immobilization of Bovine spermatozoa using tributyrin as a substrate Spermatozoa were collected, diluted, immobilized and cryoconserved ing to the procedures described above. The extender solution for the second on was added enzyme (Sigma Ll754) to a concentration of 6 g l'1 in the final solution 40 containing d spermatozoa. The extender solution for the third dilution contained 4 g 1'1 calcium carbonate and was added tributyrin to a concentration of WO 76232 0.35 g/ 100 ml. imately 3 hours after the secondary dilution a gel was formed and the straws containing the immobilized spermatozoa were frozen. The semen straws were stored in liquid N2 for several days until thawing and assessment of motility. Approximately 60 % of the spermatozoa were motile after thawing and liquefying of the gel.

Example 2 Controlled preparation of calcium alginate gel using lipase enzymes and triacetin 0r trioctanoate substrates MATERIALS AND METHODS Materials Sodium alginate (PROTANAL LF 10/60) from FMC ymer A/S (Drammen, ), enzymes L3001 and Ll754 from Sigma (St. Luis, USA), Triacetin and trioctanoate from Sigma (St. Luis, USA), calcium carbonate from KSL staubtechnik gmbh (Lauingen, y) Solutions The following solutions were used. Solution Ll.l: 10 g 1'1 solution of L3001 enzyme in distilled water, Solution Ll.2: 0.2 g l'1 triacetin in distilled water.

Solution L21: 4 g 1'1 calcium carbonate, 24 g l'1 LF10/60 sodium alginate and 0.1 g l'1 triacetin in distilled water. Solution L2.2: 4 g 1'1 m carbonate, 24 g l'1 LF10/60 sodium alginate and 0.2 g l'1 tin in distilled water. Solution L23: 4 g 1'1 calcium carbonate, 24 g l'1 LF10/60 sodium alginate and 0.3 g l'1 triacetin in distilled water. on L24: 4 g 1'1 calcium carbonate, 24 g l'1 LF10/60 sodium alginate and 10 g l'1 L3001 enzyme in distilled water. Solution L2.5: 4 g 1'1 calcium carbonate, 24 g l'1 LF10/60 sodium alginate and 0.3 g l'1 trioctanoate in distilled water.

Initiation of gelling Gelling was initialized by mixing of one of the Ll-solutions with an equal volume of one of the L2-solutions. The solutions were mixed according to table 1.

Table 1: mental trials with gelling using enzyme L3 001 and triacetin substrate. The combinations of ons investigated are shown in the table as well as concentrations of enzyme and triacetin substrate used.

Trial | Solutions | Concentrations 1 L1.1-- L2.1 10 g 1'1 enzyme L3001, 0.1 g 1'1 triacetin 2 L1.1 —— L2.2 10 g 1'1 enzyme L3001, 0.2 g 1'1 triacetin 3 L1.1 —— L2.3 10 g 1'1 enzyme L3001, 0.3 g 1'1 triacetin 4 L1.2 —— L2.4 0.2 g 1'1 triacetin, 10 g 1'1 enzyme L3001 L12 -- L2.2 0.2 g l'1 triacetin, 0.2 g l'1 triacetin (no enzyme added) 6 L13 -- L2.5 10 g l'1 enzyme Ll754, 0.4 g l'1 trioctanoate All solutions were at ambient temperature before mixing, and the solutions were left at ambient temperature for gelling. The time course of the gelling reaction was followed by Visual inspection of the solutions.

RESULTS The enzymes L3001 e from wheat germ) and Ll754 (lipase from Candida rugosa) and the substrates triacetin and trioctanoate were used for lled gelling of sodium alginate solutions. Solutions of enzyme and substrate were prepared and mixed ing to table 1. The reaction between enzyme and substrate produces H30+-ions that react with suspended calcium carbonate in the ons. This reaction releases calcium ions which interact with alginate polymer chains in the solution and a gel is formed. The observed times before a gel were formed in the experimental trials are shown in table 2. No gel was formed in experimental trial 5 in which no enzyme were added. The results show that the reaction time is ent on both type of substrate and enzyme ed as well as on the concentrations of enzyme and substrate used.

Table 2: Observed time before g occurs after mixing solutions containing enzymes (Sigma L3001 or Ll754) and/or substrate (triacetin or trioctanoate).

Trial | Solutions | Time for gel formation (h:m) 1 L1.1-- L2.1 2:00 2 L1.1-- L2.2 1:50 3 L1.1--L2.3 1:30 4 L1.2 -- L2.4 1:50 L1.2 -- L2.2 No gel was formed 6 L1.3 -- L2.5 0:30

Claims (47)

1. The use of a hydrolase and a ate being hydrolysable by the hydrolase in the preparation of an alginate hydrogel.

2. The use according to claims 1, wherein the enzyme is an esterase.

3. The use according to claim 2, wherein the esterase is a lipase. 10

4. The use according to claim 3, wherein the lipase is triacylglycerol lipase.

5. The use according to any one of the above claims, wherein the hydrolysis of the ate of the hydrolase results in production of H3O+. 15 6. The use according to claim 1, wherein the enzyme is a lipase and the substrate is a compound of formula I:

R O OR 1 3 wherein R1, R2, and R3 ndently are the same or different and represents a straight or branched, substituted or non-substituted C1-C12-alkyl carbonyl chain. 25

7. The use according to claim 6, wherein R1, R2, and R3 is selected from the group ting of methanone, ethanone, acetone, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, dodecanone.

8. The use according to claim 6, wherein R1, R2, and R3 independently are the 30 same or different and ents a C1-C3-alkyl carbonyl chain.

9. The use according to any one of claim 1-7, wherein the substrate is selected from the group consisting of tin, tripropionin and tributyrin. 35

10. The use according to claim 9, wherein the substrate is selected from tripropionin and tributyrin.

11. The use according to any one of the above claims, wherein enzyme and ate is used in the preparation of an te hydrogel embedding 40 biological material.

12. The use of claim 11, wherein the biological material is spermatozoa.

13. A process for preparation of an alginate hydrogel comprising the step of mixing a solution sing a hydrolase with a solution comprising a 5 substrate being hydrolysable by the hydrolase, and wherein the solution comprising said hydrolase or the solution sing said substrate in addition comprises alginate and divalent cation releasing compound.

14. A process according to claim 13, wherein the on comprising the 10 hydrolase comprise a nt cation releasing compound and alginate.

15. A s according to claim 13, wherein the solution comprising the substrate comprise a divalent cation releasing compound and alginate. 15

16. A process according to claim 13, wherein the hydrolase is an se.

17. A process according to claim 13, wherein the esterase is a lipase.

18. A process ing to claim 17, wherein the lipase is triacylglycerol lipase.

19. A process according to any one of claims 13-15, wherein the substrate is an ester of an organic acid.

20. A process according to any one of claims 13-15 for preparation of an 25 alginate hydrogel comprising the steps of mixing a solution comprising a lglycerol lipase with a solution comprising a ate being hydrolysable by the hydrolase, wherein said substrate is a nd of formula I: R O OR 30 1 3 wherein R1, R2, and R3 independently are the same or different and represents a straight or branched, substituted or non-substituted C1-C12-alkyl 35 carbonyl chain, and wherein the solution comprising the said lipase or the solution comprising the compound of formula I in addition comprises alginate and a divalent cation releasing compound.

21. A s according to claim 20, wherein R1, R2, and R3 is selected from the group consisting of methanone, ethanone, acetone, butanone, pentanone, hexanone, heptanone, ne, nonanone, decanone, dodecanone. 5

22. A process according to claim 20, wherein R1, R2, and R3 independently are the same or different and represents a C1-C3-alkyl carbonyl chain.

23. A process according to any one of claims 16-19, n the substrate is selected from the group consisting of triacetin, tripropionin and tributyrin.

24. A process according to claim 23, wherein the substrate is selected from tripropionin and tributyrin.

25. A s according to any one of claims 13-20, wherein the divalent cation 15 releasing compounds releases divalent cations selected from the group consisting of Ca2+, Ba2+, and Sr2+.

26. A s according to claim 21, wherein the divalent cation releasing compound is a m releasing nd.

27. A process according to claim 26, n the cation releasing compound is calcium carbonate.

28. A s according to any one of claims 13-25, wherein the first on 25 further comprises an object to be embedded in the alginate hydrogel.

29. A process according to claim 28, wherein the object to be embedded in the alginate gel is biological al.

30 30. A process according to claim 29, wherein the biological material is spermatozoa.

31. A process according to claim 23, wherein the process comprises the steps of i) forming a first solution by diluting spermatozoa with a solution 35 comprising a triacylglycerol lipase; ii) adding to the first solution obtained in step i), a second solution comprising alginate, a divalent cation releasing compound, and a compound of formula I: R O OR 40 1 3 wherein R1, R2, and R3 independently are the same or different and ents a straight or branched, tuted or non-substituted C1- 5 C12-alkyl carbonyl chain, initiating gelling; iii) transferring the solution obtained in step ii) to a container for gelling of the alginate hydrogel; and iv) optionally subjecting the alginate hydrogel of iii) to cryopreservation.

32. A process according to claim 23, wherein the process comprises the steps of i) forming a first solution by diluting spermatozoa with a solution comprising a compound of formula I: R O OR 15 1 3 wherein R1, R2, and R3 independently are the same or different and represents a straight or branched, substituted or non-substituted C1- 20 C12-alkyl yl chain; ii) adding to the on obtained in step i) a second solution comprising alginate, a divalent cation ing nd, and a triacylglycerol lipase initiating the gelling; iii) transferring the solution obtained in ii) to container for gelling of 25 the alginate hydrogel; and iv) optionally subjecting the alginate el of iii) to cryopreservation.

33. A process according to any one of claims 29 and 30, wherein the substrate is 30 selected from the group consisting of triacetin, tripropionin and tributyrin.

34. A process according to claim 33, n the substrate is selected from the group consisting of tripropionin and tributyrin.

35 35. A process according to any one of claims 31 or 32, wherein the solution obtained in iii) is subjected to cryopreservation, and wherein the first and second solution of i) and ii), tively, comprise a otectant.

36. A process according to any one of claims 16-19, wherein the alginate is 40 alginate being rich in guluronic acid.

37. An alginate el prepared according to any one of claims 1-21.

38. An alginate hydrogel comprising a) alginate; 5 b) optionally an object to be embedded in the alginate hydrogel; c) a hydrolase used in the preparation of the alginate hydrogel.

39. An alginate hydrogel kit comprising one container comprising a hydrolase 10 and a second container comprising a substrate being hydrolysable by the ase.

40. Kit according to claim 39, wherein the substrate being hydrolysable by the hydrolase is a compound of formula I: R O OR 1 3 wherein R1, R2, and R3 independently are the same or different and 20 represents a C1-C12-alkyl carbonyl chain.

41. Kit according to claim 39, wherein said kit in addition comprises alginate and a divalent cation releasing compound. 25

42. Kit according to claim 39, comprising one ner comprising a solution comprising a hydrolase, and a second ner comprising a solution comprising alginate, a divalent cation releasing compound and a substrate being hydrolysable by the hydrolase. 30

43. Kit according to claim 39, comprising one container comprising a solution comprising a substrate being hydrolysable by a hydrolase, and a second container comprising a solution comprising alginate, a divalent cation releasing compound and a hydrolase. 35

44. Use ing to claim 1, substantially as herein described with nce to any one of the anying examples thereof.

45. A process according to claim 13, substantially as herein described with reference to any one of the accompanying examples thereof.

46. An te hydrogel according to claim 37 or claim 38, substantially as herein described with reference to any one of the accompanying examples thereof. 5

47. A kit according to claim 39, substantially as herein described with reference to any one of the accompanying examples thereof.