Hydrogel materials with improved transport properties for bioencapsulation

Abstract

Embodiments of the present disclosure relate to hydrogels. The hydrogels are formed from a crosslinked network of polyanions, polycations, and carboxymethyl polysaccharides. The hydrogels have an average pore size of at least 100 nm. A method for forming the hydrogels is also disclosed. In the method, a first solution containing a polyanion and a carboxymethyl polysaccharide is formed. The first solution is added to a second solution containing a polycation to form a hydrogel having an average pore size of at least 100 nm.

Description

Priority

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Patent Application No. 63 / 428,878, filed November 30, 2022, the contents of which are incorporated by reference in their entirety. [Technical Field]

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to hydrogels, and more particularly to hydrogels having large pore sizes and methods for making the same. [Background technology]

[0003] Porous particles have been considered for use as bioreactors for the production of supernatants containing compounds of interest. For example, cell culture requires the diffusion of micro- and macromolecules for cell growth. In particular, such diffusion allows for the inflow of nutrients and the outflow of waste products. However, conventional porous particles do not have pores of sufficient size to allow the diffusion of larger macromolecules, and therefore, the transport of large particles such as extracellular vesicles and virus particles has not been fully investigated. Furthermore, conventional porous particles are fabricated using synthetic polymers, which limits their use in certain areas, such as those requiring food-grade materials. Summary of the Invention

[0004] According to aspect (1), there is provided a hydrogel, the hydrogel comprising a crosslinked network of a polyanion, a polycation, and a carboxymethyl polysaccharide, the hydrogel comprising an average pore size of at least 100 nm.

[0005] According to an aspect (2), there is provided the hydrogel of aspect (1), wherein the polyanion comprises at least one of alginate, pectinate, pectinate, amidated pectin, or gellan gum.

[0006] According to an aspect (3), there is provided the hydrogel of aspect (1) or (2), wherein the polyanion comprises a molecular weight of at least 250 kDa.

[0007] According to Aspect (4), there is provided the hydrogel of any one of Aspects (1) to (3), wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyornithine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkylammonium), poly(acrylamidoalkyl-trialkylammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride).

[0008] According to an aspect (5), there is provided the hydrogel of any one of aspects (1) to (4), wherein the polycation has a molecular weight of 10 kDa or less.

[0009] According to an embodiment (6), there is provided the hydrogel of any one of embodiments (1) to (5), wherein the polycation has a molecular weight of at least 5 kDa or less.

[0010] According to Aspect (7), there is provided the hydrogel of any of Aspects (1) to (6), wherein the carboxymethyl polysaccharide includes at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar.

[0011] According to an aspect (8), there is provided the hydrogel of any one of aspects (1) to (7), wherein the carboxymethyl polysaccharide has a molecular weight of 50 kDa to 250 kDa.

[0012] According to an aspect (9), there is provided the hydrogel of any one of aspects (1) to (8), wherein the weight ratio of polyanion to carboxymethyl polysaccharide is 3:1 to 1:1.

[0013] According to an aspect (10), there is provided the hydrogel of any one of aspects (1) to (9), wherein the weight ratio of polyanion to polycation is 2:1 to 1:1.

[0014] According to an aspect (11), there is provided the hydrogel of any one of aspects (1) to (10), wherein the hydrogel is a spherical capsule.

[0015] According to an aspect (12), there is provided the hydrogel of aspect (11), wherein the spherical capsules have an average diameter of 0.5 mm to 5 mm.

[0016] According to an aspect (13), there is provided the hydrogel of any one of aspects (1) to (10), wherein the hydrogel is a cylindrical tube.

[0017] According to aspect (14), there is provided the hydrogel of any of aspects (1) to (10), wherein the hydrogel is a ribbon having a length, a width, and a thickness, wherein the length is greater than the width and the thickness, and the width is greater than the thickness.

[0018] According to an embodiment (15), there is provided the hydrogel of any one of embodiments (1) to (14), wherein the average pore size is at most 5 μm.

[0019] According to an aspect (16), there is provided the hydrogel of any one of aspects (1) to (15), wherein a living organism is encapsulated within the pores of the hydrogel.

[0020] According to aspect (17), there is provided a method of forming a hydrogel, the method comprising: forming a first solution comprising a polyanion and a carboxymethyl polysaccharide; and adding the first solution to a second solution comprising a polycation to form a hydrogel comprising an average pore size of at least 100 nm.

[0021] According to an embodiment (18), there is provided the method of embodiment (17), wherein the first solution has a viscosity of 50 cPas to 5000 cPas.

[0022] According to an aspect (19), there is provided the method of aspect (17) or (18), wherein the living body is suspended in the first solution before adding the first solution to the second solution.

[0023] According to Aspect (20), there is provided the method of any of Aspects (17) to (19), wherein forming the first solution further comprises adding a third solution comprising 0.5 wt % to 5 wt % of a polyanion to a fourth solution comprising 0.5 wt % to 5 wt % of a carboxymethyl polysaccharide.

[0024] According to an aspect (21), there is provided the method of any one of aspects (17) to (20), wherein the weight ratio of the polyanion to the carboxymethyl polysaccharide is 3:1 to 1:1.

[0025] According to Aspect (22), there is provided the method of any one of Aspects (17) to (21), wherein the polyanion includes at least one of alginate, pectinate, pectinate, amidated pectin, or gellan gum.

[0026] According to an embodiment (23), there is provided the method of any one of embodiments (17) to (22), wherein the polyanion comprises a molecular weight of at least 250 kDa.

[0027] According to Aspect (24), there is provided the method of any one of Aspects (17) to (23), wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyornithine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkylammonium), poly(acrylamidoalkyl-trialkylammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride).

[0028] According to an embodiment (25), there is provided the method of any one of embodiments (17) to (24), wherein the polycation has a molecular weight of 10 kDa or less.

[0029] According to Aspect (26), there is provided the method of any one of Aspects (17) to (25), wherein the carboxymethyl polysaccharide comprises at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar.

[0030] According to an aspect (27), there is provided the method of any one of aspects (17) to (26), wherein the carboxymethyl polysaccharide has a molecular weight of 50 kDa to 250 kDa.

[0031] According to an embodiment (28), there is provided the method of any one of embodiments (17) to (27), wherein the weight ratio of polyanion to polycation is 2:1 to 1:1.

[0032] According to aspect (29), there is provided the method of any of aspects (17) to (28), wherein adding the first solution to the second solution further comprises adding the first solution dropwise to the second solution to form spherical capsules.

[0033] According to an embodiment (30), there is provided the method of embodiment (29), wherein the spherical capsules have an average diameter of 0.5 mm to 5 mm.

[0034] According to Aspect (31), there is provided the method of any of Aspects (17) to (28), wherein adding the first solution to the second solution includes continuously extruding the first solution into the second solution to form a cylindrical tube.

[0035] According to an aspect (32), there is provided the method of aspect (31), further comprising flattening the cylindrical tube to form a ribbon.

[0036] According to Aspect (33), there is provided the method of any of Aspects (17) to (32), further comprising sterilizing the first solution before adding the first solution to the second solution.

[0037] According to an embodiment (34), there is provided the method of embodiment (33), wherein the sterilizing comprises autoclaving the first solution.

[0038] According to an aspect (35), there is provided the method of aspect (33), wherein the sterilizing comprises filtering the first solution.

[0039] According to aspect (36), there is provided a method, comprising: encapsulating an organism in a hydrogel, the hydrogel comprising a crosslinked network of a polyanion, a polycation, and a carboxymethyl polysaccharide, and having an average pore size of at least 100 nm; and transporting extracellular vesicles secreted by the organism through the pores of the hydrogel.

[0040] According to an aspect (37), there is provided the method of aspect (36), wherein the living body comprises a cross-sectional dimension that is larger than the average pore size of the hydrogel.

[0041] According to an aspect (38), the method of aspect (36) or (37) is provided, further comprising harvesting the biological material by dissolving the hydrogel.

[0042] According to aspect (39), there is provided the method of aspect (38), wherein dissolving the hydrogel further comprises incubating the hydrogel in a solution of dextran sulfate.

[0043] According to an embodiment (40), there is provided the method of embodiment (39), wherein the dextran sulfate has a molecular weight of 5 kDa or less.

[0044] According to an embodiment (41), there is provided the method of embodiment (39), wherein the solution of dextran sulfate further comprises at least one of an anti-coagulant or alginate lyase.

[0045] Additional features and advantages will be set forth in the detailed description that follows, and in part will become readily apparent to those skilled in the art from the description, or may be learned by practicing the embodiments set forth in the written description and claims herein, as well as the accompanying drawings.

[0046] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework for understanding the nature and character of the claims.

[0047] The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. These drawings illustrate one or more embodiments and, together with the description, serve to explain the principles and operation of the various embodiments. The drawings are as follows: [Brief explanation of the drawings]

[0048] [Figure 1A] FIG. 1 shows a process flow diagram of a method for forming a porous hydrogel, according to an illustrative embodiment. [Figure 1B] FIG. 1 shows a process flow diagram of a method for forming a porous hydrogel, according to an illustrative embodiment. [Figure 2] 1 is an image of a porous hydrogel in the form of spherical capsules prepared using FITC-labeled carboxymethyl dextran, according to an exemplary embodiment. [Figure 3] 10 includes SEM images of the porosity of spherical capsules prepared using various ratios of FITC-labeled carboxymethyl dextran to alginate, comprising exemplary embodiments of the present disclosure. [Figure 4] 4 includes images of the porosity of spherical capsules of the same type as shown in FIG. 3 that have been dried using an ethanol / water gradient, according to an exemplary embodiment. [Figure 5] 1 is an image of a porous hydrogel in the form of spherical capsules prepared using carboxymethyl dextran, according to an exemplary embodiment. [Figure 6] 6 is an SEM image of the porosity of a spherical capsule of the type shown in FIG. 5, according to an exemplary embodiment. [Figure 7] 1 is an SEM image of the porosity of spherical capsules prepared using carboxymethyl cellulose, according to an exemplary embodiment. [Figure 8] 1 includes SEM images of a porous hydrogel made from a solution containing 1.5 wt. % alginate and 1 wt. % FITC-labeled carboxymethyl dextran, according to an exemplary embodiment. [Figure 9] 1 is an SEM image of a porous hydrogel made from a solution that was autoclaved prior to the formation of spherical capsules, according to an exemplary embodiment. [Figure 10] 1 illustrates a porous hydrogel microtube, according to an exemplary embodiment. [Figure 11] 1 illustrates a porous hydrogel microtube, according to an exemplary embodiment. [Figure 12] 1 illustrates a porous hydrogel microtube, according to an exemplary embodiment. [Figure 13] 13 illustrates the surface porosity of microtubes of the type shown in FIGS. 10-12, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0049] Generally, various embodiments of porous hydrogels and methods for making same are provided with reference to the following description and accompanying drawings. As described more fully below, porous hydrogels are crosslinked networks of polyanions, polycations, and carboxymethyl sugars. The inventors surprisingly and unexpectedly discovered that forming porous hydrogels using a high ratio of carboxymethyl sugars relative to polyanions (≥ 0.5:1) produces relatively large, uniform pores. Conventional hydrogels typically have pores ranging from tens to hundreds of nanometers, limiting their use in studies of transport of large molecules, extracellular vesicles (EVs), and viral particles, among others. Porous hydrogel embodiments according to the present disclosure have pores with an average size of 100 nm or greater, particularly 2 μm or greater. Advantageously, such porous hydrogels can be used in studies related to transport of larger molecules, such as EVs and viral particles. Furthermore, while conventional porous hydrogels use synthetic materials such as poly(diallyldimethylammonium chloride) (PDADMAC), which limits their use in certain contexts, porous hydrogels according to the present disclosure are prepared using natural, potentially food-grade, materials. These and other aspects and advantages of the disclosed porous hydrogels and methods of making them will be described herein and in connection with the drawings. Such exemplary embodiments are provided by way of illustration, not by way of limitation.

[0050] Embodiments of the present disclosure relate to porous hydrogels having an average pore size of at least 100 nm. In one or more embodiments, the average pore size is up to 5 μm. The porous hydrogels are formed from a crosslinked network of polyanions, polycations, and carboxymethyl polysaccharides.

[0051] In one or more embodiments, the polyanion is at least one of alginate, pectinate, pectinate, amidated pectin, or gellan gum, among others. In one or more embodiments, the polyanion comprises a molecular weight of at least 250 kDa. In one or more embodiments, the molecular weight of the polyanion is calculated after autoclaving or sterilization. In such embodiments, the molecular weight of the polyanion is selected to account for any weight loss associated with autoclaving or sterilization.

[0052] In one or more embodiments, the polycation is at least one of chitooligosaccharide, polylysine, polyarginine, or polyarnitine, among others. In one or more embodiments where the use of natural or naturally derived polycations is not required, the polycation can be at least one of poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkylammonium), poly(acrylamidoalkyl-trialkylammonium), poly(diallyldimethyl-ammonium), or PDADMAC, among others. In one or more embodiments, the polycation comprises a molecular weight of 10 kDa or less, particularly 5 kDa or less. The low molecular weight of the polycation relative to the polyanion facilitates diffusion of the polycation into the partially crosslinked polyanion membrane. If the molecular weight is too high, the crosslinked membrane will be thin and dense. In one or more embodiments, the polycation is selected to be water-soluble at neutral pH, which advantageously aids in encapsulating the organism.

[0053] In one or more embodiments, the carboxymethyl polysaccharide comprises at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar, among others. In one or more embodiments, the carboxymethyl polysaccharide has a substitution of less than 1. In one or more embodiments, the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa. While not wishing to be bound by theory, it is believed that the large porosity of the disclosed porous hydrogels is generated due to competition between the carboxymethyl polysaccharide and polyanions for crosslinking. Furthermore, polyelectrolyte complexes formed by interactions between the carboxymethyl polysaccharide and polycations have higher water solubility than polyelectrolyte complexes formed between polyanions and polycations, which may result in phase separation that unexpectedly creates large porosity.

[0054] In one or more embodiments, the weight ratio of polyanion to carboxymethyl polysaccharide ranges from 3:1 to 1:1. In one or more embodiments, the weight ratio of polyanion to polycation is from 2:1 to 1:1.

[0055] Porous hydrogels can have a variety of forms. In one or more embodiments, the porous hydrogel is in the form of spherical capsules. In one or more such embodiments, the porous hydrogel spherical capsules have an average diameter of about 0.5 mm to about 5 mm. In one or more other embodiments, the porous hydrogel is in the form of a hollow cylindrical tube having a diameter of 0.05 mm to several millimeters, such as up to 5 mm. Furthermore, in one or more embodiments, the hollow cylindrical tube can have a length of up to several meters, or even up to hundreds of meters, if desired. In one or more embodiments, the porous hydrogel is in the form of a ribbon. The ribbon has a length, a width, and a thickness. The length of the ribbon is greater than the width of the ribbon, and the width of the ribbon is greater than the thickness. In one or more embodiments, the length is at least 10 times, at least 25 times, at least 50 times, or at least 100 times the width. In one or more embodiments, the width is at least 2 times, at least 5 times, at least 10 times, or at least 20 times the thickness. In one or more embodiments, ribbons of porous hydrogen can be made by flattening cylindrical tubes of porous hydrogel.

[0056] Porous hydrogels can be used to encapsulate living organisms. In particular, living organisms can be encapsulated within an outer hydrogel membrane that forms a spherical capsule or a cylindrical tube. In one or more embodiments, living organisms can be encapsulated during the formation of the porous hydrogel or after the formation of the porous hydrogel (e.g., by growing living organisms within the hydrogel). In either case, the porous hydrogel can be loaded with living organisms, such as organoids, spheroids, or cell aggregates. In one or more embodiments, the living organisms have a size (e.g., maximum cross-sectional dimension) larger than the pores of the porous hydrogel so that the living organisms are trapped within the hydrogel. Once trapped, the living organisms release extracellular vesicles that can be transported through the relatively large pores of the disclosed hydrogel capsule membrane.

[0057] Advantageously, the organisms encapsulated within the porous hydrogel can be harvested from the hydrogel by incubating the hydrogel in low-molecular-weight (e.g., 5 kDa or less) dextran sulfate, which competes with polyanions to bind to polycations, thereby causing dissolution of the hydrogel. In one or more embodiments, the dextran sulfate solution is a 10% solution. The low-molecular-weight dextran sulfate can be used in conjunction with an anti-aggregating agent (e.g., GIBCO™, available from ThermoFisher Scientific, Waltham, MA). In one or more embodiments, alginate lyase can also be used to digest alginate polyanions.

[0058]

[0013] Embodiments of the present disclosure also relate to methods of forming porous hydrogels. Figure 1A provides a process flow diagram of a method 100 of forming a porous hydrogel. The method 100 includes a first step 101 of forming a first solution containing a polyanion and a carboxymethyl polysaccharide. In a second step 102, the first solution is added to a second solution containing a polycation to form a porous hydrogel having a large pore size according to the present disclosure.

[0059] As shown in FIG. 1B, the first step 101 can include multiple substeps. In one or more embodiments, the first step 101 includes a first substep 101a of forming a third solution containing a polyanion. In one or more embodiments, the third solution contains 0.5% to 5% by weight of the polyanion. In one or more embodiments, the first step 101 includes a second substep 101b of forming a fourth solution containing a carboxymethyl polysaccharide. In one or more embodiments, the fourth solution contains 0.5% to 5% by weight of the carboxymethyl polysaccharide. In one or more embodiments, the first step 101 further includes a third substep 101c of mixing the third solution with a fourth solution. As described above, the ratio of polyanion to carboxymethyl polysaccharide in the first solution ranges from 3:1 to 1:1, and the third and fourth solutions are selected accordingly. In one or more embodiments, the third solution and the fourth solution are combined to form a first solution having a viscosity of 50 cPas to 5000 cPas. Below 50 cPas, capsules or tubes are unlikely to form, and above 5000 cPas, it is difficult to prepare capsules or tubes with small diameters.

[0060] In one or more embodiments, the first step 101 may further include a fourth substep 101d of sterilizing the first solution before it is added to the second solution. In one or more embodiments, the first solution is sterilized by autoclaving the first solution. In one or more embodiments, the first solution is sterilized by filtering the first solution.

[0061] If it is desired to encapsulate the living organism in the porous hydrogel, the first step 101 may further include a fifth sub-step 101e of suspending the living organism in the first solution before adding the first solution to the second solution.

[0062] When the first solution is added to the second solution during the second step 102, the morphology of the hydrogel is affected by the manner in which the first solution is added. In particular, spherical capsules can be obtained by adding droplets of the first solution to the second solution. The size of the spherical capsules is based on the size of the droplets of the first solution added to the second solution. Hollow cylindrical tubes of porous hydrogel can be obtained by continuously extruding the first solution into the second solution. Furthermore, as described above, ribbons of porous hydrogel can be obtained by flattening the hollow cylindrical tubes.

[0063] Having generally described porous hydrogels and methods for making them, the following experimental examples illustrate specific embodiments.

[0064] Experimental Example Preparation of chitooligosaccharides (COS) from chitosan In this example, the polycation was selected to be COS. To prepare COS from chitosan, 250 mL of 30% hydrogen peroxide solution was combined with 250 mL of ultrapure water in a 1000 mL round-bottom, three-neck flask equipped with a thermometer and a magnetic stir bar. 20 g of chitosan was added to the flask, and the suspension was allowed to stand undisturbed for 10 minutes. The suspension was then stirred at 250 rpm in an oil bath set at 70°C. After approximately 39 minutes, the heating was stopped, and the temperature was maintained at 50°C for 7 hours and 15 minutes. The reaction mixture was transferred to a centrifuge tube and centrifuged at 3000 rpm to remove water-insoluble material. The supernatant containing the water-soluble COS was pooled and added dropwise to approximately 1.5 liters of 200-proof ethanol (in a 2-liter beaker) to precipitate the COS. The solution was stirred with a spatula to aid flocculation, and the solids were allowed to settle overnight. The supernatant was discarded by suction using a peristaltic pump, leaving a wet cake of COS. The wet cake was transferred to a 50 mL centrifuge tube and washed three times with ethanol and three times with diisopropyl ether. The solid was dried by vacuum drying. Approximately 6.838 g of COS was obtained (corresponding to a 34% yield of the original 20 g of chitosan).

[0065] Preparation of the first solution As discussed above, the first solution can be prepared by combining a third solution containing a polyanion and a fourth solution containing a carboxymethyl polysaccharide.

[0066] 1. Preparation of the Third Solution A 2 wt% aqueous alginate solution (third solution) was prepared by dissolving an appropriate amount of high molecular weight (348 kDa) alginate (J61887, available from Alfa Aesar, Tewksbury, MA) in deionized water. The mixture was stirred at 80°C for 2 hours.

[0067] 2. Preparation of the Fourth Solution To prepare the first solution, the third solution containing alginate was combined with multiple fourth solutions containing different carboxymethyl polysaccharides, the preparation of which is described below.

[0068] A. FITC-labeled CM dextran A 2 wt% aqueous solution of fluorescein isothiocyanate-labeled (FITC-labeled) carboxymethyl (CM) dextran was prepared by dissolving 0.8 g of FITC-labeled CM dextran (74817, available from Sigma-Aldrich Inc., St. Louis, MO) in 39.2 g of deionized water. The FITC-labeled CM dextran had a molecular weight of 150 kDa, a CM content of 3-7%, and a glucose content of 0.001-0.020 mol per mole of FITC. Sonication was used to facilitate dissolution.

[0069] B.CM dextran A 2 wt% aqueous solution of carboxymethyl (CM) dextran was prepared by dissolving 0.8 g of CM dextran (Carbosynth YC64861, available from Biosynth International, Inc., Louisville, KY) with a molecular weight of 150 kDa in 39.2% deionized water. Sonication was used to facilitate dissolution.

[0070] C.CM Cellulose A 2 wt% aqueous solution of carboxymethyl (CM) cellulose was prepared by dissolving 0.8 g of CM cellulose in 39.2 g of deionized water. The CM cellulose had a molecular weight of 90 kDa. Sonication was used to facilitate dissolution.

[0071] 3. Mixture of the third solution and the fourth solution To prepare the first solutions, each of the fourth solutions was mixed with the third solution to create three separate first solutions. First solution A was prepared by mixing 1 mL of the 2 wt% alginate third solution with 1 mL of the 2% FITC-labeled CM dextran fourth solution. First solution B was prepared by mixing 1 mL of the 2 wt% alginate third solution with 1 mL of the CM dextran fourth solution. First solution C was prepared by mixing 1 mL of the 2 wt% alginate third solution with 1 mL of the CM cellulose fourth solution.

[0072] Preparation of the second solution A 1 wt% COS solution was prepared by dissolving 0.4 g of COS in 39.6 g of deionized water. Sonication was used to facilitate dissolution.

[0073] Example 1 To prepare spherical capsules, a 1 wt% COS second solution was placed in a 150 mL beaker equipped with a magnetic stir bar. The stirring speed was set to 100 rpm. Using a syringe with a 34-gauge blunt needle, first solution A (alginate / FITC-labeled CM dextran) was added dropwise to the COS second solution. The drop was added dropwise from a height of approximately 3 cm. After a 5-minute crosslinking period, the spherical capsules were collected using a fiber mesh strainer and washed with pure water. The spherical capsules are shown in Figure 2. The capsules had an average diameter ranging from 1.8 mm to 2 mm.

[0074] To access surface porosity, spherical capsules were frozen and broken in liquid nitrogen. The broken capsules were kept at -80°C for 2 hours and then freeze-dried for 16 hours (using an Alpha 2-4 LD freeze dryer available from Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Freeze-dried capsules were observed using a scanning electron microscope (Phenom Pure available from ThermoFisher Scientific, Waltham, MA). Surface porosity as a function of FITC-labeled CM dextran content is shown in Figure 3. Images (a)-(e) in Figure 3 contain increasing amounts of FITC-labeled CM dextran relative to alginate. Image (a) does not contain FITC-labeled CM dextran (0:1 ratio), and image (e) contains a 1:1 ratio of FITC-labeled CM dextran to alginate. Images (b), (c), and (d) contain FITC-labeled CM dextran to alginate ratios of 0.25:1, 0.5:1, and 0.75:1, respectively. As can be seen in image (c), some porosity appears at a 5:1 ratio of FITC-labeled CM dextran to alginate, while a large amount of porosity is present in image (e), indicating a 1:1 ratio of FITC-labeled CM dextran to alginate.

[0075] To verify that porosity was not caused by thermally induced phase separation during freeze-drying, some capsules were dried through an ethanol / water gradient before vacuum drying. Specifically, capsules were successively incubated in ethanol / water solutions ranging in ratio from 0:100 to 100:0, and the water-free capsules were then dried under vacuum. Figure 4 provides SEM images of the capsules at increasing magnification. The images show that the porosity remains uniform and the pore size remains large, indicating that the porosity is not a function of thermally induced phase separation from freeze-drying.

[0076] Example 2 Similar to Example 1, capsules in Example 2 were prepared starting with a 1 wt% COS second solution provided in a 150 mL beaker equipped with a magnetic stir bar. The stirring speed was set to 100 rpm. Using a syringe with a 34-gauge blunt needle, first solution B (alginate / CM dextran) was added dropwise to the COS second solution. The drop was added from a height of approximately 3 cm. After a 5-minute crosslinking period, spherical capsules were collected using a fiber mesh strainer and washed with pure water. Figure 5 shows the spherical capsules produced. The spherical capsules were substantially identical to those produced using first solution A, but the capsules lacked the fluorescence caused by the FITC label.

[0077] The porosity was observed under magnification, and Figure 6 shows the porosity of spherical capsules prepared under the same conditions as in Example 1. As can be seen, the spherical particles also contain uniform porosity and pore sizes greater than 2 μm.

[0078] Example 3 Similar to Examples 1 and 2, capsules of Example 3 were prepared starting with a 1 wt% COS second solution provided in a 150 mL beaker equipped with a magnetic stir bar. The stirring speed was set at 100 rpm. Using a syringe with a 34-gauge blunt needle, the first solution C (alginate / CM cellulose) was added dropwise to the COS second solution. The drop was added from a height of approximately 3 cm. After a 5-minute crosslinking period, the spherical capsules were collected using a fiber mesh strainer and washed with pure water.

[0079] The porosity was observed under magnification, and Figure 7 shows the porosity of spherical capsules prepared under the same conditions as in Examples 1 and 2. As can be seen, the spherical capsules also contain uniform porosity and pore sizes greater than 2 μm.

[0080] Example 4 Example 1 was repeated, except that the first solution A of alginate / FITC-labeled CM dextran was made with 1.5 wt% alginate and 1 wt% FITC-labeled CM dextran. First solution A was added dropwise to a second solution of 1 wt% COS and allowed to crosslink for 5 minutes. As shown in Figure 8, the porosity of the resulting spherical capsules ranged from 4 μm to 40 μm.

[0081] Example 5 Example 1 was repeated again, except that the third solution of 1% alginate was dissolved at 80° C. and the first solution A (containing alginate and FITC-labeled CM dextran) was sterilized by autoclaving at 121° C. for 30 minutes. Figure 9 shows an SEM image of the porosity of the spherical capsules. As can be seen, the pores are large and uniform across the surface.

[0082] Example 6 First solution A (containing alginate and FITC-labeled CM dextran) was continuously extruded from a syringe with a 32-gauge needle into a second solution of 1 wt% COS to form microtubes. The microtubes were left in the second solution for 5 minutes to crosslink, removed from the solution using a nylon mesh, and washed with water. Figures 10, 11, and 12 show increasing magnifications of microtubes prepared according to this method, illustrating the dimensions of the microtubes. The nylon mesh can be seen in the background behind the microtubes. Figure 13 provides a further magnification of the microtubes, demonstrating the porosity of the microtube surface. As can be seen in Figure 13, the pores are substantially uniform across the surface and have a size of approximately 2 μm.

[0083] Comparative Example 1 Spherical capsules were prepared according to Example 1, except that the 1 wt% COS second solution was replaced with 4 wt% calcium chloride. The solution mixture formed capsules, but no surface porosity was observed. From this, the inventors speculated that replacing COS with calcium chloride affected the crosslinking behavior of the solution. In particular, relatively low molecular weight polycations such as COS promote the formation of large pores, whereas ionotropic crosslinking using inorganic anions does not promote large porosity.

[0084] Comparative Example 2 Example 1 was repeated again, except that the 1 wt% COS second solution was replaced with 1 wt% PDADMAC. PDADMAC is an effective cross-linking agent known to form capsules with cellulose sulfate. However, no capsules were obtained with the first solution A (alginate and FITC-labeled CM dextran). This comparative example demonstrates the importance of a low molecular weight polycation, such as COS, in cross-linking alginate / FITC-labeled CM dextran.

[0085] The foregoing discussion demonstrates the ability to form hydrogels from crosslinked networks of polyanions, polycations, and carboxymethyl polysaccharides with large pore sizes. Advantageously, the hydrogels contain naturally occurring materials, are free of synthetic components, and can be used to encapsulate living organisms. Furthermore, the pores are large enough to allow diffusion of large molecules from within the hydrogel to outside the hydrogel, allowing the hydrogel to be used, for example, as a bioreactor.

[0086] Unless otherwise expressly stated, any method set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed or specifically states in the claim or description that the steps are to be limited to a particular order, no particular order is intended to be inferred. Additionally, as used herein, the article "a" is intended to include one or more components or elements and is not intended to be construed as meaning only one.

[0087] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of the disclosed embodiments. Since combinations, subcombinations, and variations of the disclosed embodiments that incorporate the spirit and content of the embodiments may occur to those skilled in the art, the disclosed embodiments should be construed as including all within the scope of the appended claims and their equivalents.

Claims

1. A hydrogel comprising: comprising a crosslinked network of polyanions, polycations, and carboxymethyl polysaccharides; A hydrogel, wherein the hydrogel comprises an average pore size of at least 100 nm.

2. 10. The hydrogel of claim 1, wherein the polyanion comprises at least one of alginate, pectinate, pectinate, amidated pectin, or gellan gum.

3. 3. The hydrogel of claim 1 or 2, wherein the polyanion comprises a molecular weight of at least 250 kDa.

4. 4. The hydrogel of any one of claims 1 to 3, wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyornithine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkylammonium), poly(acrylamidoalkyl-trialkylammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride).

5. 5. The hydrogel of claim 1, wherein the polycation comprises a molecular weight of 10 kDa or less.

6. 6. The hydrogel of claim 1, wherein the polycation comprises a molecular weight of 5 kDA or less.

7. 7. The hydrogel of any one of claims 1 to 6, wherein the carboxymethyl polysaccharide comprises at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar.

8. The hydrogel of any one of claims 1 to 7, wherein the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa.

9. 9. The hydrogel of claim 1, wherein the weight ratio of said polyanion to said carboxymethyl polysaccharide is from 3:1 to 1:

1.

10. 10. The hydrogel of claim 1, wherein the weight ratio of said polyanion to said polycation is from 2:1 to 1:

1.

11. The hydrogel according to any one of claims 1 to 10, wherein the hydrogel is a spherical capsule.

12. 12. The hydrogel of claim 11, wherein the spherical capsules comprise an average diameter of 0.5 mm to 5 mm.

13. The hydrogel of any one of claims 1 to 10, wherein the hydrogel is a cylindrical tube.

14. 11. The hydrogel of any one of claims 1 to 10, wherein the hydrogel is a ribbon having a length, a width, and a thickness, the length being greater than the width and the thickness, and the width being greater than the thickness.

15. The hydrogel according to any one of claims 1 to 14, wherein the average pore size is at most 5 μm.

16. The hydrogel according to any one of claims 1 to 15, wherein a living organism is encapsulated within the pores of the hydrogel.

17. 1. A method of forming a hydrogel, comprising: forming a first solution comprising a polyanion and a carboxymethyl polysaccharide; adding the first solution to a second solution comprising a polycation to form a hydrogel comprising an average pore size of at least 100 nm.

18. 18. The method of claim 17, wherein the first solution comprises a viscosity of 50 cPas to 5000 cPas.

19. 19. The method of claim 17 or 18, wherein the living organism is suspended in the first solution before adding the first solution to the second solution.

20. 20. The method of any one of claims 17-19, wherein forming the first solution further comprises adding a third solution comprising 0.5% to 5% by weight of the polyanion to a fourth solution comprising 0.5% to 5% by weight of the carboxymethyl polysaccharide.

21. 21. The method of any one of claims 17 to 20, wherein the weight ratio of the polyanion to the carboxymethyl polysaccharide is from 3:1 to 1:

1.

22. 22. The method of any one of claims 17 to 21, wherein the polyanion comprises at least one of alginate, pectinate, pectinate, amidated pectin, or gellan gum.

23. 23. The method of any one of claims 17 to 22, wherein the polyanion comprises a molecular weight of at least 250 kDa.

24. 24. The method of any one of claims 17 to 23, wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyornithine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkylammonium), poly(acrylamidoalkyl-trialkylammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride).

25. 25. The method of any one of claims 17 to 24, wherein the polycation comprises a molecular weight of 10 kDa or less.

26. 26. The method of any one of claims 17 to 25, wherein the carboxymethyl polysaccharide comprises at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar.

27. 27. The method of any one of claims 17 to 26, wherein the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa.

28. 28. The method of any one of claims 17 to 27, wherein the weight ratio of said polyanion to said polycation is from 2:1 to 1:

1.

29. 29. The method of any one of claims 17 to 28, wherein adding the first solution to the second solution further comprises dropping the first solution into the second solution to form spherical capsules.

30. 30. The method of claim 29, wherein the spherical capsules comprise an average diameter of 0.5 mm to 5 mm.

31. 29. The method of any one of claims 17 to 28, wherein adding the first solution to the second solution comprises continuously extruding the first solution into the second solution to form a cylindrical tube.

32. 32. The method of claim 31, further comprising flattening the cylindrical tube to form a ribbon.

33. 33. The method of any one of claims 17 to 32, further comprising sterilizing the first solution before adding the first solution to the second solution.

34. 34. The method of claim 33, wherein sterilizing comprises autoclaving the first solution.

35. 34. The method of claim 33, wherein sterilizing comprises filtering the first solution.

36. 1. A method comprising: Encapsulating a living organism in a hydrogel, the hydrogel comprising a crosslinked network of polyanions, polycations, and carboxymethyl polysaccharides and having an average pore size of at least 100 nm; and transporting extracellular vesicles secreted by the organism through the pores of the hydrogel.

37. 37. The method of claim 36, wherein the living body comprises a cross-sectional dimension greater than the average pore size of the hydrogel.

38. 38. The method of claim 36 or 37, further comprising harvesting the living organism by dissolving the hydrogel.

39. 39. The method of claim 38, wherein dissolving the hydrogel further comprises incubating the hydrogel in a solution of dextran sulfate.

40. 40. The method of claim 39, wherein the dextran sulfate has a molecular weight of 5 kDa or less.

41. 40. The method of claim 39, wherein the solution of dextran sulfate further comprises at least one of an anti-coagulant or alginate lyase.

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