EP4626956A1 - Hydrogel material having enhanced transport properties for living organisms encapsulation - Google Patents

Abstract

Embodiments of the disclosure relate to a hydrogel. The hydrogel is formed from a cross-linked network of a polyanion, a polycation, and a carboxymethyl polysaccharide. The hydrogel has an average pore size of at least 100 nm. Also disclosed is a method of forming a hydrogel. In the method, a first solution including a polyanion and a carboxymethyl polysaccharide is formed. The first solution is added to a second solution including a polycation to form a hydrogel with an average pore diameter of at least 100 nm.

Description

HYDROGEL MATERIAL HAVING ENHANCED TRANSPORT PROPERTIES FOR LIVING ORGANISMS ENCAPSULATION

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Serial No. 63/428,878 filed on November 30, 2022, the content of which is relied upon and incorporated herein by reference in its entirety.

[0002] The disclosure relates generally to a hydrogel and, in particular, a hydrogel having large pore sizes and a method of making the same.

[0003] Porous particles have been considered for use as bioreactors for the production of supernatants containing compounds of interest. For example, cell cultures require the diffusion of micro- and macro- molecules for cell growth. In particular, such diffusion allows for the influx of nutrients and outflux of waste. However, conventional porous particles do not have pores of sufficient size to permit the diffusion of larger macromolecules, and as such, the transport of such large particles as extracellular vesicles and viral particles is not well investigated. Further, conventional porous particles are made using synthetic polymers, which limits their use in certain fields, such as fields requiring food-grade materials.

SUMMARY

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

[0005] According to aspect (2), the hydrogel of aspect (1) is provided, wherein the polyanion comprises at least one of alginic acid salt, pectic acid salt, pectinic acid salt, amidated pectin, or gellan gum.

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

[0007] According to aspect (4), the hydrogel of any of aspects (l)-(3) is provided, wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyomithine, poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkyl ammonium), poly(acrylamidoalkyl-trialkyl ammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride). [0008] According to aspect (5), the hydrogel of any of aspects (l)-(4) is provided, wherein the polycation comprises a molecular weight of 10 kDa or less.

[0009] According to aspect (6), the hydrogel of any of aspects (l)-(5) is provided, wherein the polycation comprises a molecular weight of 5 kDA or less.

[0010] According to aspect (7), the hydrogel of any of aspects (l)-(6) is provided, wherein the carboxymethyl polysaccharide comprises at least one of carboxymethyl dextran, carboxymethyl starch, carboxymethyl cellulose, carboxymethyl hyaluronic acid, carboxymethyl inulin, or carboxymethyl guar.

[0011] According to aspect (8), the hydrogel of any of aspects (l)-(7) is provided, wherein the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa.

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

[0013] According to aspect (10), the hydrogel of any of aspects (l)-(9) is provided, wherein a weight ratio of the polyanion to the polycation is from 2: 1 to 1 : 1.

[0014] According to aspect (11), the hydrogel of any of aspects (l)-(10) is provided, wherein hydrogel is a spherical capsule.

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

[0016] According to aspect (13), the hydrogel of any of aspects (l)-(10) is provided, wherein the hydrogel is a cylindrical tube.

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

[0018] According to aspect (15), the hydrogel of any of aspects (1)-(14) is provided, wherein the average pore size is up to 5 pm.

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

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

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

[0022] According to aspect (19), the method of aspect (17) or (18) is provided, wherein living organisms are suspended in the first solution prior to adding the first solution to the second solution.

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

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

[0025] According to aspect (22), the method of any of aspects (17)-(21) is provided, wherein the polyanion comprises at least one of alginic acid salt, pectic acid salt, pectinic acid salt, amidated pectin, or gellan gum.

[0026] According to aspect (23), the method of any of aspects (17)-(22) is provided, wherein the polyanion comprises a molecular weight of at least 250 kDa.

[0027] According to aspect (24), the method of any of aspects (17)-(23) is provided, wherein the polycation comprises at least one of chitooligosaccharide, polylysine, polyarginine, polyomithine, poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl-N-alkyl- pyridinium), poly(acryloyl-oxyalkyl-trialkyl ammonium), poly(acrylamidoalkyl-trialkyl ammonium), poly(diallyldimethyl-ammonium), or poly(diallyldimethylammonium chloride).

[0028] According to aspect (25), the method of any of aspects (17)-(24) is provided, wherein the polycation comprises a molecular weight of 10 kDa or less.

[0029] According to aspect (26), the method of any of aspects (17)-(25) is provided, 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 aspect (27), the method of any of aspects (17)-(26) is provided, wherein the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa.

[0031] According to aspect (28), the method of any of aspects (17)-(27) is provided, wherein a weight ratio of the polyanion to the polycation is from 2: 1 to 1 : 1.

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

[0033] According to aspect (30), the method of aspect (29) is provided, wherein the spherical capsule comprises an average diameter of from 0.5 mm to 5 mm.

[0034] According to aspect (31), the method of any of aspects (17)-(28) is provided, wherein adding the first solution to the second solution comprises continuously extruding the first solution into the second solution to form a cylindrical tube.

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

[0036] According to aspect (33), the method of any of aspects (17)-(32) is provided, further comprising sterilizing the first solution prior to adding the first solution to the second solution.

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

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

[0039] According to aspect (36), a method is provided. The method comprising: encapsulating a living organism in a hydrogel, the hydrogel comprising a cross-linked 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 living organism through pores of the hydrogel.

[0040] According to aspect (37), the method of aspect (36) is provided, wherein the living organism comprises a cross-sectional dimension larger than the the average pore size of the hydrogel. [0041] According to aspect (38), the method of aspect (36) or (37) is provided, further comprising harvesting the living organism by dissolving the hydrogel.

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

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

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

[0045] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the written description and claims hereof, as well as the appended 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 to understand the nature and character of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments. In the drawings:

[0048] FIGS. 1A and IB depict process flow diagrams of methods for forming porous hydrogels, according to an exemplary embodiment;

[0049] FIG. 2 is an image of porous hydrogels in the form of spherical capsules prepared using FITC-labeled carboxymethyl dextran, according to an exemplary embodiment;

[0050] FIG. 3 includes SEM images of the porosity of spherical capsules prepared using various ratios of FITC-labeled carboxymethyl dextran to alginate, including exemplary embodiments of the present disclosure; [0051] FIG. 4 includes images of the porosity of spherical capsules of the same type shown in FIG. 3 that were dried using an ethanol/water gradient, according to an exemplary embodiment;

[0052] FIG. 5 is an image of porous hydrogels in the form of spherical capsules prepared using carboxymethyl dextran, according to an exemplary embodiment;

[0053] FIG. 6 is an SEM image of the porosity of a spherical capsule of the type shown in FIG. 5, according to an exemplary embodiment;

[0054] FIG. 7 is an SEM image of the porosity of a spherical capsule prepared using carboxymethyl cellulose, according to an exemplary embodiment;

[0055] FIG. 8 includes SEM images of a porous hydrogel made from a solution containing 1.5% by weight alginate and 1% by weight of FITC-labeled carboxymethyl dextran, according to an exemplary embodiment;

[0056] FIG. 9 is an SEM image of a porous hydrogel made from a solution that was autoclaved before formation of spherical capsules, according to an exemplary embodiment;

[0057] FIGS. 10-12 depict porous hydrogel microtubes, according to an exemplary embodiment; and

[0058] FIG. 13 depicts the surface porosity of microtubes of the type shown in FIGS. 10- 12, according to an exemplary embodiment.

DETAILED DESCRIPTION

[0059] Referring generally to the following description and appended figures, various embodiments of a porous hydrogel and a method of making same are provided. As will be described more fully below, the porous hydrogel is a crosslinked network of a polyanion, a polycation, and a carboxymethyl saccharide. The inventor surprisingly and unexpectedly found that using a high ratio of the carboxymethyl saccharide relative to the polyanion (> 0.5:1) to form the porous hydrogel produced relatively large and uniform pores. Conventional hydrogels typically have pores that are in the range of tens of nanometers up to a few hundred nanometers, which limits the use of these hydrogels in studying transport of large molecules, extracellular vesicles (EV), and viral particles, among others. Embodiments of the porous hydrogels according to the present disclosure have pores with an average size of 100 nm or more, in particular 2 pm or more. Advantageously, such porous hydrogels can be used for studies related to the transport of larger molecules, such as EV and viral particles. Further, conventional porous hydrogels use synthetic materials, such as poly(diallyldimethylammonium chloride) (PDADMAC ), which limit their uses in certain contexts, whereas the porous hydrogels according to the present disclosure are prepared using natural and potentially even food-grade materials. These and other aspects and advantages of the disclosed porous hydrogel and method of making the same will be described herein and in relation to the figures. Such exemplary embodiments are provided by way of illustration and not by way of limitation.

[0060] Embodiments of the disclosure relate to a porous hydrogel having an average pore size of at least 100 nm. In one or more embodiments, the average pore size is up to 5 pm. The porous hydrogel is formed from a cross-linked network of a polyanion, a polycation, and a carboxymethyl polysaccharide.

[0061] In one or more embodiments, the polyanion is at least one of alginic acid salt, pectic acid salt, pectinic acid salt, amidated pectin, or gellan gum, amongst 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 taking into account any weight loss associated with autoclaving or sterilization.

[0062] In one or more embodiments, the polycation is at least one of chitooligosaccharide, polylysine, polyarginine, or polyomithine, amongst others. In one or more embodiments where the use of a natural or naturally derived polycation is not required, the polycation can be at least one of poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-oxyalkyl-trialkyl ammonium), poly(acrylamidoalkyl-trialkyl ammonium), poly(diallyldimethyl-ammonium), or PDADMAC, amongst others. In one or more embodiments, the poly cation comprises a molecular weight of 10 kDa or less, in particular 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 is thin and dense. In one or more embodiments, the polycation is selected such that it is water soluble at neutral pH. Advantageously, this helps with encapsulating living organisms.

[0063] 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, amongst others. In one or more embodiments, the carboxymethyl polysaccharide has a substitution lower than 1. In one or more embodiments, the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa. Without wishing to be bound by theory, it is believed that the large porosity of the disclosed porous hydrogel is produced because of competition between the carboxymethyl polysaccharide with the polyanion for crosslinking. Further, the polyelectrolyte complexes formed by the interaction between the carboxymethyl polysaccharide and the polycation has higher water solubility than the polyelectrolyte complexes formed between the polyanion and the polycation, potentially leading to phase separation that creates the unexpectedly large porosity.

[0064] In one or more embodiments, the weight ratio of the polyanion to the carboxymethyl polysaccharide is in a range from 3: 1 to 1 : 1. In one or more embodiments, the weight ratio of the polyanion to the polycation is from 2: 1 to 1 : 1.

[0065] The porous hydrogel can have a variety of morphologies. In one or more embodiments, the porous hydrogel is in the form of a spherical capsule. In one or more such embodiments, spherical capsules of the porous hydrogel have an average diameter of from 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. Further, in one or more embodiments, the hollow cylindrical tube can have length up to several meters or even up to several hundred 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 lOx, at least 25x, at least 50x, or at least lOOx the width. In one or more embodiments, the width is at least 2x, at least 5x, at least lOx, or at least 20x the thickness. In one or more embodiments, the ribbon of porous hydrogen can be made by flattening a cylindrical tube of the porous hydrogel.

[0066] The porous hydrogel can be used to encapsulate an organism. In particular, the organism can be encapsulated within an outer hydrogel membrane forming the spherical capsule or cylindrical tube. In one or more embodiments, the organism can be encapsulated during formation of the porous hydrogel or after formation of the porous hydrogel (e.g., by growing the organism within the hydrogel). In either case, the porous hydrogel can be loaded with organisms, such as organoids, spheroids, or cell aggregates. In one or more embodiments, the organisms have a size (e.g., maximum cross-sectional dimension) larger than the pores of the porous hydrogel such that the organism becomes trapped within the hydrogel. Once entrapped, the organisms release their extracellular vesicles, which are able to be transported through the relatively large pores of the disclosed hydrogel capsule membrane.

[0067] 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 the polyanion to link with the polycation, 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-clumping agent (such as GIBCO™ available from ThermoFisher Scientific, Waltham, MA). In one or more embodiments, alginate lyase can also be used to digest the alginic acid polyanion.

[0068] Embodiments of the present disclosure also relate to a method of forming the porous hydrogel. FIG. 1 A provides a process flow diagram of the method 100 of forming the porous hydrogel. The method 100 involves a first step 101 of forming a first solution containing the polyanion and the carboxymethyl polysaccharide. In a second step 102, the first solution is added to a second solution containing the polycation to form the porous hydrogel having the large pore size according to the present disclosure.

[0069] As shown in FIG. IB, the first step 101 can involve a plurality of substeps. In one or more embodiments, the first step 101 includes a first substep 101a of forming a third solution containing the polyanion. In one or more embodiments, the third solution comprises 0.5 wt% to 5 wt% of the polyanion. In one or more embodiments, the first step 101 includes a second substep 101b of forming a fourth solution comprising the carboxymethyl polysaccharide. In one or more embodiments, the fourth solution comprises from 0.5 wt% to 5 wt% of the carboxymethyl polysaccharide. In one or more embodiments, the first step 101 further includes a third substep 101c of blending the third solution with the fourth solution. As mentioned above, the ratio of the polyanion to carboxymethyl polysaccharide in the first solution is in the range of 3: 1 to 1: 1, and thus, the third solution and fourth solution are selected accordingly. In one or more embodiments, the third solution and the fourth solution combine to form the first solution having a viscosity of 50 cPas to 5000 cPas. Below 50 cPas, few capsules or tubes are formed, and above 5000 cPas, it is difficult to prepare small diameter capsules or tubes. [0070] In one or more embodiments, the first step 101 may further include a fourth substep lOld of sterilizing the first solution before the first solution 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.

[0071] If it is desired to encapsulate an organism in the porous hydrogel, the first step 101 may further include a fifth substep lOle of suspending the organism in the first solution prior to adding the first solution to the second solution.

[0072] When adding the first solution to the second solution during the second step 102, the morphology of the hydrogel is influenced by the manner in which the first solution is added. In particular, spherical capsules are obtained by dripping drops of the first solution into the second solution. The size of the spherical capsules is based on the size of the drops of the first solution that are dripped in the second solution. Hollow cylindrical tubes of the porous hydrogel can be obtained by continuously extruding the first solution into the second solution. Further, as mentioned above, ribbons of the porous hydrogel can be obtained by flattening the hollow cylindrical tubes.

[0073] Having generally described the porous hydrogel and method of making same, the following experimental examples illustrate particular embodiments.

[0074] Experimental Examples

[0075] Preparation of chitooligosaccharide (COS) from chitosan

[0076] In this example, the polycation was selected to be COS. To prepare the 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. To the flask, 20 g of chitosan were added, and the suspension was left undisturbed for 10 minutes. Thereafter, the suspension was stirred at 250 rpm in an oil bath set at 70 °C. After about 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 into centrifuge tubes and centrifuged at 3000 rpm to remove water insoluble matter. The supernatants containing water-soluble COS were pooled and dropped into about 1.5 liters of 200 proof ethanol (in a 2 -liter beaker) to precipitate the COS. The solution was stirred with a spatula to help flocculation, and the solid was allowed to settle overnight. The supernatant was discarded by aspiration using a peristaltic pump, leaving a moist cake of COS. The moist cake was transferred to a 50 mL centrifuge tube and was washed with ethanol three times and with diisopropylether three times. The solid was dried by vacuum drying. About 6.838 g of COS was obtained (corresponding to a yield of 34% of the original 20 g of chitosan).

[0077] Preparation o f the first solution

[0078] As discussed above, the first solution may be prepared by combining a third solution containing the polyanion with a fourth solution containing the carboxymethyl polysaccharide.

[0079] 1. Preparation o f third solution

[0080] A 2% by weight aqueous alginate solution (third solution) was prepared by dissolving the 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 two hours.

[0081] 2. Preparation o f fourth solution

[0082] To prepare the first solution, the third solution containing alginate was combined with a plurality of fourth solutions containing different carboxymethyl polysaccharides. Preparation of the fourth solutions is described below.

[0083] A, FITC-labeled CM Dextran

[0084] A 2% by weight aqueous fluorescein isothiocyanate labeled (FITC-labeled) carboxymethyl (CM) dextran solution 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 deionized water. The FITC-labeled CM dextran had a molecular weight of 150 kDa, a CM content of 3-7%, and an extent of labeling of 0.001-0.020 mol FITC per mol of glucose. Sonication was used to facilitate the dissolution.

[0085] B, CM dextran

[0086] A 2% by weight aqueous carboxymethyl (CM) dextran solution was prepared by dissolving 0.8 g of CM dextran having a molecular weight of 150 kDa (Carbosynth YC64861 available from Biosynth International, Inc., Louisville, KY) in 39.2 deionized water. Sonication was used to facilitate dissolution. [0087] C. CM cellulose

[0088] A 2% by weight aqueous carboxymethyl (CM) cellulose was prepared by dissolving 0.8 g of CM cellulose in 39.2 g deionized water. The CM cellulose had a molecular weight of 90 kDa. Sonication was used to facilitate the dissolution.

[0089] Mixture o f the third solution and the fourth solution

[0090] To prepare 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 blending 1 mL of the third solution of 2% by weight alginate with 1 mL of the fourth solution of 2% FITC-Labeled CM dextran. First solution B was prepared by blending 1 mL of the third solution of 2% by weight alginate with 1 mL of the fourth solution of CM dextran. First Solution C was prepared by blending 1 mL of the third solution of 2% by weight alginate with 1 mL of the fourth solution of CM cellulose.

[0091] Preparation o f the second solution

[0092] A 1% by weight of a solution of COS was prepared by dissolving 0.4 g of COS in 39.6 g deionized water. Sonication was used to facilitate the dissolution.

[0093] Example 1

[0094] To prepare spherical capsules, the second solution of 1% by weight of COS was provided in a 150 mL beaker equipped with a magnetic stir bar. Stirring speed was set to 100 rpm. A syringe, having a 34-gauge, blunt-end needle, was used to drip First solution A (alginate/FITC-labeled CM dextran) into the second solution of COS. The drips were dropped from a height of about 3 cm. After 5 minutes of crosslinking time, the spherical capsules were collected using a fiber mesh strainer and were washed with pure water. The spherical capsules are shown in FIG. 2. The capsules had an average diameter in the range of 1.8 mm to 2 mm.

[0095] To access the surface porosity, the 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). The 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 FIG. 3. Images (a)-(e) of FIG. 3 include increasing amounts of FITC-labeled CM dextran relative to alginate. Image (a) contains no 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 ratios of 0.25: 1, 0.5: 1, and 0.75: 1 ratios of FITC-labeled CM dextran to alginate, respectively. As can be seen in image (c), some porosity emerges at a ratio of 0.5: 1 of FITC- labeled CM dextran to alginate, and a large amount of porosity is present in image (e), depicting a ratio of 1 : 1 FITC-labeled CM dextran to alginate.

[0096] To verify that the porosity was not created by thermally inducing phase separation upon freeze drying, some capsules were dried by ethanol/water gradient before vacuum drying. In particular, the capsules were successively incubated in ethanol/water solution at ratios ranging from 0: 100 to 100:0, and then the water-free capsules were dried under vacuum. FIG. 4 provides an SEM image of the capsules at increasing magnification, and the images demonstrate that the porosity remains uniform and that the pore size remains large, indicating that the porosity is not a function of thermally induced phase separation from freeze drying.

[0097] Example 2

[0098] As with Example 1, the capsules of Example 2 were prepared starting with the second solution of 1% by weight of COS provided in a 150 mL beaker equipped with a magnetic stir bar. Stirring speed was set to 100 rpm. A syringe, having a 34-gauge, blunt- end needle, was used to drip First solution B (alginate/CM dextran) into the second solution of COS. The drips were dropped from a height of about 3 cm. After 5 minutes of crosslinking time, the spherical capsules were collected using a fiber mesh strainer and were washed with pure water. FIG. 5 depicts the spherical capsules produced. The spherical capsules are substantially the same as the spherical capsules produced using First Solution A, but the capsules lacked the fluorescence caused by the FITC labeling.

[0099] The porosity was observed under magnification, and FIG. 6 depicts the porosity of the spherical capsule prepared under the same conditions as Example 1. As can be seen, the spherical particle includes uniform porosity and pore size of greater than 2 pm.

[00100] Example 3

[00101] As with Examples 1 and 2, the capsules of Example 3 were prepared starting with the second solution of 1% by weight of COS provided in a 150 mL beaker equipped with a magnetic stir bar. Stirring speed was set to 100 rpm. A syringe, having a 34-gauge, blunt- end needle, was used to drip First solution C (alginate/CM cellulose) into the second solution of COS. The drips were dropped from a height of about 3 cm. After 5 minutes of crosslinking time, the spherical capsules were collected using a fiber mesh strainer and were washed with pure water.

[00102] The porosity was observed under magnification, and FIG. 7 depicts the porosity of the spherical capsule prepared under the same conditions as Examples 1 and 2. As can be seen, the spherical capsules also include uniform porosity and pore size of greater than 2 pm.

[00103] Example 4

[00104] Example 1 was repeated with the exception that the First Solution A of alginate/FITC-labeled CM dextran was made with 1.5% by weight alginate and 1% by weight of FITC-labeled CM dextran. First Solution A was dripped in the second solution of 1% by weight COS and allowed to crosslink for 5 minutes. As shown in FIG. 8, the porosity of the spherical capsules produced was in the range of 4 pm to 40 pm.

[00105] Example 5

[00106] Example 1 was again repeated with the exception that the third solution of 1% alginate was dissolved at 80 °C, and First Solution A (containing alginate and FITC-labeled CM dextran) was autoclaved for 30 minutes at 121 °C to sterilize it. FIG. 9 is an SEM image of the porosity of the spherical capsules. As can be seen, the pores have a large size and are uniform across the surface.

[00107] Example 6

[00108] First Solution A (containing alginate and FITC-labeled CM dextran) was continuously extruded from a syringe with a 32-gauge needle into the second solution of 1% by weight COS to form microtubes. The microtubes were left in the second solution for 5 minutes to crosslink, withdrawn from the solution using a nylon mesh, and washed with water. FIGS. 10, 11, and 12 depict increasing magnifications of a microtube prepared according to this method, showing the dimensions of the microtubes. The nylon mesh can be seen in the background behind the microtubes. FIG. 13 provides a further magnification of the microtubes showing the porosity of the microtube surface. As can be seen in FIG. 13, the pores are substantially uniform across the surface and have a size of about 2 pm.

[00109] Comparative Example 1

[00110] Spherical capsules were prepared according to Example 1 with the exception that the second solution of 1% by weight of COS was replaced with 4% by weight of calcium chloride. The mixture of the solutions formed capsules, but no surface porosity was observed. From this, the inventor surmised that the replacement of COS with calcium chloride affects the crosslinking behavior of the solutions. In particular, a relatively low molecular weight polycation, such as COS, promotes formation of large pores, whereas the ionotropic crosslinking using inorganic anions does not promote large porosity.

[00111] Comparative Example 2

[00112] Example 1 was again repeated with the exception that the second solution of 1% by weight of COS was replaced with 1% by weight PDADMAC. PDADMAC is an effective crosslinker known to form capsules with cellulose sulfate. However, no capsules were obtained with First Solution A (alginate and FITC-labeled CM dextran). This comparative example demonstrates the importance of the low molecular weight polycation, such as COS, in crosslinking the alginate/FITC-labeled CM dextran.

[00113] The foregoing discussion demonstrates the ability to form hydrogels from a crosslinked network of polyanion, polycation, and carboxymethyl polysaccharide that have large pore sizes. Advantageously, the hydrogel contains naturally derived materials and no synthetic components and can be used to encapsulate living organisms. Further, the pores are large enough to allow for diffusion of large molecules from within the hydrogel to outside the hydrogel so that the hydrogel can be used, e.g., as a bioreactor.

[00114] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred. In addition, as used herein the article “a” is intended include one or more than one component or element, and is not intended to be construed as meaning only one.

[00115] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art, the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

Claims

What is claimed is:

1. A hydrogel, comprising: a cross-linked network of a polyanion, a polycation, and a carboxymethyl polysaccharide; wherein the hydrogel comprises an average pore size of at least 100 nm.

2. The hydrogel of claim 1, wherein the polyanion comprises at least one of alginic acid salt, pectic acid salt, pectinic acid salt, amidated pectin, or gellan gum.

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

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

5. The hydrogel of any one of claims 1-4, wherein the poly cation comprises a molecular weight of 10 kDa or less.

6. The hydrogel of any one of claims 1-5, wherein the poly cation comprises a molecular weight of 5 kDA or less.

7. The hydrogel of any one of claims 1-6, 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 1-7, wherein the carboxymethyl polysaccharide comprises a molecular weight of 50 kDa to 250 kDa.

9. The hydrogel of any one of claims 1-8, wherein a weight ratio of the polyanion to the carboxymethyl polysaccharide is from 3: 1 to 1 : 1.

10. The hydrogel of any one of claims 1-9, wherein a weight ratio of the polyanion to the polycation is from 2: 1 to 1 : 1.

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

12. The hydrogel of claim 11, wherein the spherical capsule comprises an average diameter of from 0.5 mm to 5 mm.

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

14. The hydrogel of any one of claims 1-10, wherein the hydrogel is a ribbon comprising a length, a width, and a thickness, wherein the length is greater than the width and the thickness, and wherein the width is greater than the thickness.

15. The hydrogel of any one of claims 1-14, wherein the average pore size is up to 5 pm.

16. The hydrogel of any one of claims 1-15, wherein an organism is encapsulated within pores of the hydrogel.

17. 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 polycation to form a hydrogel comprising an average pore diameter of at least 100 nm.

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

19. The method of claim 17 or claim 18, wherein living organisms are suspended in the first solution prior to adding the first solution to the second solution.

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

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

22. The method of any one of claims 17-21, wherein the polyanion comprises at least one of alginic acid salt, pectic acid salt, pectinic acid salt, amidated pectin, or gellan gum.

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

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

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

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

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

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

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

30. The method of claim 29, wherein the spherical capsule comprises an average diameter of from 0.5 mm to 5 mm.

31. The method of any one of claims 17-28, 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. The method of claim 31, further comprising flattening the cylindrical tube to form a ribbon.

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

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

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

36. A method, comprising: encapsulating a living organism in a hydrogel, the hydrogel comprising a cross-linked 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 living organism through pores of the hydrogel.

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

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

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

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

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