WO2025043054A1 - Systems and methods for enzymatic reaction of modified agarose polysaccharides - Google Patents

Abstract

Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for various uses, such as for extracellular matrices, tissue fillers, regenerative implants, tissue scaffolds, wound dressings, or other applications, including biomedical applications. In some aspects, a modified agarose polysaccharide having certain secondary structures, such as a 0-sheet structure, may be reacted with an enzyme such as an agarase, e.g., to reduce its molecular weight or shear modulus, or degrade it, etc. This may be useful, for example, in certain in vivo applications where the modified agarose polysaccharide has been applied to a subject, and it is desired to alter the modified agarose polysaccharide in such a fashion. For example, an agarose implanted into a subject as a tissue filler may be altered by applying an agarase to the implant, which may allow the implant to be altered, reshaped, removed from the subject, etc. Other aspects as discussed herein are generally directed to methods of making or using such altered agaroses, other reactions involving such agarases (e.g., in vitro or in vivo), kits involving such altered agaroses, or the like.

Description

SYSTEMS AND METHODS FOR ENZYMATIC REACTION OF MODIFIED AGAROSE POLYSACCHARIDES

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/578,470, filed August 24, 2023, entitled “Systems and Methods for Enzymatic Reaction of Modified Agarose Polysaccharides,” by Sarem, et al., incorporated herein by reference in its entirety.

FIELD

Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for various uses, such as for tissue fillers or other applications, including biomedical applications.

BACKGROUND

Hydrogel forming polysaccharides such as agarose have had a tremendous impact on many areas of biomedical sciences. While historically their use was limited to the culture of bacteria, and electrophoresis, in recent years hydrogels have find applications as mammalian cell culture substrate and plant substrate. The application fields have been expanded to include the development of processing methods for the use of polysaccharide-based hydrogels as bioink (extrudable material that can encapsulate cells or serve as a carrier or substrate for cells), tissue filler, in vivo cell delivery, and drug delivery systems. In many of these applications, controlling the molecular weight of the polysaccharide is important to obtain precise mechanical properties of the hydrogel.

Due to their glycosidic bond, polysaccharides are prone to depolymerization through alkaline or acidic hydrolysis. The chemical bond between the sugar monomers can be cleaved in alkaline or acidic conditions. This process is difficult to control and lead to short oligosaccharides of low molecular’ weight which cannot form hydrogels.

Polysaccharides are natural molecules which are synthesized by plants (cellulose, lignin), algae (agarose, alginate), insect (chitin), mammalian cells (hyaluronic acid), or certain genetically modified organisms. These polysaccharides can be synthesized in living organism by enzymes and similarly, can be depolymerized by enzymes. For instance, hyaluronic acid can be depolymerized by hyaluronidase, alginate by alginate lyase, cellulose by cellulase and agarose by agarase. Each enzyme is designed by nature to be specific to the polysaccharide. Therefore, chemical modifications of the polysaccharide backbone will affect the efficacy of the enzyme to depolymerize the polysaccharide. For instance, cellulase docs not work on acetate cellulose, and a de-esterification enzyme is needed to deacetylate cellulose for the cellulase to depolymerize the polysaccharide. In addition, to be active only on a specific sugar, enzymes are actives within a specific range of condition including ion strength, type of ions, acidity, temperature, etc. For instance, hyaluronidase can depolymerize hyaluronic acid only below about 40 °C.

Among these natural polysaccharides, agarose, a polysaccharide that can be extracted from red seaweed, has been used for biomedical applications for over a century for applications such as growing prokaryotic cells, DNA electrophoresis and most recently for 3D cell culture assays. However, due to the limited physico-mechanical properties of native agarose (NA), which can only be manipulated by changing the polymer concentration or the addition of other polysaccharides such as hyaluronic acid, chemical modifications of NA have been proposed which has led to a new class of polysaccharide so called modified agarose polysaccharides (MAPs). See, e.g., Int. Pat. Apl. Pub. No. WO 2012/055596, published May 3, 2012; Int. Pat. Apl. Pub. No. WO 2013/023793 published February 21, 2013; U.S. Pat. No. 9,388,252, issued July 12, 2016; or U.S. Pat. No. 10,968,285, issued April 6, 2021. However, the development of chemical modifications of agarose raises significant questions of enzymatic depolymerization of the polysaccharide.

SUMMARY

Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for various uses, such as for tissue fillers or other applications, including biomedical applications. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

One aspect is generally drawn to a method. According to a first set of embodiments, the method comprises exposing modified agarose polysaccharide to an agarase. In some cases, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

The method, in another set of embodiments, comprises implanting a composition comprising modified agarose polysaccharide into a subject, and injecting agarase within 3 cm of the composition within the subject. In some embodiments, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties. The method, according to yet another set of embodiments, comprises injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in shear modulus (G') of at least 10%. In some cases, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

In still another set of embodiments, the method comprises injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) of at least 5 kDa. In certain cases, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

The method, in yet another set of embodiments, comprises injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) of at least 10%. In certain embodiments, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

In one set of embodiments, the method comprises liquefying modified agarose polysaccharide by exposing the modified agarose polysaccharide to an agarase. In certain cases, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

The method, in another set of embodiments, comprises liquefying modified agarose polysaccharide implanted within a subject by injecting agarase into the modified agarose polysaccharide. In some embodiments, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

Still another set of embodiments is generally directed to a method comprising exposing a first modified agarose polysaccharide to an agarase able to cause the first modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) to produce an altered agarose, and mixing the altered agarose with a second modified agarose polysaccharide exhibiting an at least partial [l-shcct structure. In some embodiments, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

The method, in yet another set of embodiments, comprises exposing modified agarose polysaccharide to an agarase able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) to produce an altered agarose, and mixing the altered agarose with hyaluronic acid. In certain embodiments, at least 5% of disaccharidc units in the modified agarose polysaccharide are carboxylate moieties.

In another set of embodiments, the method comprises injecting agarase into an occlusion of a blood vessel within a subject. In some cases, the occlusion is formed by agarose.

Yet another set of embodiments is generally directed to injecting agarase into a subject.

Another aspect is drawn to a kit. In some cases, the kit modified agarose polysaccharide, and agarase contained within solution. In some embodiments, at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

In one embodiment, the method comprises injecting, into a modified agarose polysaccharide exhibiting an at least partial p-sheet structure implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in shear modulus (G') of at least 10 Pa.

Several methods are disclosed herein of administering a subject with a composition for prevention or treatment of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the composition for use in the treatment or prevention of that particular condition, as well as use of the composition for the manufacture of a medicament for the treatment or prevention of that particular condition.

In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, modified agarose polysaccharides. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, modified agarose polysaccharides.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

Fig. 1 illustrates a reaction scheme for producing a modified agarose polysaccharide, in accordance with one embodiment;

Fig. 2 illustrates molecular weights for a modified agarose polysaccharide, in another embodiment;

Figs. 3A-3B illustrate the alteration of elastic modulus of a modified agarose polysaccharide, in still another embodiment; and

Figs. 4A-4B illustrate assays for determining carboxylation, in yet another embodiment.

DETAILED DESCRIPTION

Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for various uses, such as for extracellular matrices, tissue fillers, regenerative implants, tissue scaffolds, wound dressings, or other applications, including biomedical applications. In some aspects, a modified agarose polysaccharide having certain secondary structures, such as a P-sheet structure, may be reacted with an enzyme such as an agarase, e.g., to reduce its molecular weight or shear modulus, or degrade it, etc. This may be useful, for example, in certain in vivo applications where the modified agarose polysaccharide has been applied to a subject, and it is desired to alter the modified agarose polysaccharide in such a fashion. For example, an agarose implanted into a subject as a tissue filler may be altered by applying an agarase to the implant, which may allow the implant to be altered, reshaped, removed from the subject, etc. Other aspects as discussed herein are generally directed to methods of making or using such altered agaroses, other reactions involving such agarases (e.g., in vitro or in vivo), kits involving such altered agaroses, or the like.

As mentioned, some aspects as discussed herein are generally directed to systems and methods for altering modified agarose polysaccharides exhibiting another second structure, e.g., an at least partial P-sheet structure, for example, using enzymes such as agarases. Agarose is a polysaccharide normally having an a-helical structure, and is typically formed from repeating units of P-D-galactose and 3,6-anhydro-a-L-galactose. However, it can be modified to form a polysaccharide with another second structure, for example, an at least partial P-sheet structure, e.g., using certain reaction conditions such as those described herein. Modified secondary structures, such as P-sheet structures, can be readily determined using CD or other techniques such as those described herein. Such modified agarose polysaccharides may be useful for a variety of applications, including as extracellular matrices, regenerative implants, tissue fillers, wound dressings, or other applications. Examples of modified agarose polysaccharides include, but are not limited to, carboxylated agaroses, halogenated agaroses, sulfated agaroses, sulfonated agaroses, phosphorylated agaroses, and the like. Some modified agarose polysaccharides have previously been described in references such as Int. Pat. Apl. Pub. Nos. WO 12/55596 and WO 13/023793, or U.S. Pat. Nos. 9,388,252 and 10,968,285.

However, in some cases, after formation of the agarose, it may be desired to alter certain properties, for instance, its molecular weight, its shear modulus, or the like. In some cases, this may be performed using an enzyme such as an agarase. A variety of agarases are available commercially, and include a-agarases and p-agarases, based on whether they degrade a or p linkages in agarose. However, such agarases typically require elevated temperatures to operate, e.g., at least 45 °C (113 °F). Accordingly, such agarases often cannot be used for certain applications, e.g., for in vivo uses such as modifying agaroses implanted into a subject, as such elevated temperatures are usually detrimental to the subject.

As discussed herein, however, it has been surprisingly found that such agarases are able to react and enzymatically digest modified agarose polysaccharides, e.g., having P-sheet structures, such as those described herein. As mentioned, agarose naturally takes the form of a- helices, rather than P-sheets. Thus, while compositionally, an agarose and a modified agarose polysaccharide are similar, they have radically different secondary (and higher order) structures. Without wishing to be bound by any theory, it is believed that since agarase degrades agarose at the alpha- 1,3 or beta- 1,4 glycosidic bonds, and from the reducing ends, the modified agarose polysaccharides as discussed herein (e.g., with carboxylic acids, etc.) impede access of the agarse to such chemical bonds, and thus, the agarase is not able to react with the modified agarose polysaccharides, e.g., as discussed herein.

Thus, if a potential substrate had a radically different structure (e.g., a modified agarose polysaccharide having another secondary structure, such as a P-sheet structure, rather than an a- helical structure), the enzyme would be unable to recognize or bind to such a structure, and thus the enzyme would not be able to react with such a structure. Accordingly, such agarases would have been expected to bind to a-helical agaroses, but not modified agarose polysaccharides having different types of secondary structures. Indeed, it is believed that previously, only the action of agarase on agarose has been reported, and there have been no reports of agarases acting on modified agarose polysaccharides.

In addition, it has also been surprisingly found that under certain conditions, agarases such as those described herein are able to not only bind to and react with modified agarose polysaccharides having other secondary structures, such as P-sheet structures, they can do so under temperature conditions that are substantially lower than the temperatures under which such agarases are normally able to act. Agarases are generally derived from heat-resistant thermophilic bacteria in the deep ocean (e.g., around hydrothermal vents), and accordingly typically require elevated temperatures to operate, e.g., at least 45 °C (113 °F) or more in some cases. Such agarases cannot used in in vivo settings at those temperatures, as their use would result in, e.g., severe burns in subjects. However, as discussed herein, it has been found that certain agarases can nonetheless react with modified agarose polysaccharides, e.g., to alter properties such as its molecular weight, shear modulus, viscosity, etc., at surprisingly low temperatures.

In one aspect, the modified agarose polysaccharide contains at least one modified polysaccharide. The polysaccharide may be formed from repeating disaccharide units. Agarose is a natural, linear polysaccharide with no branching and has a backbone of 1,3-linked P-D- galactose-(l-4)-a-L-3,6 anhydrogalactose repeating units. Such dimeric repeating units may be derived from naturally occurring polysaccharides, and in some cases may be chemically modified by the specific oxidation of the primary hydroxyl, for example, to form carboxylate moieties, halogenated moieties, sulfate moieties, sulfonated moieties, phosphorylated moieties, or the like. One reaction mechanism is schematically shown in Fig. 1, although other mechanisms can also be used to form a modified agarose polysaccharide.

Fig. 1 illustrates the oxidation of a primary alcohol to a carboxylic acid, in accordance with certain non-limiting embodiments. Such oxidation can be performed, for example, using sodium hypochloride in the presence of 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO) and sodium bromide (NaBr). The reaction mechanism is shown in Fig. 1. The regioselective oxidation of the primary alcohol to the carboxylic acid can be also performed by other reactions. In addition, oxidation can also be carried out by an enzymatic process or upon using a bacteriological system. In one set of embodiments, a modified agarose polysaccharide may be prepared from native agarose by oxidizing a certain percentage of the primary hydroxyls on the native agarose, for example, to result in carboxylates, halogens, sulfates, sulfonates, phosphorylates, and the like. Examples of halogens include, but are not limited to, fluorides, chlorides, bromides, iodides, etc. In many cases, such oxidation reactions can alter the structure of the native agarose, typically converting it from an a-helical structure into another secondary structure, for example, a 0-sheet structure. Without wishing to be bound by any theory, it is believed that in part, this is because moieties such as these are larger than the native hydroxyls, and the agarose structure as a result must distort in order to fit the other moieties, thereby causing the modified agarose polysaccharide to have another second structure, for example, a P-sheet structure instead of an a- helical structure. In some cases, the beta sheet content may be a function of degree of oxidation, e.g., due to carboxylation, halogenation, sulfation, sulfonation, phosphorylation, etc.

In some embodiments, at least 5% of the modified agarose polysaccharide contains oxidized moieties, relative to native agarose (e.g., such that at least 5% of the hydroxyls on the disaccharide units are now other, oxidized moieties), and in some cases, at least 10%, at least

11%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 95%, at least 99%, or substantially all of the modified agarose polysaccharide contains oxidized moieties instead of hydroxyls. Non-limiting examples of oxidized moieties include carboxylates, halogens, sulfates, sulfonates, phosphorylates, etc. Non-limiting examples of such oxidation reactions can be seen in U.S. Pat. No. 9,441,051; other such reactions will be known to those of ordinary skill in the ait.

In some cases, no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the modified agarose polysaccharide contains oxidized moieties, relative to native agarose. Combinations of any of the following are also possible. As non-limiting examples, at least 20% to 99%, at least 50% to 95%, at least 60% to 80%, etc. of the modified agarose polysaccharide may contain oxidized moieties. These are typically determined as a percentage by number or a mole basis. Percentages such as these can be achieved, for example, by oxidizing native agarose in a controlled manner, or in some cases, oxidizing it completely so that about 100% of the hydroxyls have been oxidized.

There may be only one type of oxidized moiety present on the modified agarose polysaccharide, or there may be 2, 3, or more types of oxidized moieties present on the modified agarose polysaccharide. As non-limiting examples, all of the oxidized moieties may be carboxylates or carboxylic acids, the oxidized moieties may all be phosphorylated, or there may be a mixture of two or more moieties, such as phosphorylates and sulfonates, etc.

As mentioned, the modified agarose polysaccharide may, in certain embodiments, exhibit an at least partial P-sheet structure. The modified agarose polysaccharide may adopt an at least partial P-sheet structure due to the presence of oxidized moieties, relative to native agarose. The modified agarose polysaccharide may exhibit, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% P-sheet structure. The P-sheet structure can be determined, e.g., by circular dichroism, Raman optical activity, or by other suitable techniques known to those of ordinary skill in the art. However, it should not be assumed that this percentage is necessarily the same as the percentage of oxidized moieties within the modified agarose polysaccharide (although it can be).

In some embodiments, one or more side chains of the modified agarose polysaccharide may include a peptide or protein sequence. Non-limiting examples include the cell adhesion sequence arginine-glycine-aspartic acid (RGD), the peptide sequences IKVAV (SEQ ID NO: 1) and YIGSR (SEQ ID NO: 2) or a protein such as collagen, collagen fragments, fibronectin, hyaluronic acid, etc. In yet other embodiments, one or more side chains of the modified agarose polysaccharide may include a nucleic acid sequence. The nucleic acid sequence may comprise single- stranded DNA, double- stranded DNA, single-stranded RNA, siRNA, etc.

In addition, it should be understood that in some cases, different modified agarose polysaccharides may be present, and/or the modified agarose polysaccharides may be mixed together with native agaroses, in various embodiments. For example, a first modified agarose polysaccharide containing a first type of oxidized moiety may be mixed or blended together with a second modified agarose polysaccharide containing a second type of oxidized moiety, and/or with another first modified agarose polysaccharide containing a first type of oxidized moiety having a different molecular weight, and/or with native agarose, etc. Thus, there are a variety of different types of formulations, containing different amounts or concentrations of various modified agarose polysaccharides, that can be used.

In some embodiments, modified agarose polysaccharides such as those described herein may be present within a formulation. Such formulations may be used as implants, tissue fillers, cell or tissue scaffolds, wound dressings, and other applications, e.g., such as those described herein. As a non-limiting example, the formulation may be present as a gel, such as a hydrogel.

In certain aspects, the modified agarose polysaccharide may be exposed to an agarase, which may alter the modified agarose polysaccharide. For instance, the agarase may reduce its molecular weight or shear modulus, etc. of the modified agarose polysaccharide, and/or otherwise at least partially degrade the agarose. In addition, in some cases, applying the agarase to a modified agarose polysaccharide may cause the modified agarose polysaccharide to partially or fully liquefy. Such properties may be useful, for instance, for altering the modified agarose polysaccharide after its synthesis. Various applications are discussed in more detail herein.

As mentioned, agarases are able to react and enzymatically digest agarose, which has an a-helical structure, but are not known for reacting with substrates having another second structure, for example, a P-sheet structure, such as those described herein. Compositionally, while an agarose and a modified agarose polysaccharide are similar, they have substantially different secondary (and higher order) structures, and enzymes such as agarases can react with their substrate only if the substrate has a very specific structure, such as an a-helical structure. Accordingly, it is surprising that an agarase can enzymatically react and digest a modified agarose polysaccharide having another secondary structure, e.g., a P-sheet structure.

Examples of agarases include, but are not limited to, a-agarases and P-agarases. These agarases are identified based on whether they degrade a or P linkages in agarose, resulting in the production of oligosaccharides, e.g., ending with 3.6 anhydro-L-galactose with a-agarases and D-galactose with P-agarases.

Agarases may be derived, in some cases, from bacteria such as agarolytic bacteria or thermophilic bacteria. Such agarases typically require elevated temperatures in which to operate, e.g., at least 40 °C (104 °F) or at least 45 °C (113 °F). However, surprisingly, such enzymes may react with modified agarose polysaccharides at relatively lower temperatures. For example, the agarase may be applied to a modified agarose polysaccharide at temperatures of less than 40 °C, less than 35 °C, less than 30 °C, or less than 25 °C. In some cases, for example, the temperature may be between 35 °C and 40 °C, such as around body temperature (about 37 °C).

In one set of embodiments, the effect of the agarase on the modified agarose polysaccharide may be determined as a drop in shear modulus (G'). In some cases, the modified agarose polysaccharide may exhibit a drop in shear modulus of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc., as compared to before applying the agarase. In addition, in some embodiments, the modified agarose polysaccharide may exhibit a drop in shear modulus of at least 10 Pa, at least 20 Pa, at least 30 Pa, at least 50 Pa, at least 100 Pa, at least 200 Pa, at least 300 Pa, at least 500 Pa, at least 1 kPa, at least 3 kPa, at least 5 kPa, at least 10 kPa, etc., as compared to before applying the agarase.

In addition, in one set of embodiments, the effect of the agarase on the modified agarose polysaccharide may be determined as a drop in the molecular weight of the modified agarose polysaccharide, e.g., as measured by the weight average molecular weight, M_w. For instance, the molecular weight M_w may exhibit a drop of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc., as compared to before applying the agarase. In addition, in some embodiments, the modified agarose polysaccharide may exhibit a drop in M_w of at least 10 Da, at least 20 Da, at least 30 Da, at least 50 Da, at least 100 Da, at least 200 Da, at least 300 Da, at least 500 Da, at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 50 kDa, at least 100 kDa, etc., as compared to before applying the agarase.

In some aspects, the modified agarose polysaccharide may be present in a formulation. For instance, the modified agarose polysaccharide may be present in a gel such as a hydrogel. The agarase may thus be applied to the formulation to react with the modified agarose polysaccharide, e.g., as discussed above. Such formulations may be used for a variety of uses, including implants, tissue fillers, tissue scaffolds, wound dressings, and other applications. The formulation may also have any suitable shape, e.g., a tube, a sheet, or the like, such as those described herein. In addition, in certain embodiments, more than one such modified agarose polysaccharide may be present in a formulation. For example, in one set of embodiments, a formulation may contain two, three, or more modified agarose polysaccharides, e.g., as discussed herein. In addition, in some cases, one or more modified agarose polysaccharides in a formulation may be exposed to an agarase, e.g., as discussed herein, to alter certain properties, for instance, its molecular weight, its shear modulus, viscosity, or the like. In some embodiments, this may be combined with another polymer in the formulation, for example, hyaluronic acid, poly-L-lactic acid, poly(methylmethacrylate), another modified agarose polysaccharide including any of those discussed herein, etc.

In one set of embodiments, the formulation may have a shear modulus that can range, for example, from about 10 Pa to about 10⁷ Pa. By using agaroses and modified agarose polysaccharides with different properties, chemical modifications, etc., the structure of the formulation can be controlled, for example, to have a variety of shear moduli, depending on the particular application.

For example, the formulation containing a modified agarose polysaccharide such as discussed herein may have a shear modulus of at least 1 Pa, at least 3 Pa, at last 5 Pa, least 10 Pa, at least 20 Pa, at least 30 Pa, at least 50 Pa, at least 100 Pa, at least 300 Pa, at least 500 Pa, at least 1 kPa, at least 3 kPa, at least 5 kPa, at least 10 kPa, at least 30 kPa, at least 50 kPa, at least 100 kPa, at least 300 kPa, at least 500 kPa, at least 1 MPa, at least 3 MPa, at least 5 MPa, at least 10 MPa, etc. In addition, the formulation may have a shear modulus of no more than 10 MPa, no more than 5 MPa, no more than 3 MPa, no more than 1 MPa, no more than 500 kPa, no more than 300 kPa, no more than 100 kPa, no more than 50 kPa, no more than 30 kPa, no more than 10 kPa, no more than 5 kPa, no more than 3 kPa, no more than 1 kPa, no more than 500 Pa, no more than 300 Pa, no more than 100 Pa, no more than 50 Pa, no more than 30 Pa, no more than 10 Pa, no more than 5 Pa, no more than 3 Pa, etc. Combinations of any of these are possible. For example, the shear modulus of a formulation may be between 1 Pa and 100 kPa, between 1 Pa and 50 kPa, between 10 Pa and 10 kPa, between 100 kPa and 5 MPa, etc.

In addition, in one set of embodiments, the formulation containing a modified agarose polysaccharide such as discussed herein may have any suitable average molecular weight of the modified agarose polysaccharides within the formulation. By using agaroses and modified agarose polysaccharides with different properties, chemical modifications, etc., the overall molecular weight of the modified agarose polysaccharides within the formulation can be controlled.

For example, the average molecular weight of the modified agarose polysaccharides may be at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 30 kDa, at least 50 kDa, at least 100 kDa, at least 300 kDa, at least 500 kDa, at least 1 MDa, at least 3 MDa, at least 5 MDa, at least 10 MDa, etc. In addition, the molecular weight may be no more than 10 MDa, no more than 5 MDa, no more than 3 MDa, no more than 1 MDa, no more than 500 kDa, no more than 300 kDa, no more than 100 kDa, no more than 50 kDa, no more than 30 kDa, no more than 10 kDa, no more than 5 kDa, no more than 3 kDa, no more than 1 kDa, etc. Combinations of any of these are possible. For example, the average molecular weight of the modified agarose polysaccharides within a formulation may be between 1 Da and 100 kDa, between 1 Da to 50 kDa, between 10 Da to 10 kDa, etc.

In some embodiments, other materials may also be present in the formulation, e.g., in addition to one or more modified agarose polysaccharides. For example, the formulation may include a solute such as saline, or other components such hyaluronic acid, calcium hydroxylapatite, poly-L-lactic acid, poly(methylmethacrylate), heparin sulfate, dermatan sulfate, chondroite sulfate, alginate, chitosan, pullulan, k-carrageenan, or the like. As still other examples, pharmaceutically active agents such as growth factors, insulin, biologically active peptides, chemokines, cytokines, steroids, antibiotics, analgesics, anti-inflammatory agents, anticancer drugs, local anesthetics such as lidocaine, amino acids, antioxidants, vitamins, minerals, neurotoxic proteins such as Botulinum toxin or the like may be present within a formulation. In addition, in some embodiments, the formulation may be used for diagnostic purposes. For example, the formulation may include imaging agents such as MRI contrast agents, CT contrast agents, fluorescent imaging probes, radioactive nuclei, etc.

In one aspect, the formulation containing a modified agarose polysaccharide may be present within a subject. For example, the formulation may be implanted, injected, or otherwise placed into the subject, for example, into a body cavity within the subject. The subject may be human, or a non-human animal. Examples of subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, the subject is a non-mammal such as a bird, an amphibian, or a fish. However, it should be understood that the formulation need not be present within a subject in all embodiments.

For example, the formulation may be a cell scaffold or a tissue scaffold, e.g., for growing cells. The scaffold may be present in vitro and/or in vivo, e.g., implanted into a subject.

In some cases, the cells grown on or in the formulation may include, but are not limited to, chondrocytes, osteoblasts, osteoclast, skin epithelial cells, intestinal epithelial cells, corneal epithelial cells, astrocytes, neurons, oligodentrocytes, smooth muscle cells, endothelial cells, cardiomyocytes, pancreatic islet cells, kidney epithelial cells, embryonic stem cells, pluripotent stem cells, or naive cells obtained from umbilical cord, etc. The cells may be human or nonhuman in various embodiments.

In certain cases, the cells on the formulation may be stem cells, e.g., which have the ability to differentiate into the desired cells. This can be achieved, for example, by adding such cytokines which lead to the development of the desired tissue. The stem cells may be embryonic stem cells, or other types of stem cells such as mesenchymal stem/stromal cells. In some cases, the stem cells are somatic stem cells which may, for example, be adipose-derived. In another embodiment, reprogrammed pluripotent somatic cells can be used. In yet another embodiment, amniotic stem cells can be used.

In some cases, the formulation may be used in an artificial tissue. It is, for example, possible to grow cells as described herein to produce artificial three-dimensionally linked tissues which, in certain embodiments, can be used as an implant, for example, for the curing of various defects. It is, for example, possible to produce homologous bone structures by cultivating osteoblasts and/or osteoclasts in the formulation. As another example, artificial skin or cartilage can be produced. As yet other non-limiting examples, in some embodiments, the formulation can be used as a plastic surgery implant for reconstructive and cosmetic surgery in diverse body regions: in the facial region such as a nose, a forehead, a jaw, a cheek but also in a breast, a hip, a calf and the like.

In yet another example, the formulation can be used as a regenerative implant. Such a regenerative implant may be produced in vitro by using the formulation as a scaffold, which may grow three-dimensionally in vitro. After the implant has reached a certain structure, it can be implanted. In some cases, the form of the implant can be controlled using the appropriate stiffness and viscosity or the required modulus, e.g., by exposing the formulation to an agarase such as is discussed herein.

In still another embodiment, the formulation can be used as a wound dressing. The formulation may be applied to partially or fully cover a wound. For instance, the formulation may be formed into a bandage that is applied to the wound, or grafted into the site of a wound of a subject.

In yet another embodiment, the formulation can include pharmaceutically active components. By varying the mechanical properties of the formulation, e.g., by exposing it to a suitable agarase such as is discussed herein, the formulation can be tailored to a desired purpose. If, for example, the formulation is relatively flexible, pharmaceutical agents which may be included within the formulation may be released relatively quickly.

In some cases, the formulation may include pharmaceutically active agents such as, but not limited to, growth factors, insulin, biologically active peptides, chemokines, cytokines, steroids, antibiotics, analgesics and anti-inflammatory agents or anti-cancer drugs.

The formulation may have various forms. For example, in one embodiment, the formulation may include one or more sheets with a defined thickness. For instance, the formulation may be incubated with dermal cells to produce artificial skin or a dermal implant, etc. As another example, the formulation may comprise one or more tubes, which may be incubated with suitable cells to form blood vessels, such as artificial arteries or veins. As yet another example, the formulation may have the form of a disc. For instance, the formulation may be brought into contact with cells which form cartilage tissues. Since the mechanical properties like stiffness, rigidity and viscosity of the formulation can be controlled, for example, by selecting the appropriate ratio of modified polysaccharide: unmodified polysaccharide (e.g., modified agarose:unmodified agarose), and/or by exposing the formulation to suitable amounts of agarase (e.g., such as is described herein), the properties of the formulation (or resulting implant, etc.) can be controlled. The thickness, length or any other desired form of the formulation can be prepared, for example, by using a suitable form or mold.

In some cases, the formulation may be brought into contact with the desired cells, e.g., in an appropriate medium which may contain desired growth factors in certain embodiments. Appropriate cytokines may be included in some cases. In certain embodiments, it is possible to sequentially and/or simultaneously add two or more different types of cells. In some cases, such formulations can be used as regenerative implants. For example, it is possible to produce regenerative implants which can be used as artificial skin, as artificial blood vessels or for the replacement of nerve tissues. As another example, it is also possible to produce mucosal tissues or parts of the eye, such as artificial lenses. In some cases, the formulation may be optically clear or transparent.

In one set of embodiments, a formulation containing a modified agarose polysaccharide may be present as a tissue filler within the subject. The tissue filler may be a dermal filler or another type of filler, e.g., for use internally of a subject. In some embodiments, the formulation may be an injectable material, for example, that can be injected into the skin of a subject, or internally of a subject. For instance, the formulation may act as a dermal filler that can be used to restore volume, smooth lines, soften creases, reduce wrinkles, alter facial contours, or the like.

In some cases, however, the tissue filler introduced into a subject may have been introduction into an undesired location and/or have an undesired shape or effect, etc. In some cases, the tissue filler may contain modified agarose polysaccharide, and agarase may be introduced to partially or completely reverse the dermal filler, for example, by causing the modified agarose polysaccharide to liquefy, and/or by reducing properties such as its molecular weight, shear modulus, viscosity, etc., that may allow the dermal filler to be reshaped, removed, etc. As an example, the agarase may be injected or proximate into the formulation containing the modified agarose polysaccharide. For example, the agarase may be injected within 5 cm, within 4 cm, within 3 cm, within 2 cm, or within 1 cm of the modified agarose polysaccharide.

Yet another set of embodiments is generally directed to formulations used as food additives. Such food additives may be useful for the preparation of foods, drinks or seasonings, etc. For instance, examples of such food include the following: products of processed cereal (e.g., wheat flour products, starch products, premixed puddings, jam, buckwheat noodle, wheat- gluten bread, jelly bears, gelatin noodle and packed rice cake, etc.), products of processed fat and oil (e.g., margarine, salad oil, mayonnaise, dressing, etc.), products of processed soybeans (e.g., tofu, miso, fermented soybean, etc.), products of processed meat (e.g., brawn, sausage, etc.), processed marine products (e.g., frozen fish, fish paste, fish fingers, seafood, etc.), dairy products (e.g., raw milk, cream, yogurt, butter, cheese, condensed milk, powdered milk, ice cream, etc.), products of processed vegetables and fruits (e.g., paste, jam, pickles, etc.), and the like. Any processes including cooking, processing and the like can be used, and the formulation may be added before, during or after cooking or processing.

In still another set of embodiments, the formulation may be used as a component of cosmetic compositions such as skin care, make-up, blush, lipstick, eyeshadow, antiperspirants, deodorants, concealer, etc. In some embodiments, the cosmetic compositions are solid or semisolid at temperature of 25 °C. In some cases, the formulation may be molded into the form of a stick. In certain instances, for example, the formulation can be heated until molten and then poured into a mold and cooled. The physical properties of the cosmetic compositions can be adjusted, for example, by exposing the formulation to a suitable amount of agarase, e.g., as discussed herein.

In another set of embodiments, the formulation may be used as a material for industrial purposes, for example, for dispersion control, for conditioning of liquids, as a lubricant, etc. In some cases, properties such as the stiffness and/or temperature of gelation can be controlled, for example, by exposing the formulation to a suitable amount of agarase, e.g., as discussed herein.

U.S. Provisional Patent Application Serial No. 63/578,470, filed August 24, 2023, entitled “Systems and Methods for Enzymatic Reaction of Modified Agarose Polysaccharides,” by Sarcm. el al., is incorporated herein by reference in its entirety.

The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

EXAMPLE 1

This example illustrates enzymatic depolymerization of carboxylated agarose polysaccharide, in accordance with certain embodiments. In this example, certain experiments are used to evaluate different natural enzymes for the depolymerization of MAPs. The activity of these enzymes between NA and MAPs is compared using gel permeation chromatography and rheology to characterise the loss of average molecular weight (M_w) and gel stiffness (G'), respectively.

Materials and methods. The enzyme agarase was obtained from ThermoFischer (EO0461) and used without further purification. The agarase enzyme was supplied in a storage buffer composed of 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 % (v/v) Triton X-100 and 50 % (v/v) glycerol. Agarose was obtained from Merck (Darmstadt, Germany). Phosphate buffer saline was obtained from Lonza (Basel, Switzerland). MAP synthesis. One gram of native agarose (NA) type 1 was transferred into a threenecked round bottom flask, equipped with a mechanical stirrer and pH meter. The reactor was heated to 90 °C to dissolve the agarose and then cooled to 0 °C under mechanically stirring to prevent the solution for gelling. The reactor was then charged with TEMPO (2, 2,6,6- Tetramethylpiperidin-l-yl)oxyl) (0.160 mmol, 20.6 mg), NaBr (0.9 mmol, 0.1 g), and NaOCl (2.5 mL, 15% vol/vol solution) under vigorous stirring. The pH of the solution was adjusted to pH 10.8 throughout the duration of the reaction, and the degree of carboxylation was controlled by the addition of predetermined volumes of NaOH solution (0.5 M). At the end of the reaction NaBH4 (0.1 g) was added, and the solution was acidified to pH 8 and stirred for 1 h. The MAP was precipitated by sequential addition of NaCl (0.2 mol, 12 g) and ethanol (500 mL), and the solid was collected by vacuum filtration and extracted using ethanol. Residual ethanol was removed by extensive dialysis against water and the MAP was obtained as a white solid upon freeze-drying overnight.

Gel permeation chromatography. A hydrogel was made of 2% w/v of MAP or NA by dissolving the dry polysaccharide at 90 °C in PBS and allowing for gelation at 4 °C for 24h. The formed hydrogel (200 microliters) was added to a 1.5 mL Eppendorf container. To the hydrogel, 200 microliters of a solution of enzyme solubilized in PBS according to the concentration provided by the supplier. The container was homogenized by a 60 second vortex. The container was then incubated at 37 °C for 24 h. After incubation, the solution was heated to 90 °C for 10 min to inhibit the enzyme. The solution was then prepared for gel permeation chromatography by diluting the 250 microliters of the solution in 1750 microliters of NaCl buffer. The solution was then filtered with a 0.45 micrometer filter and injected in a Suprema column (PSS, Germany), calibrated with a Pullulan (PSS, Germany) and equipped with a UV and RI detector (Fig- 2).

Rheology. A hydrogel was made of 2% w/v MAP or NA by solubilizing the dry polysaccharide in PBS at 90 °C. The solution of hydrogel precursor (200 microliters) was loaded in 1 mL syringes. The enzyme stock solution (20 microliters) was diluted in PBS (180 microliters) to obtain a 10U solution. 200 microliters of the diluted enzyme solution were loaded in a 1 mL syringe. For a negative control, 200 microliters of PBS was loaded in a 1 mL syringe. The two syringes were connected with double female Luer connector. The enzyme solution and hydrogel were pushed back and forth 50 times by pushing alternatively each syringe piston. The PBS solution and hydrogel were pushed back and forth 50 times by pushing alternatively each syringe piston. The homogenized hydrogel was then deposited on a 25 mm Kincxus rheometer plate (Malvern, UK). Using a conical geometry (CP 1/40) with an active solvent trap filled with water, the elastic shear modulus of the hydrogel-enzyme was measured for 3 hours (180 min). The frequency was fixed at 1 Hz, the shear strain at 0.1%, and temperature at 37°C.

Agarase, agarose 4-glycanohydrolase, is a hydrolase enzyme isolated from agarolytic bacteria and cleave the P-linkage between the 3,6 anhydro-L-galactose and D-galactose. The recommended conditions of use for agarase were pH 5.5 at 42 °C. It is usually used for the digestion of agarose hydrogel after electrophoresis to recover DNA strands. For optimal digestion, it is recommended to solubilise the agarose at 70 °C before initiating the digestions. There are few protocols for the digestion of agarose at physiological conditions (pH 7.5, 37 °C).

Next the hydrolase kinetic was measured by rheology. The MAP hydrogel was mixed with the enzyme and directly deposited between the two plates of the rheometer and the elastic shear modulus was measured over a period of three hours at 37 °C. While MAP without enzyme did not exhibit a loss of elastic shear modulus over the measurement period (Fig. 3A), a drastic loss of shear modulus was observed for the MAP in PBS when mixed with 10 units of agarase. The first MAPs gel shear modulus loss happened immediately after mixing with agarose and the modulus loss intensified after 20 minutes. After 1 hour, the G’ of MAP treated by agarose decreased significantly (Fig. 3B).

Fig. 3 illustrates the elastic modulus of the MAP exposed to 10 U of agarase over 180 minutes (3 h), showing that the MAP hydrogel is digested by the enzyme.

Thus, these results demonstrate that the natural enzyme beta-agarase can depolymerize in a control manner modified agarose polysaccharides. It was demonstrated that the carboxylated agarose can be depolymerized with the enzyme, thus suggesting that other MAPs are expected to be recognized by the enzyme.

Thus, these results demonstrate that the natural enzyme beta-agarase can depolymerize in a control manner modified agarose polysaccharides (MAPs). It was demonstrated that the carboxylated agarose can be depolymerized with the enzyme, thus suggesting that other MAPs are expected to be recognized by the enzyme.

EXAMPLE 2 This example illustrates a technique for determining carboxylic acid content in sugar using spectroscopy at 530 nm after a reaction with carbazole in sulfuric acid. The assay is based on a colorimetric reaction between carbazole and uronic acids, a form of carboxylated sugar, which are present in the modified agarose polysaccharide and agarose. This reaction leads to the formation of furfural derivatives, which can be detected and quantified by measuring the color intensity using spectrophotometry.

To perform the assay, the modified agarose polysaccharide sample was first heated in a solution containing sulfuric acid and borate ions. This step facilitates the conversion of uronic acid groups into furfural derivatives, which are the compounds responsible for the color formation. The heating process promotes the reaction between the uronic acid groups and the sulfuric acid, leading to the generation of furfural derivatives.

After heating, the sample is then treated with carbazole. Carbazole reacts with the furfural derivatives produced in the previous step, resulting in the formation of colored compounds. The intensity of the color formed is directly proportional to the amount of uronic acid groups present in the agarose sample.

To quantify the carboxylation level in the agarose sample, an external calibration curve was constructed using D-glucuronic acid as a standard. D-glucuronic acid is a known compound that contains uronic acid groups. By preparing a series of standard solutions with known concentrations of D-glucuronic acid and measuring their absorbance using spectrophotometry, a calibration curve can be generated.

Next, the absorbance of the carboxylated agarose sample was measured using the same spectrophotometric method. By comparing the absorbance of the sample to the calibration curve, the concentration of uronic acid groups, and thus the level of carboxylation in the agarose, can be determined

In this example, samples containing agarose or MAP at varying concentrations of between 0 micrograms/g and 120 micrograms/g in PBS were prepared and analyzed with by spectroscopy at 530 nm OD-DO. Visible coloration and increasing intensity of the standard solutions after the acid hydrolysis step (sulfuric acid + sodium tetraborate), were observed, as shown in Figs. 4A and 4B. Thus, the degree of reaction with carbazole, producing a carboxylate, can be determined. While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A method, comprising: exposing modified agarose polysaccharide to an agarase, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

2. The method of claim 1, wherein at least 10% of the disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

3. The method of any one of claims 1 or 2, wherein at least 50% of the disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

4. The method of any one of claims 1-3, wherein at least 10% of the disaccharidc units in the modified agarose polysaccharide are halogen moieties.

5. The method of any one of claims 1-4, wherein at least 50% of the disaccharide units in the modified agarose polysaccharide are halogen moieties.

6. The method of any one of claims 1-5, wherein at least 10% of the disaccharide units in the modified agarose polysaccharide are sulfate moieties.

7. The method of any one of claims 1-6, wherein at least 10% of the disaccharide units in the modified agarose polysaccharide are sulfonate moieties.

8. The method of any one of claims 1-7, wherein at least 50% of the disaccharide units in the modified agarose polysaccharide are sulfonate moieties.

9. The method of any one of claims 1-8, wherein at least 10% of the disaccharide units in the modified agarose polysaccharide are phosphorylate moieties.

10. The method of any one of claims 1 -9, wherein at least 50% of the disaccharide units in the modified agarose polysaccharide arc phosphorylate moictics.

11. The method of any one of claims 1-10, wherein at least 10% of the modified agarose polysaccharide exhibits a P-sheet structure.

12. The method of any one of claims 1-11, wherein at least 10% of the modified agarose polysaccharide exhibits a P-sheet structure.

13. The method of any one of claims 11 or 12, wherein the P-sheet structure is determined by Raman optical activity.

14. The method of any one of claims 12 or 13, wherein the P-sheet structure is determined by circular dichroism.

15. The method of any one of claims 1-14, wherein the modified agarose polysaccharide is present within a gel.

16. The method of any one of claims 1-15, wherein the modified agarose polysaccharide is present within a hydrogel.

17. The method of any one of claims 1-16, comprising exposing the modified agarose polysaccharide to the agarase at a temperature of above 40 °C.

18. The method of any one of claims 1-17, comprising exposing the modified agarose polysaccharide to the agarase at a temperature of below 40 °C.

19. The method of any one of claims 1-18, comprising exposing the modified agarose polysaccharide to the agarase at a temperature of between 35 °C and 40 °C.

20. The method of any one of claims 1-19, comprising exposing the modified agarose polysaccharide to the agarasc at body temperature.

21. The method of any one of claims 1-20, wherein the agarase is a beta-agarase.

22. The method of any one of claims 1-21, wherein the agarase arises from thermophilic bacteria.

23. The method of any one of claims 1-22, wherein the agarase arises from agarolytic bacteria.

24. The method of any one of claims 1-23, wherein the modified agarose polysaccharide is present in a solution.

25. The method of any one of claims 1-24, comprising exposing a gel of the modified agarose polysaccharide to the agarase such that the gel liquefies.

26. The method of any one of claims 1-25, comprising exposing the modified agarose polysaccharide to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 10%.

27. The method of any one of claims 1-26, comprising exposing the modified agarose polysaccharide to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 20%.

28. The method of any one of claims 1-27, comprising exposing the modified agarose polysaccharide to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 30%.

29. The method of any one of claims 1 -28, comprising exposing the modified agarose polysaccharide exhibiting to the agarasc such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 50%.

30. The method of any one of claims 1-29, comprising exposing the modified agarose polysaccharide exhibiting to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 80%.

31. The method of any one of claims 1-30, comprising exposing the modified agarose polysaccharide exhibiting to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 5 kDa.

32. The method of any one of claims 1-31, comprising exposing the modified agarose polysaccharide exhibiting to the agarase such that the modified agarose polysaccharide exhibits a drop in weight average molecular weight (M_w) of at least 50 kDa.

33. The method of any one of claims 1-32, comprising exposing the modified agarose polysaccharide to the agarase such that the modified agarose polysaccharide exhibits a drop in shear modulus (G') of at least 10 Pa.

34. The method of any one of claims 1-33, comprising exposing the modified agarose polysaccharide to the agarase such that the modified agarose polysaccharide exhibits a drop in shear modulus (G') of at least 10%.

35. The method of any one of claims 1-34, wherein the modified agarose polysaccharide is present within a subject.

36. The method of claim 35, wherein the subject is human.

37. The method of any one of claims 35-36, wherein the modified agarose polysaccharide is present as an implant within the subject.

38. The method of any one of claims 35-37, wherein the modified agarose polysaccharide is present as an implant within a body cavity within the subject.

39. The method of any one of claims 35-38, wherein the modified agarose polysaccharide is present as a tissue filler within the subject.

40. The method of any one of claims 35-39, wherein the modified agarose polysaccharide is present as a dermal filler within the subject.

41. The method of any one of claims 35-40, wherein the modified agarose polysaccharide is present as a tissue scaffold within the subject.

42. The method of any one of claims 1-41, wherein the modified agarose polysaccharide further comprises a polysaccharide.

43. The method of any one of claims 1-42, wherein the modified agarose polysaccharide further comprises an anesthetic.

44. The method of any one of claims 1-43, wherein the modified agarose polysaccharide further comprises lidocaine.

45. The method of any one of claims 1-44, wherein the modified agarose polysaccharide further comprises a buffer.

46. The method of any one of claims 1-45, wherein the modified agarose polysaccharide further comprises a protein or a peptide.

47. The method of any one of claims 1-46, wherein the modified agarose polysaccharide further comprises an antioxidant.

48. The method of any one of claims 1 -47, wherein the modified agarose polysaccharide further comprises an osmolarity adjusting agent.

49. The method of any one of claims 1-48, wherein the modified agarose polysaccharide further comprises a chelating agent.

50. The method of any one of claims 1-49, wherein the modified agarose polysaccharide further comprises an amino acid.

51. The method of any one of claims 1-50, wherein the modified agarose polysaccharide further comprises a vitamin.

52. The method of any one of claims 1-51, wherein the modified agarose polysaccharide further comprises a mineral.

53. The method of any one of claims 1-52, wherein the modified agarose polysaccharide further comprises a neurotoxic protein.

54. The method of any one of claims 1-53, wherein the modified agarose polysaccharide prepared under conditions such that the pH varies by no more than 1 pH unit.

55. The method of claim 54, wherein the pH varies by no more than 0.5 pH unit.

56. The method of any one of claims 54 or 55, wherein the pH varies by no more than 0.3 pH unit.

57. The method of any one of claims 1-56, wherein the modified agarose polysaccharide prepared under conditions such that the pH is no more than 12.

58. A method, comprising: implanting a composition comprising modified agarose polysaccharide into a subject, wherein at least 5% of disaccharide units in the modified agarose polysaccharide arc carboxylate moictics; and injecting agarase within 3 cm of the composition within the subject.

59. The method of claim 58, comprising injecting the agarase into the modified agarose polysaccharide.

60. The method of any one of claims 58 or 59, wherein the modified agarose polysaccharide is contained within a dermal filler within the subject.

61. The method of any one of claims 58-60, wherein the modified agarose polysaccharide is contained within a tissue filler within the subject.

62. The method of any one of claims 58-61, wherein the composition further comprises hyaluronic acid.

63. A method, comprising: injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in shear modulus (G'j of at least 10%, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

64. A method, comprising: injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) of at least 5 kDa, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

65. A method, comprising: injecting, into a modified agarose polysaccharide implanted within a subject, an enzyme able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) of at least 10%, wherein at least 5% of disaccharide units in the modified agarose polysaccharide arc carboxylate moictics.

66. A method, comprising: liquefying modified agarose polysaccharide by exposing the modified agarose polysaccharide to an agarase, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

67. A method, comprising: liquefying modified agarose polysaccharide implanted within a subject by injecting agarase into the modified agarose poly saccharide, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties.

68. A method, comprising: exposing a first modified agarose polysaccharide to an agarase able to cause the first modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) to produce an altered agarose, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties; and mixing the altered agarose with a second modified agarose polysaccharide exhibiting an at least partial -sheet structure.

69. A method, comprising: exposing modified agarose polysaccharide to an agarase able to cause the modified agarose polysaccharide to exhibit a drop in weight average molecular weight (M_w) to produce an altered agarose, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties; and mixing the altered agarose with hyaluronic acid.

70. A method, comprising: injecting agarase into an occlusion of a blood vessel within a subject, wherein the occlusion is formed by agarose.

71. The method of claim 70, comprising injecting the agarasc in an amount at least sufficient to permit blood to pass the occlusion.

72. A method, comprising: injecting agarase into a subject.

73. The method of claim 72, wherein the agarase is contained within a saline solution.

74. A kit, comprising: modified agarose polysaccharide, wherein at least 5% of disaccharide units in the modified agarose polysaccharide are carboxylate moieties; and agarase contained within solution.

75. The kit of claim 74, wherein the solution is a saline solution.

76. The kit of any one of claims 74 or 75, wherein the modified agarose polysaccharide further comprises a polysaccharide.

77. The kit of any one of claims 74-76, wherein the modified agarose polysaccharide further comprises an anesthetic.

78. The kit of any one of claims 74-77, wherein the modified agarose polysaccharide further comprises lidocaine.

79. The kit of any one of claims 74-78, wherein the modified agarose polysaccharide further comprises a buffer.

80. The kit of any one of claims 74-79, wherein the modified agarose polysaccharide further comprises a protein or a peptide.

81 . The kit of any one of claims 74-80, wherein the modified agarose polysaccharide further comprises an antioxidant.

82. The kit of any one of claims 74-81, wherein the modified agarose polysaccharide further comprises an osmolarity adjusting agent.

83. The kit of any one of claims 74-82, wherein the modified agarose polysaccharide further comprises a chelating agent.

84. The kit of any one of claims 74-83, wherein the modified agarose polysaccharide further comprises an amino acid.

85. The kit of any one of claims 74-84, wherein the modified agarose polysaccharide further comprises a vitamin.

86. The kit of any one of claims 74-85, wherein the modified agarose polysaccharide further comprises a mineral.

87. The kit of any one of claims 74-86, wherein the modified agarose polysaccharide further comprises a neurotoxic protein.