EP4633678A1 - Hydrogel compositions and methods of making and using the same - Google Patents

Abstract

Described herein are compositions comprising a hydrogel such as compositions comprising a hydrogel that includes a protease inhibitor, a permeability enhancer, and/or a biologic. Also described herein are methods for making and/or using compositions of the present invention. Treating the subject may comprise administering a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and a third hydrogel comprising a biologic.

Description

HYDROGEL COMPOSITIONS AND METHODS OF MAKING AND USING THE

SAME

RELATED APPLICATION INFORMATION

This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63/387,747 filed December 16, 2022, the disclosure of which is incorporated herein by reference in its entirety.

FIELD

The present invention relates to compositions comprising a hydrogel such as compositions comprising a hydrogel that includes a protease inhibitor, a permeability enhancer, and/or a biologic. Also provided are methods for making and/or using compositions of the present invention.

BACKGROUND

Oral drug delivery can be a desired route for drug administration because it can promote patient compliance. For most therapeutics, the desired goal is to safely deliver the therapeutic agent to the gut from where it would be absorbed; thus, enhancing bioavailability. A major concern when the oral route of administration is considered for a therapeutic is the hostile environment involving gastric acid and the digestive enzymes that may degrade therapeutic biologies such as peptides and nucleic acids.

For example, meaningful oral delivery of insulin has been hampered by the susceptibility of the peptide to pH or enzymatic degradation in the stomach or other tissues, and its poor permeability across the mucus and cellular membranes in the gastrointestinal tract. Accordingly, new formulations for therapeutics may be desirable.

SUMMARY

A first aspect of the present invention is directed to a composition comprising a first hydrogel comprising a protease inhibitor, and a second hydrogel comprising a permeability enhancer. In some embodiments, the composition includes a third hydrogel comprising a biologic (e.g., a polypeptide and/or a nucleic acid).

Another aspect of the present invention is directed to a method for treating a subject in need thereof, the method comprising administering a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and a third hydrogel comprising a biologic (e.g., polypeptide and/or nucleic acid), thereby treating the subject. In some embodiments, administering the composition comprises concurrently administering the first hydrogel, the second hydrogel, and the third hydrogel to the subject. In some embodiments, the first hydrogel, the second hydrogel, and the third hydrogel are present in the same composition. In some embodiments, administering the composition comprises separately administering the first hydrogel, the second hydrogel, and/or the third hydrogel to the subject. In some embodiments, the first hydrogel, the second hydrogel, and/or the third hydrogel are present in one or more different composition(s).

It is noted that aspects of the present invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination. Applicant reserves the right to change any originally filed claim and/or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and/or incorporate any feature of any other claim or claims although not originally claimed in that manner. These and other objects and/or aspects of the present invention are explained in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a series of four images of unmodified alginate hydrogel microbeads in a solution with a pH of 2 at the initial start of incubation in the solution (time 0), and at 1 hour, 6 hours, and 24 hours from the start of incubation in the solution.

Fig. 2, panel A is a series of four images of unmodified alginate hydrogel microbeads in a solution with a pH of 6.8 at the initial start of incubation in the solution (time 0), and at 1 hour, 3 hours, and 6 hours from the start of incubation in the solution. Fig. 2, panel B is a series of three images of unmodified alginate hydrogel microbeads in a solution with a pH of 7.4 at the initial start of incubation in the solution (time 0), and at 1 hour and 2 hours from the start of incubation in the solution.

Fig. 3, panels A-D are graphs showing the percent of intact modified alginate microbeads over time in an acidic (pH 2) solution, wherein the microbeads were made with alginate materials modified by the attachment of methyl ketone (Fig. 3, panel A), benzoic acid (Fig. 3, panel B), methyl ester (Fig. 3, panel C), or phenethylamine (Fig. 3, panel D) to 2% oxidized alginate.

Fig. 4, panel A is a graph of the time until complete degradation of a group of unmodified alginate microbeads or alginate microbeads modified with different chemical compounds in a solution with a pH of 6.8. Fig. 4, panel B is a graph of the time until complete degradation of a group of unmodified alginate microbeads or alginate microbeads modified with different chemical compounds in a solution with a pH of 7.4.

Fig. 5, panel A is a graph showing the percent of intact modified alginate microbeads over time in a pH 6.8 solution. Fig. 5, panel B is a graph showing the percent of intact modified alginate microbeads over time in a pH 7.4 solution.

Fig- 6 shows graphs of the precent blood glucose level in rats at various timepoints (initial administration (TO), or 1 (Tl), 1.5 (T1.5), 2 (T2), 2.5 (T2.5), 3 (T3), 3.5 (T3.5), or 4 hour(s) (T4) after initial administration) following administration with either empty hydrogel microbeads; insulin containing hydrogel microbeads; a composition of three discrete hydrogels containing aprotinin, a cell-penetrating peptide, and insulin, respectively, where the hydrogels are formulated for sequential release of these compounds from the microbeads; or a composition of three discrete hydrogels containing aprotinin, a cell-penetrating peptide, and insulin, respectively, where the hydrogels are formulated for concurrent release of these compounds from the microbeads.

Fig- 7 shows graphs of the plasma insulin level in rats at various timepoints (initial administration (tO), or 1 (tl), 2 (t2), or 4 hour(s) (t4) after initial administration) following administration with either empty hydrogel microbeads; insulin containing hydrogel microbeads; a composition of three discrete hydrogels containing aprotinin, a cell-penetrating peptide, and insulin, respectively, where the hydrogels are formulated for sequential release of these compounds from the microbeads; a composition of three discrete hydrogels containing aprotinin, a cell-penetrating peptide, and insulin, respectively, where the hydrogels are formulated for concurrent release of these compounds from the microbeads.

DETAILED DESCRIPTION

The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and/or individual value therein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the terms "increase," "increasing," "enhance," "enhancing," "improve" and "improving" (and grammatical variations thereof) describe an elevation of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more such as compared to another measurable property or quantity (e.g., a control value).

As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof), describe, for example, a decrease of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% such as compared to another measurable property or quantity (e.g., a control value). In some embodiments, the reduction can result in no or essentially no (z.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. The abbreviations "FIG. and "Fig." for the word "Figure" can be used interchangeably in the text and figures.

It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, in “direct contact” with, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element discussed below could also be termed a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

As used herein, the term “solution” refers to a homogenous mixture of two or more substances wherein at least one gas, liquid, or solid substance (e.g., a solute) is dissolved into at least one liquid substance (e.g., a solvent). The solution may or may not be buffered so as to resist a change in the pH level when an acidic or basic component is added.

As used herein, the term “aqueous solution” refers to a solution in which the solvent is water. In some embodiments, the aqueous solution may be a saline solution (e.g., a solution of sodium chloride dissolved in water).

As used herein, the term “hydrogel” refers to a gel comprising water and a polymer network. Water may be the swelling agent for a hydrogel of the present invention. In some embodiments, a hydrogel of the present invention may contain water within the polymer network of the hydrogel, and the polymer network may be a three-dimensional (3D) polymer network. In some embodiments, a polymer network of a hydrogel of the present invention may comprise crosslinked polymers (e.g., crosslinked hydrophilic polymers). In some embodiments, a hydrogel of the present invention has a 3D structure in the presence of water and the polymer network may not dissolve in water. In some embodiments, a hydrogel of the present invention comprises a solution such that the solvent (e.g., water) and the solute (e.g., a biologic, a permeability enhancer, and/or a protease inhibitor) are distributed (e.g., uniformly distributed) within the hydrogel. A hydrogel of the present invention may be prepared to include (e.g., be loaded with) an agent (e.g., a biologic, a permeability enhancer, and/or a protease inhibitor) by contacting (e.g., soaking, submerging, and/or the like) a hydrogel and a composition comprising the agent, optionally wherein the agent is dissolved or suspended in the composition, thereby the agent is provided within the hydrogel. In some embodiments, an agent is present in an aqueous composition that is used to swell a polymer network (e.g., a modified alginate of the present invention) to thereby provide a hydrogel of the present invention comprising the agent.

A hydrogel of the present invention may be formed as or in the form of a sheet, a bead, a capsule, a cube, a cylinder, and/or a sphere. In some embodiments, a hydrogel of the present invention is a microparticle (e.g., a microbead) or a nanoparticle. In some embodiments, a hydrogel of the present invention has at least one dimension (e.g., diameter, width, or length) in a range of about 100 microns to about 500 microns. In some embodiments, a hydrogel of the present invention may be sized and/or configured for a subject to swallow with or without water. A hydrogel of the present invention may have an elastic modulus in a range of about 1 or 5 kPa to about 10, 15, or 20 kPa. In some embodiments a hydrogel of the present invention has an elastic modulus of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kPa.

A hydrogel of the present invention may be a cross-linked hydrogel (e.g., one or more polymer(s) of the polymer networks are crosslinked together). In some embodiments, a hydrogel of the present invention is cross-linked with a divalent cation (e.g., calcium). A divalent cation may be present in a hydrogel of the present invention in an amount of about 0.1, 0.2, 0.3, 0.4, or 0.5 mmol to about 0.6, 0.7, 0.8, 0.9, or 1 mmol. In some embodiments, a hydrogel of the present invention comprises an aqueous solution (e.g., a saline solution) that is optionally buffered. In some embodiments, an aqueous solution comprises one or more (e.g., 1, 2, 3, 4, 5, or more) different compound(s) that may be dissolved and/or suspended therein. For example, in some embodiments, an aqueous solution comprises a biologic, a permeability enhancer, and/or a protease inhibitor.

According to some embodiments, a hydrogel of the present invention comprises a modified carbohydrate (e.g., a modified alginate) that is optionally crosslinked. In some embodiments, a modified carbohydrate of the present invention is a modified carbohydrate as described in U.S. Patent No. 10,766,970, the contents of which is incorporated herein by reference in its entirety. In some embodiments, a modified carbohydrate (e.g., a modified alginate) of the present invention comprises at least one unit having a structure of Formula I or Formula II:

wherein

Y is absent or a C1-C4 alkyl or C1-C4 alkenyl;

Ri is each independently selected from the group consisting of -H, -OH, -NH2, -COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted -C1-C6 alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g., - COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(0H)2, wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl; n is from 1 to 1,000,000; and o is 0, 1, 2, 3, 4, or 5.

In some embodiments, a modified carbohydrate (e.g., a modified alginate) of the present invention comprises at least one unit of Formula I that has at least one Ri that is selected from the group consisting of -OH, -NH2, -COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -C1-C6 alkylhalide, unsubstituted or substituted -Ci-Ce alkyl, unsubstituted or substituted -Ci- C₆ alkenyl, -SO2H, -SO3H, -COR’ (e.g, -COCH3), -Si(OH)₃, -SO2NH2, -PO(OR’)₂, and - B(OH)2, wherein each R’ is independently selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl and aryl. In some embodiments, a modified carbohydrate (e.g., a modified alginate) of the present invention comprises at least one unit of Formula I that has at least one Ri that is hydrogen. In some embodiments, a modified carbohydrate (e.g, modified alginate) of the present invention comprises at least one unit of Formula I that has at least one Ri in the para position and is selected from the group consisting of -OH, -NH2, - COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted - Ci-Ce alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g, -COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(OH)2, and wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl. In some embodiments, a modified carbohydrate (e.g, modified alginate) of the present invention comprises at least one unit of Formula I that has at least one Ri in the para position and is selected from the group consisting of -NH2, -COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted -Ci-Ce alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g, -COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(OH)2, and wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, a modified carbohydrate (e.g, a modified alginate) of the present invention comprises at least one unit of Formula II that has at least one Ri that is selected from the group consisting of -OH, -NH2, -COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted -Ci-Ce alkyl, unsubstituted or substituted -Ci- Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g, -COCH3), -Si(OH)₃, -SO2NH2, -PO(OR’)₂, and - B(OH)2, wherein each R’ is independently selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl and aryl. In some embodiments, a modified carbohydrate (e.g, a modified alginate) of the present invention comprises at least one unit of Formula II that has at least one Ri that is hydrogen. In some embodiments, a modified carbohydrate (e.g. modified alginate) of the present invention comprises at least one unit of Formula II that has at least one Ri in the para position and is selected from the group consisting of -OH, -NH2, - COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted - Ci-Ce alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g., -COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(OH)2, and wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl. In some embodiments, a modified carbohydrate (e.g., modified alginate) of the present invention comprises at least one unit of Formula II that has at least one Ri in the para position and is selected from the group consisting of -NH2, -COOH, - C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted -Ci-Ce alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g., -COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(OH)2, and wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl.

A modified carbohydrate (e.g., a modified alginate) of the present invention may comprise at least one unit of Formula I and/or at least one unit of Formula II. In some embodiments, one or more functional groups in a modified carbohydrate (e.g., a modified alginate) of the present invention comprising at least one unit of Formula I and/or at least one unit of Formula II may be protonated or deprotonated, optionally one or more Ri in the modified carbohydrate (e.g., a modified alginate) may be protonated or deprotonated. In some embodiments, the protonation state (i.e., protonated or deprotonated) of one or more functional groups in a modified carbohydrate (e.g., a modified alginate) of the present invention may depend on the pH of the environment that the modified carbohydrate (e.g., a modified alginate) is exposed to and/or in contact with.

For simplicity, described herein are further embodiments with respect to certain carbohydrates, such as modified alginate, but it should be understood that other carbohydrates may be used.

A hydrogel of the present invention may comprise a modified alginate. A modified alginate of the present invention may be a chemically modified alginate. In some embodiments, a hydrogel of the present invention comprises an alginate that has been oxidized and/or modified (e.g., modified with a reactant to attach a moiety (e.g., a modification moiety) to the alginate). In some embodiments, about 0.1%, 0.5%, 1%, or 2% to about 5%, 10%, or 15% of the units (e.g., polysaccharide units) of a modified alginate may be modified (e.g., include a functional group (e.g., modification moiety) that is not naturally present in the alginate). In some embodiments, about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the units (e.g., polysaccharide units) of a modified alginate may be modified.

In some embodiments, a modified alginate includes a moiety that has a pKa in a range of about 3, 3.5, or 4 to about 4.5, 5, 5.5, or 6. The modified alginate may have been modified to include the moiety having a pKa in a range of about 3, 3.5, or 4 to about 4.5, 5, 5.5, or 6. In some embodiments, a modified alginate includes a moiety that has a pKa of about 3, 3.5, 4, 4.5, 5, 5.5, or 6. In some embodiments, a modified alginate includes at least one unit of Formula I and/or Formula II that includes at least one Ri that has a pKa in a range of about 3, 3.5, or 4 to about 4.5, 5, 5.5, or 6. In some embodiments, a modified alginate includes at least one unit of Formula I and/or Formula II that includes at least one Ri that has a pKa of about 3, 3.5, 4, 4.5, 5, 5.5, or 6.

Alginate used to prepare a modified alginate of the present invention may be of any type. Alginate is a polysaccharide composed of randomly oriented blocks of monomers of (1- 4)-linked P-D-mannuronic acid (M) and a-L-guluronic acid (G). In some embodiments, a modified alginate of the present invention may be prepared and/or formed using an alginate having about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total number of polysaccharide units be M units or G units. In some embodiments, an alginate used to prepare a modified alginate of the present invention may be a natural and/or unmodified alginate. In some embodiments, an alginate that is used to prepare a modified alginate of the present invention may be an oxidized alginate (e.g., alginate oxidized with an oxidizing agent (e.g., sodium periodate)). In some embodiments, an oxidized alginate may be prepared by oxidizing a vicinal diol in the alginate chain to an aldehyde.

A modified alginate of the present invention may be prepared by reacting an alginate with a reactant that is selected from the group consisting of 4-(2-aminoethyl)benzoic acid, 4- (2-aminomethyl)benzoic acid, 4-(2-aminoethyl)aniline, (2-ethylamino)4-methyl benzene, 4- (2-aminoacetyl)-benzoic acid, 4-(2-aminoethyl)salicylic acid, methyl 4-(2-aminoethyl) benzoate, phenethylamine, l-[4-(2-aminoethyl) phenyl] ethanone HC1, and/or 4-(2- aminomethyl)aniline and/or esters thereof. In some embodiments, a modified alginate of the present invention is a 2-amino ethyl aromatic (e.g., a 2-amino ethyl benzyl) modified alginate. In some embodiments, a modified alginate of the present invention is a 4-(2-aminoethyl) benzoic acid modified alginate (e.g., an alginate (optionally an oxidized alginate) that is modified (e.g., reacted) with 4-(2-aminoethyl) benzoic acid). In some embodiments, a modified alginate of the present invention is an aminomethyl aromatic (e.g., a 2-aminomethyl benzyl) modified alginate. A reactant may be incorporated into an alginate in an amount of about 1% to about 20% based on the average number of polysaccharide units in the alginate to provide a modified alginate. In some embodiments, a reactant is incorporated into an alginate in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% based on the average number of polysaccharide units in the alginate. In some embodiments, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a reactant is chemically bound (e.g., covalently bound) to an alginate to provide the modified alginate, rather than free or trapped within the polymer. In some embodiments, a reactant may be reacted with an oxidized alginate (e.g., alginate in which a vicinal diol in the alginate chain is oxidized to an aldehyde) to provide the modified alginate.

In some embodiments, a modified alginate and/or hydrogel of the present invention may be stable (e.g., may not degrade and/or may not dissolve in) in an acidic environment (e.g., an acidic composition) such as an acidic environment similar to the stomach environment. In some embodiments, a modified alginate and/or hydrogel of the present invention may disintegrate, degrade, and/or dissolve in a basic environment such as a basic environment similar to the small intestine environment. In some embodiments, a hydrogel and/or modified alginate of the present invention may degrade in about 5, 10, 15, 20, 25, or 30 minutes to about 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 minutes following exposure to (e.g., contact with) a composition (e.g., an aqueous composition) having a pH of above 7 (e.g., about 7.5 or more) and/or following administration to a subject and reaching the intestinal tract (e.g., the small intestine).

According to some embodiments of the present invention provided is a composition comprising a hydrogel of the present invention. In some embodiments, a hydrogel of the present invention may provide a barrier to a bioactive substance (e.g., a medicine, drug, enzyme, protein, peptide, nucleic acid, nucleotide, biologic, hormone, vaccine, vitamin, mineral, and/or micronutrient). The hydrogel may coat at least a portion of the bioactive substance and/or encapsulate at least a portion of the bioactive substance. In some embodiments, a hydrogel of the present invention comprises a bioactive substance and the hydrogel may prevent and/or reduce degradation and/or oxidation of the bioactive substance. In some embodiments, a modified alginate of the present invention is present in a hydrogel of the present invention in an amount of about 0.1%, 0.5%, 1%, or 2% to about 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v of the hydrogel.

In some embodiments, a composition and/or a hydrogel of the present invention comprises a biologic, a permeability enhancer, and/or a protease inhibitor. The hydrogel may coat and/or encapsulate at least a portion (e.g., about 50%, 60%, 70%, 80%, 90%, or 100%) of the biologic, permeability enhancer, and/or protease inhibitor. In some embodiments, a hydrogel of the present invention comprises a biologic, permeability enhancer, and/or protease inhibitor that is uniformly distributed within the hydrogel.

According to some embodiments, a composition and/or hydrogel of the present invention may increase the shelf life of a protease inhibitor, a permeability enhancer, and/or a biologic when present in a hydrogel of the present invention. For example, the shelf life of insulin in liquid or powder form may be increased when present in a hydrogel of the present invention. "Shelflife" as used herein refers to the length of time a substance maintains a given level of activity in an unopened package stored under recommended storage conditions. The shelf life may, for example, be evidenced by the "use by" or "best if used by" date for the product and/or the manufacturer’s expiration date of the product (i.e., the "predicted shelflife") and/or the actual product characteristics after the specified period of time (i.e., the "actual shelf life"). Accordingly, the term "shelf life" as used herein should be construed as including both the actual shelf life of the product and the predicted shelf life of the product unless stated otherwise. In some embodiments, shelf life may be determined by extrapolation of data at accelerated temperatures, such as, for example, by using the Arrhenius equation. In some embodiments, shelf life may be determined using linear regression analysis, such as, for example, when the kinetics of the protease inhibitor, permeability enhancer, and/or biologic degradation is not temperature dependent. In some embodiments, shelf life may be evaluated and/or determined by measuring the protease inhibitor, permeability enhancer, and/or biologic, such as, for example, using high pressure liquid chromatography. In some embodiments, the shelf life of a composition of the present invention that includes a hydrogel comprising a protease inhibitor, a permeability enhancer, and/or a biologic is the time that the protease inhibitor, permeability enhancer, and/or biologic in the hydrogel maintains at least 50% (e.g., about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of a given activity (e.g., the ability to deliver and/or provide a therapeutically effect amount of the protease inhibitor, permeability enhancer, and/or biologic) compared to the initial activity of the protease inhibitor, permeability enhancer, and/or biologic prior to incorporation into the hydrogel.

As used herein, the term “biologic” refers to a polypeptide (e.g., a protein), an enzyme, a hormone, a nucleotide, and/or a nucleic acid (e.g., a polynucleotide). In some embodiments, a biologic is a compound that was manufactured in, extracted from, or semisynthesized from a living cell, tissue, or organism. In some embodiments, a biologic is synthetically obtained (e.g., produced). One or more (e.g., 1, 2, 3, 4, or more) biologic(s) may be present in a hydrogel of the present invention. A biologic may be present in a hydrogel of the present invention in a concentration of about 1, 5, 50, 100, 250, or 500 ng of the biologic per mL of the hydrogel to about 0.5, 1, 2, 4, 6, 8, or 10 pg of the biologic per mL of the hydrogel. In some embodiments, a biologic is present in a hydrogel of the present invention in a total concentration of about 1 or 5 unit(s) to about 10, 15, or 20 units.

In some embodiments, a biologic is an anti-diabetes peptide or protein therapeutic and/or an anti-obesity peptide or protein therapeutic. In some embodiments, a hydrogel of the present invention comprises insulin. Exemplary insulins include, but are not limited to, human insulin, insulin glargine, insulin detemir, insulin lispro, insulin aspart, insulin glulisine, and/or a prodrug thereof. In some embodiments, an anti-diabetes and/or anti-obesity peptide or protein therapeutic increases the secretion of endogenous insulin by about 5% to about 200% (e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125% 150%, 175%, or by about 200%). In some embodiments, an anti-diabetes and/or antiobesity peptide or protein therapeutic decreases the amount of glucose in the blood (e.g., the blood sugar level) by about 5% to about 95% (e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or by about 95%). In some embodiments, an anti-diabetes and/or anti-obesity peptide or protein therapeutic is an incretin such as glucagon-like peptide 1 (GLP-1) and/or gastric inhibitory polypeptide (GIP).

In some embodiments, a biologic nucleic acid comprises one or more modified nucleotides (e.g., 1, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10000, or more modified nucleotides). In some embodiments, the one or more modified nucleotides comprise a nucleoside modification, a 2’-O-methoxyethyl (MOE) modification, a 2’-fluoro modification, a 2’-O-methyl modification, a locked nucleic acid (LNA) modification, a methyl phosphonate modification, a 5-methylcytosine modification, a methyl phosphonothioate modification, a phosphoromorpholidate modification, a phosphoropiperazidate modification, a phosphoramidate modification, or a phosphorodiamidate modification. In some embodiments, all nucleotides in a biologic nucleic acid are modified. In some embodiments, a biologic nucleic acid encodes a protein that raises an immune response in a subject (e.g., the biological nucleic acid is a vaccine). In some embodiments, a biologic nucleic acid encodes a viral protein, such as a viral surface protein (e.g., a viral spike protein), that raises an immune response in a subject to the virus (e.g., the biological nucleic acid is a viral vaccine). In some embodiments, the viral surface protein is from influenza virus, rhinovirus, respiratory syncytial virus (RSV), cytomegalovirus (CMV), epstein-barr virus (EBV), herpes simplex virus (HSV 1+2), parvovirus, adenovirus, and/or coronavirus. In some embodiments, the coronavirus causes severe acute respiratory syndrome (SARS), e.g., SARS-CoV, e.g., SARS-CoV-2. In some embodiments, a biologic nucleic acid is and/or can be encapsulated within a lipid nanoparticle (LNP). In some embodiments, a biologic nucleic acid is a messenger RNA LNP (e.g., mRNA-LNP) and/or is a messenger RNA that used in a mRNA-LNP vaccine.

As used herein, the term “protease inhibitor" describes a compound that partially or fully inhibits or blocks the activity of an enzyme that breaks apart a protein (e.g., cleaves one or more polypeptide bonds of the protein). One or more (e.g., 1, 2, 3, 4, or more) protease inhibitor(s) may be present in a hydrogel of the present invention. Exemplary protease inhibitors include, but are not limited to, Bowman-Birk inhibitors, Kunitz-type inhibitors, a- amylase inhibitors, and/or trypsin inhibitors. In some embodiments, a hydrogel of the present invention comprises aprotinin. In some embodiments, a protease inhibitor of the present invention has a molecular weight of about 500 or 1000 Daltons to about 2000, 3000, 4000, 5000, 6000, or 7000 Daltons. In some embodiments, a protease inhibitor is present in a hydrogel of the present invention in a concentration of about 0.01, 0.05, or 0.1 mg of the protease inhibitor per mL of the hydrogel to about 0.5, 1, 2, 3, 4, or 5 mg of the protease inhibitor per mL of the hydrogel.

As used herein, the term “permeability enhancer" describes a compound that improves (e.g., increases) the transport of another substance (e.g., a biologic) across a mucus and/or cellular membrane. In some embodiments, a permeability enhancer may improve the transport of another substance (e.g., a biologic) into a target cell (e.g., improved transport across a membrane to allow the substance to enter the cell). In some embodiments, a permeability enhancer may improve transport for a substance in the gastrointestinal tract of a subject and/or the target cell is in the gastrointestinal tract. One or more (e.g., 1, 2, 3, 4, or more) permeability enhancer(s) may be present in a hydrogel of the present invention. In some embodiments, a permeability enhancer is present in a hydrogel of the present invention in a concentration of about 0.01, 0.05, or 0.1 mg of the permeability enhancer per mL of the hydrogel to about 0.5, 1, 2, 3, 4, or 5 mg of the permeability enhancer per /mL of the hydrogel. In some embodiments, a permeability enhancer comprises a sugar (e.g., fructose), an antibiotic, an endocannabinoid (e.g., anandamide), and/or a cell-penetrating peptide (e.g., a cationic cell-penetrating peptide). Exemplary cell-penetrating peptides include, but are not limited to, arginine rich polypeptides, 6-aminohexanoic acid-spaced oligo-arginines, hexa-arginine penetratins, and/or nanoarginines. In some embodiments, a hydrogel of the present invention may comprise a cell- penetrating peptide that is conjugated to a nucleic acid, a peptide nucleic acid (PNA), or a phosphorodiamidate morpholino oligomer (PMO).

In some embodiments, a hydrogel of the present invention is suspended in a carrier, optionally a pharmaceutical carrier. In some embodiments, a hydrogel is suspended in water, an aqueous solution, and/or an oil. In some embodiments, a hydrogel is suspended in a carrier in which the hydrogel and/or its polymer network does not dissolve and/or degrade. As used herein, the term “pharmaceutical carrier” describes any pharmaceutically acceptable solvent, diluent, liposome, or other excipient that aids in the delivery of a composition to an organism (e.g., an animal or a human) or to a target tissue. In some embodiments, a pharmaceutical carrier is a liquid, a solid, or a semi-solid. The selection of a specific pharmaceutical carrier may depend on the desired features of the composition in which it is included, such as a need to enhance the delivery of a composition to a target tissue type, the required stability of the composition, a need to control the release schedule of the composition within the animal or human, and/or the administration method to the animal or human (e.g., orally administered capsules, orally administered liquids or gels, or other administration methods).

In some embodiments, a hydrogel and/or composition of the present invention is provided in a capsule (e.g., a biocompatible capsule). In some embodiments, a hydrogel and/or composition of the present invention are contained within a coating that optionally is configured for oral delivery and/or provides the hydrogel and/or composition in a form for oral delivery. In some embodiments, a capsule and/or coating comprises a cellulose.

A composition of the present invention may include one or more (e.g., 1, 2, 3, 4, 5, or more) hydrogel(s) of the present invention, each of the one or more hydrogel(s) may be discrete and/or separate from another hydrogel present in the composition, and the composition may optionally include a carrier. In some embodiments, a composition of the present invention includes two or more different hydrogels with each of the two or more different hydrogels including a different agent. For example, in some embodiments, a composition of the present invention comprises a first hydrogel comprising a protease inhibitor and a second hydrogel comprising a permeability enhancer. In some embodiments, a composition of the present invention comprises a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and a third hydrogel comprising a biologic. When two or more different hydrogels are present in a composition of the present invention, the hydrogels may comprise the same polymer network (e.g., modified alginate) or a different polymer network. In some embodiments, a composition of the present invention comprises two or more different hydrogels that have the same polymer network (e.g., modified alginate). In some embodiments, a composition of the present invention comprises two or more different hydrogels that have the same polymer network (e.g., modified alginate), but are modified differently (e.g., modified with different reactants) and/or have a different level of modification (e.g., have a different amount of modified polysaccharide units). In some embodiments, a composition of the present invention comprises two different hydrogels that each comprise a modified alginate that is modified with the same reactant, but one of the two hydrogels comprises a greater number of modified polysaccharide units. In some embodiments, a composition of the present invention comprises a hydrogel that comprises a modified alginate of the present invention in which about 1% of the polysaccharide units of the modified alginate are modified. In some embodiments, a composition of the present invention comprises a hydrogel that comprises a modified alginate of the present invention in which about 2% of the polysaccharide units of the modified alginate are modified.

In some embodiments, a composition of the present invention comprises a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and/or a third hydrogel comprising a biologic. A biologic (e.g., insulin) may be present in a composition of the present invention in a total concentration of about 1, 2, 3, 4, or 5 unit(s) to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 units. In some embodiments, a biologic (e.g., a GLP-1, GIP, or nucleic acid encoding a viral surface protein) may be present in a composition of the present invention in a total concentration of about 1 pg, 5 pg, 10 pg, 25 pg, 50 pg, or 100 pg, to about 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg. A protease inhibitor (e.g., aprotinin) may be present in a composition of the present invention in a total concentration of about 50 or 100 mg of the protease inhibitor per mL of the composition to about 150 or 200 mg of the protease inhibitor per mL of the composition. A permeability enhancer (e.g., a cationic cell-penetrating peptide) may be present in a composition of the present invention in a total concentration of about 100 or 200 mg of the permeability enhancer per mL of the composition to about 150 or 200 mg of the permeability enhancer per mL of the composition.

In some embodiments, a composition of the present invention comprises a first hydrogel comprising a first agent and a second hydrogel including a second agent, wherein the first and second agents are different. A composition of the present invention comprising two or more hydrogels that each include a different agent may include each of the hydrogels in the same amount or in different amounts. In some embodiments, a composition of the present invention has a ratio of hydrogel particulates (e.g., hydrogel particles such as beads) in a range of about 1 : 1 : 1 to about 3:3:2 (first hydrogel particulates : second hydrogel particulates : third hydrogel particulates). A hydrogel and/or modified alginate of the present invention may coat and/or encapsulate at least a portion of a compound (e.g., a biologic, permeability enhancer, and/or protease inhibitor), may protect the compound at pH levels that are found in the stomach (e.g., pH of about 1-3), and/or may allow and/or provide for the compound to be made available at pH levels that are found in the intestines (e.g., more basic pH levels such as about 7-9) as the alkaline environment may allow for the hydrogel and/or modified alginate to be broken down, which may allow for access to and/or release of the compound. In some embodiments, a composition and/or hydrogel of the present invention reduces or prevents exposure of a compound (e.g., a biologic, permeability enhancer, and/or protease inhibitor) to an acidic environment (e.g., to the stomach environment), which may increase and/or improve activity and/or bioavailability of the compound, optionally upon administration (e.g., oral administration) to a subject.

In some embodiments, a hydrogel and/or a modified alginate of the present invention may be able to withstand the pH of saliva (generally a pH of about 6.5-7.4). In some embodiments, a hydrogel and/or modified alginate may be able to withstand the pH of saliva for a sufficient amount of time such that, upon oral administration, the hydrogel may be able to reach the stomach and/or intestine (e.g., small intestine) with at least a portion of the compound (e.g., about 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) still encapsulated and/or coated. In some embodiments, a hydrogel and/or modified alginate of the present invention is stable for at least about 5, 10, or 15 minutes at a pH above 7. In some embodiments, upon prolonged exposure to the pH of the small intestine, a compound (e.g., a biologic, permeability enhancer, and/or protease inhibitor) present in a hydrogel may become available for its intended benefit (e.g., is released from the hydrogel).

According to some embodiments, provided is a method of treating a subject such as a subject in need thereof, the method comprising administering a hydrogel and/or a composition of the present invention to the subject. In some embodiments, a therapeutically effective amount of a hydrogel of the present invention and/or a therapeutically effective amount of a compound (e.g., a biologic, permeability enhancer, and/or protease inhibitor) that is present in a hydrogel of the present invention is administered to a subject. As used herein, the term "therapeutically effective amount" refers to an amount of a compound (e.g., a biologic, permeability enhancer, protease inhibitor, and/or hydrogel) that elicits a therapeutically useful response in a subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, a therapeutically effective amount of a hydrogel of the present invention may include delivering a therapeutically effective amount of an agent such as a biologic (e.g., insulin, an incretin, and/or a nucleic acid that encodes a viral surface protein) present in the hydrogel. In some embodiments, eliciting a useful response in a subject comprises administering a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and a third hydrogel comprising a biologic (e.g., polypeptide and/or nucleic acid). In some embodiments, a method of the present invention comprises administering a therapeutically effective amount of a first hydrogel comprising a protease inhibitor, a therapeutically effective amount of a second hydrogel comprising a permeability enhancer, and a therapeutically effective amount of a third hydrogel comprising a biologic (e.g., polypeptide and/or nucleic acid).

"Treat," "treating" or "treatment of (and grammatical variations thereof) as used herein refer to any type of treatment that imparts a benefit to a subject and may mean that the severity of the subject’s disease, disorder, or condition is reduced, at least partially improved or ameliorated, and/or that some alleviation, mitigation or decrease in at least one clinical symptom associated with a disease, disorder, or condition is achieved and/or there is a delay in the progression of a symptom. In some embodiments, the severity of a symptom associated with a subject’s disease, disorder, or condition may be reduced compared to the severity of the symptom in the absence of a method of the present invention.

In some embodiments, a hydrogel and/or an agent present therein (e.g., a biologic, permeability enhancer, and/or protease inhibitor) may be administered in a treatment effective amount. A "treatment effective" amount as used herein is an amount that is sufficient to treat (as defined herein) a subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, a treatment effective amount may be achieved by administering a composition of the present invention.

The present invention finds use in both veterinary and medical applications. Subjects suitable to be treated with a method of the present invention include, but are not limited to, mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates (e.g., simians and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) may be treated according to the present invention. In some embodiments of the present invention, the subject is a mammal and in certain embodiments the subject is a human. Human subjects include both males and females of all ages including fetal, neonatal, infant, juvenile, adolescent, adult, and geriatric subjects as well as pregnant subjects. In particular embodiments of the present invention, the subject is a human adolescent and/or adult.

A method of the present invention may also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and/or for drug screening and drug development purposes.

In some embodiments, a subject is "in need of or "in need thereof a method of the present invention, for example, the subject has findings typically associated with a disease, disorder, or condition (e.g., diabetes), is suspected to have a disease, disorder, or condition, and/or the subject has a disease, disorder, or condition. In some embodiments, a subject has diabetes (e.g., Type I Diabetes and/or Type II Diabetes). In some embodiments, a method of the present invention may treat diabetes in a subject.

A method of the present invention may comprise administering a single composition comprising one or more different hydrogel(s) of the present invention and/or administering two or more different compositions of the present invention. In some embodiments, a method of the present invention comprises administering a first composition comprising a first hydrogel comprising a protease inhibitor, a second composition comprising a second hydrogel comprising a permeability enhancer, and a third composition comprising a third hydrogel comprising a biologic, wherein first, second and third compositions are separate from each other, but two or more of the first, second and third compositions may be administered together or one or more of the first, second and third compositions may be administered separately. In some embodiments, the administering comprises separately administering at least one of the first hydrogel, the second hydrogel, and the third hydrogel to the subject from the others such that at least two of the hydrogels are administered at different times. In some embodiments, the administering comprises separately administering the first hydrogel, the second hydrogel, and the third hydrogel to the subject. In some embodiments, the administering comprises concurrently administering at least one of the first hydrogel, the second hydrogel, and the third hydrogel to the subject such that at least two of the hydrogels are administered at the same time. In some embodiments, the administering comprises concurrently administering the first hydrogel, the second hydrogel, and the third hydrogel to the subject, optionally wherein the first hydrogel, the second hydrogel, and the third hydrogel are present in the same composition.

A method and/or composition of the present invention may provide for a sequential release of at least two different agents such as at least two selected from a protease inhibitor, a permeability enhancer, and a biologic. In some embodiments, a hydrogel and/or modified alginate of the present invention may be configured to release an agent a particular time. In some embodiments, a method and/or composition of the present invention comprises a hydrogel comprises a protease inhibitor that, following administration to a subject, releases the protease inhibitor prior to a hydrogel comprising a biologic and/or a hydrogel comprising a permeability enhancer that are each administered to the subject optionally at the same time as the hydrogel comprising the protease inhibitor. In some embodiments, a composition of the present invention comprises at least one hydrogel that releases its contents and/or degrades at a different rate than another hydrogel present in the same composition. In some embodiments, a method and/or composition of the present invention may provide for a sequential release of at least two different agents such as at least two selected from a protease inhibitor, a permeability enhancer, and a biologic. In some embodiments, a method of the present invention comprises administering to a subject a composition comprising two or more different hydrogels that each release an agent concurrently.

A method of the present invention may improve bioavailability of a biologic. For example, a method of the present invention may comprise orally administering to a subject a composition comprising a hydrogel that includes insulin, wherein the hydrogel may prevent the release of the insulin in the stomach, where it could be degraded due to the pH level in the stomach, and releases insulin in the small intestine. Also, for example, a method of the present invention may comprise orally administering to a subject a composition comprising a hydrogel that includes an incretin, wherein the hydrogel may prevent the release of the incretin in the stomach, where it could be degraded due to the pH level in the stomach, and releases incretin in the small intestine. In some embodiments, a method of the present invention may comprise orally administering to a subject a composition comprising a hydrogel that includes a nucleic acid that encodes a viral surface protein, wherein the hydrogel may prevent the release of the nucleic acid in the stomach, where it could be degraded due to the pH level in the stomach, and releases nucleic acid in the small intestine

In some embodiments, a method of the present invention comprises orally administering a composition of the present invention. Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of an agent (e.g., a biologic) and/or hydrogel as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association a hydrogel of the present invention and a suitable carrier (which may contain one or more accessory ingredients). In some embodiments, a composition of the present invention may be prepared by uniformly admixing a hydrogel of the present invention with a liquid carrier.

A method of the present invention may comprise administering a biologic (e.g., insulin, an incretin, and/or a nucleic acid that encodes a viral surface protein) to a subject for a period of time so as to produce a therapeutically useful response over a time period. In some embodiments, the therapeutically useful response may be provided for a time period up to the time when an immediately subsequent dose of the biologic is administered to the subject. In some embodiments, a method of the present invention comprises administering a biologic (e.g., insulin, an incretin, and/or a nucleic acid that encodes a viral surface protein), protease inhibitor, and/or permeability enhancer that is structurally intact at the time of release from a hydrogel in which it is present and/or that has an activity that is within ± 20% of its original activity prior to incorporation in the hydrogel. In some embodiments, following administering to a subject a composition of the present invention comprising a biologic, the biologic (e.g., insulin) has a biologic peak to trough ratio of less than 2. In some embodiments, following administering to a subject a composition of the present invention comprising a biologic, the peak concentration of the biologic (e.g., insulin) is reached within 4 hours after administration of the composition.

In some embodiments, a method of the present invention provides a continuous release of a protease inhibitor, a permeability enhancer, and/or a biologic for a period of time. In some embodiments, the period of time of continuous release is about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours

In some embodiments, a method of the present invention comprises administering a composition of the present invention that includes a hydrogel of the present invention, wherein the hydrogel comprises an agent (e.g., a biologic, permeability enhancer, and/or protease inhibitor), and the hydrogel prevents release of the agent in the stomach of a subject and the hydrogel releases the agent in the small intestine of the subject. In some embodiments, the hydrogel falls apart and/or degrades in the intestines

In some embodiments, a method of the present invention comprises administering insulin to a subject via a composition of the present invention, which may prevent the subject’s blood sugar level from getting too elevated (hyperglycemia) or too low (hypoglycemia). In some embodiments, a method of the present invention comprises administering an incretin (e.g., 1 (GLP-1 and/or GIP) to a subject via a composition of the present invention, which may prevent the subject’s blood sugar level from getting too elevated (hyperglycemia) or too low (hypoglycemia). In some embodiments, a method of the present invention comprises administering a nucleic acid encoding a viral surface protein to a subject via a composition of the present invention, which may provide and/or raise an immune response in the subject to the virus.

The present invention is explained in greater detail in the following non-limiting examples.

EXAMPLES

Example 1:

Background

Oral drug delivery has been the preferred route for drug administration because it can promote patient compliance among other advantages over other routes of administration. For most therapeutics, the desired goal is to safely deliver the therapeutic agent to the gut from where it would be absorbed, thus enhancing bioavailability [1], A major concern when the oral route of administration is considered for a therapeutic agent is the hostile environment involving gastric acid and the digestive enzymes that may degrade therapeutic biologies such as peptides.

Alginate, a block copolymer consisting of (l,4)-linked P-D-mannuronate (M) and a-L- guluronate (G) monomers, is a complex polysaccharide that is typically extracted from brown algae. It has found various applications in biomedical science and engineering because of its favorable properties, including biocompatibility and ease of gelation under mild physiological conditions [2], One of the main applications of alginate hydrogels has been in drug delivery for which functionalization of the alginate polymer has been extensively investigated [3,4], We have previously described two chemical processes of modification of alginate with 4-(2- aminoethylj-benzoic acid (ABA) to achieve near neutral and weak basic pH sensitivity of the resultant [5], The products of both methods result in a hydrogel that is stable under acidic conditions as occurs in the stomach but degrades in response to near neutral and weak basic pH prevalent in the intestines.

The goal of the present study was to examine the mechanism by which the ABA- modified alginate hydrogel responds to near neutral and weak basic pH conditions. Toward this goal we wanted to better understand the chemical origin of the pH dependence of the ABA modified alginate. While not wishing to be bound by any particular theory, we had hypothesized that ABA modified alginate would be stable at acidic pH where the carboxylic acid functional group would be protonated and more unstable/water soluble at near neutral and weak basic pH where the carboxylate would be ionic.

To test this hypothesis about the effect of the added carboxylic acid functional group, we prepared a small library of compounds where the substituent on the benzene ring was varied. We added the following small molecule attachments to the alginate backbone in addition to the ABA modified alginate so that their pH dependent degradation rates could be compared: 1) phenethylamine; 2) methyl 4-(2-aminoethyl) benzoate, and 3) l-[4-(2-aminoethyl) phenyl] ethanone.

Materials

Ultra-pure low viscosity (20-200 mPa-s) sodium alginate with high guluronic acid (UPLVG) contents was purchased from Nova-Matrix (Sandvika, Norway). UPLVG alginate was reported by the manufacturer to have molecular weights 75-200kDa and guluronic acid to mannuronic acid (G/M) ratios of 1.5, and the G/M ratios were not altered by our modifications as reported previously [5],

The water suppression proton nuclear magnetic resonance (^XH NMR) and DOSY spectra were obtained using a Bruker Ascend 400 MHz spectrometer operating at 400.1 MHz. 'H NMR spectra were referenced to the residual proton or carbon signals of the respective deuterated solvents. Chemical shifts were reported in parts per million (5) relative to tetramethylsilane (TMS) or to residual resonances of the deuterated solvents: deuterium oxide (D2O). Spin multiplicities were indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), and m (multiplet). All reactions were carried out under an atmosphere of nitrogen. Deuterated solvents were purchased from Cambridge Isotope Laboratories. All ultrapure water (type 1) was acquired using a Millipore MilliQ direct water filtration system. Lyophilization was carried out using a LabConco Freezone 4.5. NaCl used in dialysis was purchased from Fisher and used as received, while cellulose acetate dialysis membrane was purchased from Sigma. Potassium phosphate monobasic and dibasic for preparing phosphate buffer were purchased from GFS and MP Pharmaceuticals, respectively. 4-(2-Aminoethyl)-benzoic acid HC1 was purchased from Sigma Aldrich. Methyl 4-(2- aminoethyl) benzoate HC1 was purchased from Combi-Blocks. l-[4-(2-aminoethyl) phenyl] ethanone HC1 was purchased from Enamine. Phenethylamine and sodium periodate were purchased from Acros Organics. 2-methylpyridine borane complex (pic-BHi) and maleic acid and all other chemicals were also purchased from Sigma-Aldrich. Methods

Preparation of Aminoethyl Benzoic acid Modified Alginate (1).

UPLVG alginate (0.180g, Immol) was dissolved in 41 mL ultrapure (type l) waterwith 10% (v/v) isopropanol and degassed with nitrogen for 1 hour. Following degassing, NalCh (7mg, 0.0327mmol) was added to the solution which was then covered with aluminum foil and stirred under a nitrogen atmosphere for 48-72 hours. The oxidized solution was transferred to a cellulose acetate dialysis membrane with a molecular cut off of 12,000 g/mol. Dialysis was carried out in 1.0M NaCl for 24 hours and then in ultrapure water for 48 hours with the bath being changed every 12 hours. The dialyzed mixture was lyophilized at -50°C and a pressure of <400 mbar for 24 hours. A cottony, white oxidized product (0.178g) was recovered. This oxidized alginate was dissolved in 15 mL ultrapure water with 12% (v/v) MeOH. Once dissolved, 4-(2-aminoethyl) benzoic acid HC1 (3.6mg, 0.020 mmol) and 2-methylpyridine borane complex (21 mg, 0.2 mmol) were added to the solution and phosphate buffer (pH=6.01) was added to adjust the pH to ~6. The solution was again covered in foil and stirred under N2 for 48 hours. The now aminated alginate solution was again dialyzed in IM NaCl followed by ultrapure water as it was after the oxidation and lyophilized. Cottony benzoic acid modified alginate (0.172g, 96%) was recovered and analyzed via water suppression. 'H NMR characteristic aromatic signals: (400 MHz, D2O) 5 7.90 (d, J = 8.1 Hz, 2H), 7.44 - 7.30 (m, 2H). 'H NMR and DOSY spectra for this material were reported [5], Preparation of Phenethylamine Modified Alginate (2),

Oxidized alginate was prepared as described above for ABA modified alginate (1) and dissolved in 15 mL ultrapure water with 12% (v/v) MeOH. Once dissolved, phenethyl amine (3.0 mg, 0.027 mmol) dissolved in 1 mL ultrapure water and 2-methylpyridine borane complex (21 mg, 0.2 mmol) were added to the solution and phosphate buffer (pH=6.01) was added to adjust the pH to ~6. Solution was again covered in foil and stirred under N2 for 48 hours. The now aminated alginate solution was again dialyzed in IM NaCl followed by ultrapure water as it was after the oxidation and lyophilized. Cottony phenethylamine modified alginate (0.176g, 99%) was recovered and analyzed via water suppression. 'H NMR characteristic aromatic signals: (400 MHz, D2O) 5 7.42 - 7.33 (m, 2H), 7.32 - 7.22 (m, 3H).

Preparation of Methyl 4-(2-aminoethyl) benzoate Modified Alginate (3),

Oxidized alginate was prepared as described above for ABA modified alginate (1) and dissolved in 15 mL ultrapure water with 12% v/v MeOH. Once dissolved, methyl 4-(2- aminoethyl) benzoate HC1 (3.8mg, 0.020 mmol) and 2-methylpyridine borane complex (21 mg, 0.2 mmol) were added to the solution and phosphate buffer (pH=6.01) was added to adjust the pH to ~6. Solution was again covered in foil and stirred under N2 for 48 hours. The now aminated alginate solution was again dialyzed in IM NaCl followed by ultrapure water as it was after the oxidation and lyophilized. Cottony methyl benzoate modified alginate (0.178g, 99%) was recovered and analyzed via water suppression 'H NMR characteristic aromatic signals: (400 MHz, D2O) 5 7.94 (d, J= 4.7 Hz, 2H), 7.44 - 7.31 (m, 2H). ¹ H NMR and DOSY spectra supplied in supplementary materials.

Preparation of l-14-(2-Aminoethyl) phenyll ethanone Modified Alginate (4),

Oxidized alginate was prepared as described above for ABA modified alginate (1) and dissolved in 15 mL ultrapure water with 12% (v/v) MeOH. Once dissolved, l-[4-(2- aminoethyl) phenyl] ethanone HC1 (3.6mg, 0.020 mmol) and 2-methylpyridine borane complex (21 mg, 0.2 mmol) were added to the solution and phosphate buffer (pH=6.01) was added to adjust the pH to ~6. Solution was again covered in foil and stirred under N2 for 48 hours. The now aminated alginate solution was again dialyzed in IM NaCl followed by ultrapure water as it was after the oxidation and lyophilized. Cottony acetophenone modified alginate (0.178g, 99%) was recovered and analyzed via water suppression. 'H NMR characteristic aromatic signals: (400 MHz, D2O) 5 8.04 - 7.84 (m, 2H), 7.51 - 7.25 (m, 2H). ID Quantitative Characterization.

Maleic acid (11.6 mg, 0.1 mmol) was dissolved in 2 mL deuterium oxide NMR solvent to give a 50 mM solution. 10 mg of the modified alginate product was added to an NMR tube with 250 pL of the maleic acid solution and 750 pL D2O. Sonication was used to fully dissolve the solid and the sample was analyzed via water suppression ¹ H NMR. Aromatic peaks in the spectrum were integrated along with the maleic acid peak at 6.25 ppm. Maleic acid integration was normalized to 1 and the corresponding integration of one aromatic peak (corresponding to 2H) was used to derive the percentage of alginate units modified.

NMR POSY,

Diffusion order spectroscopy (DOSY) spectra were obtained using a Bruker Ascend 400 MHz magnet operating at 400.1 MHz. Pertinent parameters are shown in Table 1.

Table 1. NMR DOSY parameters.

Preparation of Simulated Gastric Acid (SGF) and Simulated Intestinal Fluid (SIF),

These solutions were prepared as previously described [1], Briefly, the SGF was prepared by mixing 2 g/L of NaCl with DiffcO and adjusting the pH to 2.0. The SIF was prepared by mixing 6.8 g/L monobasic KH2PO4 with DiH20 and adjusting the pH to 6.8 or 7.4. In Vitro testing of alginate material hydrogels stability and degradation.

Unmodified or modified alginate was dissolved in Hanks Balanced Salt Solution (HBSS) at a concentration of 1.5% (w/v) overnight. 10 hydrogel beads were made by manually extruding the alginate through an 18-guage blunt needle into 3 ml of 100 mM CaCh crosslinking solution in a 6 well plate, the diameter of the hydrogel beads ranged from 2.6mm to 3.27mm with an average diameter size of 2.97mm and a standard deviation of 0.19mm. The beads were allowed to crosslink for 10-15 minutes before being incubated in either SGF for 6- 24 hours or SIF for 3 hours while being shaken at 60 RPM at 37°C. At corresponding time points, the beads were visually counted to determine the number of intact beads remaining in each respective medium. The term intact describes beads that have a complete intact structure in contrast to beads that are partially dissolved or that have lost their shell and have an opaque core.

Statistical Analysis.

Experimental data from the tests performed with the hydrogels made with the alginate materials are expressed as mean ± standard error and statistical evaluations of the data were performed using a ONE-WAY ANOVA with Tukey’s correction. Differences were considered significant if p < 0.05.

Results

Chemical modification and validation of covalent bonding of the small molecules. All the new small molecule modified alginates used in the present study were prepared by oxidizing the vicinal dialcohol backbone of alginate to a dialdehyde followed by reductive amination using one of the phenethylamine derivatives as outlined above. Following Dalheim et al’s protocol [6], oxidation of the vicinal dialcohol was carried out using 2 mol% of NalCh and the generated dialdehyde was then reacted with the appropriate substituted or unsubstituted phenethylamine followed by reduction to obtain the covalently linked small molecule in the alginate backbone (Scheme 1).

Scheme 1: Chemical modification scheme for reductive animation represented as a reaction on an alginate monomer unit.

The amount of small molecule incorporated into the polymer backbone was calculated based on ¹ H NMR data using a coaxial external standard (maleic acid) as described previously [5], Covalent bonding between the polymer backbone and the small molecule was determined using DOSY to measure the difference of diffusion coefficients between the covalently coupled product and non-coupled mixture of oxidized alginate and small molecule as described previously [5], ^JH NMR and DOSY spectra of all new modified alginates are included in supplementary material. The DOSY spectrum of benzoic acid modified alginate is contained in our prior publication [5],

Effect of pH on the stability of unmodified alginate hydrogel microbeads.

In Fig. 1, we show the stability of alginate microbeads made with unmodified alginate following 24-hour incubation at the acidic SGF medium of pH 2.0. When these unmodified alginate beads were incubated in the SIF medium at the near neutral pH 6.8, they did not degrade completely until after a 6-hour period of incubation (Fig. 2, panel A). In contrast, following incubation in the SIF medium at the weak basic pH 7.4, the beads degraded completely after 2 hours of incubation (Fig. 2, panel B).

Assessment of the stability and degradation profiles of modified alginate material hydrogels. Stability of the modified alginate material hydrogels under acidic pH.

Fig. 3, panels A-D quantitatively illustrates the stability of the chemically modified alginate microbeads at the acidic SGF pH = 2. The microbeads were made with alginate materials modified by the attachment of methyl ketone, methyl ester, benzoic acid, or phenethylamine to 2% oxidized alginate. Following incubation of the hydrogel beads in this acidic medium for 180 minutes with shaking, all the beads remained intact (Fig. 3, panels A- D), similar to our observations with the unmodified alginate beads in the current study (Fig. 1), and consistent with our previous study [5], Degradation of alginate material hydrogels under near neutral and weak basic pH conditions.

As shown in Fig. 4, panel A, it takes about 6 hours for the unmodified alginate hydrogel to degrade at the near neutral pH of 6.8 in contrast to the modified alginate microbeads, which degrade much faster. It is noteworthy that the microbeads generated with methyl ketone- and methyl ester-modified alginates degraded similarly and faster than those made with ABA and phenyl-ethylamine-modified alginates (p<0.001, n =5). It is also noteworthy that the phenyl- ethylamine-modified and ABA-modified alginates degraded in a similar manner at either pH 6.8 or pH 7.4 (Fig. 4, panels A and B, (*p=0.0153, ***p=0.0005, ****p<0.0001, n = 5). However, at the weak basic pH 7.4, the unmodified alginate microbeads were found to degrade in a little more than 1 hour, albeit its rate of degradation was still slower than the rates at which the modified alginates degraded (p<0.001, n = 5, Fig. 4, panel B).

As illustrated with the ABA-modified alginates, we found that the rate of degradation of the modified alginate microbeads was faster with an increase in the degree of modification at both pH 6.8 and pH 7.4 (Fig. 5, panels A-B). Thus, when we compared the rates of degradation at the slower degrading near neutral pH of 6.8 after 60 minutes incubation, the alginate treated with 5 mol% of reagents degraded faster than those made with lower mol% reagents (p<0.0001, n = 5, Fig. 5, panel A (****p<0.0001 and **p 0.002, n = 5)). After 30 minutes incubation at the faster degrading weak basic pH 7.4, we also found that the higher the degree of modification the faster the rate of degradation among the 3 modifications tested (p = 0.0021, n = 5, Fig. 5, panel B (****p<0.0001, n = 5)). Discussion

The degradation profiles of phenethylamine- and ABA- modified alginates were either similar or different from those of methyl 4-(2-aminoethyl) benzoate and l-[4-(2-aminoethyl) phenyl] ethanone, respectively. This result is surprising because one would expect that the benzoic acid modified alginate would be essentially completely deprotonated (ionic) at pH 6.8- 7.4, therefore more water soluble and degrade the fastest of all tested modifications, but that is not the case. While not wishing to be bound by any particular theory, since the methyl ketone and methyl ester modified alginates degrade the fastest (faster even than phenethylamine modified alginate) we suspect those benzene substituents weaken intra and inter alginate strand intermolecular interactions like hydrogen bonding, dipole-dipole, and ion-dipole interactions. While not wishing to be bound by any particular theory, it is also possible that the addition of these aromatic substituents onto the alginate backbone in all four cases studied enables polymer to polymer cation-pi interactions once Ca⁺² is introduced and that those are weaker for the alginates modified with the electron withdrawing methyl ketone and methyl ester substituents leading to faster degradation for those alginate beads [7], With respect to possible steric effects of the benzene substituents, the carboxylic acid has about the same steric bulk as the methyl ketone and the methyl ester, but its alginate is significantly more stable than those, essentially the same as phenethylamine modified alginate. In that case, while not wishing to be bound by any particular theory, we would hypothesize that while its steric bulk would weaken intra and inter alginate strand interactions its charge at these pHs would increase ion-dipole or ionhydrogen bonding interactions.

Our data has significant physiologic implications for the use of AB A-modified alginate in oral drug delivery, particularly the delivery of therapeutic peptides for the management of diseases. This oral drug delivery platform may provide protection of microbead-encapsulated peptides from acidic gastric enzymatic destruction in the stomach until entry into the small intestine, where the microbeads will be exposed to near neutral and weak basic pH conditions. The small intestine is comprised of three segments, the proximal (duodenum), the middle (jejunum) and the distal (ileum) and it is in the jejunum and ileum that most of the orally ingested substances are absorbed [8], In human subjects, it is known that while the gastric pH is highly acidic (range 1.0-2.5) the mean pH ± SD in the proximal small intestine is 6.6 (0.5) and the mean pH ± SD in the terminal ileum is 7.5 (0.4) [9], Therefore, it is clear that the ABA- modified alginate microbeads loaded with therapeutic substances would not degrade appreciably until reaching the distal small intestine where they would rapidly degrade and release their payloads. If such payloads are small molecules able to permeate the intestinal barrier against transport across the tissue, their absorption and therapeutic efficacy could be enhanced. In addition, the release of payloads in this later region of the gastrointestinal tract makes this delivery platform particularly suitable for treating diseases in that part of the intestine and beyond.

References

[1] Enck K, Banks S, Yadav H, Welker ME, Opara EC. Development of a Novel Oral Delivery Vehicle for Probiotics. Curr Pharm Des. 2020;26(26):3134-3140.

[2] Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012 Jan;37(l): 106-126.

[3] Pawar SN, Edgar KJ. Alginate derivatization: a review of chemistry, properties and applications. Biomaterials. 2012 Apr;33(l l):3279-305.

[4] Welker ME. Chemical Modifications of Alginates for Use in Drug Delivery and Regenerative Medicine Applications. In E.C. Opara Controlled Drug Delivery Systems 1st Edition. CRC Press.2020 (pp 3-28).

[5] Banks SR, Enck K, Wright M, Opara EC, Welker ME. Chemical Modification of Alginate for Controlled Oral Drug Delivery. J Agric Food Chem. 2019 Sep 18;67(37): 10481-10488.

[6] Dalheim MO, Vanacker J, Najmi MA, Aachmann FL, Strand BL, Christensen BE. Efficient functionalization of alginate biomaterials. Biomaterials. 2016 Feb;80: 146- 156.

[7] Geng H, Zhang P, Peng Q, Cui J, Hao J, Zeng H. Principles of Cation-7t Interactions for Engineering Mussel-Inspired Functional Materials. Acc Chem Res. 2022 Apr 19;55(8):1171-1182.

[8] Canadian Cancer Society: The Small Intestine [https://cancer.ca/en/cancer- information/cancer-types/small-intestine/what-is-small-intestine-cancer/the-small- intestine],

[9] Evans DF, Pye G, Bramley R, Clark AG, Dyson TJ, Hardcastle JD. Measurement of gastrointestinal pH profiles in normal ambulant human subjects. Gut. 1988 Aug;29(8): 1035-41.

Example 2:

Diabetes is one of the leading causes of chronic illness in America, and the costs associated with its treatment exceed $245 billion annually in the United States alone (Centers for Disease Control and Prevention). Patients with diabetes are unable to produce adequate amounts of insulin, an important regulator of cellular metabolic pathways involved in glucose uptake and utilization [1], The dysregulation of blood glucose that results from insufficient insulin can cause both microvascular and macrovascular pathology, with complications including retinopathy, neuropathy [2], nephropathy, and an increased risk of cardiovascular disease [3],

Oral insulin formulations face two major hurdles- degradation by gastrointestinal enzymes, and transport barriers of the GI tract mucoepithelium. The mucous layer of the GI tract varies in composition and function across different regions of the gut, yet in general, it serves as a barrier between the caustic and bacterial -laden lumen of the intestines and the surrounding epithelial brush border [5], A variety of large and highly glycosylated proteins called mucins, broadly classified as either gel-forming or transmembrane, contribute to the mucous layer and are produced in region-dependent quantities by glands, goblet epithelial cells, and enterocytes. The constant secretion of mucus generates net movement into the lumen; this “current” drags small molecules such as insulin away from their potential site of uptake at the brush border and flushes them through the remainder of the GI tract. By employing strategies of adherence to and penetration through the gastrointestinal mucous layer, oral insulin formulations increase their potential for bioavailability and efficacy.

Deep to the mucous layer is the gastrointestinal brush border and underlying epithelium. Crossing either through (transcellular movement) or around (paracellular movement) these epithelial cells allow entrance into portal circulation, from where insulin can reach various sites of its activity. Transcellular uptake can be achieved via simple diffusion, facilitated diffusion, and carrier-mediated active transport. Simple diffusion relies on compounds passing directly through the nonpolar lipid cell membrane and consequently the molecules that achieve this sort of uptake are generally small and lipophilic. Because peptides such as insulin are relatively large and typically carry surface charges, their ability to cross the cell membrane via simple diffusion is limited [6],

One of the primary activities of the GI tract is to facilitate the degradation and metabolism of various macromolecules. Because proteins are absorbed as single amino acids and dipeptides, they are readily broken down by various enzymes secreted from the pancreas and glandular epithelium. Insulin acts as a substrate for many of these enzymes including trypsin, alpha chymotrypsin, and various carboxypeptidases [6], Recent studies have also demonstrated the presence of a specific insulin degrading enzyme (termed IDE) on the enterocyte brush border [7], In concert, these enzymes severely limit the amount of intact insulin present in the GI tract, thus reducing opportunities for oral delivery and subsequent pharmacologic action.

The pH of the human GI tract ranges from strongly acidic (pH=l-1.2) to slightly alkaline (pH=7.8) owing to the regional secretions of the stomach, gallbladder, pancreas, and intestinal glandular epithelium. These local environments themselves as well as the dramatic variations between them have the capacity to cause oxidation and deamination of peptides such as insulin [8], Gastrointestinal pH also facilitates the enzymatic degradation of insulin because many of the previously discussed proteases are active only within a certain range of acidity or (more often) alkalinity. Thus, protecting insulin from local gastrointestinal pH has the potential to protect it from degradation both directly and indirectly.

Meaningful oral delivery of insulin has been hampered by the susceptibility of the peptide to enzymatic degradation and its poor permeability across the mucus and cellular membranes in the gastrointestinal tract. We have formulated an oral insulin delivery package that is comprised of insulin, a CPP (hexa-arginine) and a protease inhibitor (aprotinin) using benzoic acid modified alginate materials that enable hydrogel fabrication for timed release of encapsulated therapeutics.

Methods

Alginate preparation

Ultra-purified low-viscosity high-glucuronic acid alginate (LVG, Novamatrix, Sandvika Norway) at different levels of modification with benzoic acid solutions (1% and 2% modified alginate) were prepared with HBSS (H6648, Sigma) at 1.5% (w/v) and stirring overnight at 4°C. The modified alginate solutions were pumped through a 2-channel microfluidic device at a flow rate of 0.2 ml/min with an air pressure of .8 psi. The microspheres generated were collected in a sterile 100 mM CaCh solution and allowed to crosslink for 10 minutes prior to washing with HBSS.

Preparation of the drugs

We made fresh aprotinin (protease inhibitor) and CPP solutions with sterile Hanks’s solution at 70 mg/ml and 14 mg and dissolved in 200 pl Hank’s solution, respectively. To induce diabetes in rats, we freshly prepared Streptozotocin (STZ) dissolved in citric acid buffer, pH 4.5. The stock concentration of STZ was 10 mg/ml.

Loading up (e.g„ soaking) of alginate beads with substances We incubated groups of 10 beads in 24 pl of insulin (50IU), 15 beads in 30 pl of CPP (24.487 mg/ml) and 15 beads in 30 pl of aprotinin (15.066 mg/ml) for 24 hours at room temperature.

The summary of each of these 3 substances in the microbeads used in the experiments is as follows:

10 beads containing ~ 4.737 ng/ml insulin

15 beads containing 0.45 mg/ml aprotinin

15 beads containing 0.74 mg/ml CPP

Each animal experiment was conducted over 5 days, as follows:

Dayl : Induction of diabetes

We had 3 groups and each group had 2 rats. We obtained the body weights and got the baseline blood glucose levels in the morning using the glucometer kit. We calculated the STZ amount and prepared it at the concentration mentioned above and injected it intraperitoneally at the dose of 65 mg/kg. The injection was performed in the biosafety cabinet. Rats were diagnosed as diabetic following the standard criteria of 2 consecutive blood glucose measurements >400 mg/dl [11],

Davs 2-4: Diabetes Management

We checked the daily blood glucose in the mornings and if any were not diabetic within 72 hours, we reinjected the non-diabetic rats with a 2^nd dose of STZ. We treated the diabetic rats (>400 mg/dl glucose during 2 consecutive measurements) by injecting the rats subcutaneously with long-acting insulin (Lantus at 4IU/200g) if the blood glucose levels were above 400 mg/dl except in the last 24 hours before the oral gavage of encapsulated substances. We noted the daily body weights of the rats. On the 4^th day of the experiment we removed the food over night to reduce the amount of food in the stomach of the rats in order to facilitate the sections of the gastrointestinal examination after animal sacrifice at the end of the experiment.

Dav 5: Administration of the beads via oral gavage

We recorded the baseline blood glucose levels after venipuncture in the leg using 50 pl capillary tube with EDTA and centrifuged the blood samples at 2000 RPM for 5-10 min to get plasma to measure the insulin level in the morning. We checked the body weights by using an electronic scale. We infused the microbeads and arranged them in the oral gavage tube and put the oral gavage tube with 3 ml syringe and insert the oral gavage tube through the esophagus and pushed the beads with air. We had 3 groups and each group consisted of 2 rats each rat received 40 microbeads. Our groups were as the following:

Group 1 (insulin only) 10 beads of 1% modified alginate loaded with insulin, 15 empty beads of 1% modified alginate and 15 empty beads of 2% modified alginate.

Group 2 (empty beads)

25 beads of 1% modified alginate and 15 empty beads of 2% modified alginate all were soaked in the storage solution.

Group 3 (concurrent release)

10 beads of 2% modified alginate loaded with insulin, 15 beads of 2% modified alginate loaded with CPP and 15 beads of 2% modified alginate loaded with aprotinin.

Group 4 (sequential release)

10 beads of 1% modified alginate loaded with insulin, 15 beads of 1% modified alginate loaded with CPP and 15 beads of 2% modified alginate loaded with aprotinin.

After administration of the microbeads, we checked the blood glucose levels using the glucometer kit after 1 hour and then every 30 minutes for 4 hours with serial blood collections (50 pl). The blood samples were centrifuged to obtain plasma as was done at the time point zero before the oral gavage and afterwards at the following time points 1, 2, and 4 hours. The plasma was stored at -20°C. After the 4-hour time point, we performed CCh-asphyxia euthanasia on all animals and harvested the stomach and intestine tissues and washed them with PBS. These tissues were examined to see if there were any beads in the gastrointestinal tract. We all also harvested the liver, leg muscle and the heart and stored them in liquid nitrogen.

We did not find any microbeads in either the stomach or the intestine of any experimental animal at the end of the 4-hour follow up, indicating that all the beads had dissolved and released their contents on reaching the small intestine where the pH is neutral- basic.

Alpha Elisa

We measured plasma insulin levels in all groups at the 4 time points (0, 1, 2, and 4 hours) and degraded a group of 10 beads that had been incubated in insulin for 24 hours as baseline content to evaluate the efficiency of encapsulation using human insulin alpha Elisa kit (AL204C/F) according to the manufacturer instructions.

Blood glucose level

We measured the blood glucose levels on day 5 at 0, 1, 1.5 2, 2.5, 3, 3.5, and 4 hours after administration of the alginate compounds using the glucometer kit.

Results Quantitative data presented represent mean + standard deviation (SD). Statistical evaluation of the data was performed using a one-way analysis of variance (ANOVA) with the Tukey-Kramer post-tests (GraphPad Prism software). A value of p <0.05 was accepted as significant.

The data presented here demonstrate a -20% reduction in blood glucose 4 hours after sequential administration of the insulin, aprotinin, and CPP alginate bead compositions, which had a corresponding >2-fold increase in plasma insulin levels. The data from the other groups, i.e., the empty bead control, insulin only containing beads, and concurrent administration of beads containing insulin, aprotinin, and CPP, did not show any significant change in either blood glucose or plasma insulin level.

References

1. Clark, Matthew, et al. “Type 1 Diabetes: A Chronic Anti-Self-Inflammatory Response.” Frontiers in Immunology, vol. 8, 2017, p. 1898. PubMed, doi: 10.3389/fimmu.2017.01898.

2. Vinik, Aaron, et al. “Diabetic Neuropathies.” Endotext, edited by Kenneth R. Feingold et al., MDText.com, Inc., 2000. PubMed, http://www.ncbi.nlm.nih.gov/books/NBK279175/.

3. Arieff, Allen I. “Diabetic Nephropathy and Treatment of Hypertension.” Endotext, edited by Kenneth R. Feingold et al., MDText.com, Inc., 2000. PubMed,

4. Bliss, Michael. The Discovery of Insulin: The Twenty-Fifth Anniversary Edition. University of Toronto Press, 1982. JSTOR, JSTOR, https://www.jstor.Org/stable/10.3138/j.cttlwn0sjc.

5. Cornick, Steve, et al. “Roles and Regulation of the Mucus Barrier in the Gut.” Tissue Barriers, vol. 3, no. 1-2, Jan. 2015. PubMed Central, doi:10.4161/21688370.2014.982426.

6. Gedawy, Ahmed, et al. “Oral Insulin Delivery: Existing Barriers and Current CounterStrategies.” Journal of Pharmacy and Pharmacology, vol. 70, no. 2, 2018, pp. 197- 213. Wiley Online Library, doi : 10.1111 /j php .12852.

7. Durham, Timothy B., et al. “Dual Exosite-Binding Inhibitors of Insulin-Degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo.” The Journal of Biological Chemistry, vol. 290, no. 33, Aug. 2015, pp. 20044-59. PubMed Central, doi : 10.1074/jbc.Ml 15.638205. 8. Cikrikci, Sevil, et al. Development of pH Sensitive Alginate/Gum Tragacanth Based Hydrogels for Oral Insulin Delivery.” Journal of Agricultural andFood Chemistry, vol. 66, no. 44, Nov. 2018, pp. 11784-96. Crossref doi: 10.1021/acs.jafc.8b02525.

9. Abes R, et al. Cell-penetrating-peptide-based delivery of oligonucleotides: an overview Biochem Soc Trans Aug; 35 (Pt 4): 775 -9. 2007.

10. US Patent: Modified alginate hydrogels for therapeutic agents, their preparation and methods thereof. US Patent # US 10, 766,970 issued 9/8/2020.

11. Pareta R, et al. Long-term function of islets encapsulated in a re-designed alginate microcapsule construct in omentum pouches of immune-competent diabetic rats. Pancreas (#4): 605-613, 2014.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A composition comprising: a first hydrogel comprising a protease inhibitor; and a second hydrogel comprising a permeability enhancer.

2. The composition of claim 1, further comprising a third hydrogel, wherein the third hydrogel comprises a biologic (e.g., polypeptide and/or nucleic acid).

3. The composition of claim 1 or 2, wherein the first hydrogel encapsulates the protease inhibitor, wherein the second hydrogel encapsulates the permeability enhancer, and/or wherein the third hydrogel encapsulates the biologic.

4. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and the third hydrogel are discrete hydrogels (e.g., discrete particles or entities).

5. The composition of any one of the preceding claims, wherein the protease inhibitor, the permeability enhancer, and/or the biologic are uniformly distributed throughout the first hydrogel, the second hydrogel, and/or the third hydrogel, respectively.

6. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel are microparticles or nanoparticles, optionally wherein the first hydrogel, the second hydrogel, and/or the third hydrogel are in the form of a bead, capsule, cube, cylinder, and/or sphere.

7. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel have at least one dimension (e.g., diameter, width, or length) of about 100 microns to about 500 microns.

8. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and the third hydrogel are each individually a modified alginate hydrogel.

9. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and the third hydrogel are each individually a crosslinked hydrogel.

10. The composition of any one of the preceding claims, wherein the first hydrogel comprises a first modified alginate hydrogel, the second hydrogel comprises a second modified alginate hydrogel, and the third hydrogel comprises a third modified alginate hydrogel, wherein the first, second, and third hydrogels may be the same or different from one another.

11. The composition of any one of claims 8-10, wherein a moiety is present in about 1% to about 15% of the polysaccharide units of the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel.

12. The composition of claim 11, wherein the moiety has a pKa in a range of about 3 or 4 to about 4.5, 5, 5.5, or 6.

13. The composition of any one of claims 8-12, wherein the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel is prepared by reacting an alginate with a reactant that is selected from the group consisting of 4-(2-aminoethyl)benzoic acid, 4-(2-aminomethyl)benzoic acid, 4-(2- aminoethyl)aniline, (2-ethylamino)4-methyl benzene, 4-(2-aminoacetyl)-benzoic acid, 4-(2- aminoethyl)salicylic acid, methyl 4-(2-aminoethyl) benzoate, phenethylamine, l-[4-(2- aminoethyl) phenyl] ethanone HC1, and/or 4-(2-aminomethyl)aniline and/or esters thereof.

14. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel is/are a 2-amino ethyl or aminomethyl aromatic (e.g., a 2-amino ethyl or aminomethyl benzyl) modified alginate hydrogel, optionally wherein the first hydrogel, the second hydrogel, and/or the third hydrogel is/are a 4-(2-aminoethyl) benzoic acid modified alginate hydrogel.

15. The composition of any one of claims 8-14, wherein about 1% of the polysaccharide units of the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel are modified.

16. The composition of any one of claims 8-15, wherein about 2% of the polysaccharide units of the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel are modified.

17. The composition of any one of claims 8-16, wherein the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel comprises at least one unit having a structure of Formula I or Formula II: wherein

Y is absent or a C1-C4 alkyl or C1-C4 alkenyl;

Ri is each independently selected from the group consisting of -H, -OH, -NH2, -COOH,

-C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci-Ce alkylhalide, unsubstituted or substituted -C1-C6 alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g., - COCH3), -Si(OH)3, -SO2NH2, -PO(OR’)2, and -B(0H)2, wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl; n is from 1 to 1,000,000; and o is 0, 1, 2, 3, 4, or 5.

18. The composition of claim 17, wherein at least one Ri is in the para position and is selected from the group consisting of -NH2, -COOH, -C(O)OR’, -NO2, -CN, -Br, -Cl, -F, -Ci- Ce alkylhalide, unsubstituted or substituted -Ci-Ce alkyl, unsubstituted or substituted -Ci-Ce alkenyl, -SO2H, -SO3H, -COR’ (e.g, -COCH3), -Si(OH)₃, -SO2NH2, -PO(OR’)₂, and -B(OH)₂, and wherein R’ is unsubstituted or substituted alkyl, alkenyl, alkynyl, or aryl.

19. The composition of any one of claims 8-16, wherein the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel comprises oxidized alginate.

20. The composition of any one of claims 2-19, wherein the biologic is a peptide therapeutic (e.g., an anti-diabetes peptide) or a protein, optionally wherein the biologic is insulin.

21. The composition of any one of claims 2-20, wherein the biologic is insulin, optionally wherein the insulin is selected from human insulin, insulin glargine, insulin detemir, insulin lispro, insulin aspart, insulin glulisine, and/or a prodrug thereof.

22. The composition of any one of claims 2-21, wherein the biologic is present in the third hydrogel in a concentration of about 1 ng of the biologic per mL of the third hydrogel to about 10 pg of the biologic per mL of the third hydrogel.

23. The composition of any one of claims 2-21, wherein the biologic is present in the composition in a total concentration of about 1 or 5 unit(s) to about 10, 15, or 20 units.

24. The composition of any one of preceding claims, wherein the protease inhibitor has a molecular weight of about 500 Daltons to about 2000 or 7000 Daltons.

25. The composition of any one of preceding claims, wherein the protease inhibitor is present in the first hydrogel in a concentration of about 0.01 or 0.1 mg of the protease inhibitor per mL of the first hydrogel to about 1, 2, 3, 4, or 5 mg of the protease inhibitor per mL of the first hydrogel and/or wherein the protease inhibitor is present in the composition in a total concentration of about 50 or 100 mg of the protease inhibitor per mL of the composition to about 150 or 200 mg of the protease inhibitor per mL of the composition.

26. The composition of any one of the preceding claims, wherein protease inhibitor is a Bowman-Birk inhibitor, a Kunitz-type inhibitor, an a-amylase inhibitor, and/or a trypsin inhibitor, optionally wherein the protease inhibitor is aprotinin.

27. The composition of any one of the preceding claims, wherein the permeability enhancer comprises a sugar (e.g., fructose), an antibiotic, an endocannabinoid (e.g., anandamide), and/or a cell-penetrating peptide (e.g., a cationic cell-penetrating peptide).

28. The composition of any one of the preceding claims, wherein the permeability enhancer comprises an arginine rich polypeptide, optionally wherein the permeability enhancer comprises a 6-aminohexanoic acid-spaced oligo-arginine, hexa-arginine penetratin, or nanoarginine.

29. The composition of any one of the preceding claims, wherein the permeability enhancer is present in the second hydrogel in a concentration of about 0.01 or 0.1 mg of the permeability enhancer per mL of the second hydrogel to about 1, 2, 3, 4, or 5 mg of the permeability enhancer per /mL of the second hydrogel and/or wherein the permeability enhancer is present in the composition in a total concentration of about 100 or 200 mg of the permeability enhancer per mL of the composition to about 150 or 200 mg of the permeability enhancer per mL of the composition.

30. The composition of any one of claims 8-29, wherein the modified alginate of the first modified alginate hydrogel, the second modified alginate hydrogel, and/or the third modified alginate hydrogel is present in an amount of about 0.1% to about 10% w/v of the hydrogel.

31. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel comprises water or an aqueous solution (e.g., a saline solution) that is optionally buffered.

32. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel comprises a divalent cation in an amount of about 0.1 mmol to about 1 mmol.

33. The composition of any one of the preceding claims, wherein the first hydrogel, the second hydrogel, and/or the third hydrogel has an elastic modulus in a range of 1 or 5 kPa to about 15 or 20 kPa.

34. The composition of any one of the preceding claims, further comprising a carrier, (e.g., a pharmaceutical carrier), optionally wherein the first hydrogel, second hydrogel, and/or third hydrogel are dispersed and/or suspended in the carrier.

35. The composition of any one of the preceding claims, further comprising a capsule that encapsulates the first hydrogel, second hydrogel, and/or third hydrogel and optionally a carrier, optionally wherein the capsule is configured for oral administration.

36. The composition of claim 35, wherein the capsule comprises a cellulose.

37. A method for treating a subject in need thereof, the method comprising: administering a first hydrogel comprising a protease inhibitor, a second hydrogel comprising a permeability enhancer, and a third hydrogel comprising a biologic (e.g., polypeptide and/or nucleic acid), thereby treating the subject.

38. The method of claim 37, wherein the administering comprises separately administering at least one of the first hydrogel, the second hydrogel, and the third hydrogel to the subject, optionally wherein the administering comprises separately administering the first hydrogel, the second hydrogel, and the third hydrogel to the subject.

39. The method of claim 37 or 38, wherein the administering comprises concurrently administering at least one of the first hydrogel, the second hydrogel, and the third hydrogel to the subject, optionally wherein the administering comprises concurrently administering the first hydrogel, the second hydrogel, and the third hydrogel to the subject.

40. The method of any one of claims 37-39, wherein at least two of the first hydrogel, the second hydrogel, and the third hydrogel are present in the same composition that is administered to the subject, optionally wherein the first hydrogel, the second hydrogel, and the third hydrogel are present in the same composition that is administered to the subject.

41. The method of any one of claims 37-40, wherein at least two of the first hydrogel, the second hydrogel, and the third hydrogel are present in separate compositions that are administered to the subject, optionally wherein the first hydrogel, the second hydrogel, and the third hydrogel are each present in separate compositions that are administered to the subject.

42. The method of any one of claims 37-41, wherein the administering comprises administering the composition of any one of claims 1-36.

43. The method of any one of claims 37-42, wherein, following the administering, at least two of the protease inhibitor, the permeability enhancer, and the biologic are sequentially released from their respective hydrogel, optionally wherein, following the administering, the protease inhibitor, the permeability enhancer, and the biologic are each sequentially released from their respective hydrogel.

44. The method of any one of claims 37-43, wherein, following the administering, at least two of the protease inhibitor, the permeability enhancer, and the biologic are concurrently released from their respective hydrogel, optionally wherein, following the administering, the protease inhibitor, the permeability enhancer, and the biologic are each concurrently released from their respective hydrogel.

45. The method of any one of claims 37-44, wherein, following the administering, the protease inhibitor is released from the first hydrogel prior to the release of the permeability enhancer from the second hydrogel and the biologic from the third hydrogel, optionally wherein, following the administering, the biologic and the permeability enhancer are concurrently released from the third hydrogel and second hydrogel, respectively.

46. The method of any one of claims 37-45, wherein, responsive to the administering, the protease inhibitor, the permeability enhancer, and/or the biologic are released in the intestines of the subject.

47. The method of any one of claims 37-46, wherein the administering comprises administering to the subject the first hydrogel, the second hydrogel, and the second hydrogel in a ratio of hydrogel particulates (e.g., hydrogel particles such as beads) in a range of about 1 : 1 : 1 to about 3:3:2 (first hydrogel particulates : second hydrogel particulates : third hydrogel particulates).

48. The method of any one of claims 37-47, wherein the administering comprises orally administering the first hydrogel, the second hydrogel, and the second hydrogel to the subject.

49. The method of any one of claims 37-48, wherein, responsive to the administering, the biologic (e.g., insulin) has a biologic peak to trough ratio of less than 2.

50. The method of any one of claims 37-49, wherein, responsive to the administering, a continuous release of the biologic (e.g., insulin) is delivered to the subject for a period of time, optionally wherein the biologic is structurally intact at the time of release and/or the continuous release is provided until an immediate subsequent dose of the biologic.

51. The method of any one of claims 37-50, wherein, responsive to the administering, the protease inhibitor, the permeability enhancer, and/or the biologic are released from the first hydrogel, second hydrogel, and/or third hydrogel, respectively, for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

52. The method of any one of claims 37-51, wherein, responsive to the administering, the peak concentration of the biologic (e.g., insulin) is reached within 4 hours after administration of the third hydrogel.

53. The method of any one of claims 37-52, wherein the method comprises treating diabetes in the subj ect.