Hydrogels exhibiting boronate ester-mediated drug release

Abstract

Disclosed herein are compositions comprising a peptide domain and a boronic acid binding motif. The present compositions may optionally comprise an enzymatic cleavage signaling sequence domain, a spacer domain, or a bioactive peptide sequence domain. The present disclosure also provides hydrogels formed from such compositions and methods of use thereof.

Description

DESCRIPTIONHYDROGELS EXHIBITING BORONATE ESTER-MEDIATED DRUG RELEASESTATEMENT OF FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant Nos. R01DE021798 and R01DE030140 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on July 15, 2024, is named RICEP0134WO.xml and is 16,568 bytes in size.PRIORITY CLAIM

[0003] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 514,033, filed July 17, 2023, the entire contents of which are hereby incorporated by reference.BACKGROUND OF THE INVENTIONField of the Invention

[0004] The present disclosure relates generally to the fields of biochemistry, medicine, molecular biology, and pharmacology. The present disclosure provides compositions comprising a structural domain and a boronic acid dynamic covalent attachment motif. Compositions disclosed herein may modulate the pharmacokinetics of bioactive molecules via dynamic covalent interactions involving boronic acid.Description of Related Art

[0005] Boronic acid-containing small molecule drugs (BACSMs) are a growing class of therapeutics for the treatment of a variety of diseases, but face challenges including poor oral bioavailability, gastrointestinal side-effects following oral dosing, poor molecular specificity and relatively short in vivo half-lives. These limitations may be ameliorated using hydrogels, such asself-assembling multidomain peptide (MDP) hydrogels, as a drug delivery platform; however, controlling the release of small molecules from hydrogels is a persistent challenge in the field. More particularly, hydrogels contemplated for use as controlled drug delivery systems often exhibit release durations that are limited by rapid diffusion. Thus, improved methods for controlling the release of BACSMs or other compounds or compositions comprising a boronic acid group, particularly from hydrogel drug delivery platforms, are needed. Compositions and methods for delivering BACSMs that are able to modulate dosage with the effect of lowering, minimizing, or avoiding off-target effects, maintain effective drug concentrations for longer periods of time, improve patient adherence by reducing dosing frequency, and / or provide an alternate route for the systemic delivery of BACSMs would provide valuable alternatives to the current state of the art.

[0006] In addition to BACSMs, systems that control the release of biologies (e.g., proteins) are also very challenging to make due to their exposure to damaging microenvironments during preparation and release from many delivery systems (e.g., biodegradable microparticles). Hydrogels are generally highly compatible with proteins but release governed by diffusion alone may deliver over a shorter period than would be ideal for many applications. Therefore, extending release using additional or alternative means is often desirable due to the potential to enhance therapeutic efficacy by mitigating off-target effects, reduce the need for frequent dosing regimens, and allow for the direct local delivery to biologies to the target site in the body. Therefore, methods for the controlled release of biologies are also desired.SUMMARY OF THE INVENTION

[0007] The presently disclosed self-assembling hydrogels may, in some embodiments, alter the release of therapeutic or prophylactic small molecules or biologies via dynamic covalent bonding with a molecule containing boron. Further details are provided below and in the sections that follow.

[0008] The present disclosure provides compositions comprising a hydrogel, wherein the hydrogel comprises: a boronic acid binding motif; or a boronic acid group of the formula B(X')2; and a compound, wherein the compound comprises: a boronic acid binding motif; or a boronic acid group of the formula B(X')i; wherein each X' is an independently selected atom; and wherein if the hydrogel comprises a boronic acid binding motif, then the compound comprises a boronic acid group; and if the compound comprises a boronic acid-binding motif, then the hydrogel comprises a boronic acid group.

[0009] In some embodiments, the composition is a structure comprising one or more dynamic covalent bonds between the boronic acid binding motif and the boronic acid. In some embodiments, the boronic acid binding motif forms a B(X')2 dynamic covalent attachment with the boronic acid group, wherein each X' is independently nitrogen, oxygen, sulfur or carbon. In further embodiments, X’ is oxygen.

[0010] In some embodiments, the boronic acid binding motif is a catechol, such as a nitrocatechol or 6-nitrodopamine. In some embodiments, the boronic acid binding motif is a diol, such as a 1,2- or 1,3-diol. In other embodiments, the boronic acid binding motif is 2-hydroxy- benzhydroxamic acid or a derivative thereof, such as 2-hydroxy-benzhydroxamic acid.

[0011] In some embodiments, the hydrogel is shear-thinning. In some embodiments, the hydrogel is self-healing. In some embodiments, the hydrogel comprises a synthetic polymer such as polyethylene glycol. In other embodiments, the hydrogel comprises a naturally occurringsubstance or derivative thereof, such as alginate, mannan, dextran, gelatin, or hyaluronic acid. In some embodiments, the hydrogel comprises a synthetic polymer and a naturally occurring substance or derivative thereof.

[0012] In some embodiments, the hydrogel comprises a peptide. In further embodiments, the peptide self-assembles to form P-sheet nanofibers. In some embodiments, the sequence of the peptide comprises at least one region of alternating, independently selected hydrophilic and hydrophobic amino acid residues, wherein each region is four or more amino acids in length. In further embodiments, the hydrophilic residues are independently selected from among glycine, serine, threonine, histidine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, ornithine, citrulline, tyrosine, cysteine, and selenocysteine. In yet further embodiments, the hydrophobic residues are independently selected from among valine, leucine, threonine, phenylalanine, alanine, tryptophan, tyrosine, isoleucine, and methionine.

[0013] In some embodiments, the peptide sequence comprises: a first domain (Xi)m; a second domain (YZ)n; and a third domain (X2)m; wherein: wherein each Xi and X2 is independently selected from among amino acids that are positively charged at pH 7, amino acids that are negatively charged at pH 7, proline, and hydroxyproline;Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid; m is 1, 2, 3, 4, 5; 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and n is 1, 2, 3, 4, 5; 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0014] In some embodiments, each Xi and X2 is independently selected from among lysine, ornithine, histidine, and arginine. In some embodiments, each Xi and X2 is independently selected from among glutamic acid and aspartic acid. In some embodiments, each Xi and X2 is independently selected from among hydroxyproline and proline. In some embodiments, each Yis independently selected from among serine, threonine, histidine, lysine, citrulline, asparagine, glutamine, ornithine, tyrosine, and glycine. In some embodiments, each Z is independently selected from among leucine, isoleucine, valine, phenylalanine, alanine, methionine, and tryptophan. In some embodiments, the peptide is less than 100 amino acid residues in length. In some embodiments, the peptide has at least 95% sequence identity with SEQ No. 1, SEQ No. 2, SEQ No. 3, and / or SEQ No. 4. In some embodiments, the N-terminus of the peptide is acetylated. In some embodiments, the C-terminus of the peptide is amidated.

[0015] In some embodiments, a boronic acid-binding motif is attached to the C- or N- terminus of the peptide. In some embodiments, a boronic acid-binding motif is attached to an amino acid sidechain. In some embodiments, a boronic acid-binding motif is covalently attached to the peptide.

[0016] In some embodiments, the peptide comprises a bioactive domain. In some embodiments, the peptide comprises an enzymatically degradable domain. In some embodiments, the peptide comprises a spacer domain.

[0017] In some embodiments, the compound is a therapeutic or prophylactic compound. In some embodiments, the compound is a boronic acid-containing small molecule. In some embodiments, the compound is insulin. In some embodiments, the compound is a small molecule that has been modified to include a boronic acid. In some embodiments, the compound is a biologic that has been modified to include a boronic acid. In further embodiments, the biologic is a nucleic acid, oligo-nucleic acid, a polypeptide, or a protein. In yet further embodiments, the biologic is a protein, such as an antibody. In some embodiments, the boronic acid is an arylboronic acid, a benzoboroxole, an alkyl boronic acid, a thienyl boronic acid, a benzoxaborinine, a benzoxaborininone, a benzoxaborolone, a Wulff-type boronic acid, or any combination thereof. In some embodiments, the boronic acid is 4-carboxyphenylboronic acid (PBA). In some embodiments, the boronic acid is 4-carboxy-3-fluorobenzeneboronic acid. In some embodiments, the boronic acid is 4-(carboxymethyl)-3-fluorobenzeneboronic acid (mFluoro-PBA). In some embodiments, the boronic acid is 2-fluoro-5-nitrophenylboronic acid (Nitro-Fluoro-PBA). In some embodiments, the boronic acid is 2-aminopyrimidine-5-boronic acid (Amino-pBA). In some embodiments, the boronic acid is 3-carboxy-5-nitrophenylboronic acid (Nitro-PBA). In some embodiments, the boronic acid is 4-borono-2-nitrobenzoic acid (2-Nitro-PBA). In some embodiments, the boronic acid is 4-borono-2,6-difluorobenzoic acid (Difluoro-PBA). In someembodiments, the boronic acid is 1 -hydroxy- l ,3-dihydrobenzo[c][l ,2]oxaborole-6-carboxylic acid (BOB).

[0018] In some embodiments, the compound is useful for treating or preventing an acute disease or disorder. In other embodiments, the compound is useful for treating or preventing a chronic disease or disorder. In some embodiments, the compound is useful for treating or preventing cancer. In some embodiments, the compound is useful for treating or preventing an infectious disease or disorder. In further embodiments, the infectious disease or disorder is a viral infection. In other embodiments, infectious disease or disorder is a fungal infection. In still other embodiments, the infectious disease or disorder is a bacterial infection, such as Mycobacterium tuberculosis or methicillin-resistant Staphylococcus aureus. In some embodiments, the compound is an adjuvant. In other embodiments, the compound is a toll-like receptor agonist. In still other embodiments, the compound is a vaccine.

[0019] In another aspect, the present disclosure provides pharmaceutical compositions comprising a composition as described above and an excipient.

[0020] In another aspect, the present disclosure provides methods of treating or preventing a disease or disorder in a patient in need thereof comprising contacting one or more cells of the patient with a composition or a pharmaceutical composition as described above. In some embodiments, the patient is a non-human animal. In other embodiments, the patient is a human.

[0021] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is diabetes. In some embodiments, the disease or disorder is an infectious disease or disorder. In further embodiments, the infectious disease or disorder is a bacterial infection, such as Mycobacterium tuberculosis or methicillin-resistant Staphylococcus aureus. In some embodiments, the infectious disease or disorder is a viral infection. In other embodiments, the infectious disease or disorder is a fungal infection. In some embodiments, the disease or disorder is a chronic disease or disorder. In other embodiments, the disease or disorder is an acute disease or disorder.

[0022] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications withinthe spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and arc included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] FIGS. 1A-1H show the design and development of SABER peptides. FIG. 1A) Catechol oxidation and degradation occurred rapidly, appealing as changes in the UV-Vis spectrum of dopamine (DOPA) over time in pH 7.4 PBS. The increase in the baseline absorbance indicates increased turbidity of the sample as insoluble oxidation products form black precipitates in solution. FIG. IB) The UV-Vis spectrum of 4-nitrocatechol (nDOPA) shows that the molecule is stable over the course of 15 days. FIG. 1C) Eongitudinal analysis of the UV-Vis spectrum salicylhydroxamic acid (SHA) demonstrates that the molecule is largely stable with minor changes in absorbance observed on day 11 and day 15. FIG. ID) Boronate ester equilibrium constants between chemically distinct boronic acids (BAs) and the dynamic covalent association motifs alizarin red S (ARS), nDOPA, SHA, and DOPA. For all BAs, SHA forms the strongest boronate ester interactions. Equilibrium constants are presented as the mean value of three replicates ± 1 SD. The present disclosure therefore provides compositions which have controlled release over time at composition-dependent rates. FIG. IE- FIG. 1H) Chemical structures, mass spectra, and UPEC chromatograms of FIG. IE) Cat-K2, FIG. IF) nitroCat-K2, FIG. 1G) SHA-K2, and FIG. 1H) K2 (SEQ ID NO: 1).

[0025] FIGS. 2A-2I show characterization data of SABER hydrogels. FIG. 2A) A schematic illustrating how small molecules rapidly diffuse from peptide hydrogels due to their large mesh size (red). Without being bound by theory, the dynamic covalent attachment moieties to the hydrogel slow release, resulting in prolonged delivery (blue). FIG. 2B) Chemical structures of the three SABER peptides synthesized. FIG. 2C) FTIR of the three SABER peptides shows that they assemble into antiparallel P-sheets. FIG. 2D) CD spectra of unmodified K2 (SEQ ID NO: 1) are consistent with -sheet secondary structure. FIG. 2E- FIG. 2G) Cryo- TEM images of (FIG. 2E) Cat-K2, (FIG. 2F) nitroCat-K2, and (FIG. 2G) SHA-K2 show that all peptides self-assemble into nanofibers. The white scale bar represents 200 nm. FIG. 2H) Oscillatory rheology frequency sweeps show that the three SABER peptides form hydrogels withsimilar moduli to unmodified K2 (SEQ ID NO: 1 ). FIG. 21) Shear recovery rheology experiments where all four peptides were subjected to 200% strain for 1 min, demonstrating that all the hydrogels arc shear thinning and rapidly self-healing.

[0026] FIGS. 3A-3G show in vitro release and drug stability data. FIG. 3A- FIG. 3B) Equilibrium fluorescence release results show that (FIG. 3A) unmodified fluorescein is rapidly released from all hydrogels, but (FIG. 3B) upon modification with phenylboronic acid (FITC- PBA) SABER hydrogels significantly prolong the release of the compound. Data is the mean (n=3) ± 1 SD. FIG. 3C) The change in UPLC retention time of GFB alone and GFB released from Cat-K2 in UPLC suggests that the peptide may be degrading the drug. The new peak at 3.2 min seen in Cat-K2 + GFB is also observed when the drug is reacted with hydrogen peroxide, suggesting that this new peak corresponds to oxidation of GFB. FIG. 3D) Electrospray ionization mass spectrometry of GFB alone contains the expected mass of the drug at 258.1 m / z and an additional peak potentially due to a neutral water elimination from in- source fragmentation of the boronic acid. GFB released from Cat-K2 has a peak at 248.1 in / z. (FIG. 3E) corresponding to a loss of a boron atom due to oxidative deboronation. FIG. 3F) Stability of GFB loaded in K2 (SEQ ID NO: 1), Cat-K2, nitroCat-K2, and SHA-K2 quantified by UPLC shows that the majority of the drug remains stable over the course of 2 weeks in all hydrogels except for Cat-K2, which immediately degrades the compound. All data is represented as the mean (n=3) ± 1 SD. FIG. 3G) SHA-K2 without drug and the hydrogel maximally loaded (1:1 drug-to-peptide molar ratio) have the same -sheet secondary structure as determined by a minimum at 220 nm seen by circular dichroism, suggesting that drug loading does not perturb peptide self-assembly.

[0027] FIGS. 4A-4I show data related to the in vitro release of boronic acid-containing small molecule drugs. In vitro release of (FIG. 4A) bortezomib and (FIG. 4B) ixazomib loaded at 0.5 mg / mL shows that compositions disclosed herein are associated with prolonged delivery of chemically distinct drugs containing prototypical BAs (indicated in blue) compared to unmodified K2 (SEQ ID NO: 1). FIG. 4C) Release curves of tavaborole loaded in hydrogels formed from presently disclosed compositions at 0.25 mg / mL showed that SABER hydrogels are compatible with benzoxaborole-containing drugs (indicated in red). FIG. 4D) SHA-K2 was the only peptide able to significantly slow the release of the drug GFB loaded at 0.5 mg / mL in vitro. FIG. 4E-FIG. 4F) Altering the loading of GFB in SHA-K2 by altering the drug-to-peptidemolar ratio does not significantly alter the release rate until approaching a ratio of 1 : 1 by increasing the drug concentration. The release of GFB from unmodified K2 (SEQ ID NO: l)did not vary as a function of drug loading. FIG. 4G) Adding PBA to 1V209 did not meaningfully change the biological activity of the small molecule as determined by a TER7 reporter cell line. In vitro release curves of (FIG. 4H) 1V209-PBA and (FIG. 41) 1V209 show that the boronic acid motif (in this example, PBA) is necessary for SABER hydrogels to extend the release of the drug. The release of 1V209-PBA from Cat-K2 was identical to the unmodified K2 (SEQ ID NO: 1) hydrogel. Data points indicate mean ± 1 SD (n=3).

[0028] FIGS. 5A - 5H provide evidence that the presently disclosed compositions prolong the systemic release and local retention of BACSMs in vivo. FIG. 5A) Pharmacokinetic profile of 700 ng of BTZ administered subcutaneously without a hydrogel or loaded in 10 mg / mL K2 (SEQ ID NO: 1), nitroCat-K2, or SHA-K2 (n=4-5). FIG. 5B) Delivering BTZ from nitroCat-K2 and SHA-K2 hydrogels significantly reduces the maximum circulating concentration (Cmax) of the drug compared to BTZ alone or BTZ loaded in K2 (SEQ ID NO: 1). FIG. 5C) BTZ without a hydrogel must be administered at a 5-fold lower dose (175 ng) to match the Cmax achieved from delivering 700 ng of BTZ from nitroCat-K2 and SHA-K2. FIG. 5D) Mass spectrometry imaging analysis of BTZ at the injection site shows that MDP hydrogels retain higher local concentrations of BTZ. Darker spots in tissues are hydrogels, which suppress BTZ ionization. FIG. 5E) Pixel intensities over the 1 mm^ of tissue with the highest BTZ concentration at (top) 1 d, (middle) 7 d, and (bottom) 21 d. FIG. 5F) At early time points, the mean pixel intensity in nitroCat-K2 is the highest of all the groups while SHA-K2 is the only group statistically superior to BTZ alone at 21 d. FIG. 5G) Pharmacokinetic profile of 600 and 60 pg of GFB administered alone or in a 20 mg / mL SHA-K2 hydrogel shows that the time above the EC50 is improved by increasing drug loading (n=4). FIG. 5H) Non-compartmental pharmacokinetic analysis of the groups dosed with 600 pg revealed that the SHA-K2 improved the drug exposure (AUC) and half-life (ti / 2) of GFB while reducing the Cmax- All data points are shown as mean ± SEM.

[0029] FIGS. 6A-6D show data related to the In vivo release of Bortezomib (BTZ). FIG. 6A) Pharmacokinetics of decreasing BTZ doses administered as subcutaneous boluses without hydrogel. Data is presented as the mean (n=4) ± SEM. FIG. 6B) The maximum circulatingconcentration (Cmax) of BTZ decreases linearly with the initial dose. Dotted lines represent the Cmax for 700 ng of BTZ delivered from nitroCat- K2 (blue) and SHA-K2 (purple), indicating that a bolus BTZ dose of 175 ng yields the same Cmax as these hydrogel formulations loaded with 5- fold more drug. FIG. 6C) Chemical structure of the BTZ fragment observed in mass spectrometry imaging. FIG. 6D) K2 (SEQ ID NO: 1), nitroCat-K2, and SHA-K2 hydrogels loaded with 700 ng of BTZ imaged by mass spec imaging in vitro show very little BTZ signal, suggesting that MDP peptides suppress the ionization of BTZ within the gels.

[0030] FIGS. 7A & 7B show H&E and mass spectrometry imaging of injection site tissues. FIG. 7A) Tissue sections from mice that received subcutaneous injections of bortezomib (BTZ) alone or in a hydrogel at 1-, 7-, and 21-days stained with hematoxylin and eosin (H&E). Large dark purple sections in K2 (SEQ ID NO: 1), nitroCat-K2, and SHA-K2 are the hydrogels in the skin samples. FIG. 7B) Mass spectrometry imaging of the same tissue samples stained with H&E shows that BTZ signal does not significantly overlap with that from heme (616.178 m / z), illustrating that the drug observed in the tissue is not in circulation but in the local environment of the injection site.

[0031] FIG. 8 shows pharmacokinetic data of GFB in 50 pL of 10 mg / mL hydrogels. Longitudinal concentrations of GFB in the blood of mice after a single subcutaneous injection of 75 pg of the drug loaded into 50 pL hydrogels or PBS (n=4). GFB in the absence of a hydrogel, K2 (SEQ ID NO: 1), or nitroCat-K2 led to the rapid release of all the drug. SHA-K2 hydrogels were able to retain GFB concentrations over the EC50 for more than 200 h.

[0032] FIGS. 9A-9I show characterization data of negatively charged SABER hydrogels and their use to treat a murine model of acute tuberculosis (TB). FIG. 9 A) Chemical structure of SHA-E2. FIG. 9B) FTIR of SHA-E2 shows that the antiparallel P-sheet secondary structure of the peptide remains similar to unmodified E2 (SEQ ID NO: 2). FIG. 9C) CD spectra of both E2 (SEQ ID NO: 2) and SHA-E2 indicate the presence of P-sheet self-assembly. FIG. 9D) Rheological testing shows that both E2 (SEQ ID NO: 2) and SHA-E2 hydrogels are shear thinning and completely recover their viscoelastic properties within 10 min after being subjected to 200% strain for 1 min. FIG. 9E) Cryo-TEM images of SHA-E2 peptides in solution showing self-assembled nanofibers. Scale bar = 100 nm. FIG. 9F) SHA-E2 significantlyprolongs the release of GFB compared to E2 (SEQ ID NO: 2) in vitro when loaded at 0.5 mg / mL. Data points indicate mean ± 1 SD (n=3). FIG. 9G) Pharmacokinetic profile after dosing 600 pg of GFB shows that SHA-E2 prolongs the release of the drug in vivo compared to unmodified E2 (SEQ ID NO: 2). Data points indicate mean (n=5) ± SEM. FIG. 9H) A schematic illustrating the experimental design for the mouse model of acute TB. FIG. 91) The CFU counts from the lungs of mice with TB over the course of 14 days show that a single injection of 600 pg GFB loaded in SHA-E2 outperforms the same dose injected without the gel or the same total mass of drug given over 10 oral doses. CFU data are presented as the mean ± 1 SD (n=4).

[0033] FIGS. 10A-10E show In vitro characterization data for SHA-E2 and in vivo release of GFB for SHA-E2. FIG. 10A) Chemical structure of SHA-E2 with the mass spectrum and UPLC chromatogram of the material confirming the identity and purity of the peptide. FIG. 10B) Mass spectrum and UPLC chromatogram of the unmodified E2 (SEQ ID NO: 2) peptide. FIG. 10C) Frequency sweep collected by oscillatory rheology shows that SHA-E2 hydrogels are more frequency dependent than unmodified E2 (SEQ ID NO: 2) and form slightly weaker gels as indicated by the reduced distance between the storage (G’) and the loss (G”) moduli. FIG. 10D) Cumulative release of GFB after 24 h from SHA-K2 and SHA-E2 are statistically similar, suggesting that changing the peptide used in SABER hydrogels does not compromise its ability to control the release of BA-containing small molecules (n=3). FIG. 10E) Pharmacokinetic parameters extracted by performing a non-compartmental analysis on the in vivo release of GFB from SHA-E2 show that using the SABER hydrogel improves drug exposure (AUC), half-life (ti / 2) and reduces the maximum circulating concentration (Craax) of the compound. Pharmacokinetic parameters are presented as the mean (n=4-5) replicates ± 1 SD.

[0034] FIGS. 11A-11F illustrates local delivery of a BA-labeled model IgG antibody from SABER hydrogels. FIG. 1 1 A) A schematic illustrating dynamic covalent bonds forming between BA motifs on labeled IgG molecules and SABER hydrogels to slow down transport within the hydrogel. FIG. 11B & FIG. 11C) The normalized fluorescence intensity recovery after photobleaching a region of hydrogels loaded with fluorescently labeled IgG with 2.4 PBAs per IgG (low IgG) and 11.4 PBAs per IgG (high IgG). SHA modification of MDPs slowed down the transport of IgG within the hydrogel matrix compared to unmodified MDPs. Data is shown as the mean ± 1 SD (n=3). D) Representative in vivo fluorescence images of micesubcutaneously injected with negatively and positively charged SABER hydrogels loaded with fluorescent low and high IgG at different time points. FIG. HE & FIG. 1 IF) Quantification of changes fluorescence intensity at the injection site over time in mice receiving PBA-labeled IgG shows that SHA-K2 and SHA-E2 hydrogels prolong the release of antibody to more than 28 and 56 days, respectively. Data points indicate mean (n=4) ± SEM.

[0035] FIGS. 12A-12C illustrate local release of PBA-modified IgG. FIG. 12A) Representative confocal images illustrating fluorescence recovery and photobleaching (FRAP) experiments. Fluorescence recovery was quantified by monitoring the return of fluorescence signal in the bleached region over 10 min using a 640 nm excitation laser. The white scale bar in the bottom left represents 40 pm. FIG. 12B) FRAP data (n=3 for each group) was fit to a first- order exponential equation to extract the FRAP half-time (ti / 2) and the mobile fraction (Mf). Eoading PBA- modified IgG in SABER hydrogels reduced the Mf and increased the ti / 2, suggesting, without being bound by theory that the rate of payload diffusion in these samples is slower. Minimal differences were observed between IgG labeled with 11.4 PBAs per antibody (IgG high) and 2.4 PBAs per antibody (IgG low), demonstrating that the degree of labeling may not play a large role in controlling the rate of diffusion. All data were well-modeled by the first- order exponential equation and had R2values above 0.95 except for SHA-E2 + IgG high (denoted with an asterisk), which was poorly fit by this model and thus extracted parameters may not accurately describe the data. FIG. 12C) In vivo release data of IgG with low and high degrees of PBA modeling was modeled with a first-order exponential equation to determine the half-life (ti / 2) and burst release from the site of injection. All SABER hydrogels had a moderate burst release of 20% but significantly extended the ti / 2 of the antibody at the injection site. All fits adequately modeled the data (R^ > 0.95). All numerical data in this figure is presented as the mean (n=4) ± 95% confidence interval.

[0036] FIGS. 13A-13D shows data related to basal insulin delivery from SABER hydrogels. FIG. 13 A) Mass spectrum of PBA-modified insulin (insulin-PBA) showing that a single PBA was added to insulin. FIG. 13B) The UPEC chromatogram of the synthesized insulin-PBA confirmed the purity of the material. FIG.13C) In vitro release of unmodified insulin from SHA-E2 and E2 (SEQ ID NO: 2) illustrates that the PBA modification is necessary for the SABER peptide to delay the release of the payload. Data presented as the mean (n=3) ± 1SD. FIG. 13D) First 10 h of the initial treatment of diabetic mice with 6 IU of insulin-PBA in SHA-E2 plotted with a repeat treatment with the same formulation in the same mice 6 weeks later. The repeat dose resulted in statistically similar blood glucose levels to the initial dose at all time points. Data points for blood glucose measurements are the mean (n-5) ± SEM.

[0037] FIGS. 14A-14F illustrates delivery of PBA-modified insulin from SHA-E2 maintains normoglycemia for up to 144 h in a mouse model of type 1 diabetes. FIG. 14 A) In vitro release of 0.5 mg / mL of insulin-PBA loaded in E2 (SEQ ID NO: 2) or SHA-E2 in media with 0, 100, or 250 mg / dL of glucose. Data plotted as the mean ± 1 SD (n=3). FIG. 14B) The cumulative 24 h in vitro release of insulin-PBA from SHA-E2 reveals that the hydrogel is not likely glucose responsive over healthy blood glucose concentrations. Error bars represent ± 1 SD from the mean (n=3). FIG. 14C) Diabetic mice treated with a subcutaneous bolus of unmodified insulin without a hydrogel return to hyperglycemic blood glucose levels within 4 hours of injection while mice administered 3, 6, or 12 IU of insulin-PBA within SHA-E2 below 240 mg / dL. FIG. 14D) The duration over which insulin-PBA delivered in SHA-E2 can maintain normoglycemia in diabetic mice increases with the loading of insulin-PBA in the gel. FIG. 14E) At 4, 24, and 72 h after administration, SHA-E2 loaded with insulin-PBA results in significantly lower blood glucose levels than a single bolus of 3 IU of insulin. At 168 h, both 6 and 12 IU insulin-PBA gels are statistically superior to the 3 IU bolus control even though mice are hyperglycemic. FIG. 14F) Retreating mice with 6 IU of insulin-PBA loaded in SHA-E2 6 weeks after the initial treatment yielded statistically similar control of blood glucose levels. The blue region of blood glucose graphs represents healthy blood glucose levels in humans (80-180 mg / dL), the grey regions represent mild hyperglycemia (> 240 mg / dL) and mild hypoglycemia (54-80 mg / dL), and white regions represent critically low or high blood glucose levels. All blood glucose data points show mean (n=5) ± SEM.

[0038] FIGS. 15A-15E show the structures for (FIG. 15 A) K2 (SEQ ID NO: 1) and (FIG. 15B) D-K2 (SEQ ID NO: 12), (FIG. 15C) circular dichroism graph showing the mean residue ellipticity graphed against wavelength, and Cryo- EM images of (FIG. 15D) K2 (SEQ ID NO: 1) and (FIG. 15E) D-K2 (SEQ ID NO: 1).

[0039] FIGS. 16A-16E show the rheological properties of K2 (SEQ ID NO: 1 ) and D-K2 (SEQ ID NO: 12) showing (FIG. 16A) change in storage and loss modulus at various frequencies, (FIG. 16B) storage modulus, (FIG. 16C) loss modulus, (FIG. 16D) shear recovery over time, and (FIG. 16E) total recovery 10 min after shearing.

[0040] FIGS. 17A & 17B provide data from FRAP experiments on hydrogels of K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 1) showing (FIG. 17A) percent recovery over time and (FIG. 17B) representative images of fluorescence from labeled OVA prior to bleaching, immediately after bleaching, and 10 min after recovery (50-pixel diameter spots). The scale bar represents 20 pm.

[0041] FIGS. 18A-18C show in vivo experimental results. (FIG. 18A) Representative images of hydrogel degradation in mice (n=5). (FIG. 18B) In vivo clearance of fluorescently labeled K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12). (FIG. 18C) Representative images of histological staining of tissue surrounding the injection of K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) hydrogels harvested from mice 13 weeks after injection and stained with hematoxylin and eosin stain and Masson’s trichrome stains (n=3). The red scale bar represents 2 mm.

[0042] FIGS. 19A-19C are graphs showing in vivo (FIG. 19 A) OVA release from K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) and antibody titers shown (FIG. 19B) over time and (FIG. 19C) compared at the 12-week timepoint.

[0043] FIGS. 20A-20F provide confirmation of expected mass by (FIG. 20A) MAEDI MS positive ionization of HPEC-purified K2 (SEQ ID NO: 1) observed [M+H]+: 1774.025, expected [M]+: 1773.29. (FIG. 20B) UPEC trace of HPEC-purified K2(SEQ ID NO: 1) peptide. The presence of a single peak confirms purity. (FIG. 20C) MALDI MS positive ionization of HPLC-purified D-K2(SEQ ID NO: 12) observed [M+H]+: 1774.368, expected [M]+: 1773.29. (FIG. 20D) UPLC trace of HPLC-purified D-K2(SEQ ID NO: 12) peptide. The presence of a single peak confirms purity. (FIG. 20E) MALDI MS positive ionization of HPLC-purified K2- Atto 647 observed [M]+: 2358.658, expected [M]+: 2361.77. (I) MALDI MS positive ionization of HPLC-purified D-K2-Atto 647 observed [M]+: 2358.789, expected [M]+: 2361.77.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0044] Provided herein arc, in one embodiment, hydrogel compositions for the controlled release of prophylactic or therapeutic molecules. The present disclosure provides compositions for delivering a variety of different BACSMs as well as small molecule drugs or biologies modified with boronic acids. It should also be understood that the same dynamic covalent interaction featuring boronate esters can be achieved with boronic acid on the hydrogel and the boronic acid-binding motif on the drug. In some embodiments, the present compositions modulate the release kinetics of BACSMs. In some embodiments, the present compositions facilitate localized delivery of the BACMs, enabling the maintenance of low systemic concentration of BACSMs. In this way, the present compositions, in some embodiments, may be favorable in that off-target effects may be avoided or mitigated. In addition, compositions of the present disclosure provide an alternative for systemic delivery and may, in some embodiments, allow for effective drug concentrations to be maintained for longer periods of time. In some embodiments, the presently disclosed hydrogels facilitate a reduced need for re-administration of the therapeutic molecules. In some embodiments, the presently disclosed hydrogels facilitate reduced peak concentration of the therapeutic molecule, potentially reducing side effects. In some embodiments, the presently disclosed compositions comprise peptide hydrogels, which are favorable as drug delivery systems due to their customizability, biocompatibility, and shear thinning ability. In some embodiments, the functional group leveraging dynamic covalent bonding is on a non-peptide hydrogel. Novel delivery methods, such as those utilizing the presently disclosed compositions, may improve the utility of a variety of small molecule drugs and biologies. For example, BACSM therapeutics, such as GSK656, require daily dosing for the treatment of M. tuberculosis. The present invention could improve the effectiveness of, and patient adherence to, therapeutic regimens of such BACSMs in critical populations by providing compositions with alternative release kinetics. In addition, the hydrogel of the present compositions is attractive for delivering biologies due to its gentle aqueous preparation that minimizes potential losses in bioactivity during formulation due to heat, organic solvents, or extreme pH.I. Self-Assembling Boronate Ester Release (SABER) Hydrogels

[0045] The present disclosure provides compositions capable of engaging in dynamic covalent bonding with BAs or BABMs to extend the release, on the one hand, of BACSMs, small molecules modified with BAs, and BA-modified biologies, or alternatively small molecules modified with BABMs or BABM-modified biologies. Hydrogel-forming multidomain peptides (MDPs) that have been functionalized with BABMs, termed self-assembling boronate ester release (SABER) peptides, remain shear thinning and injectable, retain their ability to rapidly self-assemble to form nanofibrous hydrogels, and are able extend the release of a wide variety of chemically distinct BA-containing payloads for significantly longer than unmodified or catechol-modified MDPs through enhanced dynamic covalent bonding. The ability to release multiple classes of drugs from SABER hydrogels — in combination with the simplicity and modularity of the platform — make it well-suited for clinical translation, especially when long durations (e.g., weeks to months) are desired.

[0046] Supramolecular peptide hydrogels are a class of biomaterials that have been evaluated in preclinical studies and clinical trials (Zhang et al., 2021; Gelain et al., 2021). Typically composed of the same amino acids that comprise all human proteins, they offer favorable biocompatibility and readily degrade into non-toxic products. Multidomain peptides (MDPs) are a class of hydrogel-forming peptides that spontaneously assemble into -sheet-rich nanofibers (Dong et al., 2007). MDP hydrogels are shear thinning and self-healing, allowing them to be injected through small-bore needles yet reform as a hydrogel in vivo once the shear force has been removed (Farsheed et al., 2023). Minimal changes to this simple sequence pattern can give rise to hydrogels with diverse chemical and biological properties, allowing the material to be tailored to each application (Lopez-Silva et al., 2020; Aulisa et al., 2009; Leach et al., 2019). The MDPs of the presently disclosed compositions may be used to deliver diverse pay loads such as carbohydrates (Pogostin et al., 2023), proteins (Pogostin et al., 2022), small molecules (Kumar et al., 2015), and liposomes (Swain et al., 2023). However, methods known in the art involving the use of MDPs for delivering payloads have challenge associated with burst release and insufficient release lifetime. Exemplary sequences of peptides that may form the hydrogels of the compositions disclosed herein are shown in Table 1. The hydrogels of the compositions disclosed herein may be formed according to techniques known in the art. For example, methods for forming MDP hydrogels are provided in Farsheed et al., 2023.Table 1 : Exemplary domain sequences of the present disclosure.

[0047] Release of encapsulated solubilized drugs from hydrogels is primarily governed by the size of the drug and mesh size of the hydrogel network (Li & Mooney, 2016). Most proteins and small molecules are much smaller than the mesh size of low-concentration physical hydrogels, resulting in rapid drug release (Rehmann et al., 2017; Schultz & Solomon, 1961). To prolong release, affinity-based release modalities, including complementary electrostatic interactions, guest-host binding, hydrogen-bonding, ligand binding, hydrophobic interactions, and dynamic covalent interactions have been employed to add attractive interactions between the payload and the hydrogel, thereby slowing transport out of the hydrogel network (Teal et al., 2024; Nambiar & Schneider, 2022; Yaguchi et al., 2021; Ji et al., 2021; Li et al., 2016). The dynamic covalent interactions facilitated by the presently claimed compositions are much stronger than electrostatic or hydrophobic interactions but, in aqueous solution, exist at an equilibrium such that bonding is reversible (Ulrich, 2019). As a result, some fraction of the drug component of the presently disclosed composition can be covalently bonded to the hydrogelcomponent, unable to diffuse, while the unbonded drug is free to diffuse, thereby extending drug release.

[0048] The dynamic covalent bond between diols and boronic acid (BA) functional groups has been employed in the design of drug delivery systems due to its versatility and the availability of BA motifs on existing therapeutics used in the clinic (Stubelius et al., 2019; Plescia & Moitessier, 2020). There are currently five FDA-approved boronic acid-containing small molecules (BACSMs) on the market and dozens more in preclinical and late-stage clinical development (Plescia & Moitessier, 2020). BA modification is an emerging tool in medicinal chemistry as a way to improve the activity and pharmacokinetics of small-molecule therapeutics (Plescia & Moitessier, 2020; Zhang et al., 2017; Dhawan et al., 2023; Al-Omari et al., 2023). BAs readily form bidentate ester linkages with 1,2- and 1,3-diols, even in aqueous solution (Lii et al., 2013). These boronate esters and their constituent free BAs and diols exist in a dynamic equilibrium (Gosecki & Gosecka, 2022), which can be influenced by factors including the pKa of the diol and the BA (Pizer & Babcock, 1977; Barsoum et al., 2022). The presently disclosed compositions may comprise a variety of boronic acid groups. In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is PBA. In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 4-carboxy-3-fluorobenzeneboronic acid (Fluoro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 4-(carboxymethyl)-3- fluorobcnzcncboronic acid (mFluoro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 2-fluoro-5-nitrophenylboronic acid (Nitro-Fluoro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 2-aminopyrimidine-5-boronic acid (Amino-pBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 3-carboxy-5-nitrophenylboronic acid (Nitro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 4-borono-2-nitrobenzoic acid (2-Nitro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is 4-borono-2,6-difluorobenzoic acid (Difluoro-PBA). In some embodiments, the presently disclosed compositions comprise at least one boronic acid group that is l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborole-6-carboxylic acid (BOB). Any of the groups listed above or any other group comprising a boronic acid functional group according to thepresent claims may be appended to a compound or composition of interest to make the compound or composition suitable for release by a presently disclosed composition.

[0049] The present disclosure relates to compositions comprising a dynamic covalent interaction between a functionalized hydrogel and a functionalized payload. In some embodiments, the payload is a therapeutic compound, and as such the term “payload” and “therapeutic compound or composition” are used interchangeably throughout the present disclosure. Release of the payload according to the kinetics of the dynamic covalent attachment between the payload and the functionalized hydrogel is also referenced herein as the equivalent and interchangeable terms “payload delivery”, “payload release”, “delivery of therapeutic compound or composition” and any synonyms thereof.

[0050] The payload may comprise a boronic acid group or a boronic acid binding motif, The functionalization of the payload may depend upon or be selected based upon the functional group of the hydrogel. In some embodiments, the payload is a BACSM. In other embodiments, the pay load is a small molecule that has been modified to comprise a boronic acid group. In some embodiments, the payload is a biologic, such as an antibody, that has been modified to comprise a boronic acid group. In some embodiments, the pay load is a small molecule that comprises a boronic acid binding motif. In other embodiments, the payload is a small molecule that has been modified to comprise a boronic acid binding motif. In some embodiments, the payload is a biologic, such as an antibody, that has been modified to comprise a boronic acid binding motif. In this way, the present compositions may be used for the delivery of a wide variety of payloads.

[0051] Catechols are the most commonly used diol motifs in BA dynamic covalent chemistry, which form boronate esters that are orders of magnitude stronger than non-aryl diols like those found in carbohydrates (Rafiee, M. & Nematollahi, 2008; Brooks et al., 2018; Springsteen & Wang, 2002). As such, hydrogel drug delivery systems known in the art have used catechols to control the delivery of the BACSM bortezomib, clinically used to treat multiple myeoloma (Pu et al., 2014; Liu et al., 2021; Rezk et al., 2019). One limitation with catechols, however, is their facile oxidative decomposition at neutral pH (Zhang et al., 2009; Strawbridge et al., 2000). This oxidation can accelerate drug release, reduce payload stability, and lead tocrosslinked byproducts via reactive quinone intermediates (Pizer & Babcock, 1977; Graham et al., 2021; Yang et al., 2016). Catcchol-bascd delivery systems can exhibit glucosc-rcsponsivc release due to competitive BA bonding with carbohydrate polyols, which has been exploited to create glucose-responsive insulin delivery devices for the treatment of diabetes (Zhao et al., 2017). For almost every application outside of diabetes, however, it is undesirable to have drug release rate dependent on blood sugar levels, fluctuating with meals. These limitations hinder the clinical translation of catechol-containing drug delivery systems.

[0052] The present disclosure provides compositions wherein hydrogels are functionalized with at least one motif that is able bind boronic acid groups, such as a 4- nitrocatechol motif or a salicylhydroxamic acid (SHA) motif. In some embodiments, the boronic acid binding motif is SHA. In some embodiments, the boronic acid binding motif is 4- nitrocatechol. In some embodiments, the boronic acid binding motif is dopamine or 4- nitrodopamine. In some embodiments, the boronic acid binding motif of the disclosed invention has favorable characteristics or properties, such as improved oxidative stability. Oxidative stability of a boronic acid binding motif may be characterized through a lack of change in the UV-Vis spectrum over time. In some embodiments, the boronic acid binding motif is stable, for example at 25 °C at physiological pH, for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or any range derivable therein. In some embodiments, the boronic acid binding motif is stable for more than one day. In some embodiments, the boronic acid binding motif is stable for at least one week. In some embodiments, the boronic acid binding motif is stable for at least two weeks. In some embodiments, the boronic acid binding motif of the present disclosure is stable for a month or more.

[0053] The boronic acid binding motifs useful in forming the presently disclosed compositions may have a variety of association constants with boronic acid containing small molecules and / or biologies. A person of skill in the art would recognize that different payloads may require, necessitate, or benefit from release kinetics that may vary from one another. Thepresent disclosure provides a versatile platform wherein payload release kinetics may be selected or altered based upon the identity of the payload, the identity of the boronic acid binding motif of the composition, and / or the combination thereof. The boronic acid binding motifs contemplated for use in the present invention may be characterized by the binding or association constant with the boronic acid group-containing compound (such as a BACSM or a boronic-acid containing biologic). In some embodiments, the boronic acid binding motif of the present disclosure may have a higher association constant than catechol with a boronic acid group. In some embodiments, the boronic acid binding motif of the present disclosure may have a lower association constant than catechol with a boronic acid group. In some embodiments, the presently disclosed compositions may form functionalized hydrogels that have a higher association constant with a boronic acid group than catechol or a hydrogel functionalized with catechol. In some embodiments, the presently disclosed compositions may form functionalized hydrogels that have a higher association constant than the corresponding unfunctionalized hydrogel with a boronic acid group.

[0054] The self-assembly and physical properties of presently disclosed compositions may be altered according to the identity of the particular boronic acid binding motif of the composition. In some embodiments, for example, functionalized hydrogels as disclosed herein may exhibit higher propensity to cluster and form architecture. Such properties may be observed, for example, by FTIR, CD, or cryo-TEM. In some embodiments, the presently disclosed compositions form hydrogels in IX Hank’s Balanced Salt Solution at about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, or any range derivable therein. In some embodiments, the presently disclosed compositions form hydrogels in IX Hank’s Balanced Salt Solution at about 5 mg / mL, at about 10 mg / mL, or at about 15 mg / mL. In some embodiments, the presently disclosed compositions form hydrogels in IX Hank’s Balanced Salt Solution at about 10 mg / mL. In some embodiments, hydrogels formed from the presently disclosed compositions, when subjected to high deformation force, have loss moduli that exceed storage moduli, which is consistent with liquid-like flow under high shear. In some embodiments, hydrogels formed from the presently disclosed compositions recover the hydrogel character (asassessed by measurement of the storage moduli, G’) upon reduction of the shear force. In some embodiments, hydrogels formed from the present compositions recover about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the initial G’ after the removal of a high deformation force, or any range derivable therein. In some embodiments, hydrogels formed from the present compositions recover about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the initial G’. In some embodiments, hydrogels formed from the present compositions recover about 60% of the initial G’. In some embodiments, hydrogels formed from the present compositions recover more than 90% of the initial G’. The recovery of the initial G’ may occur after about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes after the removal of a high deformation force, or any range derivable therein. In some embodiments, recovery of the initial G’ may occur in less than about 15 minutes after the removal of a high deformation force. In some embodiments, recovery of the initial G’ may occur in less than about 10 minutes, after the removal of a high deformation force

[0055] In some embodiments, the presently disclosed compositions comprise hydrogels that have favorable payload release kinetics. In some embodiments, the payload is, for example, a compound or biologic comprising a boronic acid group. In some embodiments, the payload is, for example, a compound or biologic comprising a boronic acid binding motif (BABM). The duration of payload release can be measured, for example, by determining the percentage of payload (for example, a therapeutic compound comprising a BA or a BABM) released over a period of time. In some embodiments, hydrogels of the presently disclosed compositions slow the delivery of a payload. In some embodiments, functionalized hydrogels of the presently disclosed compositions have a reduced rate of payload release compared to the corresponding unfunctionalized hydrogel. In some embodiments, functionalized hydrogels of the presently disclosed compositions have a reduced rate of payload release compared to the corresponding catechol-functionalized hydrogel or other hydrogel or technology known in the art. In someembodiments, functionalized hydrogels of the presently disclosed compositions release less than 80% of the payload in the first two hours following administration. In some embodiments, functionalized hydrogels of the presently disclosed compositions release less than about 58% of the pay load in the first two hours. In some embodiments, functionalized hydrogels of the presently disclosed compositions release less than about 96% of the payload in the first two hours. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of a payload in the first two hours following administration, or any range derivable therein. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 10%-30% of a payload in the first two hours following administration. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 15%-25% of a pay load in the first two hours following administration. In some embodiments, functionalized hydrogels of the present invention release a significant portion, such as above 50% or above 80% in the first two hours following administration. Some embodiments of the present disclosure comprise functionalized hydrogels that do not exhibit this “burst release” characteristic, and as such provide a beneficial alternative payload release kinetics profile. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 20%, about 25%, about 30%, about 35%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% of a payload in the first twenty-four hours followingadministration, or any range derivable therein. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 60%-80% of a payload in the first twenty- four hours following administration. In some embodiments, functionalized hydrogels of the presently disclosed compositions release about 50%-70% of a payload in the first twenty-four hours following administration. In some embodiments, the reduced rate of release results in higher payload exposure (AUC) of payload at later timepoints than other payload delivery methods. In some embodiments, the presently disclosed compositions have about 1.2-fold, about 1.4-fold, about 1.6-fold, about 1.8-fold, about 2.0-fold, about 2.2-fold, about 2.4-fold, about 2.6- fold, about 2.8-fold, about 3.0-fold, about 3.2-fold, about 3.4-fold, about 3.6-fold, about 3.8-fold, or about 4.0-fold higher drug exposure (AUC) for payloads delivered through methods involving the presently disclosed compositions than other methods. In some embodiments, the reduced rate of release results in higher payload circulating concentration for longer periods of time than other payload delivery methods. In some embodiments, the presently disclosed compositions have about 1.2-fold, about 1.4-fold, about 1.6-fold, about 1.8-fold, about 2.0-fold, about 2.2-fold. about 2.4-fold, about 2.6-fold, about 2.8-fold, about 3.0-fold, about 3.2- fold, about 3.4-fold. about 3.6-fold, about 3.8 -fold, about 4.0-fold, about 4.2-fold, about 4.4-fold, about 4.6-fold. about 4.8-fold, or about 5.0-fold, or any range derivable therein, higher circulating pay load concentration for payloads delivered through methods involving the presently disclosed compositions than other methods. In some embodiments, the reduced rate of release of pay load and / or the more persistent circulating concentration of payload as described above allows for administration of higher doses of payload than would be tolerated if the payload were administered by other means, such as by single bolus injection. In addition, consistently achieving higher levels of circulating drug, as opposed to the peaks and troughs of oral dosing, could prove more effective, requiring a shorter duration of exposure. In this way, the present disclosure provides compositions which facilitate advantageous delivery of payload, such as therapeutic compounds, biologies, or compositions.

[0056] In some embodiments, delivery of therapeutic compounds increase the circulating half-life of a therapeutic compound or composition by compositions disclosed herein. In some embodiments, the circulating half-life of a therapeutic compound or composition is increased by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, about 5.0-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7.0-fold, about 7.5-fold, about 8.0-fold, about 8.5-fold, about 9.0-fold, about 9.5-fold, about 10-fold, about 10.5- fold, about 11-fold, about 11.5-fold about 12.0-fold, about 12.5-fold, about 13-fold, about 13.5- fold, about 14.0-fold, about 14.5-fold, about 15.0-fold, about 15.5-fold, about 16-fold, about 16.5-fold, about 17.0-fold, about 17.5-fold, about 18.0-fold, about 18.5-fold, about 19.0-fold, about 19.5-fold, about 20-fold, about 20.5-fold, about 21-fold, about 21.5-fold about 22.0-fold, about 22.5-fold, about 23-fold, about 23.5-fold, about 24.0-fold, about 24.5-fold, about 25.0-fold, about 25.5-fold, about 26-fold, about 26.5-fold, about 27.0-fold, about 27.5-fold, about 28.0-fold, about 28.5-fold, about 29.0-fold, about 29.5-fold, or about 30-fold, or any range derivable therein, in comparison to administration of the therapeutic compound or composition alone by other methods, such as bolus injection.

[0057] In some embodiments, the presently disclosed compositions facilitate maintaining therapeutic compound or composition concentrations above the EC50 of the therapeutic compound or composition for longer periods of time than administration of the drug alone. In some embodiments, the presently disclosed compositions facilitate maintaining therapeutic compound or composition concentrations above the EC50 of the therapeutic compound or composition for about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, about 5.0-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7.0- fold, about 7.5-fold, about 8.0-fold, about 8.5-fold, about 9.0-fold, about 9.5-fold, or about 10- fold longer than a therapeutic compound or composition administered on its own, such as by bolus injection. In some embodiments, concentrations above the EC50 of the therapeutic compound or composition are maintained for between about 2.0-fold and about 6.0-fold longer for delivery via presently disclosed compositions than administration of compound or composition alone. In some embodiments, concentrations above the EC50 of the therapeutic compound or composition are maintained for about 4.0-fold longer for delivery via presently disclosed compositions than administration of compound or composition alone.

[0058] As mentioned, these properties are beneficial particularly in that they persist across long periods of time after administration of the composition. In some embodiments, any of the beneficial kinetic profiles mentioned above as characteristic of the presently disclosed compositions are measured at about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, about 168 hours, about 192 hours, about 216 hours, about 240hours, about 264 hours, about 288 hours, about 312 hours, about 336 hours, about 360 hours, about 384 hours, about 408 hours, about 432 hours, about 456 hours, about 480 hours, about 504 hours after administration of the composition, or any range derivable therein. In some embodiments, these characteristics are measured between about 300 and 400 hours after administration of the composition. In some embodiments, the time period beneficial kinetic profiles mentioned as characteristic of the presently disclosed compositions is measured in animal models. In some embodiments, administration of the presently disclosed compositions produces corresponding effects in mammals, such as humans, and more particularly such as patients, over a time period which may or may not align with the time period observed in the animal model.

[0059] In some embodiments, therapeutic compounds or compositions administered delivered via the presently disclosed compositions persist at the site of injection. Retaining drug at the target site could be beneficial for some applications, such as treating solid tumors, by creating a favorable local concentration gradient at the site of the tumor (Mu et al., 2023; Wu et al., 2023). In some embodiments, therapeutic compounds or compositions delivered via presently disclosed compositions (such as, for example, according to the release kinetics of dynamic covalent attachment motifs present in the claimed compositions) persist at the site of injection for a longer period of time compared to administration of the therapeutic compound or composition alone. In some embodiments, release of therapeutic compound or composition from the presently disclosed compositions results in higher therapeutic compound or composition concentration at the site of injection for about 0.5 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or any range derivable therein, as compared to administration of the therapeutic compound or composition alone, for example by bolus injection. In some embodiments, local therapeutic compound or composition concentration is higher when administered or formulated according to the present disclosure for about one week. In some embodiments, local therapeutic compound or composition concentration is higher when administered or formulated according to the present disclosure for about two weeks. In someembodiments, local therapeutic compound or composition concentration is higher when administered or formulated according to the present disclosure for about three weeks. In some embodiments, local therapeutic compound or composition concentration is higher when administered or formulated according to the present disclosure for more than three weeks.

[0060] In some embodiments, the kinetics of payload release from a presently disclosed composition may be controlled or changed by selecting or altering the ratio of boronic acid binding motifs or BA groups of the functionalized hydrogel to the corresponding binding group (BA or BABM, respectively) of the payload. The amount of functionalized hydrogel may also be measured by the amount of functionalized peptide that forms the functionalized hydrogel of the claimed composition. The present disclosure, therefore, provides compositions formed from various ratios of functionalized peptide and payload. In some embodiments, the presently disclosed compositions have a payload:functionalized peptide molar ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1: 15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, or any molar ratio or range derivable therein.

[0061] In some embodiments, the presently disclosed compositions delivery of therapeutic compound with a lower maximum circulating drug concentration than corresponding methods known in the art. A lower maximum circulating drug concentration could, for example, result in lower toxicity and / or improved drug tolerance. In some embodiments, delivery of a payload via presently disclosed compositions results in a maximum circulating drug concentration that is about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7- fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 10.5-fold, about 11-fold, about 11.5-fold about 12.0-fold, about 12.5-fold, about 13-fold, about 13.5-fold, about 14.0-fold, about 14.5-fold, about 15.0-fold, about 15.5-fold, about 16-fold, about 16.5-fold, about 17.0-fold, about 17.5-fold, about 18.0-fold, about 18.5-fold, about 19.0- fold, about 19.5-fold, or about 20-fold, or any range derivable therein, lower than other methods of delivery. In some embodiments, delivery of a payload via presently disclosed compositionsresults in a maximum circulating drug concentration that is about 2-fold to about 12-fold lower than other methods of delivery. In some embodiments, delivery of a pay load via presently disclosed compositions results in a maximum circulating drug concentration that is about 5-fold to about 7-fold lower than other methods of delivery. In some embodiments, delivery of a payload via presently disclosed compositions results in a maximum circulating drug concentration that is about 6-fold lower than other methods of delivery. In some embodiments, delivery of a payload via presently disclosed compositions results in a maximum circulating drug concentration that is about 10-fold lower than other methods of delivery.

[0062] The disclosed compositions in some embodiments exhibit improved oxidative stability. In some embodiments, the presently disclosed functionalized hydrogel compositions facilitate an extension of the duration of the release of a payload, such as a therapeutic compound that comprises a boronic acid group. In some embodiments, the release kinetics of presently disclosed compositions are less affected by glucose concentration than similar technologies known in the art (Rafiee & Nematollahi, 2008; Zhang et al., 2009; Pang et al., 2023; Arzt et al., 2014; Stolowitz et al., 2001). The duration of release can be measured, for example, by determining the percentage of payload (for example, therapeutic compound) released over a period of time.

[0063] In some embodiments, the boronic acid group or boronic acid binding motif is on an antibody or an antibody that has been modified to include at least one boronic acid group or boronic acid binding motif. Therefore, in some embodiments the pay load is an antibody. In this way, the present disclosure provides compositions and methods of treatment relating to the delivery of antibodies. Although biotherapeutics, including antibodies, have risen in popularity owing to their high specificity and potency, their clinical translation is often limited by poor pharmacokinetics and adverse effects (Shah, 2015). For example, immune checkpoint inhibitors used in cancer therapy often cause severe immune-related adverse events, which could be mitigated by administering presently disclosed compositions comprising antibodies directly to the site of the tumor to reduce the total amount of drug needed and thereby systemic drug concentration (Heinzerling et al., 2019; Lamichhane et al., 2019).

[0064] In some embodiments, the boronic acid group or boronic acid binding motif is on an insulin that has been modified to include at least one boronic acid group or boronic acid binding motif. Therefore, in some embodiments the payload is insulin. In this way, the present disclosure provides compositions and methods related to the delivery of insulin. There is a persistent need for insulin delivery systems due to the high clinical burden of basal insulin therapy, which can require multiple subcutaneous injections per day to maintain normoglycemia in individuals with type 1 diabetes (Katsarou et al., 2017). Many groups have investigated using boronate ester chemistry to create glucose-responsive insulin delivery systems (Zhang et al., 2023; Chou et al., 2015; Dong et al., 2016; Xian et al., 2024, Yu et al., 2020) commonly by modifying the B29 lysine residue with a BA (Hoeg-Jensen et al., 2005). These materials facilitate glucose-responsive release by leveraging the ability of diols on glucose to compete with diols on the reservoir device to form boronate esters with BA-modified insulin. The interaction strength between the modified insulin and the delivery system must be near that of the weak BA- glucose boronate ester bond strength, which can limit the duration over which these systems can effectively release insulin (Ali et al., 2023; Matsumoto & Chen, 2021). Additionally, glucose responsiveness may not always be desirable for basal insulin delivery since it could complicate estimations for the dosing of fast-acting insulin around meals, leading to a higher risk of over- or under- shooting the desired blood glucose range. In some embodiments, delivery of insulin involving the presently disclosed compositions and methods results in blood glucose levels below the 240 mg / dL threshold for a longer period of time as compared to delivery of insulin via other methods of administration known in the art. In some embodiments, delivery of insulin involving the presently disclosed compositions and methods results in blood glucose levels below the 180 mg / dL threshold for a longer period of time as compared to delivery of insulin via other methods of administration known in the art. Blood glucose levels of 180 mg / dL represent the upper limit of healthy blood glucose concentrations. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5- fold, about 10-fold, about 10.5-fold, about 11-fold, about 11.5-fold about 12.0-fold, about 12.5- fold, about 13-fold, about 13.5-fold, about 14.0-fold, about 14.5-fold, about 15.0-fold, about15.5-fold, about 16-fold, about 16.5-fold, about 17.0-fold, about 17.5-fold, about 18.0-fold, about18.5-fold, about 19.0-fold, about 19.5-fold, about 20-fold, about 20.5-fold, about 21-fold, about21.5-fold about 22.0-fold, about 22.5-fold, about 23-fold, about 23.5-fold, about 24.0-fold, about24.5-fold, about 25.0-fold, about 25.5-fold, about 26-fold, about 26.5-fold, about 27.0-fold, about27.5-fold, about 28.0-fold, about 28.5-fold, about 29.0-fold, about 29.5-fold, about 30-fold, about30.5-fold, about 31-fold, about 31.5-fold about 32.0-fold, about 32.5-fold, about 33-fold, about33.5-fold, about 34.0-fold, about 34.5-fold, about 35.0-fold, about 35.5-fold, about 36-fold, about36.5-fold, about 37.0-fold, about 37.5-fold, about 38.0-fold, about 38.5-fold, about 39.0-fold, about 39.5-fold, or about 40-fold longer, or any range derivable therein, than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for between about 10-fold and about 30-fold longer than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for between about 15 -fold and about 25-fold longer than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for about 18-fold longer than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for between about 20-fold and about 40-fold longer than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for between about 30-fold and about 40-fold longer than delivery of insulin via other methods, such as bolus injection. In some embodiments, insulin delivery involving the presently disclosed compositions and / or methods results in blood glucose levels under 240 mg / dL and / or under 180 mg / dL for about 36-fold longer than delivery of insulin via other methods, such as bolus injection. In this way, the present disclosure provides compositions and methods for the basal insulin delivery to help individuals control their blood glucose levels without the need for frequent injections of insulin.IL Definitions

[0065] The present application provides, for example, compositions comprising a boronic acidbinding motif (BABM). A BABM as used herein is any compound, functional group, or chemical moiety which is known in the art to form dynamic covalent interactions or attachments with boron-containing functional groups, such as boronic acids. Non-limiting examples of BABMs that may be used to form compositions and other aspects of the present disclosure, such as hydrogels formed from the presently disclosed compositions, include diols, catechols, salicylhydroxamic acids, groups comprising one amino group and one hydroxy group, and a- hydroxycarboxylic acids.

[0066] In this disclosure, the use of the singular includes the plural, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As used herein “another” may mean at least a second or more. As used herein, “or” means “and / or”, unless specifically stated otherwise.

[0067] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) arc inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0068] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0069] All the compounds of the present invention may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. Insome embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, a prodrug, and / or a modified version of a compound known in the prior art may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such, unless explicitly stated to the contrary, all the compounds of the present invention are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0070] In some embodiments, the compounds of the present invention have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0071] In some embodiments, compounds of the present invention function as prodrugs or can be derivatized to function as prodrugs. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Non-limiting examples of suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonatcs, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, and esters of amino acids. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[0072] In some embodiments, compounds of the present invention exist in salt or non-salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0073] It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” Where the solvent is water, the complex is known as a “hydrate.” It will also be appreciated that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. All solid forms of the compounds provided herein,

[0074] As used herein, the terms “drug”, “pharmaceutical”, “therapeutic compound” “therapeutic agent”, and “therapeutically active agent” are used interchangeably to represent a compound which invokes a therapeutic or pharmacological effect in a human or animal and is used to treat a disease, disorder, or other condition.

[0075] As used herein, the term “prophylactic” is used to represent a compound which is used to prevent a disease, disorder, or other conditions, or symptoms thereof. In some embodiments, these compounds have undergone and received regulatory approval for administration to a living creature.

[0076] An “active ingredient” (Al) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologicallyactive molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.

[0077] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.

[0078] An “excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of anti- adherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0079] The term “hydrate” when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.

[0080] As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drugor other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.c., an enzyme, cell, cell receptor or microorganism) by half.

[0081] As used herein, the term “ED50” refers to a dose which is effective in 50% of the population. This quantitative measure indicates how much of a particular drug, substance, or composition is needed to produce a desired pharmacologic effect in 50% of the studied patient population that is administered the drug, substance, or composition.

[0082] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.

[0083] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0084] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which arc pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis-(3 hydroxy-2-ene-l -carboxylic acid), 4- methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4- hydroxybenzoyljbenzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tcrtiarybutylacctic acid, trimcthylacctic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).[00851 A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and / or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and / or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, hydrogels, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

[0086] A “pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).

[0087] As used in this specification, the term “significant” (and any form of significant such as “significantly”) is not meant to imply statistical differences between two values but only to imply importance or the scope of difference of the parameter.

[0088] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or experimental studies. Unless another definition is applicable, the term “about” refers to ±10% of the indicated value.

[0089] As used herein, the term “substantially free of’ or “substantially free” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of all containments, by-products, and other material is present in that composition in an amount less than 2%. The term “more substantially free of’ or “more substantially free” is used to represent that the composition contains less than 1% of the specific component. The term “essentially free of’ or “essentially free” contains less than 0.5% of the specific component.

[0090] As used herein, “treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and / or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and / or symptomatology), and / or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0091] As used herein, and unless otherwise indicated, the terms “prevent,” “preventing,” and “prevention” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0092] The term “unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, a single vial with a syringeable liquid, shear-thinning hydrogel, or other injectable formulations, or a dose that is formulated for administration in a single inhalation.

[0093] As used herein, “biocompatible” means that under physiological conditions, no harmful amounts of a substance toxic to contacted cells is released by a composition or its components, and no appreciable amount of degradation of a composition or its components occurs.

[0094] As used herein, “dynamic covalent bond” refers to a reversible covalent interaction formed between two or more components.

[0095] As used herein, “shear-thinning” means a material can behave as a liquid under shear while it behaves at a solid under static conditions.

[0096] As used herein, “self-healing” means a shear thinning material that can reform as a hydrogel once the shear force is abated.

[0097] As used herein, “hydrophilic residues” are canonical or noncanonical amino acid residues whose sidechains contain polar atoms or that can carry a net charge at any pH value between 1- 14.

[0098] As used herein, “hydrophobic residues” are canonical or noncanonical that have low polarity sidechains.

[0099] The present disclosure provides hydrogels, which in some embodiments may be formed from peptides. The amino acid residues of these peptides can be present in stereochemical configurations of the D-form, the L-form or mixtures thereof. In addition, the amino acid or peptide residue may have an asymmetric carbon atom. Examples of suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Vai, Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom.

[0100] As used herein, “enzymatically degradable domain” means a motif known to act as a substrate of an enzyme that causes the motif to be cleaved into two or more parts.

[0101] As used herein, “hydrogel” refers to material composed a network of physically or covalently crosslinked fibers and is at least 10% by weight water. Hydrogels are Theologically defined as visoelastic solids whose storage modulus is greater than its loss modulus.

[0102] As used herein, “storage modulus” is a property determined by oscillatory rheology that is a measure of the amount of energy that a viscoelastic material can store under a deformation force. As used herein, “loss modulus” is a property determined by oscillatory rheology that is a measure of the amount of energy that is dissipated when a viscoelastic solid is subjected to a deformation force.

[0103] As used herein, "bioactive domain" means a motif that can interact with biological processes through mechanisms including, but not limited to, acting as receptor ligands, enzyme substrates, or signaling molecules, that result in downstream biological effects.

[0104] As used herein, peptide self-assembly is the process by which peptides spontaneously assemble into macromolecular structures.

[0105] As used herein, P-sheet nanofibers are fiber structures that are less than 1000 nm in thickness and composed of peptides folded into a P-sheet secondary structure as characterized by circular dichroism, Fourier transform infrared spectroscopy, or an equivalent technique. The length of these fibers can extend over 1000 nm.

[0106] As used herein, the term “boronic acid”, “boronic acid group”, or synonyms thereof refers to a functional group comprising boron with the general structure BX2, wherein the two X atoms can be any atom, such as oxygen. The X atoms may be independently selected. A boronic acid may also be described by the formula R-BX2, wherein R represents the rest of a molecule to which the BX2 group is attached. R may represent, for example, a small molecule or a biologic. The term boronic acid is also used to refer herein to any compound that readily becomes or exists in equilibrium as R-BX2 with a compound in aqueous conditions at pH 1-14.

[0107] As used herein, a boronic acid-containing small molecule (BACSM) is a boron- containing compound capable of forming dynamic covalent attachments with a boronic acid binding motif.

[0108] Abbreviations as used herein include but are not limited to boronic acid (BA), self-assembling boronate ester release (SABER), Fourier transform infrared (FTIR), circular dichroism (CD), cryogenic transmission electron microscopy (cryo-TEM), maximum circulating drug concentrations (Cmax), area under curve (AUC) multidomain peptide (MDP), boronic acid containing small molecules (BACSMs), phenylboronic acid (PBA), fluorescein isothiocyanate (FITC), dopamine (DOPA), nDopa: nitrodopamine nitro-catechol (nitroCat), Alizarin Red S (ARS), tavaborole (TVB), bortezomib (BTZ), fluorescence recovery after photobleaching (FRAP), GSK3036656 also known as ganfeborole (GSK656), and salicylhydroxamic acid (SHA). The MDP referenced herein as K2 (SEQ ID NO: 1) is Ac-K2-(SL)e-K2-Am (see also, for example, Table 1). The MDP referenced herein as E2 (SEQ ID NO: 2) is Ac-EE(SL)6EE-Am (see also, for example, Table 1)

[0109] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and parameters.III. Examples

[0110] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. In no way should the following examples be read to limit or define the entire scope of the disclosure.Example 1: Synthesis and Characterization of Self-assembling Peptides and Hydrogel Compositions Thereof

[0111] To directly compare the stability of catechol, 4-nitrocatechol, and SHA motifs in dynamic covalent BA chemistry, their UV-Vis absorbance spectra were monitored to identify any changes indicative of oxidation or degradation over time (FIG. 1A- FIG. 1C). At 25 °C in pH 7.4 buffer, the model catechol dopamine rapidly oxidized within one day, as indicated by drastic changes in the UV-Vis spectrum. In contrast, SHA and 4-nitrodopamine demonstrated excellent stability over 15 d. After verifying oxidation resistance, boronatc ester association constants between catechol, SHA, and 4-nitrodopamine and several BACSMs of interest were determined via a competitive binding assay. SHA formed the strongest dynamic covalent interactions with all the BACSMs evaluated. Dopamine and 4-nitrodopamine had similar boronate ester equilibria except for in the case of ixazomib where 4-nitrodopamine displayed a 5.6-fold higher association constant (FIG. ID). These favorable stability and binding results indicated that 4-nitrocatechol and SHA motifs may be superior to catechols for drug delivery applications. Therefore, the MDP Ac-K2-(SL)e-K2-Am (referred to as K2 (SEQ ID NO: 1)), was N-terminally functionalized with catechol (Cat-K2), 4-nitrocatechol (nitroCat- K2), and SHA (SHA-K2) to yield a set of three SABER peptides with different boronate ester formation potential (FIG. 2B, FIGS. 1E-1H)

[0112] To confirm that these modified MDPs retained the ability to form nanofibrous hydrogels, their self-assembly and physical properties were assessed by Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), rheology, and cryogenic transmission electron microscopy (cryo-TEM). The FTIR spectra of all peptides assessed displayed peaks at 1618 and 1695 cm’1, consistent with the presence of the anti-parallel 0-sheet secondary structure that is characteristic of unmodified MDPs (FIG. 2C) (Dong et al., 2007; Aulisa et al., 2009). The secondary structure of these peptides in solution was further confirmed by the presence of a minimum ca. 216-220 nm in the CD spectra of all the peptides tested (FIG. 2D). Cat-K2 (FIG. 2E) and nitroCat-K2 (FIG. 2F) peptides formed long thin nanofibers as seen by cryo-TEM, morphologically similar to previous observations for unmodified MDPs (Dong et al., 2007; Aulisa et al., 2009). SHA-K2 nanofibers appeared to have a higher propensity to cluster and form nanosheet-like architectures (FIG. 2G). Rheology was employed to assess the effect of modification on the viscoelastic properties of these hydrogels. All four peptides formed hydrogels at 10 mg / mL in IX Hank’s Balanced Salt Solution, as indicated by storage moduli (G’) that exceed the loss moduli (G”) at all oscillatory frequencies tested (FIG. 2H). When subjected to a high deformation force, G” exceeded G’, suggesting, without being bound by theory, liquid-like flow under high shear. Hydrogel character rapidly recovered once shear force was reduced (FIG. 21). K2 (SEQ ID NO: 1), Cat-K2, and nitroCat-K2 each recovered over 90% of their initial G’ within 10 min of the removal of a high deformation force, whereas SHA-K2 exhibited more than 60% recovery over the same timeframe. In this way, the present disclosure provides compositions that self-assemble into shear- thinning and self-healing nanofibrous hydrogels, making them suitable for injection and drug depot formation.Example 3: In Vitro assays

[0113] To determine if boronate ester interactions could be used to slow the delivery of BACSMs, the in vivo release of five different small molecules from SABER hydrogels was evaluated. First, the release of a model fluorescent BACSM prepared was measured by conjugating phenylboronic acid (PBA) to fluorescein isothiocyanate (FITC-PBA) from K2 (SEQ ID NO: 1) and the three SABER MDPs (Cat-K2, nitroCat-K2, and SHA-K2 described in Example 1) using a fluorescence equilibrium release assay. All the SABER MDPs drastically reduced the release rate of FITC- PBA compared to K2 (SEQ ID NO: 1), as well asthe apparent point of equilibrium, whereas the presence of BA-binding motifs on hydrogels did not alter the release rate of unmodified fluorescein (FIG. 3 A, FIG. 3B). These results demonstrate in an illustrative, non-limiting fashion that both BA and the BA-binding motif are critical for extending release from peptide hydrogels.

[0114] In some embodiments, the presently disclosed compositions arc useful for the controlled release of clinically relevant therapeutics. For example, the in vitro release of the FDA-approved BACSMs bortezomib (FIG. 4A) and ixazomib (FIG. 4B), which are used clinically in the treatment of multiple myeloma, was evaluated (Plescia & Moitessier, 2020). Unmodified K2 (SEQ ID NO: 1) hydrogels released more than 80% of each drug in the first 2 h and reached maximum release over the course of 4 h. Cat-K2 hydrogels also rapidly released drug at early time points, albeit at a slightly slower rate, releasing 57.1 ± 1.5% and 58.0 ± 1.5% of their encapsulated drug in the first 2 h for bortezomib (BTZ) and ixazomib, respectively. After this burst release, Cat-K2 hydrogels released each drug more slowly over the remainder of the assay, reaching over 90% release by 24 h. In contrast, SHA-K2 and nitroCat-K2 hydrogels significantly delayed the release of BTZ and ixazomib compared to both K2 (SEQ ID NO: 1) and Cat-K2, only releasing 15-25% of the encapsulated drug within 2 h and 50-70% within 24 h. The rapid release from Cat-K2 may be attributed, without being bound by theory, to a combination of catechol oxidation and the significantly weaker boronate ester interaction relative to the other two dynamic covalent attachment motifs as previously discussed (FIG. 1A, FIG. ID).

[0115] To understand the effect of BA structure on SABER performance, the release of tavaborole (an FDA-approved topical antifungal agent) (Plescia & Moitessier, 2020) and ganfeborole (GFB, also known as GSK3036656 and in phase II clinical trials for the treatment of tuberculosis via daily dosing) ( Butler & Paterson, 2020) were assessed from these hydrogels. Both of these drugs contain benzoxaboroles, a type of BA common in BACSMs (Zhang et al., 2017; Dhawan et al., 2022). As seen with BTZ and ixazomib, K2 (SEQ ID NO: 1) rapidly released tavaborole, while the SABER hydrogels exhibited delayed release (FIG. 4C). SHA-K2 showed the slowest tavaborole release, only releasing 49.1 ± 0.3% of the payload by 24 h, followed by nitroCat-K2 and then Cat-K2, which released 64.8 ± 0.9% and 80.0 ± 4.7% of the drug, respectively. Interestingly, the release trends for GFB deviated substantially from those for tavaborole. Both K2 (SEQ ID NO: 1) and nitroCat-K2 had similarly rapid release rates,suggesting that GFB is limited in its ability to form dynamic covalent bonds with nitroCat-K2 (FIG. 4D). These results arc consistent with the low equilibrium constant for complcxation between 4-nitrodopamine and GFB (FIG. ID). The release of GFB from Cat-K2 not be quantified due to the rapid oxidative deboronation of the compound as observed by UPLC and mass spectrometry (FIGS. 3C-3E), suggesting that the catechol itself facilitates oxidative destruction of the drug (Graham el al., 2021; Bouz & Zitko, 2021). GFB was significantly more stable when loaded in the other MDPs (FIG. 3F). Only the SHA-modified peptide was able to significantly extend the release of GFB compared to unmodified K2 (SEQ ID NO: 1). In the first 2 h of the assay, K2 (SEQ ID NO: 1) released 96.1 ± 1.5% of the drug while SHA-K2 released 28.6 ± 0.5%. K2 (SEQ ID NO: 1) released more drug in 30 min (59.0 ± 1.1%) than SHA-K2 did in 24 h (48.7 ± 2.5). The results of these release assays suggest, without being bound, that the SHA motif is the most promising and versatile dynamic covalent attachment of the three tested within the SABER platform and far superior to the catechol modification.

[0116] The present disclosure provides for control of payload release rate via the ratio of boronic acid groups and boronic acid binding motif groups. For example, since any BACSM molecule bonds to a compound or composition comprising a boronic acid binding motif to avoid release via diffusion, it follows, without being bound by theory, that increasing the ratio of available boronic acid binding SHA motifs to BACSMs would increase the likelihood of boronate ester reformation after hydrolysis and thereby increase drug retention. To demonstrate the effect of a surplus of peptide-displayed SHA motifs on drug release kinetics, the molar ratio of drug-to-peptide was varied in SHA-K2 gels. K2 (SEQ ID NO: 1) and SHA-K2 hydrogels were prepared at different levels of drug loading ranging from a 1:1 to 1:10 drug-to-peptide molar ratio while keeping the peptide concentration constant at 10 mg / mL (~5 mM). The release of GFB from K2 (SEQ ID NO: 1) was fast, as expected, and did not significantly change as a function of drug loading over this range (FIG. 4E). In contrast, the release of drug from SHA-K2 was sensitive to drug loading (FIG. 4E). After 24 h, the 1:1 drug-to-peptide SHA-K2 gel loaded with the most drug released 45.4 ± 1.1%, significantly more than the 1:2 (35.1 ± 0.6%), 1:4 (31.5 ± 0.6%), and 1:10 (31.3 ± 0.7%) gels (FIG. 4F). There were no statistical differences between the three lower loadings, suggesting that drug loading has a negligible impact on release rate once the number of SHA motifs significantly exceeds the number of drug binding partners. The highest degree of drug loading was not observed to perturb self-assembly by CD analysis of the1 : 1 SHA-K2 hydrogel (FIG. 3G). SHA-K2 significantly delayed the release of GFB compared to unmodified K2 (SEQ ID NO: 1) at all molar ratios tested, which indicates that these hydrogels can be “fully” loaded with one BACSM per dynamic covalent attachment motif and still function effectively as a drug delivery vehicle.

[0117] In some embodiments, the present disclosure provides compositions comprising compounds that have been modified to comprise a boronic acid group. 1V209, a tolllike receptor 7 (TLR7) agonist, was modified with the boronic acid motif PBA, yielding 1V209- PBA, and evaluated its release from SABER hydrogels and its bioactivity. Modifying 1V209 with PBA did not impact its ability to activate TLR7 relative to the parent drug in a mouse TLR7 reporter cell line (FIG. 4G). In vitro release studies revealed 1V209-PBA exhibited dramatically slower release from nitroCat-K2 and SHA-K2 hydrogels (FIG. 4H). In the absence of the PBA modification, no altered release kinetics were observed (FIG. 41). The cumulative release of 1V209-PBA from SHA-K2 at 24 h (40.5 ± 0.5%) was lower than that of nitroCat-K2 (51.8 ± 0.4%), consistent with previous results showing that SHA is a more potent dynamic covalent attachment motif for PBA (FIG. ID). These results underscore the functional importance of the modifications of MDPs with nitroCat or SHA, which succeed at extending drug release from hydrogels when the catechol modification fails, and provide a workflow to make other smallmolecule drugs compatible with the SABER delivery platform.Example 4: In Vivo assays

[0118] To demonstrate the ability of the presently disclosed peptides to act as a platform for a wide variety of BACMs, bortezomib (BTZ) and GSK656 were used as clinically representative BACMs with diverse chemical structures.

[0119] To evaluate the controlled release of BTZ from SABER hydrogels in vivo, female BALB / c mice were subcutaneously injected with 700 ng of BTZ dissolved in 50 pL of PBS or with 700 ng of BTZ loaded in 50 pL of an MDP hydrogel composed of 10 mg / mL peptide. Subsequently, the concentration of BTZ in the blood was measured longitudinally. Mice injected with BTZ alone or BTZ loaded into K2 (SEQ ID NO: 1) hydrogels had maximum circulating drug concentrations (Cmax) of BTZ (107.3 ± 12.0 ng / mL and 75.3 ± 4.5 ng / mL, respectively) at 1 h. Mice receiving BTZ loaded in nitroCat-K2 and SHA-K2 experienced asignificantly lower Cmax(30.4 ± 2.0 ng / mL and 17.0 ± 4.3 ng / mL, respectively) and Cmax occurred at a significantly longer 34 h time point compared to the bolus injection of BTZ and K2 (SEQ ID NO: 1) alone (FIG. 5 A, FIG. 5B). A high Cmax after BTZ administration in patients has been linked to toxicity (Bilinska et al., 2013); therefore, the more than 6-fold reduction in BTZ Cmax when formulated in SABER hydrogels could improve the drug tolerance. To assess the potential magnitude of this benefit, a dose de-escalation study of BTZ alone was performed to determine the dose that results in a Cmax that matches that of 700 ng delivered from SABER hydrogels (FIG. 6A). It was observed that a dose of 175 ng of BTZ alone resulted in a Cmax of 21.2 + 3.1 ng / mL, which is statistically similar to the peak circulating concentrations observed in mice dosed with a 5-fold higher BTZ dose (700 ng) released from nitroCat-K2 and SHA-K2 (FIG. 6B). At this peak-matched dose of BTZ, mice dosed with BTZ loaded in nitroCat-K2 and SHA-K2 hydrogels had a 2.9-fold and 1.6-fold higher drug exposure (AUC), respectively and a 3-fold higher circulating BTZ concentration 336 h following administration than the peak matched dose without a hydrogel (FIG. 5C).

[0120] The presence of high local concentrations of BTZ over the course of 3 weeks was assessed by mass spectrometry imaging in BALB / c mice receiving 700 ng of BTZ subcutaneously alone or formulated in MDPs. Tissue sections collected from mice receiving BTZ loaded in MDPs showed much higher levels of signal attributable to BTZ (m / z 226.14) at the injection site (FIG. 6C), compared to mice receiving the drug alone at the same time points (FIG. 5D). Analysis was limited to portions of each section that did not contain hydrogel, because the hydrogel suppressed ionization of BTZ in mass spectrometry imaging (FIG. 6D, FIG. 7A). To focus on the areas of highest BTZ concentration in each sample while accounting for the different sizes of tissue collected, the pixel intensities were analyzed over the 1 mm2of tissue with the highest BTZ concentration (FIG. 5E). This analysis revealed that mice treated with one of the two SABER hydrogels have higher mass spectrometry imaging pixel intensities than the K2 (SEQ ID NO: 1) or bolus BTZ injection controls in these regions.

[0121] Consistent with the pharmacokinetic results that showed that nitroCat-K2 released BTZ faster than SHA-K2, at 1 and 7 d after injection, the tissue surrounding the nitroCat- K2 hydrogels was observed to have the highest BTZ concentrations (FIG. 5F). Since SHA-K2 releases BTZ more slowly, the average pixel intensity observed in SHA-K2 tissuesremains relatively constant and at 21 d post injection is the only group statistically superior to unformulatcd BTZ. Overlaying the BTZ mass spectrometry imaging signal with that of heme (616.18 m / z) shows that the BTZ being measured is largely not colocalized with blood vessels, demonstrating that BTZ being measured is primarily present in tissue immediately surrounding the injection, not from systemic circulation (FIG. 7B). These data suggest, without being bound, that SABER hydrogels maintain a high local BTZ concentration at the site of injection for at least 21 d and that the timescale of local release can be controlled by using different BA dynamic covalent attachment motifs.

[0122] To further demonstrate the compatibility of the SABER platform with multiple drugs and disease applications, the in vivo release of GFB, a drug in late-stage clinical development for the treatment of tuberculosis (TB). TB treatment typically requires months of daily oral drug dosing to effectively clear the pathogen from the body and is fraught with low patient adherence (Chimeh et al., 2020; Hirsch-Moverman et al., 2008); therefore, a controlled- release formulation using SHA-K2 to maintain GFB concentrations over the ECso for weeks or months could have a meaningful impact on clinical care (Baryakova et al., 2023). BALB / c mice were treated with 50 pL gels composed of 10 mg / mL peptide loaded with 75 pg of GFB. Consistent with in vitro release results, K2 (SEQ ID NO: 1) and nitroCat-K2 were unable to prolong GFB release in vivo compared to the drug alone (FIG. 8). In this study, the formulation in SHA-K2 maintained circulating GFB concentrations above the ECso of the drug for over 4- fold longer than other groups (Tenero et al., 2019). Since GFB has been shown to be well tolerated in humans at high doses (Tenero et al., 2019), the high levels of circulating drug achieved early on are unlikely to be toxic. Thus, the duration over the ECso was sought to be extended by increasing the peptide concentration, volume of hydrogel administered, and drug loading. In this experiment, mice were subcutaneously injected with 200 pL of 20 mg / mL SHA- K2 hydrogels loaded with 600 pg (-1:1 molar ratio of drug-to-peptide) and 60 pg (-1:10 molar ratio of drug-to-peptide) of GFB. At both drug concentrations, loading GFB into SHA-K2 significantly prolonged the release of the drug compared to the drug alone and resulted in a 5.2- fold reduction in the Cmax (FIG. 5G, FIG. 5H). Mice that received the 10-fold higher dose of GFB in SHA-K2 had circulating GFB concentrations above the ECso for over 508 h compared to under 79 h in mice that were administered the drug alone. Non-compartmental pharmacokinetic analysis of the 600 pg dose of GFB revealed that SHA-K2 improved the drug exposure (AUC)by 2.8-fold and increased the circulating half-life (ti / 2) of GFB by 6.5-fold despite the significantly reduced Cmax (FIG. 5H). Although the >500 h duration is not yet approaching the duration of TB treatment normally prescribed, it is important to note that the half-life of the drug is 10-fold longer in humans than in mice (Tenero el al., 2019; Li et al., 2017).Example 5: SABER peptide modularity and its ability to improve TB treatment in mice through prolonged delivery of GFB

[0123] To demonstrate that the SABER platform is compatible with various selfassembling peptides, we changed the base MDP in our SHA-modified SABER hydrogel from K2 (SEQ ID NO: 1) to Ac-EE(SL)eEE-Am (E2 (SEQ ID NO: 2)), a negatively charged peptide (FIG. 9A, FIG. 10A, FIG. 10B). The strongly cationic K2 (SEQ ID NO: 1) MDP is known to cause inflammation at the injection site and can act as a potent adjuvant (Lopez-Silva et al., 2020; Pogostin et al., 2022), which may be well-suited to cancer applications with BTZ but makes it a less ideal MDP for the treatment of conditions that do not benefit from inflammation, like TB. Negatively charged MDPs, in contrast, have been found to cause minimal inflammation in vivo (Lopez-Silva et al., 2020). SHA-modified E2 (SHA-E2) was found to spontaneously selfassemble into an antiparallel P-sheet secondary structure, like the base peptide, as indicated by FTIR (FIG. 9B) and CD (FIG. 10C). SHA-E2 also formed hydrogels with similar rheological properties to E2 (SEQ ID NO: 2) (FIG. 10C). Both E2 (SEQ ID NO: 2) and SHA-E2 hydrogels were found to be shear thinning and self-healing and recovered 90% and 88% of their initial G’ within 10 min, respectively, after being subjected to 200% strain (FIG. 9D). Images collected using cryo-TEM revealed the presence of thin nanofibers with similar morphology to Cat-K2 and nitroCat-K2 (FIG. 2E, FIG. 2F, FIG. 9E). SHA-E2 was found to form more robust gels with greater shear recovery than SHA-K2.

[0124] Hydrogels formed from SHA-E2 are useful for controlling release of the clinically relevant BACSM GFB. In vitro, SHA-E2 significantly reduced the release of GFB over 24 h compared to the E2 (SEQ ID NO: 2) control, which lacks the ability to form dynamic covalent bonds with the drug (FIG. 9F). SHA-E2 released 41.6 ± 6.2% of the total drug in 24 h, which is statistically similar to the cumulative release measured from SHA-K2 SABER hydrogels under the same conditions (FIG. 10D). These data suggest that altering the peptidecharge did not meaningfully interfere with the dynamic covalent bonding between SHA and GFB . An in vivo pharmacokinetic study using 200 pL of 20 mg / mL SHA-E2 loaded with 600 pg of GFB similarly showed that SHA-E2 prolongs drug release compared to the unmodified E2 (SEQ ID NO: 2) hydrogel, maintaining a circulating GFB concentration above the ECso for more than 400 h (FIG. 9G). Pharmacokinetic analysis of these mice revealed that SHA-E2 hydrogels improved the circulating ti / 2 by 10.2-fold compared to the drug alone while increasing the AUC by 33% and reducing the Cmax by 10.3-fold (FIG. 10E).

[0125] In some embodiments, delivery of a therapeutic compound by release from presently disclosed compositions is useful for treating acute tuberculosis (TB). Three days after aerosol infection with M. tuberculosis, mice were treated with 600 pg of GFB delivered as a single subcutaneous bolus or as a single subcutaneous injection of the drug in SHA-E2. Because GFB is given orally to treat TB in clinical trials, an additional group was treated orally with the same total mass of drug administered across 10 doses over the course of 14 d. Mice were euthanized 7 and 14 d after the start of the treatment and the colony forming units (CFUs) of M. tuberculosis in the lungs were quantified. Mice given the SHA-E2 hydrogel without any drug rapidly showed signs of an uncontrolled bacterial infection, exhibiting more than a 104-fold increase in CFUs over the course of two weeks. Giving the drug as a single bolus injection without the hydrogel reduced the bacterial burden of mice by 0.95 and 1.55 logw CFUs at the 7 and 14 d time points, respectively, compared to the vehicle control. The effect of the drug was dramatically improved when given as 10 oral doses, which resulted in a reduction of 1.67 logic CFUs after 7 d and 3.32 logio CFUs after 14 d compared to the vehicle control. A single injection of GFB loaded in SHA-E2 significantly enhanced the efficacy of the treatment compared to all groups. Mice that received GFB loaded in the hydrogel saw no statistically significant bacterial proliferation over the first week and minimal proliferation over the second week of the experiment, which amounted to a 2.58 logio and 4.12 logio reduction in CFUs compared to the vehicle control at 7 and 14 d, respectively. The SABER hydrogel provided a nearly 10-fold reduction in CFUs compared to mice that received standard oral dosing, suggesting that using SABER hydrogels could simultaneously reduce the frequency of GFB dosing needed to treat acute TB infection and enhance the efficacy of the drug compared to the standard oral dosing strategy.Example 6: Prolonged local delivery of a model BA-modified IgG from SABER hydrogels

[0126] In some embodiments, the presently disclosed compositions are useful for the delivery of biological pay loads. Based on the results presented elsewhere, antibodies modified with a BA-motif, without being bound by theory, may enable SABER hydrogels to retain antibodies for long periods of time in vivo (FIG. 11 A). Thus, fluorescent rabbit IgG modified with a low number of PBAs (IgG Low, 2.4 PBAs per antibody) and a high number of PBAs (IgG High, 11.4 PBAs per antibody). The diffusion of PBA-labeled IgG in SHA-E2 hydrogels was then investigated using fluorescence recovery after photobleaching (FRAP) studies. After the fluorescence signal in three distinct locations in the gel was photobleached (FIG. 12A), the diffusion of IgG into the bleached area over time was significantly higher in unmodified MDPs compared to SHA-functionalized peptides (FIG. 11B, FIG. 11C). A 50% decrease in mobile fraction of IgG, and an over 100% increase in the time it takes to recover half the final fluorescence intensity (ti / 2) was observed in SHA-K2 compared to K2 (SEQ ID NO: 1) (FIG. 12B). The decrease in the rate of IgG diffusion within SHA-modified gels was further enhanced in SABER gels based on E2 (SEQ ID NO: 2). The mobile fraction of IgG in SHA-E2 was less than 25% of the mobile fraction of IgG observed in unmodified E2 (SEQ ID NO: 2) (FIG. 12B). Interestingly, the degree of PBA labeling did not have a meaningful impact on the FRAP data, as high and low degrees of IgG labeling displayed very similar results despite the nearly 5 -fold difference in the degree of PBA labeling.

[0127] In some embodiments, the interactions of antibodies labeled with boronic acid groups in SABER hydrogels influences the local retention of hydrogel-encapsulated IgG in vivo. The dissipation of fluorescently labeled IgG functionalized with high and low degrees of PBA from the site of subcutaneous injection was quantified by in vivo fluorescence imaging (FIG. 11D). Without a hydrogel, both IgG Low and IgG High injections were rapidly cleared after administration. In the case of the positively charged MDP K2 (SEQ ID NO: 1), the local retention of IgG was improved compared to IgG delivered without a hydrogel despite the absence of dynamic covalent bonding (FIG. HE). That the extended release is not observed in the unmodified negatively charged E2 (SEQ ID NO: 2) MDP (FIG. HF), supports, without being bound by theory, that the enhanced IgG retention in unmodified K2 (SEQ ID NO: 1) is dueto electrostatic interactions between the gel and the strongly negatively charged antibody following the PBA modification of lysine residues. In the case of IgG Low, using SHA-K2 instead of K2 (SEQ ID NO: 1) more than doubled the ti / 2 of the payload from 2.2 to 5.9 d (FIG. 12C). This difference is less pronounced with IgG High, but the half-life of the protein at the injection site was still improved by over 1 d when loaded in SHA-K2 compared to unmodified K2 (SEQ ID NO: 1). Almost no IgG fluorescence remains in the positively charged peptide hydrogels 4 weeks after injection. In comparison, SHA-E2 extends the release of IgG out to 8 weeks (FIG. 1 IF). Using SHA-E2 to deliver IgG Low resulted in an almost 24-fold improvement in the ti / 2 of the antibody alone from 0.4 to 9.5 d and an almost 14-fold improvement compared to release from unmodified E2 (SEQ ID NO: 2) (FIG. 12C). The higher degree of PBA labeling led to a modest but statistically significant improvement in payload retention compared to IgG Low. The ti / 2 of IgG High release from SHA-E2 was 13 d, which is a 26-fold improvement from IgG High without a hydrogel and a more than 16-fold improvement compared to the protein delivered from unmodified E2 (SEQ ID NO: 2) (FIG. 12C). Taken together, these data suggest that minimal amounts BA labeling can significantly improve local antibody retention and that SHA-E2 is superior to SHA-K2 in releasing PBA-labelled antibodies, as shown by an approximately 2-fold increase in the duration of IgG release in vivo.Example 6: A single injection of PBA-modified insulin loaded in SHA-E2 maintains normoglycemia for days in diabetic mice.

[0128] In some embodiments, the present invention is useful for the delivery of insulin. The boronate ester association constant between SHA and PBA is over 4 x 103-fold higher than the previously reported value for the glucose-PBA interaction, and thus glucose should offer minimal competition with SHA-modified SABER peptides for PBA bonding (FIG. ID) (Brooks et al., 2018). As such, SHA-E2, without being bound by theory, could be formulated as a glucose-insensitive delivery system for PBA-modified insulin (insulin-PBA) release to maintain normal blood glucose levels in diabetic mice over the course of several days.

[0129] To assess the glucose responsiveness of insulin-PBA release from SHA- E2, in vitro release assays were performed in varying concentrations of glucose (0, 100, and 250 mg / dL). Insulin labeled with a single PBA functional group was synthesized as previouslyreported (FIG. 13A, FIG. 13B) (Hoeg-Jensen et al., 2005). SHA-E2 significantly slowed the release of insulin-PBA at all glucose concentrations compared to E2 (SEQ ID NO: 2) controls (FIG. 14A). After 24 h, all SHA-E2 groups had released less than 50% of their payload. A modest, yet statistically significant, increase in insulin release was observed at 250 mg / dL glucose compared to 0 mg / dL (FIG. 14B). Healthy blood glucose concentrations range from 80- 180 mg / dL and a blood glucose over 240 mg / dL can require urgent intervention to avoid deleterious effects; therefore, SABER hydrogels are unlikely to be meaningfully impacted by physiologically relevant glucose levels (CDC, 2021). The benefit of using SHA-E2 over E2 (SEQ ID NO: 2) was completely abrogated when unmodified insulin was released instead of insulin-PBA, demonstrating that the both the PBA-modification on insulin and SHA groups on the hydrogel are critical for prolonging payload release (FIG. 13C).

[0130] The present disclosure provides a framework wherein a single injection of insulin-PBA loaded in a SABER hydrogel could maintain normoglycemia in mice for multiple days. Without being bound by theory, in some embodiments the extended release of insulin from presently disclosed compositions, including those comprising SHA-E2, allows for the administration of higher doses of insulin than would be normally tolerated when administered as a single bolus of unmodified insulin. Additionally, again without being bound by theory, the duration of efficacy of the SABER platform is in some embodiments of the present disclosure dependent on the total amount of insulin-PBA loaded in the hydrogel. C57BL / 6J mice with streptozotocin-induced diabetes received subcutaneous injections of 10 mg / mL SHA-E2 hydrogels loaded with 3, 6, or 12 IU of insulin-PBA. Their blood glucose levels were then measured longitudinally. A bolus subcutaneous injection of 3 IU of unmodified insulin without a hydrogel was used as a control since higher insulin concentrations would likely overshoot the desired range and induce dangerous hypoglycemia. Immediately after administration, the blood glucose concentration in all mice rapidly fell, but mice that received 3 IU of insulin without a hydrogel returned to unhealthy blood glucose levels within 4 h (FIG. 14C).The average blood glucose level of diabetic mice receiving 3 IU of insulin-PBA in the SHA-E2 hydrogel remained below 240 mg / dL for 72 h (FIG. 14D), appropriately controlling blood glucose levels within the desired range 18-fold longer than 3 IU of unmodified insulin.

[0131] With 3 IU of insulin-PBA in SHA-E2, it was observed that the blood glucose concentrations of mice varied widely and consistently exceeded the ideal blood glucose level of 180 mg / dL after 48 h. Mice that received gels loaded with 6 IU remained below 180 mg / mL for 96 h and below 240 mg / dL for 144 h, while mice administered 12 IU remained below 180 mg / dL for 144 h (FIG. 14D). The 6 and 12 IU insulin-PBA gel treatment groups achieved statically significantly lower blood glucose levels than the insulin bolus control between 4 and 168 h and outperformed the 3 IU gel group at 2 h and consistently after 72 h post injection (FIG. 14E). Mice administered with 12 IU of insulin more frequently dropped into the moderately hypoglycemic blood glucose ranges between 54-80 mg / dL compared to the 6 IU mice (CDC, 2021), suggesting that 12 IU is likely near the maximum tolerable dose of insulin-PBA delivered from this system, as presently constructed. To demonstrate that this system could be used to chronically deliver basal insulin, mice previously treated with 6 IU of insulin-PBA received another dose of the same treatment 6 weeks after the initial injection to assess potential changes in treatment efficacy due to previous exposure in repeated dosing regimens. There were no statistically significant differences in blood glucose levels between the initial and second dose for all time points measured (FIG. 14F, FIG. 13D). These data establish that insulin-PBA loaded in SHA-E2 can be used to achieve normoglycemia for up to 144 h in a single injection, a 36-fold improvement over the maximum tolerated single dose of unmodified insulin without a hydrogel. We achieve similar duration of action of other boronate ester insulin release systems but without glucose sensitively (Zhang et al., 2023; Xian et al., 2024). Since this system performs consistently during repeated dosing, the SABER hydrogel platform may be a useful tool for basal insulin delivery to help individuals control their blood glucose levels without the need for frequent injections of insulin.Example 7: Hydrogel compositions formed from D-amino acid peptides.

[0132] The present compositions may in some embodiments be formed from peptides wherein one or more of the amino acid residues of the peptide are D-amino acids. In some embodiments, the functionalized hydrogels disclosed herein may be formed from peptides wherein all of the amino acid residues of the peptide are D-amino acids. Further details on such hydrogels are provided below.Materials Characterization

[0133] K2 (SEQ ID NO: 1) (FIG. 15A) and D-K2 (SEQ ID NO: 12) (FIG. 15B) hydrogels were prepared, and the secondary structure of both peptides was assessed using CD. In line with previous reports, K2 (SEQ ID NO: 1) has a maximum near 198 nm and a minimum 218 nm (FIG. 15C), presenting canonical -sheet secondary structure (Pogostin et al., 2023; El- Bakary et al., 2019). In contrast, D-K2 (SEQ ID NO: 12) shows mean residue ellipticity that is inverted compared to its enantiomeric counterpart, with a minimum near 198 nm and a maximum at 218 nm. CD is a spectroscopy technique that measures the difference in the absorption of left and right circularly polarized light, meaning that the structure of the same molecule would appear to be inverted, confirming that the secondary structure of D-K2 (SEQ ID NO: 12) is the exact enantiomer of K2 (SEQ ID NO: 1) and similarly forms P-sheets (Circular Dichroism Spectroscopy). Cryo-EM was used to determine the morphology of D-K2 (SEQ ID NO: 12) relative to K2 (SEQ ID NO: 1). Cryo-EM showed that both peptides self-assembled into high aspect ratio nanofibers, in line with previous studies of K2 (SEQ ID NO: 1) and similar MDPs (Moore & Hartgerink, 2017).

[0134] To confirm the predicted lack of differences in mechanical properties between the two peptides, oscillatory rheology experiments were performed. Both peptides formed frequency-independent hydrogels with a storage modulus (G’) greater than the corresponding loss modulus (G”) (FIG. 16A). The G’ of K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) were 117 ± 10 and 137 ± 19 Pa, respectively, which were not statistically different (FIG. 16B). The G” of K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) were 12.1 ± 1.7 and 11.9 ± 1.3 Pa, respectively, which were also not significantly different (FIG. 16C).

[0135] Shear recovery was measured by deforming the hydrogels at 200% strain for 1 min and then measuring recovery in G’ over 10 min. When the hydrogels were subjected to 200% strain, G” is greater than G’, which shows that the materials are shear thinning and behave as liquids when subjected to strong deformation forces. Once this shear force is reduced, the gels rapidly recover their viscoelastic properties, which demonstrates that both materials are self- healing. D-K2 (SEQ ID NO: 12) had a shear recovery of 92.4 ± 3.1% and K2 (SEQ ID NO: 1) had a shear recovery of 92.8 ± 0.8% (FIG. 16D, FIG. 16E). These values were not statisticallysignificantly different, indicating that both enantiomers form self-healing hydrogels. The ability of supramolccular peptide hydrogels to be both shear-thinning and self-healing is necessary to ensure that the peptides’ loss of integrity under shear stress during passage through a needle is reversible after the material has been injected. These rheological data agree with previous studies of K2 (SEQ ID NO: 1) and also show that D-K2 (SEQ ID NO: 12) has largely equivalent rheological properties (Pogostin et al., 2022; Lopez-Silva et al., 2020).In vitro payload diff usion and in vivo antigen release

[0136] While the previous experiments were expected to give identical results due to the enantiomeric nature of the peptides being compared, subsequent experiments introduce additional chirality in the form of the antigen, enzymes, or the overall cellular environment and therefore could potentially have important differences based on the diastereomeric systems generated, as some studies have shown (Hu et al., 2021). FRAP studies were conducted to evaluate the diffusivity of the OVA payload in each hydrogel in vitro (FIG. 17). In FRAP, slowed fluorescence recovery indicates the reduced mobility of a payload within the gel — sometimes due to an increased affinity between the payload and the hydrogel matrix — which implies slower release. It is known that protein release rates from peptide hydrogels can differ due to the properties of the protein, peptide primary sequence, and charge (Moore & Hartgerink, 2017). It was thought, without being bound by theory, that due to the similarities in structure, mechanical properties, and hydrogel charge, there would be minimal differences between the in vitro diffusion of OVA in D-K2 (SEQ ID NO: 12) and K2 (SEQ ID NO: 1). Ten minutes after photobleaching, the fluorescence at each photobleached location had increased due to the diffusion of unbleached, fluorophore-labeled OVA into the region. Fluorescent OVA infiltration into K2 (SEQ ID NO: 1) after photobleaching resulted in a total recovery of 27 + 1% whereas infiltration into D-K2 (SEQ ID NO: 12) resulted in a total recovery of 25 + 1%, as seen in Table 2. This difference in final percent recovery was not statistically significant, nor were the differences in r or ti / 2 (Table 2). The lack of significant differences between the two recovery profiles suggests that chirality does not affect the rate of OVA diffusion in these hydrogels in vitro.Table 2: FRAP-fitted diffusion constantsA is total percent recovery, T is the recovery time constant, and t\n is the half-recovery time

[0137] In vivo imaging studies were performed using fluorescently labeled OVA to measure antigen release over time and fluorescently labeled K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) to track hydrogel degradation over time. Hydrogel degradation was tracked over the course of 86 days. D-K2 (SEQ ID NO: 12) was observed to degrade at a significantly slower rate (p< 0.0001) than K2 (SEQ ID NO: 1) (FIG. 18A, FIG. 18B). IVIS imaging was performed until the fluorescence of the K2 (SEQ ID NO: 1) hydrogel was barely discernable from the background. Histological evaluation of tissue at the injection site was performed 13 weeks after administration using hematoxylin and eosin (H&E) stain and Masson’s trichrome (trichrome) staining (FIG. 18C). In agreement with 1V1S imaging, tissue sectioning revealed that the K2 (SEQ ID NO: 1) hydrogels had been largely cleared from the injection sites and were not observable by histology. D-K2 (SEQ ID NO: 12) hydrogels remained present and showed cellular infiltration throughout the volume of the gel. The trichrome-stained D-K2 (SEQ ID NO: 12) samples showed a collagenous fibrous capsule (blue) at the gel border as well as cellular accumulation at the hydrogel-tissue interface. Previous histological evaluation of K2 (SEQ ID NO: 1) did not reveal the formation of a fibrous capsule 6 weeks after injection, indicating that the fibrous capsule around the D-K2 (SEQ ID NO: 12) sample may be a chirality-specific biological response (Moore et al., 2018). However, D-K2 (SEQ ID NO: 12)’s very slow clearance from the injection site could result in potential negative effects, such as those observed at the injection sites of diabetic patients, where insulin can aggregate into amyloid fibrils, forming a hard subcutaneous mass that triggers an undesirable immune response (Dische et al., 1988; Shikama et al., 2010; Gupta et al., 2015; Kerr et al., 2013).

[0138] The in vivo release of OVA from K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) hydrogels occurred at rates that were not statistically significantly different, reaching100% release over the course of 11 days, demonstrating that peptide chirality does not affect OVA release, even in a biological system where differences might be expected (FIG. 19A). Interestingly, the duration of OVA release from both hydrogels was similar to the duration that has been reported for OVA desorption from alum — a traditional adjuvant (Pogostin & McHugh, 2021; Pogostin et al., 2022). Both gels extend the release of OVA dramatically compared to unadjuvanted OVA, which is cleared from the injection site within a few hours of injection (Pogostin et al., 2022). These results are consistent with our in vitro FRAP results that show similarly slow fluorescent recovery in both hydrogels. On these short time scales relative to the in vivo lifetimes of the hydrogels, it appears, without being bound by theory, that diffusion is dictating the rate of OVA release since it occurs much more rapidly than hydrogel degradation.

[0139] Finally, the potential adjuvancy of the K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) peptide hydrogel enantiomers was investigated by longitudinally quantifying the anti-OVA antibody titers in mice administered OVA adjuvanted with these two materials. Over a period of 12 weeks, blood samples were collected every two weeks from mice postvaccination, and OVA-specific IgG antibodies were quantified by ELISA (FIG. 19B). Mice receiving OVA encapsulated in either K2 (SEQ ID NO: 1) or D-K2 (SEQ ID NO: 12) exhibited rapid increases in antibody titers, reaching 13 ± 1 and 14 ± 2 on a log2 scale, respectively, two weeks after vaccination — levels that were not statistically significantly different — and the titers did not meaningfully change between 2 and 12 weeks after injection (FIG. 19C). This result suggests that humoral immunity is relatively unaffected by the chirality of the MDP adjuvant used. A previous study reported that OVA encapsulated in an alum-adjuvanted hydrogel elicited IgG antibody titers of 21.03 on a log2 scale, a greater increase in humoral immunity than is evoked by K2 (SEQ ID NO: 1) or D-K2 (SEQ ID NO: 12) hydrogels (Pogostin et al., 2022). It is important to note that IgG titers from mice receiving either peptide hydrogel with OVA are higher than the level typically achieved when administering OVA in the same dose and route, which are typically below the limit of detection for this assay (2.3 log2 titers), demonstrating that both peptides are serving as strong adjuvants (Pogostin et al., 2022).Example 8: Boronic Acid Groups

[0140] The present invention is not limited to comprising a particular boronic acid functional group. In some embodiments, the boronic acid group is 4-carboxyphcnylboronic acid (PBA). In other embodiments, compositions comprising a boronic acid group selected from among 4-Carboxy-3-fluorobenzeneboronic acid (Fluoro-PBA), 4-(Carboxymethyl)-3- fluorobenzeneboronic acid (mFluoro-PBA), 2-Fluoro-5-nitrophenylboronic acid (Nitro-Fluoro-PBA), 2- Aniinopyrimidine-5-boronic acid (Aniino-pBA), 3-Carboxy-5-nitrophenylboronic acid (Nitro-PBA), 4- Borono-2-nitrobenzoic acid (2-Nitro-PBA), 4-Borono-2,6-difluorobenzoic acid (Difluoro-PBA), or 1- Hydroxy-l,3-dihydrobenzo[c][l,2]oxaborole-6-carboxylic acid (BOB) are contemplated. A composition comprising a boronic acid group may have the advantage of improved drug release kinetics. A composition comprising any of the above-listed groups, or any other boronic acid group, may have a binding equilibrium with a boronic acid binding motif that is more in favor of the bonded state, thereby (without being bound by theory) prolonging release of an agent comprising that boronic acid binding motif. In some embodiments, the boronic acid group has a benefit of improved resistance to degradation, such as oxidation, either in the unbound state or when bound to a boronic acid binding motif.Table 3: Binding Equilibria of Boronic Acid GroupsExample 9: Materials and MethodsChemical Synthesis

[0141] All chemicals, unless otherwise specified, were purchased from Fisher Scientific (Pittsburgh, PA) or MilliporeSigma (Burlington, MA). Flash chromatography was performed on silica gel (SiliaFlash P60).Synthesis of 4-nitrodopamine

[0142] Dopamine (500 mg, 2.6 mmol) and sodium nitrate (630 mg, 9.1 mmol) were dissolved in 15 mL of water and cooled in an ice bath. Once cold, the solution was stirred vigorously and 2.5 mL of 20% sulfuric acid was added dropwise, which resulted in a yellow precipitate that was collected by vacuum filtration. The product was washed 3X with cold water and 3X with cold methanol and dried under vacuum with a 50% yield. 1HNMR (600HZ, DMF- d7) 8 = 3.35 (m, 4H), 7.27 (s, 1H), 7.53 (s, 1H). ESI-MS expected for C8H11N2O4 [M+H] 199.2, observed [M+H]+199.1.Synthesis of azido-salicylhydroxamate-OTlu 2

[0143] Azido-salicylic acid 1 was synthesized as previously reported (Pieszka et al., 2020). Solid 4- azido salicylic acid 1 (2.5 g, 14 mmol) and Hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (5.3 g, 14 mmol) were dissolved in dry DMF (50 mL) under nitrogen. D1EA (6.3 mL, 36 mmol) was added at 0 °C and the solution was stirred at rt for 10 min. Solid O-(tert-Butyl)hydroxylamine hydrochloride (1.96 g, 15 mmol) was added at 0 °C and the solution was stirred at rt for 64 h. The solution was diluted with EtOAc (200 mL) and washed with sat. KHSO4 (3 x 120 mL), 1:1 H O / brinc (3 x 120 mL), and brine (200 mL). The organic layer was dried over MgSCU, filtered, and solvent was removed under vacuum. The residue was purified by flash column (25% EtOAc / Hexane) to afford 2 as a white powder (1.29 g, 37%). 1H NMR (600 MHz, DMSO-d6): 8 7.76 (d, J = 8.50 Hz, 1H), 6.67 (m, 1H), 6.62 (s, 1H), 1.24 (s, 9H) 13C NMR (151 MHz, DMSO-d6): 8 167.6, 160.7, 144.8, 132.6, 130.3, 110.4, 107.4, 82.1, 26.8 ESI-MS expected for Cl 1H14N4O3 [M+H]+: 251.3, found 251.1.Synthesis of COOH-SHA- Q'Bu 3

[0144] Solid azido-salicylhydroxamate- O[Bu 2 (880 mg, 3.5 mmol) and 4- pcntynoic acid (380 mg, 3.9 mmol) were dissolved in THF (4.4 mL). Sodium ascorbate (850 mg, 4.2 mmol) in H2O (2.2 ml) was added followed by copper(II) sulfate pentahydrate (270 mg, 1.1 mmol) in H2O (2.2 mL). The solution was stirred vigorously at room temperature for 2 h. Solvent was removed under vacuum and the residue was suspended in MeOH, filtered, and the supernatant was concentrated under reduced pressure and then purified by flash column (20% MeOH / CH CL) to afford 3 as an off-white powder (1.08 g, 88%). 1H NMR (600 MHz, DMSO- d6): 8 8.66 (s, 1H), 7.89 (d, J = 8.56 Hz, 1H), 7.47 (s, 1H), 7.42 (d, J = 8.53 Hz, 1H), 2.93 (t, J = 7.47 Hz, 2H), 2.67 (t, J = 7.51 Hz, 2H), 1.26 (s, 9H) 13C NMR (151 MHz, DMSO-d6): 5 174, 167.0, 160.1, 147.7, 140.3, 130.4, 120.8, 115.6, 110.2, 107.9, 82.2, 33.2, 26.9, 21.1 ESLMS expected for C16H20N4O5 [M+H]+: 349.4, found 349.1.Synthesis of 3-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]- 5-yl)thioureido)phenylboronic acid (FITC-PBA)

[0145] Solid fluorescein isothiocyanate (FITC) (100 mg, 0.257 mmol) and 3- aminophenylboronic acid (Ambeed, Arlington Heights, IL) (52.8 m, 0.386 mmol) were dissolved in 0.5 mL in DMF. Diisopropylethylamine (DIEA) (44.8 pL, 0.257 mmol) was added to the reaction under agitation. The reaction was allowed to proceed overnight protected from light. Excess methanol (10 mL) was added to the reaction and the solvent was removed in vacuo. The product was redissolved in a minimal volume of methanol and the product was precipitated into chloroform. The solid was isolated by vacuum filtration and dried under vacuum for a final yield of 38%. ESLMS expected for C27HI8BN2O7S- [M-H]' 525.3, observed [M-H]' 525.21.Synthesis of (4-((4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7,8-dihydro-9H-purin- 9-yl)methyl)benzamido)methyl)phenyl)boronic acid (1V209-PBA)

[0146] Solid 4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7,8-dihydro-9H-purin-9- yl)methyl)benzoic acid (1V209) (Ambeed) (50 mg, 0.139 mmol) and HATU (P3 biosystems, Louisville, KY) (55.6 mg, 0.146 mmol) were dissolved in a minimal volume of DMF and stirred. To the solution, 144.5 uL (0.835 mmol) of DIEA was added and the reaction was allowed to stir for two min. Afterward, 52.2 mg (0.278 mmol) of 4-aminomethylphenylboronic acid (Ambeed) was added and the reaction was allowed to proceed at ambient temperature for 2 h. The reactionmixture was then purified by high-performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) on an XBridgc BEH C18 column (Waters Corp., Milford, MA) using a solvent system of water and acetonitrile with 0.05% TFA. The purified product was frozen and lyophilized for a final yield of 27%. 1H NMR (600 MHz, DMSO-d6): 5: 10.0 (s, 1H), 7.85 (d, J=7.8 Hz, 2H), 7.73 (d, J=7.8 Hz, 2H), 7.37 (d, J=8.4 Hz, 2H), 7.25 (d, J=7.8 Hz, 2H), 6.5 (s, 2H), 4.92 (s, 2H), 4.5 (d, J=6 Hz, 2H), 4.25 (t, J=4.38 Hz, 2H), 3.58 (t, J=4.86 Hz, 2H), 3.26 (s, 3H). ESI-MS expected for C23H26BN6O + [M+H]+ 493.2, observed [M+H]+ 493.2.Solid-Phase Peptide Synthesis

[0147] Peptides were synthesized manually or on an AAPPTec Focus XC autosynthesizer (AAPPTec, Louisville, KY) using standard fluorenylmethyloxycarbonyl (fmoc)- based chemistry. Fmoc-protected low-loading MBHA rink-amide and fmoc-protected amino acids were procured from Novabiochem (MilliporeSigma). Fmoc deprotection was accomplished with two steps of excess 25% v / v piperidine in DMF:DMSO 1:1 for 5 min each. Successful deprotection was verified using the ninhydrin test for primary amines. In some embodiments, each coupling step began with the pre-activation of the amino acid (4 equiv.) with hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (4 equiv.) and N,N-diisopropylethylamine (DIEA) (6 equiv.) in 10-12 mL of 1:1 DMF:DMSO for at least 1 min. The preactivated mixture was then added to the reaction vessel for 30-45 min for each coupling. After each coupling, another ninhydrin test was performed to confirm the absence of primary amines. If primary amines were found, a second coupling was performed. This process was repeated for both peptides until synthesis was completed. In other embodiments, coupling was achieved with the addition of preactivated Fmoc-protected amino acids (4 equiv.), HATU (3.99 eq.) (P3 biosystems) and DIEA (6 eq.) in a minimal amount of dimethylformamide (DMF): dimethyl sulfoxide (DMSO) 1:1 for 20-45 min at RT. The process was repeated until the peptide was complete. In the case of the K2 (SEQ ID NO: 1), D-K2 (SEQ ID NO: 12) and E2 (SEQ ID NO: 2) peptide N-terminal acetylation was performed with the addition of acetic anhydride (200 eq.) and DIEA (75 eq.) in dichloromethane (DCM) two times for 45 mins. For all other SABER peptides, the N-terminus was modified with a boronic acid binding motif instead of being acetylated. Cat-K2 and SHA-K2 / SHA-E2 were generated by coupling 4 equiv. of 3-(2,2- Dimethylbenzo[d][l,3]dioxol-5-yl)propanoic acid or 2 equiv. of COOH-SHA-OTlu, respectively, using the same coupling protocol described above. NitroCat-K2was synthesized by reacting the N-terminus of the peptide with 2 equiv. of bis(2,5- dioxopyrrolidin-l-yl) succinate (Ambccd) and 4 equiv. of DIEA for 30 min in DMF / DMSO. Successful coupling was verified by using the ninhydrin test for primary amines. The resin was then washed 3X with DCM and DMF and then reacted with 4 equiv. of nitrodopamine and 10 equiv. of DIEA in DMF / DMSO. The reaction was allowed to proceed overnight. To create fluorophore-labeled peptides, 15 mg of resin K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) was deprotected twice as described above and then dyed using a cocktail of 1 mg of Atto 647N N-hydroxy- succinimide (NHS) ester (Millipore Sigma, Burlington, MA), 1 uL of DIEA, and 1 mL of DMF overnight. The next day, both peptides were deprotected again to make K2 (SEQ ID NO: 1)-Atto 647N and D-K2 (SEQ ID NO: 12)-Atto 647N.

[0148] Peptides were cleaved from the resin by reaction with a cleavage cocktail composed of trifluoro acetic acid (TFA):Anisole:triisopropylsilane:ethylene-l,2-dithiol:H2O 90:2.5:2.5:2.5:2.5 for 3 h at RT. The t-butyl protecting group on hydroxamic acid of SHA-K2 and SHA-E2 was removed by heating the cleavage cocktail to 50 °C for an additional hour. TFA was then evaporated with a stream of nitrogen to a minimal volume ( ~ 1 mL) and peptide was precipitated with ice-cold diethyl ether.

[0149] Peptides for some embodiments of the present disclosure were purified by high performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) on an XBridge BEH OBD C4 column (Waters Corp.). Positively charged peptides were purified over a gradient of 5- 50% acetonitrile with 0.05% TFA in water with 0.05% TFA. Negatively charged peptides were purified over a gradient of 5-35% acetonitrile with an ammonium acetate buffer (5 mM ammonium and 4 mM acetic acid) in water with the same buffer with a pH of 8.5. The purity of all peptides was verified by ultraperformance liquid chromatography (UPLC) (Waters Corp.) and matrix-assisted laser desorption-ionization mass spectrometry (MALDLMS) (Bruker Daltonics, Billerica, MA).

[0150] Alternatively, in other embodiments, crude peptides were purified using high- performance liquid chromatography (HPLC) on an XBridge Protein BEH C4 OBD column (Waters Corporation, Milford, MA) with a solvent system of 0.05% TFA in MQ water (Solvent A) and 0.05% TFA in acetonitrile (Solvent B). Peptides were dissolved at a concentration of 10 mg / mL in a 20% (v / v) solution of acetonitrile in MQ water and injected on the column at a volume of 1 mL. A gradient of3% / min from 5-35% Solvent B was used to purify the solutions and the absorbance was monitored at 220 nm with a TUV detector. A Bruker (Billerica, MA) Autoflex matrix-assisted laser desorption / ionization mass spectrometer (MALDI MS) and ultra-performance liquid chromatography (UPLC) were used to confirm peptide identity and purity (FIG. 20).IgG PBA Labeling

[0151] Rabbit IgG (MilliporeSigma) was dissolved at 10 mg / mL (6.66 x 10‘5M) in pH 8.2 sodium bicarbonate buffer (0.2 M) and (4-(((2,5-Dioxopyrrolidin-l- yl)oxy)carbonyl)phenyl)boronic acid (Ambeed) was dissolved at 0.05 M in DMSO. To make the high degree of PBA-labeled IgG, 20 equivalents of (4-(((2,5-Dioxopyrrolidin-l- yl)oxy)carbonyl)phenyl)boronic acid was then added to the dissolved IgG while 5 equivalents was added for the low degree of labeling. The reaction was stirred at ambient temperature overnight. The product was then purified using a 0.5 mL size exclusion Spin-X centrifugal filter with a 50 kDa cutoff (Corning, Coming, NY). The degree of labeling was determined by quantifying the amount of unreacted (4-(((2,5-Dioxopyrrolidin-l- yl)oxy)carbonyl)phenyl)boronic acid that eluted out in the filtrate by UV-Vis. This characterization method was validated by an alizarin red S assay for the detection of BAs. These reactions generated IgG material with an average of 11.4 and 2.4 PBAs per IgG molecule, corresponding to an approximately a 50% PBA labeling efficiency. The IgG was then subsequently labeled with the NHS ester-functionalized AZDye 647 fluorophore (Vector Laboratories, Newark, NJ) using the protocol published by the supplier to achieve a degree of labeling of 0.2 fluorophores per IgG. The product was then purified by size exclusion using a 15 mL 50 kDa Amicon centrifugal filter (MilliporeSigma) against water and stored as a lyophilized powder.Synthesis of insulin-PBA

[0152] Insulin labeled with a single phenylboronic acid on the B29 Lys residue (insulin-PBA) was prepared following previously published protocols (Hoeg- Jensen et al., 2005; Chou et al., 2015). In brief, 5.6 mg of (4-(((2,5-Dioxopyrrolidin-l- yl)oxy)carbonyl)phenyl)boronic acid (Ambeed) was dissolved in 1 mL of acetonitrile (21.12 mM; 1.2 equiv.) and 100 mg of recombinant human insulin (Thermo Fisher Scientific, Waltham,MA) was dissolved in 1 mL of 0.1 M sodium carbonate buffer set to pH 10.2 (17.6 mM; 1 cquiv.). After complete dissolution of the insulin, the pH was adjusted back to pH 10.2 with NaOH. The two solutions were then mixed 1:1 and stirred for 1 h at ambient temperature. The reaction was then quenched through the addition of 88 p.L of 0.2 M methylamine (MilliporeSigma). The pH of the reaction solution was then adjusted to pH 5.5 with 6 M HO at which point the product crashed out of solution as a white precipitate. The solution was then cooled at 4 °C for 30 min and the precipitate was isolated by centrifugation at 10,000 ref for 2 min. The product was then dissolved in 4 mL of DMSO with 0.1% TFA and purified by HPLC on an XSelect OBD BEH C18 column (Waters Corp.) using a solvent system of water and acetonitrile both with 0.5% TFA. The purity and identity of the product was assessed by UPLC and MALDI-MS using sinapic acid (MilliporeSigma) as the matrix. Expected [M+H]+: 5957.9; observed [M+H-B(0H)2]+: 5914.9. The loss of 43 Da is consistent with in-source fragmentation of the boronic acid. The purified product was stored as a lyophilized solid until use.UV-Vis Oxidation Study

[0153] Oxidation of dopamine, 4-nitrodopamine, and SHA were analyzed by qualitative changes in absorbance spectra from 200 to 800 nm with a Cary 60 UV Vis Spectrophotometer (Agilent Technologies, Santa Clara, CA) using a quartz cuvette. Compounds of interest were prepared in 1 X PBS at concentrations such that the maximum absorbance of the sample would not exceed 1 absorbance unit. Samples were stored at ambient temperature in the dark at 25 °C for the duration of the experiment. Spectra were collected at 0, 1, 4, 6, 11, and 15 d after preparation to monitor the changes in the samples over time.Boronate Ester Association Constant Determination

[0154] The determination of boronate ester equilibrium by competitive binding assay were determined by adapting previously described protocols (Brooks et al., 2018; Springsteen & Wang, 2002). To determine the BACSM-alizarin red S (ARS) binding constants (KARS) for aryl BACSMs, stock solutions of ARS and BACSM were prepared in IX PBS and corrected to pH 7.4 as needed. Titrations of decreasing BACSM concentration (starting at 2 mM) in constant ARS concentration (9 pM) were prepared in triplicate. Fluorescence was measured (468 / 572 nm Ex / Em) with a Tecan Infinite M200 Pro Plate Reader (Zurich, Switzerland) usingblack 96-well plates. The ARS association constant (KARS) was determined using equations previously described (Brooks et al., 2018). KARS was then used to determine the boronatc ester equilibrium constant between the BACSM and dopamine (DOPA) and SHA by preparing serial dilutions in triplicate of these two compounds (starting at 5 mM) with constant concentrations of ARS (9 pM) and BACSM (2 mM). The fluorescence (468 / 572 nm Ex / Em) of these dilutions was measured using a microplate reader and KDOPA and KSHA were calculated as previously reported (Brooks et al., 2018).

[0155] The absorbance spectrum of 4-nitrodopamine (nDOPA) overlaps with the excitation wavelength of ARS; however, its / .max is sensitive to boronate ester formation (observed as a blue-shift from 420 nm). Thus, to determine the BACSM-nDOPA binding constants (KnDOPA), serial dilutions of each BACSM (starting at 2 mM) were prepared in triplicate with a constant concentration of nDOPA (0.1 mM) in pH 7.4 IX PBS. The absorbance at 420 nm was measured by microplate reader using a quartz 96 well plate. KnDOPA was determined with the same equations used to calculate KARS, where 1-absorbance was substituted for fluorescence intensity. Since ARS fluorescence changes only occur with aryl BAs, KnDOPA was used to determine binding constants between Ixazomib (1XB) and DOPA (KDOPA) or SHA (KSHA). In brief, serial dilutions of DOPA or SHA (starting at 2 mM) were prepared in triplicate with constant IXB (2 mM) and nDOPA (0.1 mM). The absorbance at 420 nm of these solutions was measured and used to calculate KDOPA or KSHA using the previously calculated KnDOPA for IXB with 1-absorbance substituted for fluorescence intensity in the original equation previously published (Brooks et al., 2018).Hydrogel Preparation

[0156] For Examples 1-7, MDP hydrogels were prepared by dissolving the MDP at 2X the final desired peptide concentration (20 mg / mL or 40 mg / mL) in MilliQ water. The desired BACSM or boronic acid-labeled biologic was dissolved at twice the final concentration in 2X Hank’s balanced salt solution (HBSS). The pH of the 2X HBSS solution was adjusted to pH 7.5-8.5 to assist in the dissolution of BACSMs. These two stock solutions were then mixed in a 1:1 ratio to yield a hydrogel with IX HBSS, and the final IX peptide and payloadconcentrations (10 mg / mL or 20 mg / mL). The pH of the gel was adjusted to pH 7-8 as necessary with microliter additions of NaOH or HC1 and the gel was vortexed to ensure homogeneity.

[0157] For Example 8, K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) were prepared the day before experiments by combining equal amounts of a 20 mg / mL peptide in MQ water solution with 2X Hank’s Balanced Salt Solution (HBSS) and titrated to pH 7.2-7.4 through the addition of 0.1 M NaOH and HC1. The resulting solution’s pH was confirmed using a Seven Excellence S400 pH meter (Mettler Toledo, Columbus, OH). For fluorescence recovery after photobleaching (FRAP) experiments, hydrogels were prepared similarly by combining a 20 mg / mL peptide solution in MQ water with a 0.4 mg / mL OVA-fluorescein solution in 2X HBSS and then pH-corrcctcd. FRAP hydrogels were stored in the dark to allow for gelation overnight without light interference.Fourier Transform Infrared Spectroscopy (FTIR)

[0158] Attenuated total reflectance FTIR was performed using a NicoletTM iS20 FTIR spectrometer (Thermo Fisher Scientific). Before use, a stream of nitrogen was used to purge the instrument to avoid signals from atmospheric molecular vibrations. Hydrogel samples at a peptide concentration of 10 mg / mL were plated (10 LIL) and allowed to dry until all water had evaporated. Absorbance spectra were collected as an accumulation of 30 measurements. The amide I FTIR peak was visualized by background subtraction and plotting the area normalized absorbance from 1575-1705 cm-1.Circular Dichroism ( CD) Spectroscopy

[0159] Circular dichroism spectra were obtained on a Jasco J-810 spectropolarimeter (Easton, MD). Hydrogel samples (10 pL) with or without drug were loaded into a 0.1 mm quartz cuvette and analyzed at ambient temperature. All spectra were obtained as an average of 5 accumulations between 190 and 250 nm. Millidegrees (mdeg) of rotation were converted to mean residual ellipticity (MRE) for ease of comparison using the equation [9] = 0 x 106l(c x I x nr), wherein c is concentration (pmol / L), / is cuvette pathlength (mm), nr is the number of amino acid residues, and 9 is the ellipticity reading in mdeg.Rheology

[0160] Oscillatory rheology was performed on an AR-G2 rheometer (TA Instruments, New Castle, DE). Rheology measurements were collected from 75 pL hydrogel samples using a stainless steel 12 mm parallel plate geometry. In some embodiments, hydrogel sample was pipetted onto the center of the rheometer stage and mineral oil was applied to the sample to prevent evaporation. After plating, samples were allowed to equilibrate at 1 rad / s and 1% strain for 30 min. After equilibration, a frequency sweep was performed over the range of 0.1-10 rad / s at a constant 1 % strain. Subsequently, shear recovery experiments were conducted by equilibrating the hydrogels at 1 rad / s and 1 % strain for 2 min and then sheared for 1 min at 200% strain. The recovery of G’ and G” was subsequently monitored for 10 min at the starting conditions. Percent recovery was quantified by comparing the G’ values after 10 min equilibration with the G ’ values after 2 min of initial equilibration.Cryogenic Transmission Electron Microscopy (cryo-TEM)

[0161] For Examples 1-6, hydrogels were prepared as previously described at 10 mg / mL in IX HBSS buffer. Before plunging, samples were diluted to 1 mg / mL in fresh MQ water. Samples (5 pL) were placed on 200 mesh lacey carbon grids and then were plunged into liquid ethane with a Leica EM GP automatic plunge freezer (Leica Microsystems, Wetzlar, Germany). The plunging chamber was set to a temperature of 20 °C and a humidity of 90%. Samples were analyzed at the National Center for High-Resolution Electron Microscopy at Lund University, Sweden, on a JEM-2200FS (JOEL, Tokyo, Japan) transmission electron microscopy instrument equipped an F416.0 camera (TVIPS, Gauting, Germany) using an accelerator voltage of 200 kV. Serial EM in a low-dose mode were used to acquire zero-loss images.

[0162] For Example, 8, lacey carbon grids were glow discharged twice for 2 min with 30 mA discharges. All hydrogels were prepared according to the above description, and samples were diluted 1: 10 in lx HBSS. Diluted sample (3 pL) was pipetted onto the grid, blotted, and plunged into liquid ethane using a Vitrobot instrument. Grids were then transferred to liquid nitrogen for storage and imaged within 1 week of blotting. Imaging was performed using an FEI Tecnai F20 transmission electron microscope (FEI Company, Hillsboro, OR).In Vitro Release AssaysPlate Reader Release Assay

[0163] Hydrogels at a final peptide concentration of 10 mg / mL were loaded with 1.5 mM fluorescein or FITC-PBA were prepared as previously described and allowed to equilibrate overnight at ambient temperature in the dark. To start the experiment, 50 pL of each hydrogel was plated in triplicate on to a custom 3D-printed 48-well plate designed previously for plate reader release assays, 17 and the gels were allowed to recover for 10 min. The gels were then covered gently with 600 pL of IX phosphate buffer (PBS), sealed with a transparent plate sticker to prevent evaporation, and loaded into a microplate reader (Tecan, Zurich, Switzerland). The microplate reader was set to heat the plate to 37 °C while orbitally shaking it at 142 rpm, and it was set to read the fluorescence (490 / 525 nm Ex / Em) of the release supernatant every 45 min. The percentage of payload released was calculated by dividing the fluorescence intensity at each time point to the total fluorescence of a solution of 0.115 mM of fluorescein or FITC-PBA, representing the concentration of cargo at 100% release.BACSM UPLC Release Assay

[0164] All BACSMs were purchased from Ambeed except GFB, which was purchased from MedChemExpress (Monmouth Junction, NJ). Hydrogels at a final peptide concentration of 10 mg / mL were loaded with 500 pg / mL BACSM, were prepared as previously described earlier in the methods and equilibrated in the dark at ambient temperature overnight. Tavaborole (250 pg / mL), 1V209 (50 pg / mL), and 1V209-PBA (50 pg / mL) gels were prepared with lower concentrations due to solubility limitations in the hydrogels. Additionally, hydrogels with 1V209 and 1V209-PBA were prepared with 25% DMSO to improve the solubility of these hydrophobic small molecules. Gels (50 pL) were then plated in triplicate on a custom 3D-printed 48-well plate as described in previous publications. The gels were then allowed to recover for 10 min before 450 pL of IX PBS was gently added to each well to start the experiment. The plate was then covered with a 96-well plate cover sticker to prevent evaporation and orbitally agitated at 100 rpm at 37 °C. At the desired sampling time points, 400 pL of the release media was removed and refreshed. The amount of BACSM in the removed release medium was then assessed by UPLC using a Poroshell Cl 8 (Agilent Technologies, Santa Clara, CA) UPLC column and adjusting for the volume remaining in the well after each sampling (Table 4). Thepercentage of released BACSM was determined by dividing the cumulative mass of drug released by the total loaded in the hydrogel.Table 4: UPLC methods for quantification of in vitro drug releaseInsulin Release Assay

[0165] Unmodified insulin and insulin-PBA were loaded at 500 g / mL in hydrogels containing 10 mg / mL peptide as described above using IX PBS instead of HBSS to avoid the addition of glucose. Each hydrogel (50 pL) was then pipetted into low protein bind Eppendorf tubes (Hamburg, Germany) in triplicate and centrifuged to settle the gel in the bottom of the tube. After the gels equilibrated for 10 min, 450 pL of IX PBS 0, 100, and 250 mg / dL of glucose was gently pipetted on top of each gel and incubated at 37 °C. At the desired sampling time point, 200 uL of release media was removed and replaced, and the concentration of insulin and insulin-PBA in the release medium was quantified by UPLC using a BEH C14 column (Waters Corp.) accounting for the volume remaining in the tube after each sampling (Table 4). The percentage of released Insulin and insulin-PBA was determined by dividing the cumulative mass of the protein released by the total loaded in the hydrogel.Pharmacokinetic AssaysBortezomib

[0166] All animal work was performed in compliance with an lACUC-approved protocol. Hydrogels loaded with 700 ng of BTZ were prepared as previously described at a finalpeptide concentration of 10 mg / mL. Female BALB / c mice (16-18 g) were injected subcutaneously with 50 pL of drug-loaded hydrogel or BTZ dissolved in HBSS. Blood (10 pL) was collected using an untreated Safe-T-Fill™ plastic hematocrit capillaries (RAM Scientific, Austell, GA) and spotted on a Whatman 903 Proteinsaver Card (Cytiva, Marlborough, MA). Blood cards were dried overnight protected from light and then stored with desiccant at 4 °C for up to 1 week or -80 °C for up to one month. Extractions were performed following a previously published protocol for BTZ (Harrold & Abraham, 2014). The compound was extracted off the blood card by using a 1 / 8-inch hole puncher to remove the center of each blood spot, corresponding to 2.4 pL of blood. The blood spots were then submerged in 40 pL of methanol containing 1 ng / mL apatinib (APExBIO, Houston, TX) as the internal standard. Extractions proceeded for 1 h at ambient temperature on an orbital shaker set to 100 rpm. The extraction solution was then removed and diluted 1:2 with water and analyzed by LC-MS.

[0167] Multiple Reaction Monitoring (MRM) LC-MS analysis of BTZ was carried out on an Agilent 6470B Triple Quadrupole (QqQ) mass spectrometer using apatinib as an internal standard. The MS system was interfaced to an Agilent 1290 Infinity ii LC system through an Agilent Jet Spray (AJS) electrospray ionization (ESI) source that was operated in the positive mode. Separations were carried out using a Water’s ACUITY Premier HHS T3 100 mm x 2.1 mm ID, 1.8 um column that was operated at 0.4 mL / min. Mobile phase A (MPA) was 0.1% formic acid in water, and mobile phase B (MPB) was 0.1 % formic acid in acetonitrile. Initial LC conditions were 20%B up to 80%B over 4.0 min. The column was flushed at 80%B for 2.0 min and then re-equilibrated at 20%B for 2 min prior to the next injection.

[0168] The AJS Source and MS data acquisition settings were optimized to meet the needed sensitivity requirements. Briefly, the AJS source conditions were as follows, Gas Temp: 320 °C, Gas Flow: 8 L / min, Nebulizer Gas Pressure: 25 psi, Sheath Gas Temp: 400 °C, Sheath Gas Flow: 11 L / min, Capillary Voltage: 3800 V. QqQ MRM data acquisitions settings were as follows, Cell Accelerator Voltage: 5 V, Dwell Time: 50 ms. The MRM ion transitions for bortezomib and apatinib are shown in Table 5.Table 5: Bortezomib and apatinib MRM ion transitions used to quantify blood concentrations by QqQ LC-MS.Compound Precursor Product Fragmentor CollisionName Ion (Da) Ion (Da) Voltage (V) Energy (eV)0 eApatinib 398.2 92 152 46 QualifierApatinib 398.2 80.1 152 62 QualifierApatinib 398.2 212 152 30 PrimaryBrotezomib 367.2 226 136 19 PrimaryBrotezomib 367.2 208 136 31 QualifierBrotezomib 367.2 79.1 136 54 QualifierGanfeborole

[0169] Pharmacokinetic analysis of GFB was performed nearly identically to the procedure described above for bortezomib with minor alterations. Hydrogels loaded with 60 or 600 pg GFB were prepared as previously described at a final peptide concentration of 10 mg / mL or 20 mg / mL depending on the experiment. Female BALB / c mice (16-18 g) were injected subcutaneously with the drug loaded hydrogel or GFB dissolved in PBS. Blood (10 pL) was collected using an untreated plastic capillary and spotted on a Whatman 903 Proteinsaver Card. Blood cards were dried overnight protected from light and then stored with desiccant at 4 °C for up to 1 week or -80 °C for up to one month. The compound was extracted off the blood card by using a 1 / 8-inch hole puncher to remove the center of each blood spot, corresponding to 2.4 pL of blood. The punches were then submerged in 40 pL of 90:10 methanol: water containing 5 ng / mL apatinib as an internal standard. The submerged blood spots were extracted at 37 °C for 1 h while being shaken at 100 rpm. The extraction solution was then removed and diluted 1:2 with water and analyzed by LC-MS. MRM LC-MS analysis of GFB was carried out on an Agilent 6470B QqQ mass spectrometer using apatinib as an internal standard. The MS system was interfaced to an Agilent 1290 Infinity ii LC system through an AJS ESI source that was operated in the positive mode. Separations were carried out using a Water’s ACUITY Premier HHS T3 100 mm x 2.1 mm ID, 1.8 um column that was operated at 0.4 mL / min. MPA was 0.1% formic acid in water, and MPB was 0.1% formic acid in acetonitrile. Initial LC conditions were 10%B up to 60%B over 5.0 min from 5.0 min to 5.5 min the gradient was increased to 80%B. The column was flushed at 80%B for 2.5 min and then re-equilibrated at 10%B for 3 min prior to the next injection. The AJS Source and MS data acquisition settings were optimized to meet theneeded sensitivity requirements. Briefly, the AJS source conditions were as follows, Gas Temp: 320 °C, Gas Flow: 5 L / min, Nebulizer Gas Pressure: 30 psi, Sheath Gas Temp: 400 °C, Sheath Gas Flow: 12 L / min, Capillary Voltage: 3800 V. QqQ MRM data acquisitions settings were as follows, Cell Accelerator Voltage: 5 V, Dwell Time: 100 ms, MSI (QI) resolution: Unit, MS2 (Q3) resolution: Wide. The MRM ion transitions for GFB and apatinib are shown in Table 6. The data collected was corrected for dilutions during sample preparation and plotted in GraphPad Prism 10. Pharmacokinetic parameters were determined by noncompartmental analysis using Ubiquity in RStudio (Harrold & Abraham, 2014).Table 6: Ganfeborole and apatinib MRM ion transitions used to quantify blood concentrations by QqQ LC-MSCompound Precursor Product Fragmentor Collision . .Name Ion (Da) Ion (Da) Voltage (V) Energy (eV)NoteApatinib 398.2 92 152 46 QualifierApatinib 398.2 212 152 30 PrimaryGanfeborole 258.1 222 106 13 PrimaryGanfeborole 258.1 187 106 25 QualifierMass Spectrometry Imaging

[0170] Female BALB / c mice (16-18 g) were subcutaneously injected with 700 ng of BTZ loaded in 50 pL of IX HBSS, K2 (SEQ ID NO: 1), nitroCat-K2, or SHA-K2. Hydrogels were prepared as previously described. Mice were then sacrificed 1, 7, and 21 d post administration and the tissue at the injection sites were collected. The tissue was cryopreserved with liquid nitrogen without the use of any fixative. Mouse skin was cross sectioned at 12 pm thickness using at Thermo NX50 cryostat (Epredia, Kalamazoo, MI) and collected onto standard plus slides. Optical images of the slides were acquired at 4800 dpi using an Epson Perfection V600 Photo flatbed document scanner (Epson US, Los Alamitos, CA). Sections were coated with 10 mg / mL a-cyano-4-hydroxycinnamic acid matrix in 70% ACN, 0.1% TFA using an HTX M5 Robotic Reagent Sprayer (HTX Technologies, LLC, Chapel Hill, NC) as follows: 4 passes, nozzle temperature of 75 °C, flow rate of 100 pL / min, track speed of 1200 mm / min, track spacing of 3 mm, an HH track pattern, and a nozzle height of 40 mm. Serial sections were collected for H&E staining and were digitized using a Hamamatsu NanoZoomerSQ Digital SlideScanner (Hamamatsu Photonics, Bridgewater, NJ). Mass spectrometry images were acquired at 50 pm resolution in positive ion mode using a Brukcr timsTOF flcX QTOF mass spectrometer (Bruker Daltonics) over the m / z range 50-1000 with a summation of 700 shots per pixel. Instrument tuning was as follows: a Funnel 1 RF of 100.0 Vpp, a Funnel 2 RF of 150.0 Vpp, a Multipole RF of 200.0 Vpp, a Collision Energy of 5.0 eV, a Collision RF of 600.0 Vpp, a Quadrupole Ion Energy of 5.0 eV, a Transfer Time of 60.0 ps, and a Pre Pulse Storage of 6.0 ps. BTZ was detected in tissue as the in source generated fragment at m / z 226.09 that was confirmed through MALDI analysis of a standard. Image files loaded into SCiLS Lab Pro 2023b (Bruker Daltonics) for visualization and analysis. Data were root mean square normalized. H&E images were annotated using Hamamatsu NDP.view2 software for regions of gels in the sections. These annotations were transferred to SCiLS to create ROIs corresponding to areas of gel and non-gel within each sample. Intensities of BTZ from each pixel outside the gels were exported to a .CSV file using the SCiLS Lab API for R. The 400 pixels from each sample with the highest intensity were then further analyzed in GraphPad Prism 10.Fluorescence Recovery After Photobleaching (FRAP)

[0171] For Examples 1-6, hydrogels loaded with 2 mg / mL of AZ647-labeled IgG-PBA were prepared as previously described, and 10 uL of each hydrogel was pipetted onto a 25 x 75 x 1 mm Diamond White glass microscope slide (Globe Scientific Inc., Mahwah, NJ) with a secure seal imaging spacer with a 9 mm diameter and 0.12 mm depth (Electron Microscopy Sciences, Hatfield, PA). The sample in the spacer was then covered with 24 x 40 mm cover glass (Coming Inc.). TRAP experiments were conducted on a Nikon Al Confocal microscope utilizing the NIS- Elements AR 5.21.03 software (Nikon, Tokyo, Japan) Galvano mode with an 20X objective lens following a previously published protocol.80 Total field of view was 512 x 512 pixels. The field of view was focused until the fluorescence signal from the 640 nm laser line was maximized. Bleaching was performed on three 50 pm diameter spots at a scan speed of 1 frame per second using 486 nm, 561 nm, and 640 nm lasers at 100% intensity for 15.16 sec with a 10 min recovery imaging period using the 640 nm laser. Images were taken every 2 sec for the first min of recovery and then every 10 sec for the following 9 min. Three different bleach spots were measured and recorded for each sample. The fluorescence intensity at each time point during the recovery was normalized to the pre-bleached fluorescence intensity ofthe spot and the data was fit to a first-order exponential equation using a least-squares regression in Prism 10 to extract the fluorescence recovery half-time (ti / 2) and the mobile fraction (Mf) as previously described (Zheng el al., 2011). Error for all parameters were reported as 95% confidence intervals.

[0172] For Example 7, 10 pL of each peptide sample was pipetted onto a 25x75x1 mm Diamond White glass microscope slide (Globe Scientific Inc., Mahwah, NJ) into the center of a secure seal imaging spacer 9 mm in diameter and 0.12 mm in depth (Electron Microscopy Sciences, Hatfield, PA) and covered with a 24x40 mm cover glass (Corning Inc., Corning, NY). NIS-Elements AR 5.21.03 software in Galvano mode was used for the experiment with a 20X objective lens and 512 x 512-pixel images at a scan speed of 1 frame per second on a Nikon Al Confocal microscope (Nikon, Tokyo, Japan). 400 nm, 486 nm, 561 nm, and 640 nm lasers at 100% intensity were used to bleach three 50-pixel diameter spots for 15.16 seconds to achieve a 70% bleach depth followed by a ten-minute recovery imaging period at 488 nm. To quantify percent recovery after bleaching, fluorescence intensity measurements were divided by pre-bleach fluorescence intensity measurements. The average percent recovery readings were quantified and fitted to F(t) = A * (1 —where F(t) is the fitted fluorescence recovery, A is the plateau intensity, t is the time since bleach, and r is the disassociation parameter. The time to half recovery was found by dividing In (0.5) by the negative disassociation parameter (— T).In Vivo Fluorescence Animal Imaging IgG Release Assay

[0173] Sterile 10 mg / mL MDP hydrogels containing 2 mg / mL of AZ647-labeled IgG-PBA were prepared one day before the assay and stored at 4 °C overnight protected from light. SKH1 -Elite mice obtained from Charles River Laboratories (Wilmington, MA) and tissue background autofluorescence was quantified at Ex / Em 640 / 700 nm for each mouse using an In Vivo Imaging System (IVIS) small animal imager (PerkinElmer, Waltham, MA). Background fluorescence intensity was subtracted from all subsequent images. Mice were subcutaneously injected bilaterally in the flank with 50 pL of sample or control and imaged longitudinally. The percentage of material released from the injection site was calculated by drawing equally sized region-of-interest rectangles around the injection sites and dividing the observed total radiant efficiency in that region by the maximum total radiant efficiency measured for that injection onthe first day of the experiment. The data were fit to first-order exponential equation in GraphPad Prism 10 using a least-squares regression to model the release from the injection site. The error for parameters extracted from the model arc reported as 95% confidence intervals.Mouse Model of Acute TB

[0174] M. tuberculosis H37Rv was grown in 7H9 broth supplemented with 10% oleic acid, albumin, dextrose, catalase ([OADC] Difco Laboratories, Detroit, MI) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO) before infection. A log phase growth (acute infection) model infection was used for this experiment. In brief, 6-week-old female BALB / c mice (Charles River Laboratories) were infected with a log-phase culture of M. tuberculosis (optical density at 600 nm of approximately 1.0) using an inhalation exposure system (Glas-Col, Terre Haute, IN), aiming to implant approximately 4.5 logio CFUs in the lungs. Mice were infected with approximately 4.5 logio CFU. After infection, mice were randomized into treatment groups (12 mice per group). Untreated mice were sacrificed at the initiation of treatment to determine pretreatment CFU counts. Mice were initiated on treatment 3 d post- inf ection with one of four different regimens. The vehicle control group received a 200 pL subcutaneous injection of 20 mg / mL SHA-E2 without any drug. The second cohort of mice received a single 200 pL subcutaneous injection containing 600 pg of GFB (MedChemExpress). An additional cohort of mice received 10 doses of 60 pg of GFB administered by oral gavage once per day over the course of 14 days (5 doses for every 7 d). The experimental group received a 200 pL subcutaneous injection of 20 mg / mL SHA-E2 loaded with 600 pg of GFB. Lung CFU counts were assessed after 1 and 2 weeks of treatment by performing quantitative cultures of lung homogenates on OADC-enriched 7H11 agar (Difco Laboratories).Mouse Type 1 Diabetes Model

[0175] For in vivo studies with insulin, male C57BL / 6J mice aged 6 to 8 weeks were purchased from Charles River Laboratories. After one week of acclimatation, mice were treated with 50 mg / kg of streptozotocin (STZ) (MilliporeSigma) for five consecutive days. STZ was dissolved at a concentration of 7.5 mg / ml in pH 4.5 sodium citrate buffer immediately before injection. Then, the mice's blood glucose (BG) levels and weights were monitored. For blood glucose measurement, a drop of blood was collected from the tail and tested using a-liOneTouch UltraMini glucometer (LifeScan, Malvern, PA). Mice with blood glucose levels that exceeded 350 mg / dL for several sequential days were deemed diabetic and suitable for inclusion in the study. Insulin-PBA was prepared from commercially purchased Insulin (35 pg / IU) as described in the chemical synthesis section of the methods. SHA-E2 hydrogels were prepared with Insulin-PBA with minor modifications to the protocol previously described. Insulin-PBA was dissolved at 8.4 mg / mL for 12 and 6 IU gels and 4.2 mg / mL for 3 IU gels in 2X PBS. PBS was used instead of HBSS to avoid injecting diabetic mice with glucose, which is part of the HBSS buffer. These solutions were then mixed 1:1 with a stock solution of 20 mg / mL of SHA- E2) dissolved in MQ water to form Insulin-PBA loaded hydrogels. Diabetic mice were then injected with 100 pL of 12 IU gels, 50 pL of 6 and 3 IU gels or with 3 IU of unmodified Insulin dissolved in 50 pL of IX PBS. Right after treatment administration, blood glucose levels were measured every hour until 4 hours and every 2 hours until 10 hours. Blood glucose levels for mice that received insulin-PBA gel injections were subsequently monitored daily for 9 d. Mice that had blood glucose levels below 240 mg / dL were considered normoglycemic.Fluorophore Labeling and Purification of Ovalbumin

[0176] NHS -fluorescein (ThermoFisher Scientific, Waltham, MA) dye and Dylight 755 NHS Ester (ThermoFisher Scientific, Waltham, MA) were covalently attached to OVA (Millipore Sigma, Burlington, MA) at 0.1 equivalents following a modified version of the manufacturer’s recommended protocol. In brief, 10 mg of OVA was dissolved in 3 mL of a 0.2 M sodium bicarbonate solution. 2.7 uL of a 10 mM solution of fluorescein / DyLight 755 in DMSO was added to 495 uL of 0.2 M sodium bicarbonate and added dropwise to the dissolved OVA solution on a stir plate. The vial was covered in aluminum foil and the reaction proceeded overnight. Excess dye was removed from both solutions using a size-exclusion PD-10 Sephadex G-25 M gel filtration column (Cytiva, Marlborough, MA). Once the labeled protein was isolated from both solutions, the fluorescein-labeled OVA solution was frozen and lyophilized. The DyLight 755-labeled OVA was purified using a HiLoad 16 / 600 Superdex 200 pg column (Cytiva, Marlborough, MA) to remove both unbound fluorophore and endotoxin and then sterilized using a 0.2 um PTFE syringe filter (CELLTREAT, Pepperell, MA).Endotoxin Test

[0177] Before vaccine preparation, all materials were tested for endotoxin and confirmed to be cndotoxin-frcc (<0.25 EU per mg) using the HEK-Bluc mTLR4 reporter cell line (InvivoGen, San Diego, CA), following the manufacturer’s protocol. 20 pL of sterile 1 mg / mL peptide or OVA in endotoxin-free water (Cytiva, Marlborough, MA) was deposited in a 96-well tissue culture-treated plate. A standard curve of lipopolysaccharide was plated across a range of 10-0.015 Eu / mL. 180 pL of HEK-Blue mTLR4 cells at a 2.2 x 105cells / mL density were added to the samples and incubated at 37 °C and 5% CO2 for 20-24 hours. Following incubation, 20 pL of supernatant was collected from each well and mixed with 180 pL of QUANTI-Blue detection buffer (InvivoGen, San Diego, CA). The detection solution was then incubated at 37 °C for 15 min to 6 h until the lowest standard turned blue. The absorbance at 630 nm was measured using a microplate reader (Tecan, Mannedorf, Switzerland), and the endotoxin content of the samples was determined based on the standard curve.In Vivo Ovalbumin Release and Hydrogel Degradation

[0178] All animal experiments in this study were carried out in accordance with an lACUC-approved protocol (Rice University protocol 22-246). The day before injection, vaccines were prepared using sterile endotoxin-free 20 mg / mL K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12) solutions and 0.4 mg / mL OVA-Dylight 755 as described for FRAP (Example 7). Vaccine solutions were loaded into 300 pL insulin syringes (BD Biosciences, Franklin Lakes, NJ) and refrigerated at 4 °C overnight. Immediately before administration, the inherent tissue autofluorescence (Ex / Em 640 / 700 nm) was captured for each female SKH-1 mouse (Charles River Laboratories, 5-7 weeks of age) using a PerkinElmer (Waltham, MA) Spectrum in vivo imaging system (IVIS). Samples were then subcutaneously injected into the flank with 50 pL of the hydrogel solution containing 20 pg of OVA and IVIS imaging was performed regularly for up to 86 days. The percentage of OVA released was determined using equally sized regions of interest around the injection site and dividing the observed total background- subtracted radiant efficiency by the maximum total background-subtracted radiant efficiency.Histology

[0179] For Example 7, the skin encompassing the injection site in SKHl-Elite mice was collected 13 weeks after vaccination. Explanted skin was pinned to a wax tray andfixed overnight in formalin at 4 °C. The formalin was replaced with a 30% sucrose solution in DI water and the samples were again stored overnight at 4 °C. After patting the samples dry, they were frozen in Optimal Cutting Temperature (OCT) compound on dry ice. Samples were stored at -80 °C until being sectioned at 5 pm by a cryotome.

[0180] For one round of staining, sections were treated with Scytek Laboratories' Hematoxylin and Eosin (H&E) Stain Kit following the manufacturer's protocol with some adjustments. Sections were stained by dipping the samples in different reagents as follows: 15 sec in hematoxylin, washes in two changes of DI water, 15 sec in Bluing Reagent, washes in two changes of DI water, 2 sec in eosin, and then a rinse in ethanol. Sections were then dehydrated using three changes of ethanol, clearing in three changes of xylenes, and then mounting using Shandon Mount (Epredia, Kalamazoo, MI).

[0181] A second round of staining on additional sections was done using the Masson Trichrome Stain Kit (Epredia, Kalamazoo, MI) following the manufacturer’s protocol with adaptations for frozen sections. Slides were incubated in Bouin’s Fluid at 56 °C for 1 h, rinsed in tap water, and placed in Weigert’s Iron Hematoxylin for 3.5 min. After rinsing, slides were stained in Biebrich Scarlet- Acid Fuchsin for 2.5 min, followed by another rinse. Finally, slides were stained with Aniline Blue for 30 sec, dipped in 1% acetic acid, and rinsed. Dehydration took place for the H&E staining, using three changes of ethanol, clearing in three changes of xylenes, and then mounting using Shandon Mount (Epredia, Kalamazoo, MI).Assessment of Humoral Immunity

[0182] Submandibular bleeds were performed on female SKH-1 mice using a 5 mm lancet (Medipoint Inc., Mineola, NY) one day before injection to obtain serum prior to treatment. 200 pL of blood was collected in microvettes (Sarstedt, Numbrecht, Germany) using the supplier’s recommended protocol. Filled microvettes were stored on ice until the completion of all submandibular bleeds and then serum was separated from red blood cells using centrifugation at 4 °C at 10,000 relative centrifugal force (RCF) for 10 min. 20 pL aliquots of serum were stored in LoBind Eppendorf tubes (Eppendorf, Hamburg, Germany) at -20 °C. Injections were performed as described in the previous section and blood was collected 2, 4, 6, 8, 10, and 12 weeks after injection.

[0183] OVA-specific TgG antibody titers were quantified using an enzyme-linked immunosorbent assay (ELISA). Every well in Nunc Maxisorp 96-wcll plates (ThermoFisher Scientific, Waltham, MA) was coated with 100 pL of 1 pg / mL OVA in a pH 9.6 carbonatebicarbonate buffer and left overnight on an orbital shaker at 4 °C. Plates were then washed three times with 0.5% Tween in PBS (PBST) using a 96-well automatic BioTek Microplate Washer 405 (Agilent, Santa Clara, CA). Afterward, each well was blocked using 5% Blotto non-fat dry milk (Rockland Immunochemicals, Limerick, PA) in PBST solution (blocking solution) for 2 h on an orbital shake at room temperature. The blocking solution was removed from the plates and serial two-fold dilutions of serum were prepared in the blocking solution. 50 pL of each serum dilution was added to its corresponding well and the plates were placed on an orbital shake for 2 h at room temperature. Plates were then washed three times with PBST using the BioTek Microplate Washer before adding 100 pL of anti-mouse IgG HRP-conjugated secondary antibodies in blocking solution to each well. Plates were covered and placed on an orbital shaker for 2 h at room temperature. The secondary antibody was then removed from the plates by washing five times with PBST using the BioTek Microplate Washer. 100 pL of SureBlue TMB substrate solution (SeraCare Life Sciences Inc., Milford, MA) was added to wells and incubated for 5 min. To stop the reaction, 1 M hydrochloric acid was added and then the absorbance at 450 nm and 650 nm was read using a Tecan M1000 microplate reader (Mannedorf, Switzerland). Titers were calculated per mouse by calculating the lowest serum dilution by absorbance that was at least two-fold higher than the background signal from the pre-injection serum of the same mouse.Statistical Analysis

[0184] For Examples 1-6, multiple group comparisons were calculated by one- or two-way ANOVA with Tukey’s multiple comparisons test. Statistical calculations were performed in GraphPad Prism 10. Statistical significance is denoted with asterisks as follows: *p <0.05; **p <0.01; ***p <0.001; ****p <0.0001. Error bars and reported error represents standard error of the mean (SEM) unless otherwise specified as standard deviation (SD) or as a 95% confidence interval.

[0185] For Example 7, all data were reported as mean ± standard deviation unless otherwise specified. Independent two-sample Student’s T-tests were performed for pairwise comparisons between K2 (SEQ ID NO: 1) and D-K2 (SEQ ID NO: 12). The threshold for significance was set at p<0.05.[001861 All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.REFERENCES

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Claims

WHAT IS CLAIMED IS1. A composition comprising:A. a hydrogel, wherein the hydrogel comprises: i. a boronic acid binding motif; or ii. a boronic acid group of the formula B(X')2; andB. A compound, wherein the compound comprises: i. a boronic acid binding motif; or ii. a boronic acid group of the formula B(X')2; wherein each X' is an independently selected atom; and wherein if the hydrogel comprises a boronic acid binding motif, then the compound comprises a boronic acid group; and if the compound comprises a boronic acid binding motif, then the hydrogel comprises a boronic acid group.

2. The composition according to claim 1, wherein composition is a structure comprising one or more dynamic covalent bonds between the boronic acid-binding motif and the boronic acid.

3. The composition according to either claim 1 or claim 2, wherein the boronic acid-binding motif forms a B(X')i dynamic covalent attachment with the boronic acid group, wherein each X' is independently nitrogen, oxygen, sulfur or carbon.

4. The composition of claim 3, wherein X' is oxygen.

5. The composition according to any one of claims 1-4, wherein the boronic acid-binding motif is a nitrocatechol or 6-nitrodopamine.

6. The composition according to any one of claims 1-4, wherein the boronic acid-binding motif is a diol.

7. The composition according to any one of claims 1-4, wherein the boronic acid-binding motif is 2-hydroxybenzhydroxamic acid or a derivative thereof.

8. The composition according to any one of claims 1-7, wherein the hydrogel comprises a peptide.

9. The composition according to claim 8, wherein the peptide sequence comprises: a first domain (Xi)m;a second domain (YZ)n; and a third domain (X2)m; wherein: wherein each Xi and Xi is independently selected from among amino acids that arc positively charged at pH 7, amino acids that arc negatively charged at pH 7, proline, and hydroxyproline;Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid; m is 1, 2, 3, 4, 5; 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and n is 1, 2, 3, 4, 5; 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

10. The composition of claim 9, wherein each Xi and X2 is independently selected from among lysine, ornithine, histidine, arginine, glutamic acid, aspartic acid, hydroxyproline and proline.

11. The composition of claim 9, wherein each Y is independently selected from among serine, threonine, histidine, lysine, citrulline, asparagine, glutamine, ornithine, tyrosine, and glycine.

12. The composition of claim 9, wherein each Z is independently selected from among leucine, isoleucine, valine, phenylalanine, alanine, methionine, and tryptophan.

13. The composition according to any one of claims 9-12, wherein Xi and Xiis lysine or glutamic acid, Y and Z are selected from serine and leucine.

14. The composition of either claim 8 or claim 9, wherein the peptide has at least 95% sequence identity with SEQ No. 1, SEQ No. 2, SEQ No. 3, SEQ No. 4 and / or SEQ No. 12.

15. The composition according to any one of claims 8-14, wherein a boronic acid-binding motif is covalently attached to the peptide.

16. The composition according to any one of claims 1-15, wherein the compound is a therapeutic or prophylactic compound.

17. The composition according to any one of claims 1- 16, wherein the compound is a boronic acid-containing small molecule, a small molecule that has been modified to include a boronic acid, or a biologic that has been modified to include a boronic acid.

18. The composition according to any one of claims 1-17, wherein the compound is useful for treating or preventing an acute disease or disorder or a chronic disease or disorder.

19. A pharmaceutical composition comprising:A. a composition according to any one of claims 1-18; andB . an excipient.

20. A method of treating or preventing a disease or disorder in a patient in need thereof comprising contacting one or more cells of the patient with the composition according to any one of claims 1-18 or the pharmaceutical composition of claim 19.

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