Dynamic hydrogels for delivery of therapeutic peptides

Abstract

Compositions and methods for delivery of therapeutic peptides are disclosed herein. In some embodiments, for example, the composition comprises a dynamic hydrogel, an incretin mimetic encapsulated by the dynamic hydrogel, and an antioxidant encapsulated by the dynamic hydrogel, wherein the antioxidant is configured to stabilize the incretin mimetic.

Description

DYNAMIC HYDROGELS FOR DELIVERY OF THERAPEUTIC PEPTIDESCROSS REFERENCE TO RELATED APPLICATION(S)|0001] The present application claims the benefit of priority to U. S. Provisional Application No. 63 / 748,880, filed January 23, 2025, and U. S. Provisional Application No.63 / 783,116, filed April 3, 2025, the disclosures of each of which are incorporated by reference herein in their entireties.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application contains an electronic Sequence Listing in XML file format named “APL_015WO_SL.xml,” created on November 12, 2025, and having a size of 2,643 bytes, the contents of which are incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] The present technology generally relates to drug delivery, and in particular, to dynamic hydrogels for delivery of therapeutic peptides.BACKGROUND

[0004] Diabetes or pre-diabetes affects approximately 500 million people worldwide, including an estimated 130 million individuals in the U. S. In the U. S. alone, the annual spendings directly related to diabetes and pre-diabetes amounts to roughly $400 billion, making it the tenth most costly disease in the U. S. Type 2 diabetes (T2D), which accounts for 90-95% of all diabetes cases, is a metabolic disorder characterized by insulin resistance, deterioration of pancreatic β-cell function, and impaired regulation of hepatic glucose production eventually leading to β-cell failure. Patients with poorly managed T2D are at risk of serious micro- and macrovascular complications, including cardiovascular disease, nephropathy, retinopathy, neuropathy, and stroke.

[0005] Current insulin treatments for T2D are highly burdensome, resulting in poor patient compliance, and can sometimes cause dangerous hypoglycemic events. In contrast, treatment strategies based on incretin mimetics, which mimic natural incretin hormones that are secreted following carbohydrate intake, eliminate the risk of hypoglycemia. Thesetreatments lower endogenous glucose production and drive expansion of insulin-secreting β-cells to restore the native ability of patients to regulate glycemia. However, conventional treatments with incretin mimetics involve daily or weekly injections, which is a significant patient burden and results in poor compliance.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating clearly the principles of the present disclosure.

[0007] FIGS. 1A and 1B illustrate PNP hydrogels for the prolonged delivery of therapeutic peptides. FIG. 1A is a schematic illustration of a polymer nanoparticle (PNP) hydrogel prepared by mixing of hydrophobically-modified hydroxypropylmethylcellulose (HPMC) with poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles that allows for facile encapsulation of a therapeutic peptide. FIG. 1B is a schematic illustration of formation of a localized depot in the subcutaneous space following subcutaneous injection of the PNP hydrogel which can provide a tunable platform for sustained release of a therapeutic peptide.

[0008] FIG. 2A is a graph indicating that once weekly dosing frequency does not significantly improve patient compliance compared to a once daily dosing frequency.

[0009] FIG. 2B is a graph of clinical data showing the release profile of conventional incretin mimetic treatments, where the dotted line in the bottom graph represents repeated weekly injections that patients take every week for four months to reach therapeutic concentrations of the incretin mimetic. Conventional treatment strategies involve daily or weekly subcutaneous injections with significant ramp-up time to achieve therapeutic concentrations. In contrast, the solid line in the top graph represents the target delivery profile of a single PNP hydrogel depot injection that sustains release of the incretin mimetic for 120 days. The dashed line in both graphs indicates the therapeutic threshold.[00101 FIG. 3 A is a graph showing a size exclusion chromatogram (SEC) trace of PEG- PLA polymer. Gel permeation chromatography characterization of the PEG-PLA polymer showed a single peak corresponding to the PEG-PLA block copolymer (Mn = 22 kDa; Đ = 1.08).

[0011] FIG. 3B is a graph showing dynamic light scattering (DLS) data of PEG-PLA nanoparticles after nanoprecipitation (DH = 33.2, PDI = 0.038).

[0012] FIG. 4 shows a series of photographs illustrating preparation of PNP hydrogels. PNP hydrogels were prepared by mixing a solution of hydrophobically-modified HPMC (“polymer solution,” right syringe) with a solution of PEG-PLA nanoparticles and therapeutic peptide (e.g., semaglutide) (“NP solution,” left syringe) using a Luer lock mixer. After mixing, a homogenous, bubble-free, solid-like PNP hydrogel was formed. Owing to their dynamic crosslinking, PNP hydrogels are injectable through clinically relevant, high gauge needles and rapidly self-heal after injection.

[0013] FIGS. 5A and 5B are graphs showing rheological characterization of PNP- 1-10 hydrogel formulations with varying concentrations of semaglutide: frequency-dependent oscillatory shear sweep (FIG. 5A) and stress-dependent oscillatory shear sweep (FIG. 5B).

[0014] FIGS. 6 A and 6B are graphs showing rheological characterization of PNP-2-10 hydrogel formulations with varying concentrations of semaglutide: frequency-dependent oscillatory shear sweep (FIG. 6A) and stress-dependent oscillatory shear sweep (FIG. 6B).

[0015] FIGS. 7A-7C are graphs showing rheological characterization of PNP-1-10 hydrogel formulations with varying concentrations of tirzepatide: frequency-dependent oscillatory shear sweep (FIGS. 7A and 7C) and stress-dependent oscillatory shear sweep (FIG.7B).

[0016] FIG. 8A is a schematic illustration of an in vitro release assay of semaglutide from PNP hydrogels immersed in saline over two weeks. In vitro release assays were designed to minimize hydrogel erosion by minimizing the surface area-to-volume of the hydrogel.

[0017] FIG. 8B is a graph of in vitro release profiles showing the % cumulative release of semaglutide from PNP-2-10 formulations at low, medium, and high semaglutide loadings, over the course of two weeks.

[0018] FIG. 8C is a graph of in vitro release profiles showing the % cumulative release of semaglutide from PNP-1-10 formulations at low, medium, and high semaglutide loadings, over the course of two weeks.

[0019] FIG. 8D is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks,showing the effect of Tween® 20 addition on semaglutide release in the presence of propylene glycol and saline.

[0020] FIG. 8E is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks, showing that there is negligible effect of alpha-cyclodextrin addition on semaglutide release in the presence of propylene glycol and saline.

[0021] FIG. 8F is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks, showing the effect of Tween® 20 addition on semaglutide release in the presence of propylene glycol and absence of saline.[00221 FIG. 8G is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations, showing the effect of bovine serum albumin (BSA) in the release buffer and in the hydrogel.

[0023] FIG. 9 is a graph of in vitro release profiles showing the % cumulative release of liraglutide from 1.8 mg / mL PNP-1-10 and PNP-2-10 hydrogel formulations, over the course of two weeks.

[0024] FIG. 10 is a graph illustrating in vitro release profiles showing the % cumulative release of liraglutide and insulin glargine (Lantus) from PNP-1-10 hydrogel formulations, over the course of two weeks.

[0025] FIG. 11 is a schematic illustration of a treatment schedule and timing of blood glucose measurements and serum collection for analysis. Diabetic rats received either a single subcutaneous injection of a semaglutide-loaded PNP hydrogel (PNP-1-10 with 1.8 mg / mL semaglutide and 0.05 wt% Tween® 20) or a tirzepatide-loaded PNP hydrogel (PNP-1-10 with 4.5 mg / mL tirzepatide and 0.05 wt% Tween® 20), or daily subcutaneous bolus injections of PBS, 20 pg semaglutide, or 50 pg tirzepatide.

[0026] FIGS. 12A and 12B are graphs illustrating the results of oral glucose tolerance testing before treatment (FIG. 12A) and after 6 weeks of treatment (FIG. 12B). An oral glucose tolerance test (OGTT) was conducted to group diabetic rats into treatment groups. Rats were fasted before administration of a glucose load by oral gavage. Baseline (fasting) blood glucose measurements were taken before glucose administration and measurements were made at regular intervals thereafter. Blood glucose was measured at -5, 0, 5, 15, 30, 45, 60, and 120min. Using the area under the curve (AUC), rats with similar glucose tolerance were paired and then randomized into treatment groups.

[0027] FIG. 13 is a graph illustrating the percent change in blood glucose (BG) levels for rats treated with PNP hydrogels versus bolus injections. A single administration of semaglutide PNP or tirzepatide PNP hydrogel reduced the BG of type 2-like diabetic male rats over the course of 6 weeks, compared to daily PBS bolus injections. The plot shows change in BG over 6 weeks following each treatment group regimen (n = 6).

[0028] FIG. 14 is a graph illustrating the percent change in weight for rats treated with PNP hydrogels versus bolus injections. A single administration of semaglutide PNP or tirzepatide PNP hydrogel reduced the overall weight gain in type 2-like diabetic male rats over the course of 6 weeks post treatment, compared to daily PBS bolus injections. The plot shows change in weight over 6 weeks of each treatment group (n = 6).

[0029] FIG. 15 is a graph illustrating pharmacokinetics of a 20 pg daily bolus injection of semaglutide versus a PNP hydrogel (PNP-1-10 with a 1.8 mg / mL semaglutide loading and 0.05 wt% Tween® 20) in male diabetic rats (n = 6) over the course of 6 weeks post treatment.

[0030] FIG. 16 is a graph illustrating pharmacokinetics of a 20 pg daily bolus injection of semaglutide versus a PNP hydrogel (PNP-1-10 with a 1.8 mg / mL semaglutide loading and 0.05 wt% Tween® 20) over the first 48 hours, overlaid with the 24 hour pharmacokinetics of a 20 pg intravenous (I V.) and subcutaneous (S. C.) bolus injection in male diabetic rats (n = 6).

[0031] FIG. 17 shows graphs illustrating an assessment of treatment biocompatibility using blood chemistry to look for negative effects on the liver or kidney and evaluating the effect of treatment on HbAlc. Blood was collected pre- and post-treatment (after 6 weeks). Liver toxicity was assessed through measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin. Kidney toxicity was evaluated by examining creatinine and blood urea nitrogen (BUN) levels. Values for ALT, AST, creatine, and BUN were within the range of healthy rats (defined as the mean ± 2 standard deviations) for both the treatment and control groups.

[0032] FIGS. 18 A-l 8E illustrate the results of material characterization of Sangelose® and Tween® 80 hydrogels. FIG. 18A shows that a hydrophobically-modified hydroxypropyl methylcellulose polymer (Sangelose®) forms a modular and dynamic hydrogel when mixed with the surfactant Tween® 80. FIG. 18B illustrates oscillatory frequency sweeps of hydrogelswith increasing Tween® 80 weight percent (1.5 wt% Sangelose® (SI.5), 1.5 wt% Sangelose® and 0.1 wt% Tween® 80 (S1.5TwsoO.l), 1.5 wt% Sangelose® and 0.5 wt% Tween® 80 (S1.5TW8OO.5), and 1.5 wt% Sangelose® and 1 wt% Tween® 80 (S1.5Twsol)). FIG. 18C shows oscillatory amplitude sweeps of various hydrogels with increasing Tween® 80 concentrations. FIG. 18D plots static yield stress of hydrogels determined by stress-controlled flow sweeps (n=4). FIG. 18E plots step shear measurements of high (10 s-1) and low (0.1 s-1) shear rates in 30-s steps.

[0033] FIGS. 19A and 19B illustrate extensional rheology of Sangelose® and Tween® 80 hydrogels. FIG. 19A shows extensional strain-to-break measurements of Sangelose® hydrogels with increasing Tween® 80 weight percent (SI.5, S1.5TwsoO.l, S1.5TwsoO.5, and S1.5Tw8ol) at four different strain rates. Strain-to-break shows a positive correlation with Tween® 80 concentration. One-way ANOVA analysis with post hoc Tukey test was used to measure p-values (n=4). FIG. 19B provides images of Sangelose® and Tween® 80 hydrogels before the break at 0.3 s-1strain rate. Strain-to-break measurements were performed as denoted in the images.

[0034] FIGS. 20A-20F illustrate the results of material characterization of Sangelose® and a-cyclodextrin (aCD). FIG. 20A shows that Sangelose® forms a modular and dynamic hydrogel when mixed with the cyclic polysaccharide aCD. FIG. 20B illustrates oscillatory sweeps of a high weight percent Sangelose® aCD hydrogel (3 wt% Sangelose® and 0.02 wt% aCD (S3aCD0.02)) and a low weight percent Sangelose® aCD hydrogel (1.8 wt% Sangelose® and 0.023 wt% aCD (S1.8aCD0.023)). FIG. 20C shows flow sweeps of S3aCD0.02 and S1.8aCD0.023 hydrogels. FIG. 20D plots static yield stress of hydrogels determined by stress-controlled flow sweeps. FIG. 20E shows oscillatory sweeps at 0.1 rad·s-1. FIG. 20F plots step shear measurements of high (10 s'1) and low (0.1 s'1) shear rates in 30 s steps.

[0035] FIGS. 21A–21F illustrate the results of material characterization of Sangelose® and Tween® 20 hydrogels. FIG. 21A shows that Sangelose® forms a modular and dynamic hydrogel when mixed with the surfactant Tween® 20. FIG. 21B illustrates oscillatory frequency sweeps of a Sangelose® only hydrogel (S3) and Sangelose® hydrogels with Tween® 20 (STw2oO.75). FIG. 21C shows flow sweeps of S3 and STw2oO.75 hydrogels. Both hydrogels displayed shear-thinning behavior with decreasing viscosity at high shear rates. FIG.21D plots static yield stress of hydrogels determined by stress-controlled flow sweeps (n=3). FIG. 21E shows oscillatory sweeps of S and STw200.75 hydrogels at 0.1 rad·s-1. FIG. 21F plots step shear measurements of high (10 s-1) and low (0.1 s-1) shear rates in 30-s steps. Bothhydrogels displayed a viscosity decrease of two orders of magnitude at a high shear rate, and restored mechanical properties when the shear stress was removed.

[0036] FIG. 22A is a schematic illustration of micelle formation of the hydrophobic side chains and hydrophilic backbone of hydrophobically modified hydroxypropyl methylcellulose (HPMC-C18, Sangelose®) (top) and mixed micelle formation after addition of a second agent, which has the ability to disperse within the micelle and influence the crosslink dynamic of the Sangelose® hydrophobic side chains (bottom).

[0037] FIGS. 22B-22D are plots showing rheological measurements of hydrogels with varying concentrations of Sangelose® (1.5 wt% (SI.5), 2 wt% (S2), 2.5 wt% (S2.5), and 3 wt% (S3)): frequency-dependent oscillatory shear sweep (FIG. 22B), storage modulus (G') (FIG.22C), and tan(delta) (FIG. 22D) measured at 10-1rad·s-1from 6B.

[0038] FIGS. 22E-22H are plots showing the frequency-dependent oscillatory shear rheology of hydrogel compositions with Sangelose® combined with various second agents: Tween® 20 (S3Tw2oO.75) (FIG. 22E), Tween® 80 (S3TwsoO.75) (FIG. 22F), a-cyclodextrin (S3aCD0.05) (FIG. 22G), and Span® 20 (S3Span2oO.75) (FIG. 22H).

[0039] FIGS. 221 and 22J are plots of Storage modulus (G') (FIG. 22I) and tan(delta) (FIG. 22J) of Sangelose® based hydrogels with Tween® 20 (S3Tw2oO.75), Tween® 80 (S3Tw800.75), aCD (S3aCD0.05), Span® 20 (S3 SpamoO.75), or no second agent (S3).

[0040] FIGS. 23A-23H are plots showing rheological measurements of Sangelose®-based hydrogel compositions with varying second agents: frequency-dependent oscillatory shear sweep of compositions comprising Tween® 20 (S3Tw2oO.75) (FIG. 23A), Tween® 40 (S3TW4OO.75) (FIG. 23B), Tween® 60 (S3Tw6oO.75) (FIG. 23C), Tween® 80 (S3TwsoO.75) (FIG. 23D), Tween® 65 (S3Tw650.75) (FIG. 23E), and Tween® 85 (S3Tw850.75) (FIG. 23F). Comparison of the storage modulus (G') (FIG. 23G) and tan(delta) (FIG. 23H) values for each formulation measured at 10-1rad·s-1from the data shown FIGS. 23A–23F.

[0041] FIGS. 24A-24F are plots showing rheological measurements of Sangelose®-Tween® 80 hydrogel compositions with 3 wt% Sangelose® and increasing concentrations of Tween® 80 (0.1 wt% (S3Tw800.1), 0.5 wt% (S3Tw800.5), 1 wt% (S3Tw801), and 2 wt% (S3Tw802): frequency-dependent oscillatory shear sweep (FIG. 24A), oscillatory amplitude sweep measured at a frequency of 10-1rad·s-1(FIG. 24B), storage modulus G' (FIG. 24C) and tan(delta) (FIG. 24D) values measured at 10-1rad·s-1, relaxation time (FIG. 24E), and yield stress (FIG. 24F).

[0042] FIG. 24G is a plot of extensional strain data of hydrogel compositions comprising Sangelose® and varying concentrations of Tween® 80.

[0043] FIG. 24H shows representative images of hydrogel compositions comprising Sangelose® and varying concentrations of Tween® 80 under strain at a strain rate of 0.3 s-1.

[0044] FIGS. 25A-25E are plots showing rheological measurements of Sangelose®-Tween® 20 hydrogel compositions with 3 wt% Sangelose® and increasing concentrations of Tween® 20 (0.1 wt% (S3Tw₂₀0.1), 0.75 wt% (S3Tw2oO.75), 1 wt% (S3Tw2ol), and 2 wt% (S3Tw₂₀2)): frequency-dependent oscillatory shear sweep (FIG. 25A), oscillatory amplitude sweep measured at a frequency of 10-1rad·s-1(FIG. 25B), storage modulus G' (FIG. 25C) and tan(delta) (FIG. 25D) values measured at 10-1rad·s-1, and yield stress (FIG. 25E).[00451 FIGS. 25F and 25G are plots of rheological data on a Sangelose®-Tween® 20 hydrogel composition (S3Tw2oO.75) and a Sangelose® only hydrogel (S3), showing sheardependent viscosity (FIG. 25F) and step-shear measurements taken over three cycles of alternating low shear and high shear rates (FIG. 25G).

[0046] FIG. 26A is a plot showing frequency-dependent oscillatory shear sweep rheological characterization of Sangelose® hydrogel formulations (SANG-2-0.6) with and without semaglutide cargo (1.8 mg / mL).

[0047] FIG. 26B is a plot showing frequency-dependent oscillatory shear sweep rheological characterization of Sangelose® hydrogel formulations (SANG-2-0.6) with and without tirzepatide cargo (4.5 mg / mL).

[0048] FIG. 26C is a plot showing frequency-dependent oscillatory shear sweep rheological characterization of Sangelose® hydrogel formulations (SANG-2-0.6) with and without liraglutide cargo (1.8 mg / mL).

[0049] FIG. 27A is a plot of the % cumulative in vitro release of semaglutide over the course of one week from Sangelose®-Tween® 20 hydrogel compositions.

[0050] FIG. 27B is a plot of the % cumulative in vitro release of liraglutide over the course of one week from Sangelose®-Tween® 20 hydrogel compositions.

[0051] FIG. 28 is a schematic illustration of a timeline of semaglutide administration to diabetic rats and blood collection. Glucose measurements and serum collection were taken 5 days before injection of semaglutide and continued over a period of 6 weeks from injection.

[0052] FIG. 29 is a plot of data from an oral glucose tolerance test in rats, showing the area under the curve (AUC) of blood glucose after administration of 1) semaglutide as a bolus injection, 2) semaglutide in a Sangelose® hydrogel composition, or 3) PBS.

[0053] FIG. 30 is a plot of the change in blood glucose (BG) of diabetic rats 5 days after administration of 1) semaglutide as bolus injection, 2) semaglutide in a Sangelose® hydrogel composition, or 3) PBS.

[0054] FIG. 31 is a plot of the change in weight of diabetic rats 5 days after administration of 1) semaglutide as a bolus injection, 2) semaglutide in a Sangelose® hydrogel composition, or 3) PBS.

[0055] FIG. 32 is a plot of semaglutide serum concentration in diabetic rats over the course of the first 5 days post administration of a daily bolus injection of semaglutide or a semaglutide-loaded Sangelose® hydrogel composition.

[0056] FIG. 33 A is a schematic illustration of an experimental scheme for investigating in vivo release of GLP-1 hydrogel formulations in mice.

[0057] FIG. 33B is a graph illustrating in vivo release profiles of GLP-1 serum concentrations in mice over 2 weeks for Sangelose®-semaglutide hydrogels formulated at two different pH values (pH 7 and pH 4).

[0058] FIG. 33C is a graph illustrating in vivo release profiles of GLP-1 serum concentrations in mice over 2 weeks for Sangelose®-semaglutide hydrogels formulated with three different concentrations of zinc chloride additives at neutral pH.

[0059] FIG. 33D is a graph illustrating in vivo release profiles showing the GLP-1 serum concentrations in mice for Sangelose®-semaglutide hydrogels formulated with 6 molar equivalents of zinc chloride additives at pH 7 and 4.

[0060] FIG. 33E is a graph illustrating in vivo release profiles of GLP-1 serum concentrations in mice over 2 weeks for Sangelose®-semaglutide hydrogels formulated with 6 molar equivalents of zinc chloride additives at pH 4, using a double injection volume while maintaining a constant dosage.

[0061] FIG. 33F is a graph illustrating in vivo release profiles of GLP-1 serum concentrations in mice over 2 weeks for Sangelose®-semaglutide hydrogels formulated with calcium chloride and strontium chloride.

[0062] FIG. 33G is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for PNP hydrogel formulations containing albumin or hydroxypropyl-beta-cyclodextrin (hp-bCD).

[0063] FIG. 33H is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for Sangelose® and PNP hydrogel formulations.

[0064] FIG. 34A is a graph illustrating an in vivo release profile for a therapeutic peptide that results from a typical daily / weekly bolus injection regimen (arrows indicate injection timing).

[0065] FIG. 34B is a graph illustrating an in vivo release profile for a therapeutic peptide delivered via a dynamic hydrogel (arrows indicate timing of hydrogel delivery).

[0066] FIG. 34C is a graph illustrating an in vivo release profile for a therapeutic peptide delivered via a dynamic hydrogel (arrows indicate timing of hydrogel delivery).

[0067] FIG. 35 A is a schematic of how lipidated GLP-1 receptor agonists interact with hydrophobically modified HPMC-C18 to assemble into a long-acting subcutaneous depot.

[0068] FIG. 35B is a graph illustrating oscillatory frequency sweeps of HPMC-C18 hydrogel compositions with and without semaglutide.

[0060] FIG. 35C is a graph illustrating the plateau modulus (G'o) of HPMC-C18 hydrogel compositions with and without semaglutide.

[0070] FIG. 35D is a graph illustrating the relaxation time (τR) of HPMC-C18 hydrogel compositions with and without semaglutide extracted from rheological measurements.

[0071] FIG. 35E is a graph illustrating Herschel -Bulkley-fit dynamic yield stress for HPMC-C 18 hydrogel compositions with and without semaglutide.

[0072] FIG. 35F is a graph illustrating injection forces for HPMC-C18 hydrogel compositions.

[0073] FIG. 36A is a schematic illustrating surfactant-mediated micelle stabilization of semaglutide in the presence of Tween-20.

[0074] FIG. 36B is a graph illustrating dynamic light scattering (DLS) size distributions of semaglutide with and without Tween-20.

[0075] FIG. 36C is a graph illustrating frequency sweeps of HPMC-C 18 hydrogel compositions.

[0076] FIG. 36D is a schematic of albumin-semaglutide complexation.

[0077] FIG. 36E is a graph illustrating dynamic light scattering (DLS) size distributions of semaglutide mixed with albumin.

[0078] FIG. 36F is a graph illustrating frequency sweeps of albumin-loaded HPMC-C18 hydrogel compositions.

[0079] FIG. 36G is a schematic of divalent cation (Sr2+)-mediated multimerization of semaglutide.

[0080] FIG. 36H is a graph illustrating dynamic light scattering (DLS) size distributions showing Sr2+-induced peptide multimer formation.

[0081] FIG. 36I is a graph illustrating frequency sweeps of Sr2+-loaded HPMC-C18 hydrogel compositions.

[0082] FIG. 37A is a graph illustrating the plateau modulus (G'0) of HPMC-C18 hydrogel compositions.

[0083] FIG. 37B is a graph illustrating the relaxation time (T_R) of HPMC-C18 hydrogel compositions.

[0084] FIG. 37C is a graph illustrating the yield stress of HPMC-C18 hydrogel compositions.

[0085] FIG. 38A is a schematic of an in vitro assay for semaglutides sustained release in a capillary tube.

[0086] FIG. 38B is a graph illustrating cumulative release example profiles for HPMC-C18 with Korsmeyer-Peppas fits highlighting differences in diffusional behavior.

[0087] FIG. 39A is a schematic of a mice pharmacokinetics study design.

[0088] FIG. 39B is a graph illustrating semaglutide serum concentration time-curves for HPMC-C18 hydrogel compositions.

[0089] FIG. 39C is a graph illustrating semaglutide serum concentration time-curves for HPMC-C18 hydrogel compositions.

[0090] FIG. 39D is a graph illustrating semaglutide serum concentration time-curves for HPMC-C18 hydrogel compositions.

[0091] FIG. 39E is a graph illustrating the elimination half-life (t1 / 2,elim) for HPMC-C18 hydrogel compositions.

[0092] FIG. 39F is graph illustrating the release half-life (t1 / 2,rel) for HPMC-C18 hydrogel compositions.

[0093] FIG. 39G is a graph illustrating Cmax for HPMC-C18 hydrogel compositions.

[0094] FIG. 39H is a graph illustrating the area under the curve (AUC) for HPMC-C18 hydrogel compositions.

[0095] FIG. 40A is a schematic of the structure of semaglutide highlighting oxidation-prone residues and schematic of antioxidant incorporation into HPMC-C18 hydrogel compositions.

[0096] FIG. 40B is a schematic of a capillary burst-release assay used to quantify early-stage release of semaglutide and antioxidants from HPMC-C18 hydrogel compositions.

[0097] FIG. 40C is a graph illustrating in vitro burst release across HPMC-C18 hydrogel compositions.

[0098] FIG. 40D is a graph illustrating short-timescale release kinetics for semaglutide and antioxidant-modified HPMC-C18 hydrogel compositions.

[0099] FIG. 40E is a graph illustrating short-timescale release kinetics for semaglutide and antioxidant-modified HPMC-C18 hydrogel compositions.

[0100] FIG. 40F is a graph illustrating short-timescale release kinetics for semaglutide and antioxidant-modified HPMC-C18 hydrogel compositions.

[0101] FIG. 40G is a graph illustrating serum pharmacokinetics in healthy mice for HPMC-C18 hydrogel compositions, fit with a two-compartment model.

[0102] FIG. 40H is a graph illustrating serum pharmacokinetics in healthy mice for HPMC-C18 hydrogel compositions, fit with a two-compartment model.

[0103] FIG. 41A is a graph illustrating extracted pharmacokinetic parameters for various HPMC-C18 hydrogel compositions.

[0104] FIG. 4 IB is a graph illustrating extracted pharmacokinetic parameters for various HPMC-C18 hydrogel compositions.

[0105] FIG. 41C is a graph illustrating extracted pharmacokinetic parameters for various HPMC-C18 hydrogel compositions.

[0106] FIG. 4 ID is a graph illustrating extracted pharmacokinetic parameters for various HPMC-C18 hydrogel compositions.

[0107] FIG. 4 IE is a graph illustrating extracted pharmacokinetic parameters for various HPMC-C18 hydrogel compositions.

[0108] FIG. 42A is a schematic of a 42-day design for in vivo studies of HPMC-C18 hydrogel compositions in Type 2 diabetic (T2D) rat model.

[0109] FIG. 42B is a schematic of a two-mode, two-compartment model for interpretation of pharmacokinetics profiles in T2D rat model.

[0110] FIG. 42C is a graph illustrating serum concentration time curves for HPMC-C18 hydrogel compositions in T2D rat model.

[0111] FIG. 42D is a graph illustrating serum concentration time curves for HPMC-C18 hydrogel compositions in T2D rat model.

[0112] FIG. 42E is a graph illustrating 2-mode, 2-compartment model-derived halflives for HPMC-C18 hydrogel compositions.

[0113] FIG. 42F is a graph illustrating 2-mode, 2-compartment model-derived halflives for HPMC-C18 hydrogel compositions.

[0114] FIG. 42G is a graph illustrating peak-to-trough analysis for HPMC-C18 hydrogel compositions.

[0115] FIG. 42H is a graph illustrating area under the curve (AUC) measurements for HPMC-C18 hydrogel compositions.

[0116] FIG. 43 A is a graph illustrating blood glucose profiles for T2D rats over 6 weeks comparing daily bolus and HPMC-C18 hydrogel compositions.

[0117] FIG. 43B is a graph illustrating body -weight trajectories showing weight stabilization comparing Sprague-Dawley rat standard growth curve, daily bolus and HPMC-C18 hydrogel compositions.

[0118] FIG. 43 C is a graph illustrating the percent weight change at study end comparing daily bolus and HPMC-C18 hydrogel compositions.

[0119] FIG. 43D is a graph illustrating HbAlc before treatment and after 47 days for daily bolus and HPMC-C18 hydrogel compositions.

[0120] FIG. 43E shows representative whole-pancreas H&E sections from untreated mice and mice treated with HPMC-C18 compositions.

[0121] FIG. 43F is a graph illustrating quantified islet-to-pancreas area ratios for HPMC-C18 compositions.]0122| FIG. 43G shows the local skin response at the injection site on T2D rat, excised depot images, and corresponding H& E and Masson’s tri chrome histology.DETAILED DESCRIPTION10123] Therapeutic peptides that mimic the activity of naturally occurring peptides may be used for treating a wide variety of diseases and conditions. For example, glucagon-like peptide-1 (GLP-1) is an incretin hormone and neurotransmitter secreted from intestinal L-cells in response to nutrients to stimulate insulin and block glucagon secretion in a glucosedependent manner. GLP-1 in itself is rapidly degraded, but long-acting GLP-1 receptor agonists (GLP-1 RAs) have been developed for the treatment of type 2 diabetes because of the beneficial effects extending also beyond glucose control. However, patient adherence to antihyperglycemic treatment medications is surprisingly low, described for GLP-1 RA to fall between 29% and 54%, resulting in suboptimal type 2 diabetes management which carries an increased risk of stroke, heart and kidney disease, amputations, and blindness. For drugs with short half-lives, poor compliance with prescribed treatment regimens reduces plasma concentrations to unsuitable levels, and multiple doses are often required to return to therapeutic plasma concentrations. Complex and / or frequent administration of treatment is one hurdle to adherence. Conventionally, GLP-1 RA therapeutics are injected either daily or weekly or taken daily orally, leaving room for technological innovations that enable less frequent administrations, which can reduce patient burden and increase patient compliance.

[0124] To address these and other challenges, the present technology provides composition and methods for delivery of therapeutic peptides, such as incretin mimetics (e.g., GLP-1 RAs) and / or acylated peptides. In some embodiments, the present technology provides an advanced injectable hydrogel depot technology for long-acting delivery of incretin mimics, addressing the significant challenges in glycemic control for the 130 million individuals in the US living with Type II diabetes. Current treatment options, such as insulin therapy, are burdensome, lead to poor patient compliance, and pose risks of hypoglycemia. Incretin mimics,which stimulate insulin secretion and [3-cell expansion while reducing endogenous glucose production, provide a safer alternative without the risk of hypoglycemia. However, the frequent injection schedules required by current incretin mimics, ranging from daily to weekly, remain a significant barrier to patient adherence. The present technology aims to overcome these limitations by creating a sustained-release formulation capable of delivering continuous therapy, e.g., for up to four months from a single administration, aligning with the typical cadence of physician visits and / or significantly improving patient compliance and therapeutic outcomes.

[0125] In some embodiments, for example, the disclosure provides a composition for treating a disease or condition (e.g., diabetes and / or obesity), where the composition includes a dynamic hydrogel composed of a polymer (e.g., a hydrophobically modified cellulose derivative) and a plurality of nanoparticles (e.g., amphiphilic polymeric nanoparticles). The polymer can be non-covalently crosslinked with the plurality of nanoparticles, thus conferring shear-thinning, self-healing, and / or viscoelastic properties to the dynamic hydrogel. The composition can further include an acylated peptide encapsulated by the dynamic hydrogel, such as an incretin mimetic. In some embodiments, the acylated peptide is encapsulated via hydrophobic interactions between a fatty acid side chain of the acylated peptide and hydrophobic surfaces of the nanoparticles. The acylated peptide can be gradually released from the dynamic hydrogel via erosion of the dynamic hydrogel in vivo. Accordingly, upon administration of the composition to the subject, the composition can provide sustained, controlled release of the acylated peptide over a desired treatment period at a rate that is effective for treating the disease or condition.

[0126] In some embodiments, the present technology provides a composition, the composition including a dynamic hydrogel that includes a first agent, where the first agent is octadecyl modified hydroxypropyl methylcellulose (HPMC-C18), and one or more second agents that non-covalently interact with the hydrophobic chains of the first agent, where the dynamic hydrogel exhibits shear-thinning, self-healing, and dynamic viscoelastic properties, and optionally one or more therapeutic agents. For instance, the second agent can have reversible, non-covalent interactions with a hydrophobic chain of the first agent. In some embodiments, the first agent forms micellar structures, and the one or more second agents modulate the micellar structures. The second agent can be a therapeutic agent, such as an acylated peptide (e.g., an incretin mimetic such as semaglutide). The second agent can be propylene glycol. The second agent can be a surfactant or cyclic polysaccharide.

[0127] The embodiments of the present technology can provide numerous advantages compared to conventional therapeutic products and treatment approaches. For instance, conventional hydrogel-based depot technologies typically exhibit several critical shortcomings, including complicated manufacturing, poor formulation stability, challenging administration, burst release that can contribute to poor tolerability of the therapy, and insufficiently slow release to enable appropriately long-acting therapies. In contrast to conventional covalently crosslinked hydrogels, the dynamic hydrogels of the present technology are formed through strong yet dynamic physical interactions. As a result, these materials can address the shortcomings of other hydrogel-based depot technologies by exhibiting: (i) mild formulation requirements favorable for facile formulation with therapeutic peptides such as incretin mimetics, and maintaining drug stability during manufacturing and storage; (ii) shear-thinning properties allowing for straightforward injectability through standard syringes and needles, thus improving patient convenience; (iii) rapid self-healing of hydrogel structure and depot formation to avoid burst release of the drug cargo, thus providing excellent tolerability by maintaining consistent slow release to circumvent undesirable side effects (e.g., gastrointestinal discomfort); (iv) sufficiently high yield stress to form a robust depot that persists under the normal stresses of the subcutaneous space following administration; (v) prolonged delivery of therapeutic cargo allowing for continuous delivery over clinically desirable timeframes; (vi) biodegradability; and / or (vii) non-immunogenicity, as well as not promoting immune responses to the encapsulated cargo.

[0128] Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples set forth herein are non-limiting examples and are merely examples among other possible examples.

[0129] The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology. Embodiments under any one heading may be used in conjunction with embodiments under any other heading.I. Dynamic Hydrogels

[0130] The present technology utilizes dynamic hydrogels that can serve as a versatile platform for controlled release of therapeutic cargo, such as the therapeutic peptides described in Section II below. In some embodiments, the dynamic hydrogels exhibit dynamic behavior,such as shear-thinning behavior, self-healing behavior, and / or highly tunable viscoelastic mechanical properties. The shear-thinning, self-healing, and / or viscoelastic properties of the dynamic hydrogels can result from non-covalent, supramolecular interactions between the components of the hydrogel. The non-covalent interactions can include physical crosslinking, which may encompass various types of crosslinking arising from weak physical interactions such as hydrogen bonding, hydrophobic interactions, ionic interactions, van der Waals interactions, host-guest interactions, crystal formation, physical entanglement, or combinations thereof. The non-covalent interactions can allow for the formation of dynamic, reversible crosslinks between components of the hydrogel that are capable of dissociating and reforming, e.g., spontaneously and / or in response to applied stress.

[0131] The dynamic hydrogels described herein can provide many advantages for therapeutic applications. For instance, the dynamic hydrogels described herein can exhibit high drug loading capacity, gentle conditions for encapsulation of biologic cargo, sustained delivery of cargo, and / or mechanical tunability. However, unlike traditional covalently crosslinked hydrogels, the dynamic hydrogels herein can be easily administered via techniques such as direct injection, catheter delivery, spreading, or spraying, due to their shear-thinning and / or self-healing properties. Additionally, the dynamic hydrogels herein can exhibit unique dynamic network rearrangements that provide highly tunable release characteristics for the therapeutic cargo. The dynamic hydrogels provided herein can also be synthesized in a straight-forward, cost-effective manner that is easily scalable.A. Polymer Nanoparticle Hydrogels

[0132] In some embodiments, the dynamic hydrogels described herein are polymer nanoparticle (PNP) hydrogels. PNP hydrogels are a type of supramolecular hydrogel formed from non-covalent interactions between polymers and nanoparticles. A PNP hydrogel can selfassemble rapidly upon mixing of a polymer solution with a nanoparticle solution. Selfassembly of the PNP hydrogel network can occur when polymers are linked together by adsorption of segments of the polymer chains onto the surfaces of the nanoparticles through multivalent, transient interactions. PNP hydrogel formation can be an entropy-driven process in which solvent molecules (e.g., water) solvating the polymer chains and nanoparticle surfaces are released into the bulk solution upon binding of the polymer chains to the nanoparticle surfaces, thus producing large gains in translational entropy. The interactions between the polymers and nanoparticle surfaces can be transient and reversible, thus allowing the PNPhydrogel to flow under applied shear stress, followed by rapid self-healing when the stress is relaxed.

[0133] The PNP hydrogels described herein can be composed of any suitable combination of polymers and nanoparticles that are capable of interacting non-covalently with each other to form crosslinks with the desired dynamic behavior. In some embodiments, the nanoparticle and polymer are selected to have a sufficiently strong affinity to produce efficient crosslinking. That is, the free energy gain (c) resulting from the adsorption of a polymer chain to the surface of a nanoparticle can be greater than or comparable to the thermal energy (kBT). In addition, the average number of interactions per polymer chain and particle can be greater than 2 to achieve percolation of the hydrogel network. Moreover, to favor polymer bridging of multiple nanoparticles (as opposed to polymer wrapping around individual particles), the nanoparticle diameter can be comparable to or less than the persistence length of the polymer chains. When some or all of these criteria are met, the nanoparticles can serve as crosslinkers between the polymer chains, while the polymer chains can bridge many different particles, thus enabling hydrogel formation. In some embodiments, the modulus (G) of the PNP hydrogel is related to the number of dynamic hydrogel interactions per unit volume (n) and the energy associated with each interaction (αkBT) according to the following relation: G ~ nαkBT.

[0134] In some embodiments, the nanoparticle surfaces are hydrophobic, such that the adsorption of the polymer chains to the nanoparticle surfaces are at least partially influenced by the general level of hydrophobicity along the polymer chain (e.g., the size and / or number of hydrophobic groups attached to the polymer chain). For example, the PNP hydrogels described herein can utilize polymer-nanoparticle interactions between hydrophobically-modified cellulose derivatives and nanoparticles, such as dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12) and biodegradable polymeric nanoparticles composed of poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA). Additional examples of nanoparticles and polymers suitable for use in the PNP hydrogels herein are provided in Sections I. A.l and I. A.2 below, respectively.

[0135] The PNP hydrogels described herein can be differentiated from conventional drug delivery systems that include nanoparticles embedded in a covalently crosslinked hydrogel. Such conventional systems typically include gel-forming polymers that are covalently crosslinked with each other to form the gel network, while the nanoparticles serve as an optional additive that plays no role in gel formation, and thus can be freely substituted with other additives or omitted altogether. In contrast, the PNP hydrogels herein may bespecifically formed through the interactions between the nanoparticles and polymers. In some embodiments, the polymers and nanoparticles used in the PNP hydrogels herein each independently do not form a gel alone, or are not used at concentrations where the polymer alone or the nanoparticle alone form a gel, such that gel formation occurs only when the polymer and nanoparticle are combined.

[0136] In some embodiments, the PNP hydrogels herein include one or more polymers combined with one or more nanoparticles, such that the loss modulus of a solution of the one or more polymers and the loss modulus of a solution of the one or more particles are each greater than their respective storage moduli at an angular frequency within a range from 0.1 rad / s to 100 rad / s (e.g., 10 rad / s) as measured by oscillatory shear rheometry in the linear viscoelastic region. The storage modulus of the PNP hydrogel produced by combining the one or more polymers with the one or more particles may be greater than the loss modulus of the PNP hydrogel at an angular frequency within a range from 0.1 rad / s to 100 rad / s (e.g., 10 rad / s) as measured by oscillatory shear rheometry in the linear viscoelastic region. In some embodiments, the dynamic shear viscosity of the PNP hydrogel at a shear rate within a range from 0.1 s-1to 100 s-1(e.g., 10 s-1) is greater than the sum of the dynamic shear viscosity of the solution of the one or more polymers and dynamic shear viscosity of the solution of the one or more nanoparticles at the shear rate within the range from 0.1 s-1to 100 s-1. For example, the dynamic shear viscosity of the PNP hydrogel can be greater than the sum of the dynamic shear viscosities of the polymer solution and the nanoparticle solution by a multiplicative factor within a range from 2 to 100,000, 2 to 1000, 2 to 100, or 2 to 10.

[0137] The PNP hydrogels described herein can include any concentration of polymers and nanoparticles suitable for providing desired hydrogel properties. For instance, higher polymer concentrations can produce PNP hydrogels with a higher stiffness and / or slower degradation rate. Higher nanoparticle concentrations can produce PNP hydrogels with a higher viscosity, stiffness, and yield stress, and / or slower degradation rate. The hydrogel properties may depend not only on the overall amount of solid content in the hydrogel, but also on the stoichiometry of polymer content to nanoparticle content. For example, increasing the nanoparticle concentration at a constant polymer concentration can produce a hydrogel having a more solid-like rheological response (e.g., lower tan delta), increased strain-to-yield, and increased yield stress. Increasing the polymer concentration at a constant nanoparticle concentration can produce a hydrogel having a more liquid-like rheological response (e.g.,higher tan delta and greater frequency dependency of the storage modulus) and reduced strain-to-yield.

[0138] In some embodiments, the PNP hydrogels described herein include at least 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt % polymer; and / or at least 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, or 15 wt% nanoparticles. Alternatively or in combination, the concentration of polymer within the PNP hydrogel can be within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%; and / or the concentration of nanoparticles within the PNP hydrogel can be within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%. The nomenclature “X-Y hydrogel” or “X: Y hydrogel” is used herein to refer to a hydrogel having X wt% polymer and Y wt% nanoparticles.

[0139] In some embodiments, the PNP hydrogels herein are prepared by simple mixing of the polymers, nanoparticles, therapeutic cargo, and any optional additives. For example, the PNP hydrogel can be prepared by forming a polymer solution (e.g., by dissolving the polymer in an aqueous solvent such as water or a buffered solution such as phosphate-buffered saline (PBS)), forming a nanoparticle solution (e.g., by suspending the nanoparticles in an aqueous solvent), and forming a solution containing the therapeutic cargo (e.g., by dissolving or suspending the therapeutic cargo in an aqueous solvent). The solutions can then be combined, optionally with external agitation, to form the PNP hydrogel including the therapeutic cargo.1. Nanoparticles

[0140] The PNP hydrogels described herein can include a plurality of nanoparticles. The nanoparticles can be any suitable shape, such as spheres, cubes, rods, tubes, plates, fibers, etc. The nanoparticles can have a mean particle size (e.g., diameter) within a range from 1 nm to 1000 nm, 1 nm to 500 nm, 1 nm to 250 nm, 1 nm to 150 nm, 1 nm to 100 nm, 1 nm to 50 nm, 1 nm to 25 nm, 1 nm to 10 nm, 10 nm to 1000 nm, 10 nm to 500 nm, 10 nm to 250 nm, 10 nm to 150 nm, 10 nm to 100 nm, 10 nm to 50 nm, 10 nm to 25 nm, 25 nm to 1000 nm, 25 nm to 500 nm, 25 nm to 250 nm, 25 nm to 150 nm, 25 nm to 100 nm, 25 nm to 50 nm, 50 nm to1000 nm, 50 nm to 500 nm, 50 nm to 250 nm, 50 nm to 150 nm, 50 nm to 100 nm, 100 nm to 1000 nm, 100 nm to 500 nm, 100 nm to 250 nm, 100 nm to 150 nm, 150 nm to 1000 nm, 150 nm to 500 nm, 150 nm to 250 nm, 250 nm to 1000 nm, 250 m to 500 nm, or 500 nm to 1000 nm. In some embodiments, the nanoparticles have a mean particle size less than or equal to 500 nm, 250 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. As described herein, to facilitate hydrogel formation, the mean particle size of the nanoparticles can be similar to or less than the persistence length of the polymer in the PNP hydrogel, such as less than or equal to 125%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the persistence length of the polymer. As used herein, “mean particle size” may refer to the statistical mean particle size (e.g., diameter) of the particles in the PNP hydrogel composition. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer to the hydrodynamic diameter or to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering.

[0141] The nanoparticles can be made out of a single material or can be made out of a combination of multiple different materials (e.g., two, three, four, five, or more different materials). The material(s) can be biodegradable and / or biocompatible. For example, in some embodiments, the nanoparticles are made partially or entirely out of one or more biodegradable and / or biocompatible polymers. Generally, biodegradable polymers can degrade by enzymatic hydrolysis, exposure to water in vivo, surface erosion, and / or bulk erosion. Biodegradable polymers can include synthetic polymers, naturally occurring polymers, or combinations thereof. Examples of synthetic biodegradable polymers include polyhydroxy acids (e.g., poly(lactic acid), poly(glycolic acid)), polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide), poly(lactide-co-caprolactone), poly(ethylene-co-maleic anhydride), poly(ethylene maleic anhydride-co-L-dopamine), poly(ethylene maleic anhydride-co-phenylalanine), polyethylene maleic anhydride-co-tyrosine), poly(butadiene-co-maleic anhydride), poly(butadiene maleic anhydride-co-L-dopamine) (pBMAD), poly(butadiene maleic anhydride-co-phenylalanine), poly(butadiene maleic anhydride-co-tyrosine), and combinations (e.g., mixtures, copolymers) thereof. Examples of naturally occurring biodegradable polymers include polysaccharides (e.g., cellulose, alginate, collagen, chitosan, hyaluronic acid, starch, agarose, agar, xanthan gum),proteins (e.g., collagen, fibrin, albumin, zein, gelatin), and derivatives thereof (e.g., derivatives of cellulose such as cellulose nanocrystals, cellulose nanofibers), and combinations thereof.

[0142] Alternatively or in combination, the nanoparticles can be made partially or entirely out of one or more non-biodegradable polymers. Examples of non-biodegradable polymers include polystyrenes, polyalkylene glycols, poly(meth)acrylates, poly (meth)acrylamides, polyalkylenes (e.g., polyethylene, polyvinyls, poly(vinyl acetate), poly(ethylene terephthalate)), and combinations thereof.10143] The polymer(s) used to form the nanoparticles herein can have any suitable molecular weight, such as a molecular weight (e.g., number-average molecular weight (Mn)) within a range from 500 Da to 10,000 kDa, 1 kDa to 1000 kDa, or 10 kDa to 100 kDa. As used herein, “molecular weight” may refer to the relative average chain length of the bulk polymer, and can be estimated or characterized in various ways including gel permeation chromatography (GPC) and capillary viscometry. GPC molecular weights are reported as the number-average molecular weight (Mn) as opposed to the weight-average molecular weight (Mw). Capillary viscometry provides estimates of molecular weight (Mv) as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.10144] In some embodiments, the nanoparticles are made partially or entirely out of one or more inorganic materials, such as clays (e.g., silicates) or other types of minerals (e.g., sulfides, oxides, halides, carbonates, sulfates, phosphates, apatites), or combinations thereof. Alternatively or in combination, the nanoparticles can be made partially or entirely out of one or more metals, such as gold, silver, copper, platinum, palladium, ruthenium, or combinations thereof. Optionally, the nanoparticles can be made partially or entirely out of carbon nanotubes (e.g., single-walled or multi -walled nanotubes), graphene, graphene oxide, or other ultrathin single crystals, including black phosphorous and boron based nanosheets.

[0145] In some embodiments, the nanoparticles are core-shell particles (also known as “core-corona particles”). A core-shell particle can have a core containing or formed from a first material, and a shell or corona containing or formed from a second, different material. For example, a core-shell particle can include at least two polymers, such that the core is made from a first polymer, and the shell or corona is made from a second, different polymer. As another example, the core-shell particle can include a single block copolymer, such that the core is made from a first block of the block copolymer, and the shell or corona can be madefrom a second block of the block copolymer. In some embodiments, one or both of the components of the core-shell particle is a non-polymeric material.

[0146] A core-shell particle can be composed of two compositionally disparate phases, of which one (either the core or shell / corona) is hydrophobic and the other (core or shell / corona) is hydrophilic. Suitable hydrophobic components include polyamides (e.g., poly(amino acids)), polyesters (e.g., poly(lactic acid), poly(caprolactone)), polypropylene oxides, polystyrenes, and combinations thereof. Suitable hydrophilic components include polysaccharides, proteins, polyamides (e.g., poly(amino acids)), naturally occurring polymers, synthetic polymers, and combinations thereof. Suitable block copolymers include combinations of polyethylene glycol and polyesters (e.g., PEG-PLA, poly(ethylene glycol)-block-poly(caprolactone) (PEG-PCL)) and combinations of polyethylene glycol and polypropylene glycol (e.g., poloxamers). In some embodiments, the core-shell particle is composed of an amphiphilic polymer including (1) one or more hydrophobic polymers selected from polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, and / or copolymers thereof, and (2) one or more hydrophilic polymers selected from polysaccharides, proteins, poly(amino acids), and / or polyalkylene oxides.

[0147] Alternatively, the nanoparticles can be homogenous nanoparticles. A homogenous nanoparticle can be uniformly formed from a single material, or can be formed from multiple materials that are not separated into disparate phases within the particle as in core-shell particles.

[0148] The nanoparticles can be prepared using techniques known in the art. The technique to be used can depend on a variety of factors, including the materials used to form the nanoparticles, the desired size range of the resulting nanoparticles, and suitability for the material to be encapsulated. Examples of suitable techniques include, but are not limited to, solvent evaporation, solvent removal, hot melt microencapsulation, spray drying, phase inversion, polyelectrolyte condensation, single and double emulsion (e.g., probe sonication), nanoparticle molding, and electrostatic self-assembly.

[0149] The concentration of the nanoparticles in the PNP hydrogel can be varied to produce the desired hydrogel properties. In some embodiments, for example, the concentration of the nanoparticles in the PNP hydrogel is within a range from 1 wt% to 15 wt%, 2 wt% to 12 wt%, 3 wt% to 10 wt%, 5 wt% to 8 wt%, 5 wt% to 15 wt%, 5 wt% to 10 wt%, 10 wt% to 15 wt%, or 10 wt% to 12 wt%. The concentration of the nanoparticles in the PNP hydrogel can beabout 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, or about 15 wt%. In some embodiments, the concentration of the nanoparticles in the PNP hydrogel can be greater than or equal to 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, or 14 wt%. Alternatively or in combination, the concentration of the nanoparticles in the PNP hydrogel can be less than or equal to 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, or 15 wt%.2. Polymers

[0150] The PNP hydrogel can be formed when the nanoparticles are mixed with and interact with one or more polymers. The shear-thinning and / or self-healing properties of the PNP hydrogel can be derived from reversible, non-covalent interactions between the nanoparticles and the polymer chains, as described herein. The PNP hydrogel can include a single type of polymer or can include a combination of multiple different polymers (e.g., two, three, four, five, or more different polymers). The polymer(s) can be biodegradable and / or biocompatible. The polymer(s) can include naturally occurring polymers, synthetic polymers, or derivatives or combinations thereof. Examples of naturally occurring polymers include polysaccharides (e.g., cellulose, alginate, collagen, chitosan, hyaluronic acid, starch, agarose, agar, xanthan gum), proteins (e.g., collagen, fibrin, albumin, zein, gelatin), and combinations thereof. Examples of synthetic polymers include polyacrylamide, poly(lactic acid), polyethylene glycol, polyethylene glycol-co-propylene glycol (PEO-PPO), poly(acrylates) (e.g., poly(2-hydroxy ethyl methacrylate)), and combinations thereof. In some embodiments, the PNP hydrogel includes a derivative of a naturally occurring polymer, such as a cellulose derivative. Examples of cellulose derivatives include hydroxypropylmethylcellulose (HPMC), hydroxyethyl cellulose (HEC), hydroxypropylcellulose (HPC), ethylcellulose (EC), methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), carboxymethylcellulose (CMC), carboxymethyl ethyl cellulose (CMEC), and combinations thereof.

[0151] In some embodiments, the PNP hydrogels herein include at least one polymer that is modified with a hydrophobic moiety. Hydrophobic modification of polymers may increase the energy associated with each polymer nanoparticle interaction (αkBT), thereby increasing the modulus of the dynamic hydrogel given the same number of interactions per unit volume. Such modification may facilitate favorable interactions between the hydrophobicmoiety on the polymer chain and the hydrophobic core of the nanoparticle, thereby enhancing the adsorption energy of the polymer to the nanoparticles. The hydrophobic moiety can include a plurality of carbon atoms (e.g., from 2 to 50 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 18 carbon atoms), and can be a saturated molecule or an unsaturated molecule. Examples of hydrophobic moieties that may be used include, but are not limited to, alkyl moi eties (e.g., C4 to Cl 8 alkyls, such as butyl (-C4), hexyl (-C6), octyl (-C8), decyl (-C10), dodecyl (-C12), tetradecyl (-C14), pentadecyl (-C15), hexadecyl (-C16), heptadecyl (-C17), octadecyl (-C18)), alkenyl moieties (e.g., oleyl, linoleyl), aryl moieties (e.g., phenyl, benzyl, pyryl, naphthyl, anthracene), and cycloalkyl moieties (e.g., adamantyl, cyclohexyl, cholesterol). In some embodiments, the degree of modification of the polymer (e.g., percentage of reactive groups on the polymer have been functionalized with the hydrophobic moiety) is within a range from 1% to 50%, 5% to 30%, 5% to 25%, or 10% to 15%. For example, the degree of modification can be about 5%, 10%, 15%, 20%, or 25%.[0152| The concentration of the polymer(s) in the PNP hydrogel can be varied to produce the desired hydrogel properties (e.g., stiffness, storage modulus, degradation rate). In some embodiments, for example, the concentration of the polymer(s) in the PNP hydrogel is within a range from 0.25 wt% to 10 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 2 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%. The concentration of the polymer(s) in the PNP hydrogel can be about 0.1 wt%, 0.25 wt%, 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. In some embodiments, the concentration of the polymer(s) in the PNP hydrogel can be greater than or equal to 0.25 wt%, 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, or 4.5 wt%. Alternatively or in combination, the concentration of the polymer(s) in the PNP hydrogel can be less than or equal to 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%.3. Additional Components

[0153] The PNP hydrogels herein can optionally include one or more additional components to facilitate gel formation and / or modify the properties of the hydrogel. For example, the PNP hydrogels herein can include at least one enhancer compound that enhances the interactions between the polymers and nanoparticles, e.g., by providing bridging-type non-covalent interactions between the polymers and nanoparticles. In some embodiments, a portionof an enhancer compound interacts non-covalently with the polymer and a second portion of the enhancer compound interacts non-covalently with the nanoparticle. Non-limiting examples of such interactions include ionic interactions such as cationic / anionic interactions, electrostatic interactions, and hydrogen bonding interactions.[0154| For example, in embodiments where the polymer is negatively charged at physiological pH (e.g., hyaluronic acid, carboxymethyl cellulose), a cationic surfactant can be used to enhance adsorption of the anionic polymer to the nanoparticles via electrostatic interactions. Examples of positively charged surfactants include cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium iodide, cetyltrimethylammonium fluoride, and cetyltrimethylammonium chloride. Conversely, in embodiments where the polymer is positively charged at physiological pH (e.g., chitosan, aminopolysaccharides, poly(lysine), cationic acrylate polymers, cationic vinyl polymers), an anionic surfactant can be used to enhance adsorption of the cationic polymer to the nanoparticles via electrostatic interactions. Examples of negatively charged surfactants include sodium dodecyl sulfate, sodium stearate, and charged fatty acid surfactants.

[0155] In some embodiments, molecular recognition between at least two compounds can provide the enhancement. For example, the adsorption of polymers, such as polysaccharides, to nanoparticles can be enhanced by an enhancer compound which includes a carbohydrate in one portion of the enhancer and a polymer tail that interacts with the nanoparticle.

[0156] The concentration of the enhancer compound can be varied to produce the desired effect on hydrogel formation. In some embodiments, for example, the concentration of the enhancer compound in the PNP hydrogel is within a range from 0.25 wt% to 10 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 2 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%. The concentration of the enhancer compound in the PNP hydrogel can be about 0.1 wt%, 0.25 wt%, 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. In some embodiments, the concentration of the enhancer compound in the PNP hydrogel can be greater than or equal to 0.25 wt%, 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, or 4.5 wt%. Alternatively or in combination, the concentration of the enhancer compound in the PNP hydrogel can be less than or equal to 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. Optionally, the PNP hydrogel may not include any enhancer compounds.B. PXY Hydrogels

[0157] In some embodiments, the dynamic hydrogels described herein are PXY hydrogels, which refer to hydrogels that include a first agent (“P”), where the first agent is a hydrophobically modified polysaccharide; one or more second agents (“XY”), where the second agent has a high hydrophilic / hydrophobic ratio and / or non-covalently interacts with hydrophobic chains; and optionally one or more therapeutic agents (which may or may not also serve as the second agent). The non-covalent interactions between the hydrophobically modified polysaccharide (e.g., HPMC-C18) and the second agent influences the dynamic character of the hydrogel.

[0158] For instance, in the polymer hydroxypropyl methylcellulose (HPMC) modified with long hydrocarbon side chains, hydrophobic interactions between the hydrocarbon chains may result in areas of side chain packing and / or organized assembly, resulting in a hydrogel having a relatively static character rather than a hydrogel with dynamic properties. The hydrophobic side chains may form micelles, aggregates of hydrophobic hydrocarbon chains surrounded by the hydrophilic backbone of HPMC, contributing to a relatively static character. Introduction of a second agent capable of interacting with the hydrocarbon side chains may modulate the organized interactions of hydrocarbon side chains and form free volume (e.g., cavities) within the hydrocarbon chain packed regions of the micelles, thus producing a hydrogel with desirable dynamic properties.[0159| FIG. 22A provides schematic illustration of hydrogels formed from Sangelose®, an HPMC modified with a Cl 8 hydrocarbon (top), and hydrogels formed from Sangelose® and a second agent (bottom). Sangelose® has a hydrophilic cellulose backbone from which hydrophobic Cl 8 side chains are attached. The hydrophobic side chains of Sangelose® interact with each other forming hydrophobic aggregates surrounded by the hydrophilic backbone of the cellulose, in what can be described as a micelle. Micelle formation may occur along a single polymer chain and / or between different polymer chains to form crosslinks. When enough interactions between different polymer chains are formed, a hydrogel will form. The resulting hydrogel composition can have a relatively static character (e.g., the crossover frequency of the storage modulus (G') and the loss modulus (G") of the hydrogel composition can be less than 0.01 rad·s-1).

[0160] To form a hydrogel composition with a dynamic character, a second agent is added to modify the network dynamics of the Sangelose® polymer chains. In someembodiments, addition of a second agent (e.g., a surfactant, an acylated peptide), represented by the star in FIG. 22A, modulates the hydrophobic interactions within the micelle to form a “mixed micelle.” For instance, the second agent can include a hydrophobic chain having a length that is different than the length of the Sangelose® side chains, can include an unsaturated hydrocarbon group that disrupts alignment between neighboring chains, and / or can include sterically bulky moieties (e.g., large hydrophilic headgroups) that reduce packing density. Unsaturated hydrocarbon groups contain alkenes (-CH=CH-) or alkynes (-C=C-) in their structure. For instance, oleyl and linoleyl are mono- and di- unsaturated hydrocarbon groups, respectively. In such embodiments, the second agent may be dispersed within the micelle to create free volume (e.g., cavities) within the micelle.

[0161] Alternatively or in combination, addition of a second agent (e.g., alphacyclodextrin) can modulate the micelle by binding to the hydrophobic side chains of Sangelose®, thereby preventing or at least hindering the hydrophobic side chains from entering the micelles and creating free volume within the micelles. In such embodiments, the second agent may be dispersed within the micelles, may be located only partially within the micelles, or may be located entirely outside of the micelles.10162] The addition of the second agent modifies the micellar character, promotes reversible crosslinking of the polymer, increases the dynamic nature, and / or reduces the static nature of the hydrogel composition (e.g., the crossover frequency of the storage modulus (G') and the loss modulus (G") of the hydrogel composition can be greater than 0.01 rad·s-1). The increased dynamic nature of the hydrogel may permit, for instance, penetration of immune cells into the hydrogel, while retaining therapeutic agents in the hydrogel, as compared to hydrophobically modified HPMC in the absence of a second agent. Additional examples of first and second agents suitable for use in the PXY hydrogels herein are provided in Sections I. B.l and I. B.2 below, respectively.1. First Agent - Hydrophobically Modified Polysaccharide |0163] The first agent refers to a hydrophobically modified polysaccharide. Examples of polysaccharides include cellulose, alginate, chitosan, hyaluronic acid, starch, agarose, agar, and xanthan gum. In some embodiments, the polysaccharide is a derivative of a naturally occurring polymer, such as a cellulose derivative. Examples of cellulose derivatives include hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), ethylcellulose (EC), methylcellulose (MC), hydroxyethyl methylcellulose(HEMC), carboxymethylcellulose (CMC), carboxymethyl ethyl cellulose (CMEC), and combinations thereof. In some embodiments, the polysaccharide is HPMC.

[0164] Hydrophobic modification of the polysaccharide provides a hydrophilic polymer backbone with hydrophobic side chains. The hydrophobic side chain can be or include a hydrophobic group having a plurality of carbon atoms (e.g., from 2 to 50 carbon atoms, 2 to 30 carbon atoms, or 2 to 18 carbon atoms), and can be a saturated molecule or an unsaturated molecule. Examples of hydrophobic groups that may be used include, but are not limited to, alkyl groups (e.g., C4 to Cl 8 alkyls, such as butyl (-C4), hexyl (-C6), octyl (-C8), decyl (-C10), dodecyl (-C12), tetradecyl (-C14), pentadecyl (-C15), hexadecyl (-C16), heptadecyl (-C17), octadecyl (-C18), alkenyl groups (e.g., oleyl, linoleyl), aryl groups (e.g., phenyl, benzyl, pyryl, naphthyl, anthracene), and cycloalkyl groups (e.g., adamantyl, cyclohexyl, cholesterol). In some embodiments, the degree of modification of the polysaccharide (e.g., percentage of reactive groups on the polysaccharide have been functionalized with the hydrophobic side chain) is within a range from 0.1% to 5%, 0.1% to 1%, 0.1 % to 3%, 1% to 50%, 5% to 30%, 5% to 25%, or 10% to 15%. For example, the degree of modification can be about 5%, 10%, 15%, 20%, or 25%.

[0165] In some embodiments, the hydrophobically modified polysaccharide is hydrophobically modified HPMC. In some embodiments, the hydrophobically modified polysaccharide has a plurality of hydrocarbon groups, each having 14 to 22 carbon atoms. In some embodiments, the first agent is modified HPMC having a plurality of hydrocarbon groups, each having 14 to 22 carbon atoms. In some embodiments, the hydrophobically modified polysaccharide is octadecyl modified HPMC (HPMC-C18).

[0166] Sangelose® is a tradename of HPMC-C18, of which the chemical formula is shown below:C’H, ORR - -fl, -CIE.or -CH?CH(OWCH> OR‘( i = -C:SH5? )

[0167] The term “Sangelose®” may be used herein to refer to the commercial product as well as to HPMC-C18 generally, regardless of source.

[0168] The hydrophobic side chains of the hydrophobically modified polysaccharide can interact noncovalently with each other, thereby forming noncovalent crosslinks between different polysaccharide chains to form a hydrogel. For instance, the hydrophobic side chains can aggregate with each other to form micellar structures that serve as the noncovalent crosslinks of the hydrogel. These crosslinks can be relatively static (e.g., due to tight packing of the chains), such that hydrogels formed from the hydrophobically modified polysaccharide only are static rather than dynamic hydrogels.2. Second Agent

[0169] In some embodiments, the second agent can be an additive that is capable of non-covalently interacting with the hydrophobic side chains of the first agent, such that the interaction modulates the hydrophobic side chain packing or organization, thereby increasing the dynamic character of the hydrogel crosslinks to produce a dynamic hydrogel. For instance, the second agent can be dispersed within the micellar structures of the first agent, thereby creating free volume within the micellar structures. Alternatively or in combination, the second agent can bind non-covalently to the hydrophobic side chains of the first agent to prevent the hydrophobic side chains from interacting with other hydrophobic side chains within the micellar structure. In some embodiments, the second agent is a surfactant or a cyclic polysaccharide.

[0170] In some embodiments, the second agent is a surfactant. Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and / or zwitterionic surfactants. A surfactant can include at least one hydrophobic tail and a hydrophilic head group. The hydrophobic tail can be or include a hydrophobic group having a plurality of carbon atoms (e.g., from 2 to 50 carbon atoms, 2 to 30 carbon atoms, or 2 to 18 carbon atoms), and can be a saturated group or an unsaturated group, and can be a branched group or an unbranched molecule. Examples of hydrophobic groups that may be used include, but are not limited to, alkyl groups (e.g., C4 to Cl 8 alkyls, such as butyl (-C4), hexyl (-C6), octyl (-C8), decyl (-C10), dodecyl (-C12), tetradecyl (-C14), pentadecyl (-C15), hexadecyl (-C16), heptadecyl (-C17), and octadecyl (-C18), alkenyl groups (e.g., oleyl, linoleyl), aryl groups (e.g., phenyl, benzyl, pyryl, naphthyl, anthracene), and cycloalkyl groups (e.g., adamantyl, cyclohexyl, cholesterol). In some embodiments, the length of the hydrophobic tail is selected to be different than (e.g., longer or shorter than) the length of the hydrophobic side chain of the first agent. In some embodiments, the hydrophobic tail is unsaturated and / or branched. In some embodiments, the surfactant includes a single hydrophobic tail, while in other embodiments,the surfactant includes multiple hydrophobic tails (e.g., two, three, or more). The hydrophilic head group can be a nonionic group, an anionic group, a cationic group, or a zwitterionic group. In some embodiments, relatively large hydrophilic head groups (e.g., polymers, polyethoxylated sorbitan) are advantageous for modulating micellar structures due to steric hindrance.[01711 In some embodiments the surfactant is a non-ionic surfactant. An example of a non-ionic surfactant is the family of polysorbates, which are derived from ethoxylated sorbitan esterified with fatty acids. For instance, polyoxyethylene (20) sorbitan monooleate is derived from polyethoxylated sorbitan and oleic acid. The number 20 following the 'polyoxyethylene' part refers to the total number of oxyethylene (–CH2CH2O–) groups found in the molecule. Polyoxyethylene (20) sorbitan monooleate is more commonly referred to as polysorbate 80 or Tween® 80. The number following the 'polysorbate' part is related to the type of major fatty acid associated with the molecule, where monooleate is indicated by 80. The chemical formula of polyoxyethylene (20) sorbitan monooleate (Tween® 80 or polysorbate 80) is:

[0172] Other examples of polysorbate include polyoxyethylene (20) sorbitan monolaurate (polysorbate 20 or Tween® 20), polyoxyethylene (20) sorbitan monopalmitate (polysorbate 40 or Tween® 40), and polyoxyethylene (20) sorbitan monostearate (polysorbate 60 or Tween® 60). Monolaurate is indicated by 20, monopalmitate is indicated by 40, monostearate by 60, and monooleate by 80.

[0173] In some embodiments, the second agent is a non-ionic surfactant (e.g., a polysorbate or a polyoxyethylene fatty ether). In some embodiments, the second agent is a polysorbate. In some embodiments the polysorbate is polyoxyethylene (20) sorbitan monooleate (Tween® 80 or polysorbate 80), or polyoxyethylene (20) sorbitan monolaurate (polysorbate 20 or Tween® 20).|0174] Another example of a non-ionic surfactant is the family of polyoxyethylene fatty ethers, commonly referred to by their tradename Brij® surfactants. Brij® surfactants contain a polyethylene oxide polar head group and a hydrocarbon hydrophobic chain derived from astraight or branched hydrocarbon chain alcohol. Examples of alcohols used to prepare Brij® surfactants include: oleyl alcohol (O, Cl 8, one unsaturation), stearyl alcohol (S, Cl 8, saturated), cetyl alcohol (C, Cl 6, saturated), and lauryl alcohol (L, Cl 2, saturated). Some Brij® surfactants are derived from a mixture of fatty alcohols, for example cetyl alcohol and stearyl alcohol.

[0175] Brij® surfactants have the general chemical formula:R–(OCH2CH2)n–OHwhere R represents the hydrocarbon chain of the parent alcohol and n is the number of ethylene oxide units. In some embodiments, R may have a chain length of 12 to 18 carbons and / or n may be 2 to 100. Brij® molecules as described herein may be referred to by the parent alcohol and the number of ethylene oxide units. For instance, Brij® 010, refers to a Brij® molecule derived from oleyl alcohol (O), and having 10 ethylene oxide units. Brij® 020 refers to a Brij® molecule derived from oleyl alcohol (O), and having 20 ethylene oxide units. Brij® C20 refers to a Brij® molecule derived from cetyl alcohol (C), and having 20 ethylene oxide units. In some embodiments, the second agent is a polyoxyethylene fatty ether.[0176| Other examples of the second agent include poloxamers (e.g., P188, P237, P338, P407), alkyl sulfates (e.g., dodecyl sulfate) and salts thereof (e.g., sodium dodecyl sulfate (SDS)), fatty acids (e.g., lauric acid, myristic acid, palmitic acid, stearic acid), fatty alcohols (e.g., lauryl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol), phospholipids, and other lipids or derivatives thereof exhibiting surfactant behavior.

[0177] In some embodiments, the second agent can be the therapeutic peptide, such as one or more incretin mimetics (e.g., GLP-1 RAs) and / or other therapeutic peptides disclosed herein. In such embodiments, the second agent may not include a surfactant and / or a cyclic polysaccharide. While in some formulations disclosed herein the use of a surfactant (such as Tween® 20 or others) to disrupt Sangelose® polymer networks can be beneficial, certain therapeutic peptides, including lipidated peptides such as semaglutide, have a similar effect and thus obviate the need to include such surfactants. The mechanism by which lipidated peptides can modulate network viscoelasticity is discussed in greater detail below.10178] The hydrophilic-lipophilic balance (HLB), often used to describe surfactants or emulsifiers, may be used to describe the amphiphilic character of a molecule and is mainly based on the respective sizes / structure of the hydrophobic and hydrophilic groups of the molecule. For instance, the size and length of the fatty acid or polyethylene oxide groupsinfluence the amphiphilic character and corresponding HLB value. HLB is a measure of a molecule’s degree of hydrophilicity or lipophilicity and is calculated from the weight percentage of the hydrophilic groups to the hydrophobic groups in a molecule, with values ranging from 1–20. Molecules with low HLB values are more oil-soluble (lipophilic), while those with higher values are more water-soluble (hydrophilic).

[0179] Table 1 lists the HLB values of non-limiting, exemplary Span®, Tween®, and Brij® surfactants.10180] Table 1.Exemplary surfactants HLBSpan® 20 8.6Span® 40 6.7Span® 80 4.3Tween® 20 16.7Tween® 40 15.6Tween® 80 15.0Brij® C2 5.3Brij® C20 12.9Brij® 05 9.1Brij® O10 12.4Brij® 020 15.3

[0181] In some embodiments, the second agent has a hydrophilic-lipophilic balance (HLB) value of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, the HLB value is at least 9. In some embodiments, the HLB value is at least 14.

[0182] In some embodiments, the second agent is a non-ionic surfactant having a HLB value of at least 12, at least 13, or at least 14. In some embodiments, the second agent is a polysorbate having a HLB value of at least 12, at least 13, or at least 14. In some embodiments, the second agent is a Brij® molecule having a HLB value of at least 10, at least 11, at least 12, at least 13, or at least 14.

[0183] In some embodiments, the second agent is a cyclic polysaccharide, and the cyclic polysaccharide is a cyclodextrin. As used herein, a cyclodextrin consists of a macrocyclic ring of glucose subunits joined by alpha-1,4 glycosidic bonds. Cyclodextrins havefive or more glucose monomers linked in a ring forming the shape of a hollow truncated cone. The exterior of the cone is hydrophilic and the cavity of the cone is hydrophobic or lipophilic. This structure permits the cyclodextrin to be water soluble while permitting the ability to accommodate hydrophobic molecules / groups in the cavity; for instance, hydrophobic hydrocarbon chains of the first agent. An example of a cyclodextrin derived from 6 glucose subunits, an a-cyclodextrin (aCD) is depicted below:10184] Examples of cyclodextrins include but are not limited to a-cyclodextrin (aCD, 6 glucose monomers), P-cyclodextrin (PCD, 7 glucose monomers), and y-cyclodextrin (yCD, 8 glucose monomers). In some embodiments, the second agent is water soluble while having some affinity for hydrophobic groups such as hydrocarbon side chains of the first agent. Such affinity permits interaction between the first and second agent. For instance, the second agent may physically disrupt hydrocarbon chain packing. In some embodiments, the cyclic polysaccharide comprises a ring of five to ten glucose monomers. In some embodiments, the cyclic polysaccharide comprises a ring of five to eight glucose monomers. In some embodiments, the cyclic polysaccharide comprises a ring of five to six glucose monomers. In some embodiments, the cyclic polysaccharide comprises a ring of six glucose monomers. In some embodiments, the cyclic polysaccharide is aCD. In some embodiments, the cyclic polysaccharide is PCD. In some embodiments, the cyclic polysaccharide is yCD.[01851 In some embodiments, the second agent is an organic solvent that is miscible with water but also has sufficient hydrophobicity to interact with the hydrophobic chains of the first agent. Example of such organic solvents include propylene glycol (PG), ethanol, glycerol,triacetin, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAC), and N-methyl-2-pyrrolidone (NMP).

[0186] In some embodiments, the second agent is an acylated peptide that is attached to a lipophilic substituent that interacts with the hydrophobic chains of the first agent. In such embodiments, the second agent may also serve as a therapeutic agent in the hydrogel, e.g., the acylated peptide may be the only therapeutic agent or there may be additional therapeutic agents besides the acylated peptide. The lipophilic substituent of the acylated peptide can have a plurality of carbon atoms, such as at least 10, 15, 20, 25, 30, 35, or 40 carbon atoms. In some embodiments, the lipophilic substituent is an acyl group of a fatty acid, such as a straight chain fatty acid or a branched fatty acid. The fatty acid can be a fatty monoacid, e.g., an aliphatic monocarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a fatty diacid, e.g., an aliphatic dicarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a C4 to C38 fatty acid, such as a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a CIO fatty acid, a C12 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.

[0187] In some embodiments, the second agent has a hydrophilic-lipophilic balance (HLB) value of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15, and interacts with the hydrophobic chains of the first agent and modulates the hydrophobic chain interactions. In some embodiments the second agent forms a mixed micelle with the first agent. In some embodiments, the first agent forms micellar structures and the second agent is dispersed within the micellar structures to form at least one mixed micelle. In some embodiments, the second agent non-covalently interacts with the hydrophobic side chains of the first agent (e.g., HPMC-C18). In some embodiments, the non-covalent interaction modulates organization or packing of the hydrophobic side chains of the first agent.

[0188] In some embodiments, the first agent is Sangelose® (hydrophobically modified hydroxypropyl methylcellulose, HPMC-C18) and the second agent is a lipidated peptide such as semaglutide. The Sangelose®-based dynamic hydrogel system can be designed to deliver lipidated peptides such as semaglutide through reversible molecular interactions and amphiphilic self-assembly. Sangelose® contains long alkyl (Cl 8) chains that impart hydrophobic domains within the otherwise hydrophilic polymer network. These hydrophobicregions enable noncovalent and dynamic interactions, primarily hydrophobic association and van der Waals forces, with the lipid moiety of semaglutide.

[0189] Upon mixing, semaglutide molecules can self-assemble into micellar aggregates stabilized by Sangelose® chains that act as both steric stabilizers and structural scaffolds. The resulting network exhibits dynamic and reversible cross-linking, allowing controlled diffusion of peptide molecules while maintaining gel integrity. This amphiphilic association not only enhances drug loading and uniform dispersion but also contributes to the modulation of burst release and prolonged sustained release through equilibrium between bound and free semaglutide within the hydrogel matrix. Together, these molecular interactions underpin the controlled pharmacokinetic behavior of the Sangelose®-based depot system, offering a tunable platform for the long-acting delivery of lipidated peptide therapeutics.

[0190] In some embodiments, the first agent is a hydrophobically modified polysaccharide (e.g., HPMC-C18) and the second agent is a surfactant (e.g., Tween® 20 or Tween® 80), a cyclic polysaccharide (e.g., cyclodextrin), an organic solvent (e.g., PG), or an acylated peptide. In some embodiments, the first agent is a hydrophobically modified polysaccharide (e.g., HPMC-C18) and the second agent includes an organic solvent (e.g., PG) and an acylated peptide. In some embodiments, the first agent is a hydrophobically modified polysaccharide (e.g., HPMC-C18); and the second agent includes a surfactant (e.g., Tween® 20 or Tween® 80), an organic solvent (e.g., PG), and an acylated peptide.[0191| The concentration of the first agent in the hydrogel composition can be varied to produce the desired hydrogel properties (e.g., stiffness, storage modulus, degradation rate). In some embodiments of the hydrogel compositions described herein, the concentration of the first agent is at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.75 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%; and / or the concentration of the second agent is at least 0.02 wt%, at least 0.1 wt%, at least 0.5 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, or at least 5 wt%.

[0192] Alternatively or in combination, the concentration of the first agent is 0.1 wt% to 5 wt%, 0.1 wt% to 4 wt%, 0.1 wt% to 3 wt%, 0.1 wt% to 2 wt%, 0.1 wt% to 1 wt%, 0.1 wt% to 0.5 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5wt%; and / or the concentration of the second agent is 0.02 wt% to 5 wt%, 0.02 wt% to 4 wt%, 0.02 wt% to 3 wt%, 0.02 wt% to 2 wt%, 0.02 wt% to 1 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 4 wt%, 0.1 wt% to 3 wt%, 0.1 wt% to 2 wt%, 0.1 wt% to 1 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, or 0.5 wt% to 1 wt%.[01931 Alternatively or in combination, a ratio of the concentration of the first agent to the concentration of the second agent can be greater than or equal to 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, or 30:1; and / or less than or equal to 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, or 1: 1. The ratio of the concentration of the first agent to the concentration of the second agent can be 1:1 to 10:1, 1.5:1 to 5:1, or 2:1 to 4:1.C. Hydrogel Properties

[0194] The dynamic hydrogels described herein (e.g., the PNP hydrogels of Section I. A, the PXY hydrogels of Section I. B) can exhibit favorable physical and biological properties that contribute to their efficacy as drug delivery platforms. The properties of the dynamic hydrogels herein can be tuned in various ways, such as by modifying the types of components used to form the hydrogel (e.g., polymers, nanoparticles, and / or additional components as previously described in Section I. A; first agent and second agent as previously described in Section I. B., and / or the therapeutic cargo carried by the hydrogel as described below in Section II), the concentrations of the components, and / or the chemical functionalities of the components. Accordingly, the properties of the dynamic hydrogels herein can be adapted to the particular therapeutic application, such as forming a stable and / or persistent depot when delivered in vivo, providing a desired release profile for the therapeutic cargo (e.g., short-term release versus long-term release), providing a desired release mechanism for the therapeutic cargo (e.g., diffusion-based release versus erosion-based release), compatibility with a desired route of administration (e.g., injecting, infusing, spraying, spreading), biodegradability, biocompatibility, and / or allowing for cellular infiltration. Any reference herein to a property of a dynamic hydrogel may refer to the property of the dynamic hydrogel without any therapeutic cargo (e.g., a PNP hydrogel composed only of polymers and nanoparticles; a PXY hydrogel composed only of the first agent and the second agent, where the second agent is not a therapeutic agent), the property of the dynamic hydrogel including the therapeutic cargo (e.g., a PNP hydrogel including polymers, nanoparticles, and the encapsulated therapeutic cargo; a PXY hydrogel including the first agent, the second agent, and the encapsulated therapeuticcargo (which may also serve as the second agent)), or both, unless otherwise stated or otherwise evident from the context.

[0195] The storage modulus (G') of the dynamic hydrogel can correlate to the overall stiffness of the hydrogel, which in turn can dictate the time scale of degradation of the hydrogel (e.g., hydrogels having a higher storage modulus may be stiffer and degrade more slowly than gels having a lower storage modulus). Accordingly, in embodiments where the therapeutic cargo of the dynamic hydrogel is released primarily or entirely via an erosion-based mechanism, the release rate of the therapeutic cargo can be tuned by adjusting the storage modulus of the hydrogel (e.g., a higher storage modulus can produce a slower degradation rate and thus a slower release rate of the therapeutic cargo, while a lower storage modulus can produce a higher degradation rate and thus a faster release rate of the therapeutic cargo). For example, in embodiments where the dynamic hydrogel is a PNP hydrogel, the storage modulus of the PNP hydrogel can be increased or decreased by increasing or decreasing the polymer concentration, and / or by increasing or decreasing the nanoparticle concentration. As another example, in embodiments where the dynamic hydrogel is a PXY hydrogel, the storage modulus of the PXY hydrogel can be increased or decreased by increasing or decreasing the concentration of the first agent (e.g., HPMC-C18).

[0196] In some embodiments, the dynamic hydrogels herein have a storage modulus within a range from 1 Pa to 10,000 Pa, 1 Pa to 5000 Pa, 1 Pa to 2500 Pa, 1 Pa to 1000 Pa, 1 Pa to 500 Pa, 1 Pa to 200 Pa, 1 Pa to 10 Pa, 10 Pa to 10,000 Pa, 10 Pa to 5000 Pa, 10 Pa to 2500 Pa, 10 Pa to 1000 Pa, 10 Pa to 500 Pa, 10 Pa to 200 Pa, 10 Pa to 100 Pa, 10 Pa to 50 Pa, 50 P to 10,000 Pa, 50 Pa to 5000 Pa, 50 Pa to 2500 Pa, 50 Pa to 1000 Pa, 50 Pa to 500 Pa, 50 Pa to 200 Pa, 50 Pa to 100 Pa, 100 Pa to 10,000 Pa, 100 Pa to 5000 Pa, 100 Pa to 2500 Pa, 100 Pa to 1000 Pa, 100 Pa to 500 Pa, 100 Pa to 200 Pa, 200 Pa to 10,000 Pa, 200 Pa to 5000 Pa, 200 Pa to 2500 Pa, 200 Pa to 1000 Pa, 200 Pa to 500 Pa, 500 Pa to 10,000 Pa, 500 Pa to 5000 Pa, 500 Pa to 2500 Pa, 500 Pa to 1000 Pa, 1000 Pa to 10,000 Pa, 1000 Pa to 5000 Pa, 1000 Pa to 2500 Pa, 2500 Pa to 10,000 Pa, 2500 Pa to 5000 Pa, or 5000 Pa to 10,000 Pa. The storage modulus can be measured, for example, using an oscillatory shear test in a parallel plate rheometer at an angular frequency of 10 rad / s, a strain within the linear viscoelastic region of the hydrogel (e.g., 1% strain), and a temperature of 25 °C.

[0197] The yield stress (τy) of the dynamic hydrogel can correlate to the ability of the hydrogel to form and maintain a cohesive depot in vivo (e.g., materials lacking a yield stress may flow rather than forming a cohesive depot). The dynamic hydrogels herein can exhibitlittle or no flow when subjected to stresses below the yield stress. When subjected to stresses above the yield stress, the dynamic hydrogels can flow, corresponding to a significant drop in observed viscosity (e.g., a decrease of at least one or two orders of magnitude). In embodiments where the dynamic hydrogel is a PNP hydrogel, the yield stress can be increased or decreased by increasing or decreasing the nanoparticle concentration, respectively. In embodiments where the dynamic hydrogel is a PXY hydrogel, the yield stress can be by increasing the concentration of the first agent (e.g., HPMC-C18) and / or by decreasing the concentration of the second agent (e.g., surfactant, cyclic polysaccharide, or therapeutic peptide); conversely, the yield stress can be decreased by decreasing the concentration of the first agent (e.g., HPMC-C18) and / or by increasing the concentration of the second agent (e.g., surfactant, cyclic polysaccharide, and / or therapeutic peptide).10198] In some embodiments, the dynamic hydrogels herein have a yield stress within a range from 0.1 Pa to 1000 Pa, 0.1 Pa to 500 Pa, 0.1 Pa to 200 Pa, 0.1 Pa to 100 Pa, 0.1 Pa to 50 Pa, 0.1 Pa to 20 Pa, 0.1 Pa to 10 Pa, 0.1 Pa to 1 Pa, 1 Pa to 1000 Pa, 1 Pa to 500 Pa, 1 Pa to 200 Pa, 1 Pa to 100 Pa, 1 Pa to 50 Pa, 1 Pa to 10 Pa, 10 Pa to 1000 Pa, 10 Pa to 500 Pa, 10 Pa to 200 Pa, 10 Pa to 100 Pa, 10 Pa to 50 Pa, 10 Pa to 20 Pa, 20 Pa to 1000 Pa, 20 Pa to 500 Pa, 20 Pa to 200 Pa, 20 Pa to 100 Pa, 20 Pa to 50 Pa, 50 Pa to 1000 Pa, 50 Pa to 500 Pa, 50 Pa to 200 Pa, 50 Pa to 100 Pa, 100 Pa to 500 Pa, 100 Pa to 200 Pa, 200 Pa to 1000 Pa, 200 Pa to 500 Pa, or 500 Pa to 1000 Pa. The yield stress can be measured, for example, using a stress ramp or stress sweep (e.g., from 1 Pa to 100 Pa, or from 1 Pa to 1000 Pa) in a parallel plate rheometer at a temperature of 25 °C to identify the stress at which the hydrogel exhibits a drop in viscosity.

[0199] The tan delta of the dynamic hydrogel (the ratio of the loss modulus (G") over the storage modulus (G1) (tan(6) = G" / G')) can describe the overall viscoelasticity of the hydrogel (e.g., lower tan delta values correspond to more solid-like behavior, higher tan delta values correspond to more liquid-like behavior), and can correlate to the degradation rate of the hydrogel. In some embodiments, the dynamic hydrogels herein have a tan delta less than or equal to 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. The tan delta can be within a range from 0.1 to 1, 0.1 to 0.5, 0.1 to 0.3, 0.2 to 1, 0.2 to 0.5, or 0.5 to 1. The tan delta can be measured, for example, using an oscillatory shear test in a parallel plate rheometer at an angular frequency of 10 rad / s, a strain within the linear viscoelastic region of the hydrogel (e.g., 1% strain), and a temperature of 25 °C.

[0200] In some embodiments, the dynamic hydrogels described herein exhibit viscoelastic behavior, wherein the storage modulus (G') of the hydrogel is equal to the lossmodulus (G") at a crossover point, for example, a crossover frequency as observed in an oscillatory frequency sweep measurement in a range from 0.1 rad·s-1to 100 rad·s-1on an oscillatory rheometer performed in the linear viscoelastic region at a temperature of 25 °C. In embodiments where the dynamic hydrogel is a PXY hydrogel, the crossover point of the PXY hydrogel including both the first agent and the second agent is greater than the crossover point of a hydrogel including the first agent only (e.g., the crossover frequency of a hydrogel with the first and second agents is shifted toward higher frequencies in an oscillatory frequency sweep measurement compared to the crossover point of a composition with the first agent but not the second agent). In some embodiments, the crossover frequency of a PXY hydrogel including both the first agent and the second agent is greater than or equal to 0.001 rad·s-1, 0.01 rad·s-1, or 0.1 rad·s-1, e.g., as measured in an oscillatory frequency sweep measurement in a parallel plate rheometer in the linear viscoelastic region at a temperature of 25 °C. The crossover frequency is inversely correlated to the relaxation time of the hydrogel, which is indicative of the dynamic or static nature of the composition (e.g., longer relaxation times correspond to more static materials, while shorter relaxation times correspond to more dynamic materials).

[0201] In some embodiments, the dynamic hydrogels herein exhibit shear-thinning behavior, in that the viscosity of the dynamic hydrogel decreases with increasing shear rate and / or shear stress. Shear-thinning behavior can be advantageous, for example, to allow the dynamic hydrogel to be administered via injection. In some embodiments, the viscosity of the gel decreases with increasing shear rate at a shear rate within a range from 0.1 s-1to 1000 s-1, for example, as observed on an oscillatory rheometer (e.g., a parallel plate rheometer) at 25 °C. In some embodiments, the dynamic hydrogels herein have a viscosity within a range from 10 mPa-s to 2000 mPa-s, 10 mPa-s to 1000 mPa-s, 10 mPa-s to 500 mPa-s, 10 mPa-s to 200 mPa-s, 10 mPa-s to 100 mPa-s, 10 mPa-s to 50 mPa-s, 50 mPa-s to 2000 mPa-s, 50 mPa-s to 1000 mPa-s, 50 mPa-s to 500 mPa-s, 50 mPa-s to 200 mPa-s, 50 mPa-s to 100 mPa-s, 100 mPa-s to 2000 mPa-s, 100 mPa-s to 1000 mPa-s, 100 mPa-s to 500 mPa-s, 100 mPa-s to 200 mPa-s, 200 mPa-s to 2000 mPa-s, 200 mPa-s to 1000 mPa-s, 200 mPa-s to 500 mPa-s, 500 mPa-s to 2000 mPa-s, 500 mPa-s to 1000 mPa-s, or 1000 mPa-s to 2000 mPa-s at a shear rate of 1000 s-1. The viscosity can be measured, for example, using steady shear measurements in a parallel plate rheometer at a temperature of 25 °C.

[0202] In some embodiments, the dynamic hydrogels herein exhibit self-healing behavior. Self-healing may refer to a process in which a gel that exhibits reduced resistance toflow when subjected to an external stress regains some or all of its rigidity and / or strength after the external stress is removed. Self-healing behavior can be advantageous, for example, to allow the dynamic hydrogel to form a cohesive depot after administration via injection and / or to limit burst release. In some embodiments, the dynamic hydrogels herein stop flowing and recover their mechanical properties in no more than 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or 10 minutes after the external stress is removed. Optionally, the modulus and / or viscosity of the dynamic hydrogel can recover to at least 90% of the initial value before application of the external stress within 5 minutes in a step-strain measurement (conducted with strains of 0.5% and 500%) or step-shear measurement (conducted with shear rates of 0.1 s-1and 100-1), respectively, on an oscillatory rheometer.

[0203] In some embodiments, the dynamic hydrogels herein exhibit viscoelastic behavior, in that the storage modulus (G’) of the hydrogel is dominant over the loss modulus (G”) at some point, for example, as observed in an oscillatory frequency sweep measurement in a range from 0.1 rad / s to 100 rad / s on an oscillatory rheometer performed in the linear viscoelastic region, yet the hydrogel exhibits complete stress relaxation following application of a constant strain of 500% within 15 minutes.

[0204] In some embodiments, the dynamic hydrogels described herein are biocompatible. A biocompatible material can be a material that is, along with any metabolites or degradation products thereof, generally non-toxic to the subject, and do not cause any significant adverse effects to the subject, at concentrations resulting from the degradation of the administered materials. A biocompatible material can be a material that does not elicit a significant inflammatory or immune response when administered to a subject.

[0205] In some embodiments, the dynamic hydrogels described herein are biodegradable. A biodegradable material can be a material that will degrade or erode under physiological conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. For example, upon in vivo administration to a subject, the dynamic hydrogel can dissolve as the non-covalent bonds dissociate. The degradation rate of the dynamic hydrogel can be varied as desired, e.g., depending on the desired release profile for the therapeutic cargo. In some embodiments, following in vivo administration, the dynamic hydrogels are designed to persist at the administration site (e.g., remain as a cohesive depot) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months. Alternatively or incombination, the dynamic hydrogels herein can persist at the administration site for no more than 12 months, 9 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 28 days, 21 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.II. Compositions for Delivery of Therapeutic Peptides and Associated Methods

[0206] In some embodiments, the present technology provides compositions for delivery of therapeutic peptides for treating a disease or condition in a subject. The composition can include a dynamic hydrogel and at least one therapeutic peptide carried by the dynamic hydrogel. The dynamic hydrogel can encapsulate the therapeutic peptide and provide sustained, controlled release of the therapeutic peptide when the composition is administered to a subject. In some embodiments, the dynamic hydrogel exhibits shear-thinning behavior that allows for facile administration via injection, as well as self-healing behavior that allows for formation of a cohesive depot that delivers the therapeutic peptide over a prolonged treatment period.

[0207] For example, FIG. 1 A is a schematic illustration of a PNP hydrogel prepared by mixing of hydrophobically-modified HPMC with PEG-PLA nanoparticles that allows for facile encapsulation of a therapeutic peptide, and FIG. 1B is a schematic illustration of formation of a localized depot in the subcutaneous space following subcutaneous injection of the PNP hydrogel, thus providing a tunable platform for sustained release of the therapeutic peptide. Although FIGS. 1A and IB illustrate a depot formed from a PNP hydrogel, it will be appreciated that a depot may formed from other types of dynamic hydrogels described herein, such as a PXY hydrogel (e.g., a Sangelose® hydrogel).A. Therapeutic Peptides1. Incretin Mimetics

[0208] In some embodiments, the therapeutic peptide is an incretin mimetic. Incretin mimetics are compounds that mimic the activity of incretin hormones. Incretin hormones are peptides that are secreted by the gut in response to nutrient ingestion. The primary incretin hormones are GLP-1 and gastric inhibitory peptide (GIP). GLP-1 contributes to the regulation of glucose homeostasis within the body through its interaction with the GLP-1 receptor. GLP-1 is secreted from intestinal L-cells in response to nutrients and lowers blood glucose by stimulating insulin and suppressing glucagon secretion in a glucose-dependent manner, reducing the risk of hypoglycemia. In addition, GLP-1 is also a neurotransmitter synthesizedby preproglucagon neurons in the brain and acts via central pathways to lower energy intake through an effect on satiety, hunger, and reward-related measures, leading to a lowering of body weight. Metabolic effects of GLP-1 include glucose-dependent stimulation of insulin secretion, inhibition of glucagon secretion, inhibition of food intake, decrease of gastric emptying, and increase of natriuresis and diuresis. GLP-1 has also been shown to influence learning, memory, reward behavior, and palatability, as well as to exhibit neuroprotective, cardioprotective, and anti-inflammatory effects. However, the therapeutic applicability of native GLP-1 is limited by its short half-life in vivo (approximately 2 to 3 minutes) and inactivation by the enzyme dipeptidyl peptidase 4 (DPP4). GIP is secreted by enteroendocrine K-cells in response to nutrients and also exhibits an insulinotropic effect via binding to the GIP receptor. However, unlike GLP-1, GIP stimulates glucagon secretion. GIP also influences appetite, fat accumulation, memory, and bone formation. Native GIP also exhibits a short halflife (approximately 4 to 5 minutes) and is inactivated by DPP4.

[0209] In some embodiments, the incretin mimetic is a GLP-1 RA. GLP-1 RAs (also known as “GLP-1 analogues”) are a class of drugs that interact with the GLP-1 receptor and display structural similarities to native GLP-1, but with modifications to extend the in vivo halflife and thus provide improved bioavailability. GLP-1 RAs can be categorized as either shortacting or long-acting compounds. Short-acting GLP-1 RAs have been rendered resistant to cleavage by DPP4 by altering the amino acids at the second and third N-terminal positions, but are still subject to renal elimination and thus generally have a half-life from approximately 2 to 5 hours. Examples of short-acting GLP-1 RAs include exenatide and lixisenatide. Long-acting GLP-1 RAs implement mechanisms to reduce renal elimination, such as acylation with fatty acids to facilitate binding to serum albumin or conjugation to a larger molecule / component, and thus can have a half-life from 12 hours to several days. Examples of long-acting GLP-1 RAs include liraglutide (acylation with C16 fatty monoacid), semaglutide (acylation with Cl 8 fatty diacid), tirzepatide (acylation with C20 fatty diacid), retatrutide (acylation with C20 fatty diacid), albiglutide (conjugation to albumin), dulaglutide (conjugation to Fc fragment of IgG), and exenatide-LAR (long-acting release) (coupled to biodegradable polymer microspheres).

[0210] The GLP-1 RA can include a peptide that binds to the GLP-1 receptor. The peptide can be an analogue of a native GLP-1 peptide, such as the endogenous human GLP-1 peptide (e.g., GLP-1 (7-36) or GLP-1 (7-37)). For example, the peptide of the GLP-1 RA can include a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or100% sequence identity to any one of SEQ ID NO: 1 or SEQ ID NO: 2. The peptide can be produced in suitable host cells via recombinant DNA technology, can be produced in a cell-free system, or can be produced synthetically via solid phase synthesis.

[0211] Table 2: GLP-1 PeptidesPeptide SEQ ID NO SequenceEndogenous SEQ ID NO: 1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR human GLP-1peptide (7-36)Endogenous SEQ ID NO: 2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG human GLP-1peptide (7-37)

[0212] In some embodiments, the GLP-1 RA is a mono-receptor agonist that binds exclusively to the GLP-1 receptor. Examples of GLP-1 mono-receptor agonists include exenatide, exenatide-LAR, lixisenatide, liraglutide, semaglutide, albiglutide, dulaglutide, efpeglenatide, and ecnoglutide. The peptide of the GLP-1 mono-receptor agonist can be an analogue of a native GLP-1 peptide, as previously described.

[0213] In some embodiments, the GLP-1 RA is a dual-receptor agonist that binds to the GLP-1 receptor and an additional receptor. The additional receptor can be any of the following receptors: a glucagon receptor, a GIP receptor, a cholecystokinin receptor, a xenin receptor, a secretin receptor, a neuropeptide Y receptor, or a neurotensin receptor. For instance, the GLP-1 RA can be a dual GLP-l / glucagon receptor agonist that binds to the GLP-1 receptor and the glucagon receptor. Examples of dual GLP-l / glucagon receptor agonists include efinopegdutide, cotadutide, mazdutide, and BI 45690. In such embodiments, the peptide of the dual GLP-l / glucagon receptor agonist can be an analogue of oxyntomodulin, which is a gut hormone that activates both the GLP-1 receptor and the glucagon receptor. As another example, the GLP-1 RA can be a dual GLP-l / GIP receptor agonist that binds to the GLP-1 receptor and the GIP receptor. Examples of dual GLP-l / GIP receptor agonists include tirzepatide, LY3493269, VK2735, CT-868, and AMG133. In such embodiments, the peptide of the dual GLP-l / GIP receptor agonist can be an analogue of GIP, which has high sequence similarity to GLP-1 in the N-terminal part of the peptide.

[0214] In some embodiments, the GLP-1 RA is a triple-receptor agonist that binds to the GLP-1 receptor and two additional receptors. The additional receptors can be any two of the following receptors: a glucagon receptor, a GIP receptor, a cholecystokinin receptor, a xenin receptor, a secretin receptor, a neuropeptide Y receptor, or a neurotensin receptor. For example, the GLP-1RA can be a triple GLP-l / glucagon / GIP receptor agonist that binds to the GLP-1 receptor, the glucagon receptor, and the GIP receptor. Examples of triple GLP-l / glucagon / GIP receptor agonists include retatrutide.

[0215] The peptide of the GLP-1 RA can be attached to at least one substituent (also referred to herein as a “side chain”). The substituent can prolong the half-life of the peptide in vivo, such as by binding of the substituent to serum albumin. Optionally, the substituent can provide other beneficial effects, such as enhancing solubility, promoting cellular uptake, and / or reducing immunogenicity. The substituent of the GLP-1 RA can be attached to the peptide of the GLP-1 RA via any suitable mechanism, such as acylation, alkylation, ester formation, amide formation, coupling to a cysteine residue, and / or other conjugation chemistries known to those of skill in the art. For example, the substituent can be covalently attached to the peptide via an amide bond between a carboxyl group of the substituent and an amino group of the peptide. The amino group of the peptide can be the N-terminal amino group of the peptide or can be a side chain amino group of an amino acid residue of the peptide (e.g., an amino group of a lysine residue of the peptide). The substituent can be attached to the peptide directly, or can be attached to the peptide indirectly via a linker, which may also be referred to herein as a spacer. For instance, the substituent can be attached to the peptide via an amide bond between a carboxyl group of the linker and an amino group of an amino acid residue of the peptide. The linker can be any suitable linker known to those of skill in the art, such as a peptide linker (e.g., a y-glutamate linker), a hydrophilic spacer (e.g., 8-amino-3,6-dioxaoctanoic acid), a hydrophobic spacer, or combination thereof.|0216] For example, the GLP-1 RA can be an acylated peptide that is attached to a lipophilic substituent having a plurality of carbon atoms, such as at least 10, 15, 20, 25, 30, 35, or 40 carbon atoms. In some embodiments, the lipophilic substituent is an acyl group of a fatty acid, such as a straight chain fatty acid or a branched fatty acid. The fatty acid can be a fatty monoacid, e.g., an aliphatic monocarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a fatty diacid, e.g., an aliphatic dicarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a C4 to C38 fatty acid, such as a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a CIO fatty acid,a C12 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a Cl 8 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.

[0217] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as CH3(CH2)4CO-, CH3(CH2)6CO-, CH3(CH2)8CO-, CH3(CH2)10CO-, CH3(CH2)12CO-, CH3(CH2)14CO-, CH3(CH2)16CO-, CH3(CH2)18CO-, CH3(CH2)20CO-, CH3(CH2)22CO-, or CH3(CH2)24CO-.

[0218] In some embodiments, the lipophilic substituent is an acyl group having the formula HOOC(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as HOOC(CH2)14CO-, HOOC(CH2)16CO-, HOOC(CH2)18CO-, HOOC(CH2)20CO-, or HOOC(CH2)22CO-.

[0219] In some embodiments, the lipophilic substituent is an acyl group of a straightchain or branched alkane a, co-dicarboxylic acid.

[0220] In some embodiments, the lipophilic substituent is an acyl group having the formula HOOC(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as HOOC(CH2)16CO-, HOOC(CH2)18CO-, HOOC(CH2)2OCO-, or HOOC(CH2)22CO.

[0221] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-NHCH(COOH)(CH2)2CO-, where n is an integer from 10 to 24.

[0222] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-NHCH((CH2)2COOH)CO-, where n is an integer from 8 to 24.

[0223] In some embodiments, the lipophilic substituent is an acyl group having the formula COOH(CH2)nCO-, where n is an integer from 8 to 24.

[0224] In some embodiments, the lipophilic substituent is an acyl group having the formula -NHCH(COOH)(CH2)4NH-CO(CH2)nCH3, where n is an integer from 8 to 18.

[0225] The lipophilic substituent can be attached to the peptide of the GLP-1 RA via a linker, as described herein. In some embodiments, the lipophilic substituent interacts with a component of the dynamic hydrogel to promote encapsulation and controlled release of the GLP-1 RA from the dynamic hydrogels, as described further below.

[0226] In some embodiments, a composition of the present technology includes a dynamic hydrogel and an incretin mimetic encapsulated by the dynamic hydrogel. The incretinmimetic can be a GLP-1 RA, such as one or more of exenatide, exenatide-LAR, lixisenatide, liraglutide, semaglutide, albiglutide, dulaglutide, efpeglenatide, ecnoglutide, efinopegdutide, cotadutide, mazdutide, BI 45690, tirzepatide, LY3493269, VK2735, CT-868, AMG133, or retatrutide. In some embodiments, the incretin mimetic is a GLP-1 RA that is an acylated peptide, such as one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide. Additional examples of GLP-1 RAs and incretin mimetics are provided in International Patent Application Publication Nos. WO 2005 / 027978 and WO 2014 / 005858, the disclosures of each of which are incorporated by reference herein in their entirety. Optionally, the composition can include a combination of two or more different incretin mimetics, such as two or more of any of the incretin mimetics disclosed herein.

[0227] Optionally, the compositions herein can include other therapeutic cargo carried by the dynamic hydrogel, in addition to the incretin mimetic. The other therapeutic cargo can include one or more therapeutic agents that produce a desired therapeutic effect, such as small molecule drugs, peptides, proteins, polysaccharides, nucleic acids, cells, or combinations thereof. In some embodiments, the therapeutic agent(s) act in concert with the incretin mimetic to treat the disease or condition, such as antidiabetic agents, antiobesity agents, appetite suppressants, and / or antihypertensive agents. Examples of such therapeutic agents include alpha-glucosidase inhibitors, amylin analogues (e.g., cagrilintide), biguanides, DPP4-inhibitors, glucagon antagonists, insulin and insulin analogues (e.g., insulin degludec, insulin detemir, insulin icodec, insulin glargine), meglitinides, SGLT-2 inhibitors, sulfonylureas, thiazolidinediones, and combinations thereof. Such therapeutic agents can be encapsulated in the dynamic hydrogel via physical entrapment, interactions with hydrogel components (e.g., hydrophobic interactions), or suitable combinations thereof. For instance, therapeutic agents that precipitate at physiological pH (e.g., insulin glargine) can be physically entrapped within the hydrogel. Optionally, the therapeutic agent(s) can be administered to the subject separately from the incretin mimetic via any suitable administration route (e.g., parenteral or non-parenteral administration).2. Acylated Peptides

[0228] Although certain embodiments of the compositions herein are described in connection with incretin mimetics such as GLP-1 RAs, this is not intended to be limiting, and the compositions of the present technology can be used to deliver other types of therapeutic peptides, such as acylated peptides. Acylation of peptides with a lipophilic substituent (e.g., afatty acid moiety) can produce improved pharmacokinetics compared to the native peptide via binding to serum albumin, while maintaining the activity of the native peptide. Other beneficial effects of acylation can include enhancing solubility, promoting cellular uptake, and / or reducing immunogenicity.

[0229] In some embodiments, a composition of the present technology includes a dynamic hydrogel and at least one acylated peptide encapsulated by the dynamic hydrogel. The acylated peptide can include a peptide that exhibits a therapeutic effect when administered to the subject, and at least one substituent that is attached to the peptide via acylation. The peptide can be a native peptide (e.g., having 100% sequence identity to the sequence of the endogenous human peptide), or can be an analogue with one or more modifications relative to the native peptide (e.g., having less than 100% sequence identity to the sequence of the endogenous human peptide). For instance, the native peptide sequence can be modified by substitution of one or more amino acids (e.g., with a natural or non-natural amino acid), addition of one or more amino acids (e.g., a natural or non-natural amino acid), deletion of one or more amino acids, or suitable combinations thereof. The peptide can be produced in suitable host cells via recombinant DNA technology, can be produced in a cell-free system, or can be produced synthetically via solid phase synthesis.

[0230] The lipophilic substituent of the acylated peptide can have a plurality of carbon atoms, such as at least 10, 15, 20, 25, 30, 35, or 40 carbon atoms. In some embodiments, the lipophilic substituent is an acyl group of a fatty acid, such as a straight chain fatty acid or a branched fatty acid. The fatty acid can be a fatty monoacid, e.g., an aliphatic monocarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a fatty diacid, e.g., an aliphatic dicarboxylic acid having 4 to 38 carbon atoms, which may be saturated or unsaturated. The fatty acid can be a C4 to C38 fatty acid, such as a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a CIO fatty acid, a C12 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a Cl 8 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.

[0231] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as CH3(CH2)4CO-, CH3(CH2)6CO-, CH3(CH2)8CO-, CH3(CH2)IOCO-, CH3(CH2)12CO-, CH3(CH2)14CO-, CH3(CH2)16CO-, CH3(CH2)18CO-, CH3(CH2)20CO-, CH3(CH2)22CO-, or CH3(CH2)24CO-.

[0232] In some embodiments, the lipophilic substituent is an acyl group having the formula HOOC(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as HOOC(CH2)14CO-, HOOC(CH2)16CO-, HOOC(CH2)18CO-, HOOC(CH2)20CO-, or HOOC(CH2)22CO-.

[0233] In some embodiments, the lipophilic substituent is an acyl group of a straightchain or branched alkane a, co-dicarboxylic acid.

[0234] In some embodiments, the lipophilic substituent is an acyl group having the formula HOOC(CH2)nCO-, where n is an integer from 4 to 38, or from 4 to 24, such as HOOC(CH2)16CO-, HOOC(CH2)18CO-, HOOC(CH2)20CO-, or HOOC(CH2)22CO.

[0235] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-NHCH(COOH)(CH2)2CO-, where n is an integer from 10 to 24.

[0236] In some embodiments, the lipophilic substituent is an acyl group having the formula CH3(CH2)nCO-NHCH((CH2)2COOH)CO-, where n is an integer from 8 to 24.

[0237] In some embodiments, the lipophilic substituent is an acyl group having the formula COOH(CH2)nCO-, where n is an integer from 8 to 24.

[0238] In some embodiments, the lipophilic substituent is an acyl group having the formula -NHCH(COOH)(CH2)4NH-CO(CH2)nCH3, where n is an integer from 8 to 18.

[0239] In some embodiments, the lipophilic substituent interacts with a component of the dynamic hydrogel to promote encapsulation and controlled release of the acylated peptide from the dynamic hydrogels, as described further below.

[0240] In some embodiments, the lipophilic substituent is covalently attached to the peptide via an amide bond between a carboxyl group of the substituent and an amino group of the peptide. The amino group of the peptide can be the N-terminal amino group of the peptide or a side chain amino group of an amino acid residue of the peptide (e.g., an amino group of a lysine residue). The lipophilic substituent can be attached to the peptide directly, or can be attached to the peptide indirectly via a linker. For instance, the lipophilic substituent can be attached to the peptide via an amide bond between a carboxyl group of the linker and an amino group of an amino acid residue of the peptide. The linker can be any suitable linker known to those of skill in the art, such as a peptide linker (e.g., an amino acid, a y-glutamate linker), a hydrophilic spacer (e.g., PEG, 8-amino-3,6-dioxaoctanoic acid (OEG)), a hydrophobic spacer, or combination thereof.

[0241] In some embodiments, the acylated peptide is an acylated analogue of a proglucagon-derived peptide. Proglucagon-derived peptides are a family of peptides that are derived from differential processing of a common prohormone, proglucagon, and include glucagon, GLP-1, glucagon-like peptide-2 (GLP-2), oxyntomodulin (OXM), glicentin, glicentin-related pancreatic peptide (GRPP), intervening peptide- 1 (IP-1), intervening peptide-2 (IP-2), and major proglucagon fragment (MPGF). These peptides exhibit a wide variety of physiological effects, including metabolism, energy regulation, cardioprotection, bone health, renal function, liver function, and cognition. The acylated analogue of the proglucagon peptide can include a peptide including a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the native proglucagon peptide, and a lipophilic substituent attached to the peptide, as described herein.

[0242] In some embodiments, the acylated peptide is an acylated amylin analogue, such as cagrilintide. Amylin is a hormone that is secreted by pancreatic P-cells in response to nutrient ingestion. Amylin signaling plays a role in the regulation of blood glucose by delaying gastric emptying, suppressing food intake, and inhibiting meal-related glucagon secretion, and is thus complementary to the activity of the incretin hormones. The acylated amylin analogue can include a peptide including a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the native amylin peptide, and a lipophilic substituent attached to the peptide, as described herein.

[0243] In some embodiments, the acylated peptide is an acylated insulin analogue, such as insulin degludec, insulin detemir, or insulin icodec. An acylated insulin analogue can be codelivered with an incretin mimetic for treatment of diabetes. The acylated insulin analogue can include a peptide including a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the native insulin peptide, and a lipophilic substituent attached to the peptide, as described herein.B. Compositions and Methods

[0244] In some embodiments, the present technology provides compositions including a dynamic hydrogel and one or more therapeutic peptides (e.g., an incretin mimetic and / or acylated peptide) encapsulated by the dynamic hydrogel. The dynamic hydrogel carrying the therapeutic peptide can be any of the dynamic hydrogels described in Section I above. For example, the dynamic hydrogel can be a PNP hydrogel composed of a polymer and a plurality of nanoparticles that interact non-covalently with each other, as previously discussed in SectionI. A. As another example, the dynamic hydrogel can be a PXY hydrogel composed of a first agent and a second agent that interact non-covalently with each other, as previously discussed in Section LB. The dynamic hydrogel can exhibit shear-thinning, self-healing, and / or viscoelastic properties resulting from non-covalent, supramolecular interactions between the hydrogel components, as described above in Section I. C.

[0245] The composition can include any suitable amount of the therapeutic peptide for providing the desired therapeutic effect. For example, the composition can include at least 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, or 400 mg of the therapeutic peptide. Alternatively or in combination, the composition can include no more than 500 mg, 400 mg, 300 mg, 275 mg, 250 mg, 225 mg, 200 mg, 175 mg, 150 mg, 125 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, or 2 mg of the therapeutic peptide. The amount of the therapeutic peptide in the composition can be within a range from 1 mg to 500 mg, 1 mg to 250 mg, 1 mg to 150 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 40 mg, 1 mg to 30 mg, 1 mg to 20 mg, 1 mg to 10 mg, 10 mg to 500 mg, 10 mg to 250 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, 10 mg to 40 mg, 10 mg to 30 mg, 10 mg to 20 mg, 20 mg to 500 mg, 20 mg to 250 mg, 20 mg to 150 mg, 20 mg to 100 mg, 20 mg to 50 mg, 20 mg to 40 mg, 20 mg to 30 mg, 30 mg to 500 mg, 30 mg to 250 mg, 30 mg to 150 mg, 30 mg to 100 mg, 30 mg to 50 mg, 30 mg to 40 mg, 40 mg to 500 mg, 40 mg to 250 mg, 40 mg to 150 mg, 40 mg to 100 mg, 40 mg to 50 mg, 50 mg to 500 mg, 50 mg to 250 mg, 50 mg to 150 mg, 50 mg to 100 mg, 100 mg to 500 mg, 100 mg to 250 mg, 100 mg to 150 mg, 150 mg to 500 mg, 150 mg to 250 mg, or 250 mg to 500 mg. The amount of the therapeutic peptide in the composition can be approximately 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 400 mg, or 500 mg.

[0246] In some embodiments, the therapeutic peptide is present in the composition at a concentration of at least 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 100 mg / mL, 120 mg / mL, or 150 mg / mL. Alternatively or in combination, the concentration of the therapeutic peptide in the composition is no more than 200 mg / mL, 150 mg / mL, 120 mg / mL, 100 mg / mL, 50 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, or 0.5 mg / mL. The concentration of the therapeutic peptide in the composition can be within a range from 0.5 mg / mL to 200 mg / mL, 0.5 mg / mL to 150 mg / mL,0.5 mg / mL to 120 mg / mL, 0.5 mg / mL to 100 mg / mL, 0.5 mg / mL to 50 mg / mL, 0.5 mg / mL to 20 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 0.5 mg / mL to 2 mg / mL, 0.5 mg / mL to 1.5 mg / mL, 0.5 mg / mL to 1 mg / mL, 1 mg / mL to 200 mg / mL, 1 mg / mL to 150 mg / mL, 1 mg / mL to 120 mg / mL, 1 mg / mL to 100 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 10 mg / mL, 1 mg / mL to 5 mg / mL, 1 mg / mL to 2 mg / mL, 1 mg / mL to 1 mg / mL to 1.5 mg / mL, 1.5 mg / mL to 200 mg / mL, 1.5 mg / mL to 150 mg / mL, 1.5 mg / mL to 120 mg / mL, 1.5 mg / mL to 100 mg / mL, 1.5 mg / mL to 50 mg / mL, 1.5 mg / mL to 20 mg / mL, 1.5 mg / mL to 10 mg / mL, 1.5 mg / mL to 5 mg / mL, 1.5 mg / mL to 2 mg / mL, 2 mg / mL to 200 mg / mL, 2 mg / mL to 150 mg / mL, 2 mg / mL to 120 mg / mL, 2 mg / mL to 100 mg / mL, 2 mg / mL to 50 mg / mL, 2 mg / mL to 20 mg / mL, 2 mg / mL to 10 mg / mL, 2 mg / mL to 5 mg / mL, 5 mg / mL to 200 mg / mL, 5 mg / mL to 150 mg / mL, 5 mg / mL to 120 mg / mL, 5 mg / mL to 100 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 10 mg / mL, 10 mg / mL to 200 mg / mL, 10 mg / mL to 150 mg / mL, 10 mg / mL to 120 mg / mL, 10 mg / mL to 100 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 20 mg / mL, 20 mg / mL to 200 mg / mL, 20 mg / mL to 150 mg / mL, 20 mg / mL to 120 mg / mL, 20 mg / mL to 100 mg / mL, 20 mg / mL to 50 mg / mL, 50 mg / mL to 200 mg / mL, 50 mg / mL to 150 mg / mL, 50 mg / mL to 120 mg / mL, 50 mg / mL to 100 mg / mL, or 100 mg / mL to 200 mg / mL.

[0247] In some embodiments, the size of the therapeutic peptide is smaller than the mesh size of the dynamic hydrogel. For example, the mesh size of the dynamic hydrogel can be greater than or equal to 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, or 5 nm; while the size of the therapeutic peptide (e.g., hydrodynamic diameter) can be less than 2 nm, 1.5 nm, 1 nm, or 0.5 nm. Accordingly, physical entrapment of the therapeutic peptide by the hydrogel network may be ineffective for producing controlled release, in that the therapeutic peptide may be able to diffuse freely out of the dynamic hydrogel, resulting in uncontrolled burst release in vivo. For example, physical entrapment may be insufficient for controlling the release of a therapeutic peptide having a molecular weight less than or equal to 5 kDa, 4.5 kDa, 4 kDa, or 3.5 kDa.

[0248] In such embodiments, the dynamic hydrogel can include at least one component that binds to the therapeutic peptide to control the release of the therapeutic peptide from the dynamic hydrogel. The interaction can be a non-covalent interaction, such as a hydrophobic interaction. For instance, in embodiments where the therapeutic peptide is acylated with a fatty acid, the lipid in the fatty acid can interact with one or more hydrophobic components of the dynamic hydrogel (e.g., for a PNP hydrogel, the hydrophobic surfaces of the nanoparticlesand / or hydrophobic moieties on the polymer chain; for a PXY hydrogel, the hydrophobic side chains of the first agent). These hydrophobic interactions can cause the therapeutic peptide to adhere to the hydrophobic components of the dynamic hydrogel, thus inhibiting uncontrolled diffusion of the therapeutic peptide out of the dynamic hydrogel. In such embodiments, the therapeutic peptide can be released from the dynamic hydrogel primarily or entirely via erosion of the dynamic hydrogel in vivo. The release kinetics of the therapeutic peptide can thus be adjusted by tuning the degradation rate of the dynamic hydrogel (e.g., by controlling the storage modulus of the dynamic hydrogel).[G24S>] In some instances, therapeutic peptides may exhibit aggregation behavior that may interfere with binding of the therapeutic peptide to the dynamic hydrogel, thus resulting in a significant fraction of “free” peptide that is released diffusively over undesirably short timeframes from the hydrogel. For example, semaglutide has been shown to form a dimer species. When dimerized, the fatty acid side chain of semaglutide may be unavailable for hydrophobic interactions with the hydrogel, but the semaglutide dimers may still be too small for physical entrapment by the hydrogel network. Thus, when administered in vivo, the semaglutide may be rapidly released over a short timeframe, resulting in excessively high Cmax values that may produce undesirable gastrointestinal side effects.

[0250] Accordingly, the compositions herein can include at least one dispersing agent that inhibits aggregation of the therapeutic peptide (e.g., by weakening or otherwise disrupting interactions of the therapeutic peptide with other therapeutic peptides) in order to enhance binding of the therapeutic peptide to the dynamic hydrogel. For example, the dispersing agent can include one or more surfactants, such as a nonionic surfactant, an anionic surfactant, a cationic surfactant and / or a zwitterionic surfactant. Examples of surfactants that may be used include polysorbates (e.g., polysorbate 20 (Tween® 20), polysorbate 40 (Tween® 40), polysorbate 60 (Tween® 60), polysorbate 80 (Tween® 80)), sorbitan fatty acid esters (e.g., sorbitan monolaurate (Span® 20), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65)), poloxamers (e.g., P188, P237, P338, P407), polyoxyethylene alkyl ethers (e.g., Brij® surfactants), alkyl sulfates (e.g., dodecyl sulfate) and salts thereof (e.g., sodium dodecyl sulfate (SDS)), fatty acids (e.g., lauric acid, myristic acid, palmitic acid, stearic acid), fatty alcohols (e.g., lauryl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol), phospholipids, and other lipids or derivatives thereof exhibiting surfactant behavior. As another example, the dispersing agent can include one or more tonicity agents, such as alcohol-related tonicity agents (e.g., PG, glycerol, mannitol). Optionally, the compositions herein can includea combination of two or more dispersing agents, such as a surfactant and a tonicity agent (e.g., Tween® 20 and polysorbate). The dispersing agent(s) can be mixed with the therapeutic peptide before the therapeutic peptide is combined with the components of the dynamic hydrogel.

[0251] In some embodiments, the concentration of the dispersing agent is sufficiently to inhibit aggregation of the therapeutic peptide, but sufficiently low to avoid interfering with the interactions between the therapeutic peptide and the dynamic hydrogel, and / or to avoid interfering with hydrogel formation. For example, the concentration of the dispersing agent in the composition can be no more than 50 mg / mL, 40 mg / mL, 30 mg / mL, 25 mg / mL, 20 mg / mL, 15 mg / mL, 10 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, or 0.5 mg / mL. The concentration of the dispersing agent in the composition can be within a range from 0.5 mg / mL to 50 mg / mL, 0.5 mg / mL to 25 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 0.5 mg / mL to 2 mg / mL, 0.5 mg / mL to 1 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 25 mg / mL, 1 mg / mL to 15 mg / mL, 1 mg / mL to 10 mg / mL, 1 mg / mL to 5 mg / mL, 1 mg / mL to 2 mg / mL, 2 mg / mL to 50 mg / mL, 2 mg / mL to 25 mg / mL, 2 mg / mL to 15 mg / mL, 2 mg / mL to 10 mg / mL, 2 mg / mL to 5 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 25 mg / mL, 5 mg / mL to 15 mg / mL, 5 mg / mL to 10 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 25 mg / mL, 10 mg / mL to 15 mg / mL, 15 mg / mL to 50 mg / mL, 15 mg / mL to 25 mg / mL, or 25 mg / mL to 50 mg / mL.

[0252] The concentration of the dispersing agent in the composition can depend on the type of dispersing agent used. For instance, in embodiments where the dispersing agent is or includes a surfactant (e.g., Tween® 20), the surfactant can be present in the composition at a concentration less than or equal to 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1.5 mg / mL, 1.2 mg / mL, 1.1 mg / mL, 1 mg / mL, or 0.5 mg / mL. The concentration of the surfactant can be within a range from 0.1 mg / mL to 5 mg / mL, 0.5 mg / mL to 1.5 mg / mL, or 1 mg / mL to 2 mg / mL. As another example, in embodiments where the dispersing agent is or includes a tonicity agent (e.g., propylene glycol), the tonicity agent can be present in the composition within a range from 1 mg / mL to 50 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 16 mg / mL to 26 mg / mL.

[0253] In some embodiments, the compositions herein include little or no saline. Saline can increase hydrophobic effects that promote aggregation of the therapeutic peptide. Accordingly, removal of saline from the therapeutic peptide solution can reduce aggregation and enhance binding of the therapeutic peptide to the dynamic hydrogel. In some embodiments,the concentration of sodium chloride in the composition is no more than 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, or 0.01 mg / mL. In some embodiments, the compositions herein do not include any sodium chloride.[02541 In other embodiments, however, the size of the therapeutic peptide can be larger than the mesh size of the dynamic hydrogel, such as if the therapeutic peptide is conjugated to, binds to, or otherwise attached to a larger molecule or component (e.g., a protein or microparticle), and / or if the therapeutic peptide precipitates under formulation conditions to form larger aggregates. For example, albiglutide and dulaglutide are conjugated to proteins or protein fragments, and exenatide-LAR is encapsulated in biodegradable microparticles. In such embodiments, the therapeutic peptide can be encapsulated in the dynamic hydrogel via physical entrapment by the hydrogel network.

[0255] In embodiments where the therapeutic peptide includes a lipophilic substituent (e.g., a fatty acid side chain), the composition can further include a binding agent that binds to and / or otherwise interacts with the lipophilic substituent (e.g., via non-covalent interactions such as hydrophobic interactions). For example, the binding agent can be albumin or hydroxypropyl-beta-cyclodextrin (hp-bCD). The binding agent can be sufficiently large to be physically entrapped within the hydrogel network of the dynamic hydrogel. Accordingly, when the larger molecule is present in the composition, the therapeutic peptide can be bound to the binding agent via interactions between the lipophilic substituent and the binding agent, and thus be physically entrapped within the dynamic hydrogel along with the binding agent. In some embodiments, the binding agent is present in the composition in an amount within a range from 0.1 molar equivalents to 1 molar equivalents relative to the amount of the therapeutic peptide in the composition. In some embodiments, there is at least 1 molar equivalent of the therapeutic peptide per binding site on the binding agent (e.g., albumin has two binding sites for lipophilic substituents). The binding agent may be used in combination with a dispersing agent or without a dispersing agent.

[0256] In some embodiments, physical entrapment of the therapeutic peptide is achieved and / or enhanced through the addition of a divalent cation, such as one or more of Zn2+, Ca2+, Mg2+, Ba2+, or Sr2+. The divalent cation may be provided on its own or with a corresponding counterion, such as Cl-. Without wishing to be bound by theory, it is hypothesized that divalent cations may neutralize negatively charged functional groups (e.g., carboxylic acid groups) that may be present on the lipophilic substituent of the therapeuticpeptide (e.g., semaglutide has two carboxylic acid groups that are negatively charged at physiological pH), and thus can increase hydrophobic interactions between therapeutic peptides to create aggregates that are larger than the mesh size of the hydrogel network and thus are physically entrapped in the dynamic hydrogel. The aggregates formed due to the presence of the divalent cations may be larger than aggregates that form in the absence of the divalent cations (e.g., semaglutide dimers). In some embodiments, the divalent cation is present in the composition in an amount within a range from 0.1 molar equivalents to 10 molar equivalents relative to the amount of the therapeutic peptide in the composition.

[0257] In some embodiments, physical entrapment of the therapeutic peptide is achieved and / or enhanced by controlling the pH of the dynamic hydrogel. Without wishing to be bound by theory, it is hypothesized that changes in pH (e.g., decreasing the pH below physiological or neutral pH) may result in protonation and neutralization of negatively charged functional groups (e.g., carboxylic acid groups) that may be present on the lipophilic substituent of the therapeutic peptide (e.g., semaglutide has two carboxylic acid groups that are negatively charged at physiological pH), and thus can increase hydrophobic interactions between therapeutic peptides to create aggregates that are larger than the mesh size of the hydrogel network and thus are physically entrapped in the dynamic hydrogel. The aggregates formed due to the lower pH may be larger than aggregates that form at neutral pH (e.g., semaglutide dimers). For instance, the dynamic hydrogel may be formulated with a pH that is less than or equal to 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2.

[0258] In some embodiments, the dynamic hydrogel includes a buffer compound that forms a complex with the therapeutic peptide to achieve and / or enhancement physical entrapment of the therapeutic peptide. Examples of buffer compounds that may be used include acetate, citrate, Tris, arginine, histidine, lysine, tricine, bicine, TES, MOPS, PIPES, MES, TAPS, and HEPES.

[0259] In some embodiments, the dynamic hydrogel includes an antioxidant that stabilizes the therapeutic peptide to reduce or prevent degradation of the therapeutic peptide when exposed to in vivo conditions. The presence of the antioxidant can allow more of the therapeutic peptide payload to be delivered in an intact and / or functional form, and / or can also ensure therapeutic efficacy over longer treatment periods. Examples of antioxidants that may be used include natural antioxidants, such as vitamins and derivatives thereof (e.g., Vitamin E (tocopherols such as a-tocopherol), Vitamin C (ascorbic acid)), peptides (e.g., glutathione, methionine, cysteine, histidine); and synthetic antioxidants, such as sodium bisulfite, sodiummetabisulfite, ethylenediaminetetraacetic acid (EDTA), and butylated hydroxytoluene (BHT). In some embodiments, the antioxidant is a hydrophobic antioxidant (e.g., Vitamin E, which is a hydrophobic lipid-like molecule), which may exhibit improved retention in the dynamic hydrogel to provide longer term stabilization of the therapeutic peptide. In some embodiments, the antioxidant is a hydrophilic antioxidant, such as methionine (e.g., L-methionine) or histidine (e.g., L-histidine).

[0260] In some embodiments, oxidative stabilization of the therapeutic peptide includes the addition of sacrificial antioxidants to the dynamic hydrogel. Such an approach can be especially beneficial, for example, when the therapeutic peptide is semaglutide. Semaglutide contains oxidation-prone residues, including His7, TyrlO, and Trp31, which are susceptible to reactive oxygen species (ROS) in vivo. Histidine (His7) is highly reactive toward ROS, forming oxo-histidine or imidazolone derivatives that can distort binding-site geometry and impair receptor activation. Tyrosine (Tyr10) undergoes oxidation to form tyrosyl radicals via phenol oxidation, which may influence receptor binding affinity and peptide activity. Tryptophan (Trp31) shows very high susceptibility to oxidation through cleavage or modification of the indole ring, which can alter protein stability and fluorescence properties.

[0261] The inclusion of antioxidants in the dynamic hydrogel formulas of the present technology neutralize ROS, thereby mitigating degradative pathways and preserving the native chemical structure and bioactivity of semaglutide throughout its sustained release period. When the dynamic hydrogel is first implanted, ROS-producing cells (e.g., macrophages) are recruited to the site by the depot as part of the body’s normal foreign body response. The antioxidants act as a first responder, scavenging the initial burst of ROS and protecting the therapeutic peptide during this vulnerable early period. The depot is biocompatible and biodegradable in the sense that it does not trigger a sustained immune activation and the macrophage recruitment decreases over time (typically after a few days). ROS production at the site drops accordingly. In sum, the antioxidants counter a biological source of ROS (immune cells) during the initial implantation period, after which the depot reaches a non-inflammatory steady state. Although most antioxidants are released from the dynamic hydrogel within the initial 2-3 day period, this early release is sufficient to protect the therapeutic peptide throughout its substantially longer sustained release period (e.g., 30 days).

[0262] In some embodiments, the hydrophobicity and / or strength of the antioxidant may be tuned to modulate interaction of the antioxidant with the hydrogel and thus control the retention time of the antioxidant within the hydrogel matrix. Without being bound by theory,it is believed that increasing antioxidant strength and / or hydrophobicity enhances interaction of the antioxidant with the hydrogel matrix, thus extending its retention. Acetyl methionine (e.g., N-acetylmethionine), for example, is a hydrophobically modified antioxidant suitable for use with the compositions of the present technology. Others are possible. In some cases, incorporation of more hydrophobic antioxidants may require optimization of the formulation parameters to achieve desired solubility and compatibility with the hydrogel matrix.

[0263] The compositions herein can be administered to the subject via any suitable route, such as a parenteral route. For example, in some embodiments, the composition is administered to the subject via injection (e.g., subcutaneous injection or intramuscular injection). The shear-thinning properties of the dynamic hydrogel can allow for delivery via injection, while the self-healing properties of the dynamic hydrogel can allow for formation of a depot at the injection site that produces controlled release of the therapeutic peptide over the desired treatment period. Injection of the composition can be performed using any suitable tubular device having a lumen configured for delivery of a hydrogel, such as needles (e.g., hypodermic needles, surgical needles, infusion needles), injector pens, catheters, trocars, cannulas, tubing, etc. The composition can be injected into any suitable site in the subject’s body, such as an arm, thigh, abdomen, or buttock. The composition can be formulated to have a volume that is sufficiently small for injection, such as a volume less than or equal to 2 mL, 1.75 mL, 1.5 mL, 1.25 mL, 1 mL, 0.75 mL, 0.5 mL, or 0.25 mL; and / or a volume within a range from 0.25 mL to 1 mL, 1 mL to 1.25 mL, 1.25 mL to 1.5 mL, or 1.5 mL to 2 mL. In some embodiments, the composition is administered as a single injection at a single injection site, while in other embodiments, the composition can be administered as multiple injections at the same or different injection sites. The composition can be administered to the subject at any suitable frequency, such as once per week, once per 2 weeks, once per 4 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once 9 months, or once per year.

[0264] The compositions herein can be configured to deliver a therapeutically effective amount of the therapeutic peptide over a desired treatment period, which can be an amount that is effective to ameliorate or prevent a symptom of a disease or condition in a subject. For example, the treatment period can be at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year. Thetreatment period can be approximately 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months. The treatment period can be modulated by tuning the degradation rate of the hydrogel, as described elsewhere herein.

[0265] During the treatment period, the composition can deliver the therapeutic peptide at a rate of approximately 0.1 mg / week, 0.25 mg / week, 0.5 mg / week, 1 mg / week, 1.5 mg / week, 2 mg / week, 2.5 mg / week, 3 mg / week, 4 mg / week, 5 mg / week, 6 mg / week, 7 mg / week, 8 mg / week, 9 mg / week, 10 mg / week, 11 mg / week, 12 mg / week, 15 mg / week, 20 mg / week, or 25 mg / week. The delivery rate can be within a range from 0.1 mg / week to 25 mg / week, 0.25 mg / week to 25 mg / week, 0.5 mg / week to 20 mg / week, 0.5 mg / week to 2 mg / week, 0.5 mg / week to 1.5 mg / week, 1 mg / week to 2 mg / week, 2 mg / week to 5 mg / week, 5 mg / week to 15 mg / week, 5 mg / week to 10 mg / week, 10 mg / week to 20 mg / week, 10 mg / week to 15 mg / week, 12 mg / week to 25 mg / week, 12 mg / week to 15 mg / week, 15 mg / week to 25 mg / week, 15 mg / week to 20 mg / week, or 20 mg / week to 25 mg / week. Alternatively or in combination, the composition can deliver the therapeutic peptide at a rate of approximately 0.5 wt% / day, 0.6 wt% / day, 0.7 wt% / day, 0.8 wt% / day, 0.9 wt% / day, 1 wt% / day, 1.25 wt% / day, 1.5 wt% / day, 1.75 wt% / day, 2 wt% / day, 2.5 wt% / day, 3 wt% / day, 4 wt% / day, or 5 wt% / day (the wt% of the GLP-1 RA can be measured relative to the total amount of the therapeutic peptide initially present in the composition).

[0266] In some embodiments, when administered to a subject in vivo, the composition produces a steady state concentration (Csteady-state) and / or mean concentration at steady state of the therapeutic peptide in serum of approximately 10 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL, 1500 ng / mL, or 2000 ng / mL. The Csteady-state of the therapeutic peptide in serum produced by the composition can be within a range from 10 ng / mL to 2000 ng / mL, 10 ng / mL to 1000 ng / mL, 10 ng / mL to 500 ng / mL, 10 ng / mL to 100 ng / ml, 10 ng / mL to 50 ng / mL, 10 ng / mL to 25 ng / mL, 50 ng / mL to 2000 ng / mL, 50 ng / mL to 1000 ng / ml, 50 ng / mL to 500 ng / mL, 50 ng / mL, to 100 ng / mL, 100 ng / mL to 2000 ng / mL, 100 ng / mL to 1000 ng / mL, 100 ng / mL to 500 ng / mL, 100 ng / mL to 300 ng / mL, 150 ng / mL to 250 ng / mL, 500 ng / mL to 2000 ng / mL, 500 ng / mL to 1000 ng / mL, or 1000 ng / ml to 2000 ng / mL. In situations where there is no steady state concentration, “Csteady-state” may be understood to refer to the mean concentration over the time period between Cmax and Cmin at the end of the delivery period. The Csteady-state of the therapeutic peptide can be achieved within the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of administeringthe composition to the subject. In some embodiments, when administered to a subject in vivo, the composition produces a maximum concentration (Cmax) of the therapeutic peptide in serum that is less than or equal to 2000 ng / mL, 1500 ng / mL, 1000 ng / mL, or 500 ng / mL. In some embodiments, when administered to a subject in vivo, the composition produces a Cmax of the therapeutic peptide in serum that is no more than 1000X, 500X, 200X, 100X, 50X, or 10X of the Csteady-state and / or mean concentration at steady state of the therapeutic peptide in serum. In some embodiments, when administered to a subject in vivo, the composition produces a Cmax to Csteady-state ratio that is less than or equal to 1000:1, 500:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, or 1:1.

[0267] In some embodiments, the present technology provides methods for treating a subject by administering a composition as described herein. The composition can treat a disease or condition of the subject by producing a desired therapeutic effect in the subject, such as alleviation of symptoms, a reduction in the severity of the disease or condition, inhibiting an underlying cause of the disease or condition, steadying the disease or condition in a nonadvanced state, delaying the progress of a disease or condition, and / or improvement or alleviation of the disease or condition. Examples of diseases and conditions that may be treated using the compositions described herein include diabetes and / or related conditions (e.g., prediabetes, type 1 diabetes, type 2 diabetes, hyperglycemia, impaired glucose tolerance), obesity or excessive body weight, eating disorders (e.g., bulimia nervosa, binge eating disorder), obstructive sleep apnea, cardiovascular disease (e.g., hypertension, atherosclerosis, myocardial infarction, coronary heart diseases), liver disease (e.g., non-alcoholic fatty liver disease), neurological and / or neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, peripheral neuropathy, ischemia, stroke, multiple sclerosis), inflammatory diseases (e.g., asthma, psoriasis, inflammatory bowel disease), renal diseases, bone diseases (e.g., bone fragility, osteoporosis), hormonal diseases (e.g., polycystic ovary syndrome), and gastrointestinal diseases (e.g., short bowel syndrome).

[0268] In some embodiments, a method of treating diabetes and / or a diabetes-related condition (e.g., prediabetes, type 1 diabetes, type 2 diabetes, hyperglycemia, impaired glucose tolerance) includes administering a composition of the present technology to a subject in need thereof. The composition can be administered to the subject via a single injection (e.g., a subcutaneous or intramuscular injection). The composition can include a dynamic hydrogel (e.g., a PNP hydrogel or a PXY hydrogel) encapsulating a therapeutically effective amount of a therapeutic peptide (e.g., an incretin mimetic such as a GLP-1 RA) for treating the diabetesand / or diabetes-related condition. For example, the therapeutically effective amount can be an amount of the therapeutic peptide that results in the sustained reduction and / or regulation of the subject’s blood glucose levels over the treatment period. The therapeutically effective amount can be within a range from 0.1 mg / week to 25 mg / week, 0.1 mg / week to 5 mg / week, 0.5 mg / week to 20 mg / week, 0.5 mg / week to 2 mg / week, 0.75 mg / week to 1.5 mg / week, 1 mg / week to 2 mg / week, 5 mg / week to 10 mg / week, 10 mg / week to 20 mg / week, or 10 mg / week to 15 mg / week. The therapeutically effective amount may vary depending on the type of therapeutic peptide, e.g., the therapeutically effectively amount may be approximately 1 mg / week for semaglutide; approximately 12.6 mg / week (1.8 mg / day) for liraglutide; approximately 2.5 mg / week, 5 mg / week, 7.5 mg / week, 10 mg / week, 12.5 mg / week, or 15 mg / week for tirzepatide; and approximately 1 mg / week, 4 mg / week, 8 mg / week or 12 mg / week for retatrutide. The components of the dynamic hydrogel can be selected to provide injectability, formation of a cohesive depot in vivo, and controlled release of the therapeutic peptide over a sufficiently long period for treatment of the diabetes and / or diabetes-related condition. For example, the treatment period can be at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months.

[0269] For example, the dynamic hydrogel can be a PNP hydrogel (e.g., composed of a hydrophobically modified cellulosic derivative (e.g., HPMC-C12) and a plurality of amphiphilic nanoparticles (e.g., PEG-PLA nanoparticles). The PNP hydrogel can be a 0.5-5 hydrogel, a 0.5-8 hydrogel, a 0.5-10 hydrogel, a 0.5-12 hydrogel, a 0.5-15 hydrogel, a 0.8-5 hydrogel, a 0.8-8 hydrogel, a 0.8-10 hydrogel, a 0.8-12 hydrogel, a 0.8-15 hydrogel, a 1-5 hydrogel, a 1-8 hydrogel, a 1-10 hydrogel, a 1-12 hydrogel, a 1-15 hydrogel, a 1.5-5 hydrogel, a 1.5-8 hydrogel, a 1.5-10 hydrogel, a 1.5-12 hydrogel, a 1.5-15 hydrogel, a 2-5 hydrogel, a 2-8 hydrogel, a 2-10 hydrogel, a 2-12 hydrogel, a 2-15 hydrogel, a 3-5 hydrogel, a 3-8 hydrogel, a 3-10 hydrogel, a 3-12 hydrogel, or a 3-15 hydrogel.

[0270] Optionally, the dynamic hydrogel can include at least one dispersing agent that inhibits aggregation of the therapeutic peptide, such as a surfactant (e.g., Tween® 20) and / or a tonicity agent (e.g., propylene glycol). The dynamic hydrogel can include a combination of multiple different tonicity agents (“high entropy solvent”) to increase the entropy of the formulation which may reduce retention of the therapeutic peptide within the hydrogel following administration.

[0271] Optionally, the dynamic hydrogel can be formulated to increase physical entrapment of the therapeutic peptide within the hydrogel, such as by adding a binding agent (e.g., albumin), a divalent cation (e.g., Zn2+), and / or formulating with a lower pH (e.g., pH 4).

[0272] In some embodiments, a method of treating obesity and / or reducing body weight includes administering a composition of the present technology to a subject in need thereof. The composition can be administered to the subject via a single injection (e.g., a subcutaneous or intramuscular injection). The composition can include a dynamic hydrogel (e.g., a PNP hydrogel or a PXY hydrogel) encapsulating a therapeutically effective amount of a therapeutic peptide (e.g., an incretin mimetic such as a GLP-1 RA) for treating obesity and / or reducing body weight of the subject. For example, the therapeutically effective amount can be an amount of the therapeutic peptide that results in loss of a desired amount of body weight and / or maintenance of body weight within a desired range. The therapeutically effective amount can be within a range from 0.1 mg / week to 25 mg / week, 0.1 mg / week to 5 mg / week, 0.5 mg / week to 20 mg / week, 0.5 mg / week to 2 mg / week, 0.75 mg / week to 1.5 mg / week, 1 mg / week to 2 mg / week, 5 mg / week to 10 mg / week, 10 mg / week to 20 mg / week, or 10 mg / week to 15 mg / week. The therapeutically effective amount may vary depending on the type of therapeutic peptide, e.g., the therapeutically effectively amount may be approximately 1 mg / week for semaglutide; approximately 12.6 mg / week (1.8 mg / day) for liraglutide; approximately 2.5 mg / week, 5 mg / week, 7.5 mg / week, 10 mg / week, 12.5 mg / week, or 15 mg / week for tirzepatide; and approximately 1 mg / week, 4 mg / week, 8 mg / week or 12 mg / week for retatrutide. The components of the dynamic hydrogel can be selected to provide injectability, formation of a cohesive depot in vivo, and controlled release of the therapeutic peptide over a sufficiently long period for the treatment of obesity and / or reduction of body weight. For example, the treatment period can be at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months.

[0273] For example, the dynamic hydrogel can be a PNP hydrogel that is composed of a hydrophobically modified cellulosic derivative (e.g., HPMC-C12) and a plurality of amphiphilic nanoparticles (e.g., PEG-PLA nanoparticles). The PNP hydrogel can be a 0.5-5 hydrogel, a 0.5-8 hydrogel, a 0.5-10 hydrogel, a 0.5-12 hydrogel, a 0.5-15 hydrogel, a 0.8-5 hydrogel, a 0.8-8 hydrogel, a 0.8-10 hydrogel, a 0.8-12 hydrogel, a 0.8-15 hydrogel, a 1-5 hydrogel, a 1-8 hydrogel, a 1-10 hydrogel, a 1-12 hydrogel, a 1-15 hydrogel, a 1.5-5 hydrogel, a 1.5-8 hydrogel, a 1.5-10 hydrogel, a 1.5-12 hydrogel, a 1.5-15 hydrogel, a 2-5 hydrogel, a 2-8 hydrogel, a 2-10 hydrogel, a 2-12 hydrogel, a 2-15 hydrogel, a 3-5 hydrogel, a 3-8 hydrogel,a 3-10 hydrogel, a 3-12 hydrogel, or a 3-15 hydrogel. Optionally, the PNP hydrogel can include at least one dispersing agent that inhibits aggregation of the therapeutic peptide, such as a surfactant (e.g., Tween® 20) and / or a tonicity agent (e.g., propylene glycol). Optionally, the PNP hydrogel can be formulated to increase physical entrapment of the therapeutic peptide within the hydrogel, such as by adding a binding agent (e.g., albumin), a divalent cation (e.g., Zn2+), a buffer compound, and / or a high entropy solvent, and / or by formulating with a lower pH (e.g., pH 4).

[0274] As another example, the dynamic hydrogel can be a PXY hydrogel that is composed of a first agent (e.g., a hydrophobically modified polysaccharide such as HPMC-C18) and a therapeutic peptide. The concentration of the hydrophobically modified polysaccharide in the dynamic hydrogel can be from 0.1 wt% to 5 wt%, 0.1 wt% to 4 wt%, 0.1 wt% to 3 wt%, 0.1 wt% to 2 wt%, 0.1 wt% to 1 wt%, 0.1 wt% to 0.5 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%. Optionally, the PXY hydrogel can include a surfactant (e.g., Tween® 20 or Tween® 80). Optionally, the PXY hydrogel can include an organic solvent (e.g., PG). Optionally, the PXY hydrogel can be formulated to increase physical entrapment of the therapeutic peptide within the hydrogel, such as by adding a binding agent (e.g., albumin), a divalent cation (e.g., Zn2+), and / or formulating with a lower pH (e.g., pH 4).

[0275] In some embodiments, the present technology provides methods for preparing a composition for treating a disease or condition as described herein. The method can include combining the components of a dynamic hydrogel (e.g., polymer and nanoparticles for a PNP hydrogel, first agent and optional second agent(s) for a PXY hydrogel) with the therapeutic peptide, thus forming a dynamic hydrogel encapsulating the therapeutic peptide. The combining of the hydrogel components and therapeutic peptide can be performed using simple mixing under gentle conditions, such as physiological pH (e.g., pH 7.0 to 7.4) or a lower pH (e.g., pH 4) at room temperature (e.g., 25 °C) or physiological temperature (e.g., 37 °C). Optionally, the method can include combining the hydrogel components and therapeutic peptide with other components, such as a dispersing agent, a binding agent, a divalent cation, and / or an additional therapeutic agent (e.g., an antidiabetic agent or antiobesity agent). In some embodiments, the composition is prepared no more than 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes, or 1 minute before administering the composition to the subject.Alternatively or in combination, the composition can be prepared at least 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour before administering the composition to the subject. The dynamic hydrogel can be mostly or fully formed before the composition is administered to the subject. For example, the dynamic hydrogel can be sufficiently crosslinked (e.g., non-covalently crosslinked) to exhibit the shear-thinning, self-healing, and / or viscoelastic properties described herein before the composition is administered to the subject.

[0276] In some embodiments, the present technology provides kits for preparing a composition as described herein. The kit can include a solution containing the therapeutic peptide and one or more solutions containing the components of a dynamic hydrogel (e.g., for PNP hydrogels, a solution containing a polymer and a solution containing nanoparticles, or a single solution containing a polymer and nanoparticles; for PXY hydrogels, a solution containing a first agent and optionally a solution containing a second agent, or a single solution containing both the first and second agents). The therapeutic peptide solution can include a dispersing agent (e.g., a surfactant and / or a tonicity agent), a binding agent, and / or a divalent cation.

[0277] Optionally, the kit can include a solution containing an additional therapeutic agent. The solutions can be provided in tubes, bottles, ampoules, syringes, or any other suitable storage container. In some embodiments, the solutions each independently include a suitable pharmaceutically acceptable diluent. The pharmaceutically acceptable diluent can be any diluent that does not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. Examples of pharmaceutically acceptable diluents include, but are not limited to, saline, Ringer’s solution, dextrose solution, phosphate buffered saline, water, or a combination thereof. The pharmaceutically acceptable diluent can include an isotonicity imparting agent, such as sodium chloride, potassium chloride, or monosodium phosphate. The pharmaceutically acceptable diluent can include a buffer, such as bicarbonate, TRIS, HEPES, MOPS, CHES, CHAPS, or phosphate buffered saline. The pharmaceutically acceptable diluent can include stabilizers and / or preservatives, as appropriate. Additional examples and details of pharmaceutically acceptable diluents can be found in Martin, Remington’s Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), which is incorporated herein by reference in its entirety.Examples

[0278] The present technology is further illustrated by the following non-limiting examples.Example 1: Preparation of Incretin Mimetic Loaded PNP Hydrogels

[0279] This example describes a process for preparing incretin mimetic loaded PNP hydrogels. Briefly, clinically used incretin mimics are dissolved in a buffer and mixed with polymeric nanoparticles, which are subsequently mixed with a hydrophobically modified cellulosic polymer to form a shear-thinning, self-healing hydrogel. Different incretin mimics can be used in the hydrogels, and additives can be included to stabilize the cargo and / or tune the release properties.

[0280] Semaglutide, liraglutide, and tirzepatide are GLP-1 RAs carrying a single optimized fatty acid and linker modification enabling reversible binding to albumin to extend the circulating half-life while maintaining optimal potency. While reducing treatment frequency from daily to weekly is associated with improved patient adherence, there is still room for improvement to reduce treatment burden and improve patient compliance (FIG. 2A). To address this challenge, long-acting formulations of semaglutide and tirzepatide were developed to provide continuous therapy for upwards of four months from a single administration to coincide with the typical cadence with which type 2 diabetes patients visit their endocrinologist or primary care provider (FIG. 2B).

[0281] As shown in FIG. 2B, in conventional treatment strategies the drug rapidly disperses (dotted line) and is cleared within days, requiring regular weekly bolus injections to maintain therapeutic levels. In contrast, the hydrogel depot forms a localized, semi-solid reservoir under the skin that slowly releases (solid line) the encapsulated drug over months, maintaining plasma concentrations above the therapeutic threshold with a single injection. This sustained-release approach reduces injection frequency, dampens peak-trough fluctuations in serum drug levels, improves patient compliance, and enhances overall therapeutic efficacy through stable and prolonged exposure.

[0282] HPMC (meets USP testing specifications), N, N-diisopropylethylamine (Hunig's base), hexanes, diethyl ether, N-methyl-2-pyrrolidone (NMP), di chloromethane (DCM), lactide (LA), 1-dodecylisocynate, and diazobicylcoundecene (DBU) were purchased from Sigma-Aldrich and used as received. Monomethoxy-PEG (5 kDa) was purchased from Sigma-Aldrich and was dried under vacuum prior to use. Glassware and stir bars were oven-dried at 180 °C. When specified, solvents were degassed by three cycles of freeze, pump, and thaw.

[0283] Dodecyl-modified (hydroxypropyl)m ethyl cellulose (HPMC-C12) was prepared as follows. HPMC (1.0 g) was dissolved in NMP (40 mL) by stirring at 80 °C for 1 h. Once the solution reached room temperature (RT), 1-dodecylisocynate (105 mg, 0.5 mmol) and N, N-diisopropylethylamine (catalyst, ~3 drops) were dissolved in NMP (5.0 mL). This solution was added dropwise to the reaction mixture, which was then stirred at RT for 16 h. This solution was then precipitated from acetone, decanted, redissolved in water (~2 wt%), and placed in a dialysis tube for dialysis for 3-4 days. The polymer was lyophilized and reconstituted to a 60-mg / mL solution with sterile PBS.]0284| PEG-PLA nanoparticles (PEG-PLA NPs) were prepared as follows. Monomethoxy -PEG (5 kDa; 0.25 g, 4.1 mmol) and DBU (15 pL, 0.1 mmol; 1.4 mol% relative to LA) were dissolved in anhydrous dichloromethane (1.0 mL). LA (1.0 g, 6.9 mmol) was dissolved in anhydrous DCM (3.0 mL) with mild heating. The LA solution was added rapidly to the PEG / DBU solution and was allowed to stir for 10 min. The reaction mixture was quenched and precipitated by a 1: 1 hexane and ethyl ether solution. The synthesized PEG-PLA was collected and dried under vacuum. Hydrogel permeation chromatography (GPC) was used to verify that the molecular weight and dispersity of polymers met quality control (QC) parameters (FIG. 3 A). A 1-mL solution of PEG-PLA in DMSO (50 mg / mL) was added dropwise to 10 mL of water at RT under a high stir rate (600 rpm). Nanoparticles were purified by centrifugation over a filter (molecular weight cutoff of 10 kDa; Millipore Amicon Ultra- 15) followed by resuspension in PBS to a final concentration of 200 mg / mL. Nanoparticles were characterized by dynamic light scattering (DLS) to determine the nanoparticle diameter, 35 ± 4 nm (FIG. 3B).

[0285] Hydrogel formulations contained 1 wt% HPMC-C12 and 10 wt% PEG-PLA nanoparticles, and are denoted as PNP-1-10. These hydrogels were made by mixing a weighted ratio of 6 wt% HPMC-C12 polymer solution, 20 wt% nanoparticle solution, and PBS or water containing GLP-1 RAs (semaglutide, liraglutide, or tirzepatide). Semaglutide, liraglutide, and tirzepatide were obtained from the Stanford University Hospital Formulary as the drug products Ozempic® (Novo Nordisk), Victoza® (Eli Lilly), and Mounjaro® (Lilly), respectively. These drug products were provided as aqueous formulations including phosphate buffer, tonicity agents such as propylene glycol (Ozempic® and Victoza®) or saline (Mounjaro®), and preservatives such as phenol. These formulations were lyophilized and thenresuspended in water to formulate the hydrogels. For formulations including Tween® 20 and / or a-cyclodextrin (aCD), these additives were mixed with the GLP-1 RAs, and the resulting solution was mixed with the nanoparticles. The nanoparticles and aqueous components were loaded into one syringe, the HPMC-C12 was loaded into a second syringe and components were mixed using an elbow connector (FIG. 4). After mixing, the elbow was replaced with a 21 -gauge needle for injection. The semaglutide, liraglutide, and tirzepatide hydrogel formulations and their corresponding components are listed in Tables 3A and 3B, 4A and 4B, and 5A and 5B below, respectively.

[0286] Table 3A: Semaglutide PNP hydrogel formulationsSemaglutide HPMC-C12 Tween® 20 PEG-PLA NPs (µg / mL) Propylene Glycol (mg / mL) Phenol (mg / mL)PNP-S-1.2 mg 2-10 1200 20 0 100 0 0 PNP-S-1.8 mg 2-10 1800 20 0 100 0 0 PNP-S-2.4 mg 2-10 2400 20 0 1000 0 PNP-S-1.2 mg 1-10 1200 10 0 100 0 0 PNP-S-1.8 mg 1-10 1800 10 0 100 0 0 PNP-S-2.4 mg 1-10 2400 10 0 100 0 0 PNP-1-10-S1 1800 10 0 100 18.9 7.425 PNP-1-10-S2 1800 10 0.55 100 18.9 7.425 PNP-1-10-S3 1800 10 1.1 100 18.9 7.425 PNP-1-10-S4 1800 10 0 100 18.9 7.425 PNP-1-10-S5 1800 10 0 100 18.9 7.425 PNP-1-10-S6 1800 10 0 100 18.9 7.425 PNP-1-10-S7 1800 10 0.55 100 18.9 7.425 PNP-1-10-S8 1800 10 0.825 100 18.9 7.425 PNP-1-10-S9 1800 10 1.1 100 18.9 7.425 PNP-1-10-S10 1800 10 0.55 100 18.9 7.425

[0287] Table 3B: Semaglutide PNP hydrogel formulationsSodium Sodium Potassium. Chloride P mh,osphate P mh,osphate, aC, DT,, BS, AT, (mg / mL) (mg / mL) (mg / mL)(mg / mL) (mg / mL)PNP-S-1.2 mg 2-10 9.72 0.859 0.156 0 0 PNP-S-1.8 mg 2-10 9.27 0.819 0.148 0 0 PNP-S-2.4 mg 2-10 9.18 0.811 0.147 0 0 PNP-S-1.2 mg 1-10 9.81 0.867 0.157 0 0 PNP-S-1.8 mg 1-10 9.36 0.827 0.150 0 0 PNP-S-2.4 mg 1-10 9.27 0.819 0.148 0 0 PNP-1-10-S1 8.01 2.625 0.128 0 0 PNP-1-10-S2 8.006 2.624 0.128 0 0 PNP-1-10-S3 8.001 2.624 0.128 0 0 PNP-1-10-S4 8.006 2.624 0.128 0.521 0 PNP-1-10-S5 8.001 2.624 0.128 1.042 0 PNP-1-10-S6 0 1.917 0 0 0 PNP-1-10-S7 0 1.917 0 0 0 PNP-1-10-S8 0 1.917 0 0 0 PNP-1-10-S9 0 1.917 0 0 0 PNP-1-10-S10 0 1.917 0 0 3.59

[0288] Table 4A: Liraglutide PNP hydrogel formulationsLiraglutide HPMC-C12 Tween® 20 PEG-PLANPs(pg / mL) (mg / mL) (mg / mL) (pg / mL)PNP-1-10-L1 1800 10 0 100PNP-2-10-L1 1800 20 0 100

[0289] Table 4B: Liraglutide PNP hydrogel formulationsPropylene,., Sodium Potassium. Phenol Sodium ChlorideGlycol / / Tx / / Tx Phosphate Phosphate (mg / mL)(mg / mL) (mg / mL)(mg / mL) (mg / mL) PNP-1-10-L1 0 0 9.36 0.827 0.150 PNP-2-10-L1 0 0 9.27 0.819 0.148

[0290] Table 5A: Tirzepatide PNP hydrogel formulationsTirzepatide HPMC-C12 Tween® 20 PEG-PLANPs(pg / mL) (mg / mL) (mg / mL) (pg / mL)PNP-1-10-T1 280 10 0 100PNP-1-10-T2 1800 10 0 100PNP-1-10-T3 1800 10 0.55 100PNP-1-10-T4 1800 10 1.1 100PNP-1-10-T5 280 10 0 100PNP-1-10-T7 1800 10 0.55 100PNP-1-10-T8 1800 10 1.1 100PNP-1-10-T9 4500 10 0 100PNP-1-10-T10 4500 10 0 100PNP-1-10-T11 4500 10 0.55 100PNP-1-10-T12 4500 10 1.1 100

[0291] Table 5B: Tirzepatide PNP hydrogel formulationsPropylene Sodium Sodium PotassiumGlycol Chloride Phosphate Phosphate(mg / mL) (mg / mL) (mg / mL) (mg / mL)PNP-1-10-T1 0 8.201 0.740 0.128PNP-1-10-T2 0 9.240 0.918 0.128PNP-1-10-T3 0 9.236 0.917 0.128PNP-1-10-T4 0 9.231 0.917 0.128PNP-1-10-T5 0 0.191 0.033 0PNP-1-10-T7 0 1.230 0.210 0PNP-1-10-T8 0 1.230 0.210 0PNP-1-10-T9 21.9 1.230 0.210 0PNP-1-10-T10 39.2 1.230 0.210 0PNP-1-10-T11 0 1.230 0.210 0PNP-1-10-T12 0 1.230 0.210 0Example 2: Rheological Characterization of Incretin Mimetic Loaded PNP Hydrogels

[0292] This example describes rheological characterization of PNP hydrogels loaded with incretin mimetics. Rheological testing was performed using a 20-mm-diameter serrated parallel plate at a 600-pm gap on a stress-controlled TA Instruments DHR-2 rheometer. All experiments were performed at 25 °C. Frequency sweeps were performed from 0.1 to 100 rad / s with a constant oscillation strain within the linear viscoelastic regime (1%). Amplitude sweeps were performed at a constant angular frequency of 10 rad / s from 0.01% to 10000% strain with a gap height of 500 pm. Flow sweeps were performed from low to high stress with steady-state sensing. Steady shear experiments were performed by alternating between a low shear rate (0.1 s⁻¹) and high shear rate (10 s⁻¹) for 60 s each for three full cycles. Shear rate sweep experiments were performed from 10 to 0.001 s⁻¹. Stress controlled yield stress measurements (stress sweeps) were performed from low to high stress with steady-state sensing and 10 points per decade.

[0293] FIGS. 5 A and 5B are graphs showing rheological characterization of PNP- 1-10 hydrogel formulations with varying concentrations of semaglutide: frequency-dependent oscillatory shear sweep (FIG. 5A) and stress-dependent oscillatory shear sweep (FIG. 5B).

[0294] FIGS. 6 A and 6B are graphs showing rheological characterization of PNP-2-10 hydrogel formulations with varying concentrations of semaglutide: frequency-dependent oscillatory shear sweep (FIG. 6A) and stress-dependent oscillatory shear sweep (FIG. 6B).

[0295] FIGS. 7A-7C are graphs showing rheological characterization of PNP-1-10 hydrogel formulations with varying concentrations of tirzepatide: frequency-dependentoscillatory shear sweep (FIG. 7A and 7C) and stress-dependent oscillatory shear sweep (FIG.7B). In FIG. 7C, PNP-TZP-4.5 mg 1-10 corresponds to PNP-1-10-T9, PNP-TZP-0.28 mg 1-10 corresponds to PNP-1-10-T5, PNP-TZP-4.5 mg 0.1 wt% tween 1-10 corresponds to PNP-1-10-T11, and PNP-TZP-4.5 mg 0.1 wt% tween 1-10 corresponds to PNP-1-10-T12.[0296| These results demonstrate that addition of the GLP-1 RAs and other formulation excipients did not affect the mechanical behaviors of the hydrogels.Example 3: In Vitro Release Kinetics of Semaglutide from PNP Hydrogel Formulations

[0297] This example describes in vitro characterization of release of semaglutide from PNP hydrogel formulations. An in vitro release assay was used to study release behaviors of semaglutide from PNP hydrogels (FIG. 8 A). 100 pL of each hydrogel formulation was loaded into four-inch capillaries and 400 pL of PBS medium was added slowly on top. The surrounding PBS was removed for analysis after 1, 3, 6, 12, 24, and 48 hours and at one week and two weeks after injection into the capillary, and fresh PBS was replaced after each aliquot removal. Semaglutide was quantified by ELISA to determine release kinetics over time.

[0298] FIGS. 8B and 8C are graphs of in vitro release profiles showing the % cumulative release of semaglutide from PNP-2-10 (FIG. 8B) and PNP- 1-10 (FIG. 8C) hydrogel formulations at low, medium, and high semaglutide loadings, over the course of two weeks. For all formulations, a significant proportion of the semaglutide cargo was released over the two-week period. These results suggest that the semaglutide-loaded hydrogels may contain a significant fraction of “free” cargo (not adhered to the PNP hydrogel matrix) that undergoes fast, diffusion-based release. Semaglutide may form robust dimeric species at formulationrelevant concentrations, and these dimers were hypothesized to constitute the “free” fraction of the drug cargo. Semaglutide dimers are sufficiently small (RH < 2 nm) to be released over relatively short timeframes from the PNP hydrogels on account of its comparatively large mesh size (ξ~3.5 nm).[0299J FIG. 8D is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks, showing the effect of Tween® 20 addition on semaglutide release in the presence of propylene glycol and saline. All formulations exhibited low levels of burst release and high cargo retention over the two-week period attributable primarily to the presence of propylene glycol. The release rate was further reduced with increasing amounts of Tween® 20. It is hypothesized that propylene glycol and Tween® 20 act as dispersing agents to inhibit the formation ofsemaglutide dimers, thus promoting stronger binding of the fatty acid side chain of semaglutide to the PNP hydrogel matrix.

[0300] FIG. 8E is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks, showing that there is negligible effect of a-cyclodextrin (aCD) addition on semaglutide release in the presence of propylene glycol and saline. All formulations exhibited low levels of burst release and high cargo retention over the two-week period.

[0301] FIG. 8F is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations over the course of two weeks, showing the effect of Tween® 20 addition on semaglutide release in the presence of propylene glycol and absence of saline. All formulations exhibited low levels of burst release and high cargo retention over the two-week period attributable primarily to the presence of propylene glycol. The release rate was further reduced with increasing amounts of Tween® 20. Formulations including Tween® 20 in the absence of saline exhibited modest improvements in cargo retention, compared to formulations including Tween® 20 in the presence of saline (FIG. 8D). The presence of saline may increase hydrophobic effects that promote dimerization of semaglutide. Thus, removal of saline prior to formulation into to the hydrogel may facilitate breaking up of semaglutide dimers and improve binding of semaglutide to the hydrogel.

[0302] FIG. 8G is a graph of in vitro release profiles showing the % cumulative release of semaglutide from various PNP-1-10 hydrogel formulations, showing the effect of bovine serum albumin (BSA) in the release buffer and in the hydrogel. The PNP-1-10-S7 hydrogel formulation was prepared without BSA in the hydrogel, and with or without 1% BSA in the PBS release buffer. The PNP-1-10-S10 hydrogel formulation was prepared with 3.59 mg / mL BSA in the hydrogel and without BSA in the release buffer. The PNP-1-10-S16 formulation was prepared without BSA in the release buffer or in the hydrogel. No significant differences in release rate were observed between the various groups, thus indicating that the presence of BSA around the hydrogels does not affect semaglutide release.Example 4: In Vitro Release Kinetics of Liraglutide from PNP Hydrogel Formulations

[0303] This example describes in vitro characterization of release of liraglutide from PNP hydrogel formulations. The in vitro release assay of Example 3 was used to study release behaviors of liraglutide from PNP hydrogels.

[0304] FIG. 9 is a graph illustrating in vitro release profiles showing the % cumulative release of liraglutide from 1.8 mg / mL PNP-1-10 and PNP-2-10 hydrogel formulations, over the course of two weeks. The formulations exhibited negligible and high cargo retention over the two-week period, even in the absence of propylene glycol and Tween® 20. It was hypothesized that essentially all of the liraglutide was “bound” cargo that was adhered to the PNP hydrogel structure and would be primarily released by hydrogel erosion, which is severely limited in this capillary release model. The fatty acid side chain of liraglutide may drive the formation of more disorganized heptameric structures at micromolar concentrations, thus allowing the liraglutide to associate more strongly with the structural motifs within the PNP hydrogels even in the absence of a dispersing agent.Example 5: In Vitro Release Kinetics of Tirzepatide from PNP Hydrogel Formulations

[0305] This example describes in vitro characterization of release of tirzepatide from PNP hydrogel formulations. The in vitro release assay of Example 3 is used to study release behaviors of tirzepatide from PNP hydrogels. The formulations exhibit low levels of burst release and high cargo retention over the two-week period.Example 6: In Vitro Release Kinetics of PNP Hydrogels Formulated with Liraglutide and Insulin Glargine

[0306] This example describes in vitro characterization of release of liraglutide and insulin glargine from PNP hydrogel formulations.

[0307] Insulin glargine was obtained from the Stanford University Hospital Formulary as the drug product Lantus® (Sanofi). PNP-1-10 hydrogels with and without liraglutide and / or insulin glargine were prepared according to the protocol of Example 1. The in vitro release assay of Example 3 was used to study release behaviors of liraglutide and insulin glargine from PNP hydrogels. The hydrogel formulations and their corresponding components are listed in Tables 6 A and 6B below.

[0308] Table 6A: Liraglutide and Lantus PNP hydrogel formulationsLiraglutide Lantus HPMC-C12 PEG-PLA NPs Propylene Glycol Phenol Zinc (µg / mL) (µg / mL) (mg / mL) (µg / mL) (mg / mL) (mg / mL) (mg / mL)PNP-1-10-LIRA 1800 0 10 100 4.32 1.697 0 PNP-1-10-LIRA-LAN 1800 1004 10 100 4.32 1.697 0.828 PNP-1-10-LAN 0 1004 10 100 0 0 0.828

[0309] Table 6B: Liraglutide and Lantus PNP hydrogel formulations,,, Glycerol Tween® Sodium Sodium Potassium Metacresol. (85%, 20 Chloride Phosphate Phosphate(mg / mL) (mg / mL) (mg / mL) (mg / mL) (mg / mL) PNP-1-10-LIRA 0 0 0 8.010 1.146 0.128 PNP-1-10-LIRA-LAN 0.075 0.552 0.552 5.526 0.926 0.088 PNP-1-10-LAN 0.075 0.552 0.552 5.526 0.488 0.088

[0310] FIG. 10 is a graph illustrating in vitro release profiles showing the % cumulative release of liraglutide and Lantus from PNP-1-10 hydrogel formulations measured via ELISA, over the course of two weeks. All formulations exhibited low levels of burst release and high cargo retention over the two-week period, indicating that both liraglutide and insulin glargine could be successfully encapsulated in PNP hydrogels.Example 7: In Vivo Characterization of Incretin Mimetic Loaded PNP Hydrogel Formulations

[0311] This example describes characterization of the in vivo pharmacokinetics, pharmacodynamics, and biocompatibility of incretin mimetic loaded PNP hydrogel formulations in diabetic rats.

[0312] Male Sprague Dawley rats 160-230 g (8-10 weeks, Charles River) were weighed and fasted in the morning 6-8 h prior to treatment with nicotinamide (NA) and streptozotocin (STZ). NA was dissolved in IX PBS and administered intraperitoneally at 110 mg / kg. STZ is diluted to 10 mg / mL in sodium citrate buffer immediately before injection. STZ solution was injected intraperitoneally at 65 mg / kg into each rat. Rats were provided with water containing 10% sucrose for 24 h after injection with STZ. Rat blood glucose (BG) levels were tested for hyperglycemia daily after the STZ treatment via tail vein blood collection using a handheld blood glucose monitor (Bayer Contour Next). Type 2 diabetes (T2D) is defined as having three consecutive BG measurements in the range of 130-200 mg / dL in non-fasted rats.

[0313] Diabetic rats received either a) a single subcutaneous injection of a PNP hydrogel loaded with semaglutide (1.85 mg / mL) (PNP-1-10-S7 in Tables 2A and 2B) or tirzepatide (4.5 mg / mL) (PNP-1-10-T11 in Tables 4A and 4B); orb) daily subcutaneous bolus injections of either PBS, 20 pg semaglutide, or 50 pg tirzepatide. For each of the treatment groups, baseline blood was collected from the tail vein at day zero and daily blood glucose measurements are taken from the tail vein using a handheld blood glucose monitor (Bayer Contour Next) for 42 days following treatment. Blood glucose was measured, immediately followed by blood samples collected from the tail vein, every day for the first seven days of the study to measure serum semaglutide or tirzepatide concentrations using ELISA, and twotimes a week thereafter. Plasma GLP-1 RA concentrations were measured by ELISA at each time-point and total bioavailability of semaglutide or tirzepatide were determined at the endpoint of the study. An oral glucose tolerance test was used to group rats according to the glucose tolerance before treatment (at day -1).

[0314] FIG. 11 is a schematic illustration of the treatment schedule and timing of blood glucose measurements and serum collection for analysis. Diabetic rats received either a single subcutaneous injection of a semaglutide- or tirzepatide-loaded PNP hydrogel, or daily subcutaneous bolus injections of PBS, 20 pg daily semaglutide, or 50 pg tirzepatide.

[0315] FIGS. 12A and 12B are graphs illustrating the results of oral glucose tolerance testing before treatment (FIG. 12A) and after 6 weeks of treatment (FIG. 12B). An oral glucose tolerance test (OGTT) was conducted to group diabetic rats into treatment groups. Rats were fasted before administration of a glucose load by oral gavage. Baseline (fasting) blood glucose measurements were taken before glucose administration and measurements were made at regular intervals thereafter. Blood glucose was measured at -5, 0, 5, 15, 30, 45, 60, and 120 min. Using the area under the curve (AUC), rats with similar glucose tolerance were paired and then randomized into treatment groups.

[0316] FIG. 13 is a graph illustrating the percent change in blood glucose (BG) levels for rats treated with PNP hydrogels versus bolus injections. A single administration of semaglutide PNP or tirzepatide PNP hydrogel reduced the BG of type 2-like diabetic male rats over the course of 6 weeks, compared to daily PBS bolus injections. The plot shows change in BG over 6 weeks following each treatment group regimen (n = 6).

[0317] FIG. 14 is a graph illustrating the percent change in weight for rats treated with PNP hydrogels versus bolus injections. A single administration of semaglutide PNP or tirzepatide PNP hydrogel reduced the overall weight gain in type 2-like diabetic male rats over the course of 6 weeks post treatment, compared to daily PBS bolus injections. The plot shows change in weight over 6 weeks of each treatment group (n = 6).

[0318] FIG. 15 is a graph illustrating pharmacokinetics of a 20 pg daily bolus injection of semaglutide versus a PNP hydrogel (PNP-1-10 with a 1.8 mg / mL semaglutide loading and 0.05 wt% Tween® 20) in male diabetic rats (n = 6) over the course of 6 weeks post treatment.[G31S>] FIG. 16 is a graph illustrating pharmacokinetics of a 20 pg daily bolus injection of semaglutide versus a PNP hydrogel (PNP-1-10 with a 1.8 mg / mL semaglutide loading and 0.05 wt% Tween® 20) over the first 48 hours, overlaid with the 24 hour pharmacokinetics ofa 20 pg intravenous (I V.) and subcutaneous (S. C.) bolus injection in male diabetic rats (n = 6).

[0320] FIG. 17 shows graphs illustrating an assessment of treatment biocompatibility using blood chemistry to look for negative effects on the liver or kidney and evaluating the effect of treatment on hemoglobin A1C (HbAlc). Blood was collected pre- and post-treatment (after 6 weeks). Liver toxicity was assessed through measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin. Kidney toxicity was evaluated by examining creatinine and blood urea nitrogen (BUN) levels. Values for ALT, AST, creatine, and BUN were within the range of healthy rats (defined as the mean ± 2 standard deviations) for both the treatment and control groups.

[0321] These results indicate that the hydrogel formulations effectively maintained therapeutically relevant concentrations of semaglutide and tirzepatide throughout the duration of the six-week-long study. Hydrogel-based treatments resulted in a significant reduction in average BG and body weight over the course of the study relative to PBS bolus injection controls. The reduction in average BG and body weight was comparable to the reduction achieved by treatments with daily semaglutide and tirzepatide bolus injections. Hydrogel -based treatments were shown to be well tolerated, exhibiting no observable differences in liver and kidney compared to untreated animals.Example 8: Examples of Hydrogel Compositions, Rheological Studies, and Vaccine Delivery

[0322] This example describes preparation and characterization of hydrogel compositions.

[0323] A hydrogel is a crosslinked hydrophilic polymer network that can absorb high content of water (over 90% of the total weight). Hydrogels are useful in various biomedical-related applications due to their unique properties, such as high water content, biocompatibility, and injectability. A physically crosslinked hydrogel network may have tunable characteristics with reversible crosslinks that react to external stress such as temperature, pressure, and / or stress. Such properties may give rise to shear-thinning behavior — the viscosity of the material decreases under shear stress — that enables the hydrogel network to be injectable. Reversible crosslinking may allow the hydrogel network to self-heal once the shear stress is removed. These distinctive features, as well as biodegradability and biocompatibility through high watercontent, motivated the investigation of hydrogels for forming a drug cargo niche post-injection for sustained drug delivery.

[0324] Sangelose® 90L (Daido Chemical Corporation) is a hydrophobically-modified hydroxypropyl methylcellulose with a Cl 8 stearyl group side chain. The polymer is a white powder that forms a water-swollen polymer network (hydrogel) when dissolved in water with high water content (over 90 wt% water). Sangelose® can provide a cost-effective and facile hydrogel network with shear-thinning and self-healing behavior, which may be important characteristics of an injectable hydrogel system.

[0325] The rheological properties of Sangelose® hydrogels can be easily tuned when mixed with surfactants such as Tween® 20 or Tween® 80, or with aCD. Oscillatory frequency sweeps of Sangelose®-only hydrogels showed long relaxation times, and flat plateaus of storage and loss modulus. Following addition of aCD or Tween® surfactants, the crossover point between storage and loss modulus shifted to the right, thus creating a hydrogel with more dynamic properties. Results of material characterization of Sangelose® hydrogels with Tween® 80, aCD, and Tween® 20 are shown in FIGS. 18A-21F, respectively. Some of the procedures used herein were performed according to the techniques described in U. S. Patent Publication No. 2017 / 0319506, the disclosure of which is incorporated herein by reference in its entirety. The results indicate that hydrogel properties were easily tunable by simply controlling the weight percent of Sangelose® and the surfactants or aCD. These Sangelose® hydrogels with Tween® or aCD can be easily formed by simply mixing Sangelose® powder with water that contains either the surfactant or aCD.

[0326] The hydrogels described herein can outperform existing hydrogel methods in ease of production, cost-effectiveness, and short preparation time. These advantages are ideal for mass production of the product and commercialization, among other benefits.Example 9: Preparation of Unloaded Hydrogel Compositions

[0327] This example describes a process for preparing hydrogel compositions comprising hydrophobically modified hydroxypropyl methylcellulose (e.g., HPMC-C18) as a first agent, and a second agent (e.g., Tween® 80, Tween® 20, Tween® 40, Tween® 60, aCD, and Span® 20).

[0328] The first agent, hydrophobically modified hydroxypropyl methylcellulose (e.g., HPMC-C18), also known by the tradename Sangelose®, was dissolved in phosphate-buffered saline at 6 wt% and loaded into a 1.5 mL Eppendorf tube. The second agent (Tween® 80,Tween® 20, Tween® 40, Tween® 60, aCD, or Span® 20) was dissolved in either phosphate-buffered saline or ethanol, depending on the solubility of the second agent, then added to the Sangelose® solution. The contents were mixed thoroughly in the Eppendorf tube using a long spatula until homogeneous. The tubes were placed on a tabletop centrifuge for 5 minutes to remove bubbles, then stored at 25° C overnight before testing. All hydrogel compositions were composed of Sangelose® and a second agent in phosphate-buffered saline unless otherwise specified.

[0329] Table 7 lists the hydrogel compositions prepared using the above method.

[0330] Table 7.Sangelose® Second Wt% of Second(wt%) Agent Agent3 Tween® 80 0.1, 0.5, 0.75, 1, 21.5 Tween® 80 0.1, 0.5, 13 Tween® 20 0.1, 0.75, 1, 23 Tween® 40 0.753 Tween® 60 0.753 aCD 0.053 Span® 20 0.75

[0331] The hydrogel compositions herein may be referred to in an abbreviated form. For instance, the weight percent of each component is noted after the component name. For instance, S3Tw200.75 refers to the hydrogel formulation with 3 wt% of Sangelose® and 0.75 wt% of Tween® 20. Abbreviations: Sangelose® (HPMC-C18) = S; Tween® 80 = Twso; Tween® 60 = Tweo; Tween® 40 = Tw4o; Tween® 20 = Tw2o; Span® 20 = Span20; a-cyclodextrin = aCD or aCD.Example 10: Rheological Characterization of Hydrogel Compositions

[0332] This example describes methods to measure rheological properties of the hydrogel compositions described in Example 2 as well as Sangelose® compositions without a second agent.

[0333] Rheological characterization was performed using a 20-mm diameter serrated parallel plate at a 500-µm gap on a TA Instruments DHR-2 stress-controlled rheometer. All measurements were performed at 25 °C. Frequency sweep measurements were performed at aconstant 1% strain. Steady shear flow sweeps were performed from high to low shear rates. Stress sweeps were performed from low to high with steady-state sensing and yield stress values defined as the stress at which the viscosity decreases 10% from the maximum. Amplitude sweep measurements were performed at a frequency of 0.1 rad·s-1. P values are calculated with a one-way ANOVA followed by post hoc Tukey multiple comparison test unless otherwise noted.

[0334] Extensional rheology was performed on a TA Instruments ARES-G2 rheometer in axial mode using an 8-mm parallel plate geometry (R = 4 mm) at a H = 4 mm gap, resulting in an aspect ratio of H / R=l. A sample volume of 400 pl was loaded for each of the three replicated measurements. All experiments were performed at 25 °C. Samples of the hydrogel compositions were loaded and tested immediately within seconds to minimize dehydration. Hencky (exponential) strain rates were applied at three different strain rates.

[0335] Sangelose®-only compositions. Rheological measurements were taken of hydrogel compositions comprising Sangelose® without a second agent with varying amounts of Sangelose® (1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%). FIG. 22B shows the results of frequencydependent oscillatory shear sweep of the hydrogel samples. The crossover frequency (the frequency at which the storage modulus equals the loss modulus) was lower than the lowest angular frequency tested (0.1 rad·s-1), which indicates that the hydrogels comprising only Sangelose® have a static character (crossover frequency is inversely correlated to the relaxation time of the material; longer relaxation times correspond to more static materials, while shorter relaxation times correspond to more dynamic materials).[03361 The storage modulus (G') (FIG. 22C), and tan(delta) (FIG. 22D) was measured at 0.1 rad·s-1from FIG. 22B. In FIG. 22C, a decrease in Sangelose® concentration was observed to correlate with a decrease in storage modulus (G'), which relates to the stiffness of the material (a lower storage modulus correlates to reduced stiffness). However, as shown in FIG. 22D, changes in Sangelose® concentration did not significantly affect the tan(delta) values, which relate to the viscoelasticity of the material. This data demonstrates that changing the weight percent of Sangelose® in a Sangelose® only hydrogel can tune the stiffness but not the viscoelasticity of the gel.

[0337] Varying second agents. The frequency-dependent oscillatory shear rheology was measured for hydrogel compositions comprising Sangelose® and various second agents:Tween® 20 (FIG. 22E), Tween® 80 (FIG. 22F), aCD (FIG. 22G), and Span® 20 (FIG. 22H). The compositions tested include:S3 (Sangelose® only),S3Tw200.75,S3Tw800.75,S3aCD0.05, andS3Span2oO.75.

[0338] As shown in FIGS. 22E-22G, the addition of Tween® 20, Tween® 80, and aCD caused the crossover frequency (the frequency at which the storage modulus equals the loss modulus) of the hydrogel to shift rightward (toward higher angular frequencies) compared to the Sangelose® only hydrogel. Accordingly, these results indicate that the addition of Tween® 20, Tween® 80, and aCD produced hydrogels with increased dynamic behavior compared to Sangelose® only hydrogels. However, as shown in FIG. 22H, the addition of Span® 20 did not change the crossover frequency of the hydrogel compared to the Sangelose® only hydrogel, thus indicating that this second agent did not produce a dynamic hydrogel. Without being bound by theory, it is hypothesized that Tween® 20, Tween® 80, and aCD inserted into and created free volume within the micelles formed by the C 18 side chains of the Sangelose® polymer, thereby producing more dynamic crosslinks between the polymer chains. This effect may be due to chain length mismatch (e.g., Tween® 20 has a saturated C12 tail which is shorter than the C18 side chain), presence of unsaturated bonds preventing tight packing (e.g., Tween® 80 has an unsaturated C18 tail), and / or steric hindrance (e.g., Tween® 20 and Tween® 80 have large polyethoxylated sorbitan head groups, aCD is a sterically bulky molecule). In contrast, without being bound by theory, it is hypothesized that insertion of Span® 20 into the micelles did not produce free volume due to reduced steric hindrance from its smaller sorbitan head group, such that the crosslinking between Sangelose® polymer chains retained a relatively static character.

[0339] For each sample, the storage modulus (G1) and tan(delta) data was measured at 0.1 rad·s-1from FIGS. 22E-22H. As shown in FIG. 221, the storage modulus of the hydrogels including Tween® 20, Tween® 80, and aCD was lower than the storage modulus of the Sangelose® only hydrogels and hydrogels with Span® 20. As shown in FIG. 22J, the tan(delta) values of the hydrogels including Tween® 20, Tween® 80, and aCD was higher than the tan(delta) values of the Sangelose® only hydrogels and hydrogels with Span® 20. These resultsdemonstrate that varying the type of second agent influences both the stiffness and the viscoelasticity of the Sangelose®-based hydrogel composition.

[0340] Comparison of Tween® second agents. Additional Tween®-type molecules were screened as second agents in hydrogel compositions. In addition to Tween® 20 and Tween® 80 discussed above, Tween® 40, Tween® 60, Tween® 65, and Tween® 85 were also used in hydrogel preparations. The Tween® agents share the same hydrophilic head group. Their hydrophobic tails vary in chain length, saturation, and / or number, which can be seen in their chemical structure as shown in FIGS. 23 A-23F. The hydrophobic tails shown are derived from lauric acid (Cl 2, Tween® 20), palmitic acid (Cl 6, Tween® 40), stearic acid (Cl 8, Tween® 60, Tween® 65), and oleic acid (monounsaturated Cl 8, Tween® 80, Tween® 85). The compositions tested include:S3 (Sangelose® only),S3Tw200.75 (C12),S3Tw400.75 (C16),S3Tw600.75 (C18),S3Tw800.75 (monounsaturated C18),S3Tw650.75 (trisubstituted C18), andS3Tw850.75 (trisubstituted monounsaturated C18).

[0341] The frequency-dependent oscillatory shear rheology was measured for hydrogel compositions and shown in FIGS. 23A-23F. A shift of the crossover point of G' and G" to the right was observed to correlate with decrease in hydrophobic tail length, as shown in FIGS.23 A-23C. A shift of the crossover point of G' and G" to the right was also observed to correlate with the introduction of an unsaturated site, as shown in FIGS. 23C and 23D. However, tri substituted molecules did not significantly shift the crossover point compared to the Sangelose® only hydrogel, as shown in FIGS. 23E and 23F, indicating that the hydrogels with tri substituted molecules retained their relative static character. These results indicate that the chain length, saturation, and / or number of hydrophobic tails in Tween®-type molecules affect their ability to produce dynamic behavior when combined with Sangelose®.

[0342] The storage modulus G' (FIG. 23 G) and tan(delta) values (FIG. 23H) were measured at 0.1 rad·s-1from FIGS. 23A-23F. As shown in FIG. 23G, hydrogels formulatedwith Tween® 20, Tween® 40, and Tween® 80 exhibited lower G' values at a frequency of 10 rad-s'1compared to the hydrogels comprising only Sangelose® and hydrogels formulated with Tween® 80, Tween® 65, and Tween® 85. These data corroborate the increases in the observed crossover frequency described above as a shift of the crossover to higher frequencies results in a decrease in the observed modulus at a single low-to-intermediate frequency. As shown in FIG. 23H, hydrogels formulated with Tween® 20, Tween® 40, and Tween® 80 exhibited significantly higher tan(delta) values compared to the hydrogels comprising Sangelose® only, with the most significant change in tan(delta) value observed with Tween® 20.

[0343] Varying the concentration of Tween® 80. Hydrogel compositions comprising 3 wt% Sangelose® and varying amounts of monounsaturated C18 Tween® 80 (0.1 wt%, 0.5 wt%, 1 wt%, or 2 wt%) were prepared and the rheological measurements recorded. The compositions tested include:S3 (Sangelose® only),S3Tw₈₀0.1,S3Tw₈₀0.5,S3Tw₈₀1, andS3Tw₈₀2.

[0344] The frequency-dependent oscillatory shear sweep data of the four compositions are shown in FIG. 24A. A shift of the crossover point of G' and G" to the right was observed to correlate with increasing concentrations of Tween® 80, thus indicating increased dynamic character of the hydrogels with higher Tween® 80 concentrations.|0345] The oscillatory amplitude sweep of the four compositions was measured at a frequency of 0.1 rad·s-1and depicted in the plots of FIG. 24B. The storage modulus G' and tan(delta) values were measured at 0.1 rad·s-1and data shown in FIG. 24C and FIG. 24D, respectively. The storage modulus G', which is an indicator of stiffness, was observed to decrease with increasing concentrations of Tween® 80. The tan(delta), an indicator of viscoelasticity, generally increased with increasing concentrations of Tween® 80. This data suggests that as the weight percent of the Tween® 80 increases, the hydrogel becomes less stiff and more liquid-like.

[0346] Relaxation time of the hydrogel was calculated by fitting the frequency sweep with the continuous relaxation spectrum. FIG. 24E shows that the gel relaxation time decreasedas the weight percent of the Tween® 80 increased, thus indicating increased dynamic character of the hydrogels with higher Tween® 80 concentrations.

[0347] Yield stress was determined through stress-controlled flow sweep with increasing stress. The plot of yield stress in FIG. 24F showed a general correlation between reduced yield stress and increased Tween® 80 concentrations.[0348| FIG. 24G is a plot of extensional strain data of hydrogel compositions comprising Sangelose® and varying concentrations of Tween® 80. FIG. 24H shows representative images of hydrogel compositions comprising Sangelose® and varying concentrations of Tween® 80 under strain at a strain rate of 0.3 s-1. As shown in FIGS. 24G and 24H, hydrogels with higher Tween® 80 concentrations exhibited higher extensibility.[03491 Varying the concentration of Tween® 20. Similar to the experiment described above, studies were conducted to investigate the effect of increasing the concentration of Tween® 20 on the rheology of the hydrogel compositions. Hydrogel compositions comprising 3 wt% Sangelose® and varying amounts of monounsaturated C12 Tween® 20 (0.1 wt%, 0.75 wt%, 1 wt%, or 2 wt%) were prepared and the rheological measurements recorded. The compositions tested were:S3 (Sangelose® only),S3Tw₂₀0.1,S3Tw200.75,S3Tw₂₀1, andS3Tw₂₀2.

[0350] The frequency-dependent oscillatory shear sweep data of the four compositions are shown in FIG. 25 A. A shift of the crossover of G' and G" to the right is observed to correlate with increasing concentrations of Tween® 20, thus indicating increased dynamic character of the hydrogels with higher Tween® 20 concentrations.

[0351] The oscillatory amplitude sweep of the four compositions was measured at a frequency of 0.1 rad·s-1and depicted in the plots of FIG. 25B. The storage modulus G' and tan(delta) values were measured at 0.1 rad·s-1and data shown in FIG. 25C and FIG. 25D, respectively. The storage modulus G', which is an indicator of stiffness, was observed to decrease with increasing concentrations of Tween® 20. The tan(delta), an indicator ofviscoelasticity, generally increased with increasing concentrations of Tween® 20. This data suggests that as the weight percent of the Tween® 20 increases, the hydrogel becomes less stiff and more liquid-like.

[0352] Yield stress was measured through a stress-controlled flow sweep with increasing stress and is shown for each of the compositions in FIG. 25E. The yield stress was similar for compositions with 0.1 wt%, 0.75 wt%, and 1 wt% Tween® 20. The S3Tw₂₀2 composition containing 2 wt% of Tween® 20 had a lower yield stress than the other compositions.

[0353] FIGS. 25F and 25G are plots of rheological data for a Sangelose®-Tween® 20 hydrogel composition (S3Tw2oO.75) and a Sangelose® only hydrogel (S3). The viscosity of S3Tw200.75 and S3 as a function of shear rate was measured and the data presented in a plot shown in FIG. 25F. Both hydrogels showed shear-dependent viscosity and displayed shearthinning behavior. Step-shear measurements taken over three cycles of alternating low shear (0.1 rad·s-1) and high shear (10 rad-s'1) rates in 50 s steps are shown in FIG. 25G. These results indicate that both hydrogels displayed self-healing behavior.Example 11: Preparation of Hydrogel Compositions with GLP-1 RAs

[0354] This example describes the preparation of hydrogel compositions comprising a GLP-1 RAs (semaglutide or tirzepatide) and various amounts of Sangelose® and Tween® 20.

[0355] Sangelose® was purchased from Daido Chemical Corporation and polysorbate 20 was purchased from Sigma-Aldrich; both were used as received. Glassware and stir bars were oven-dried at 180 °C.[0356| Hydrogel compositions contained either 1.8 wt%, 2 wt% or 3 wt% Sangelose® and 0.5 wt%, 0.6 wt% or 0.4 wt% Tween® 20, and are denoted SANG-1.8-0.5, SANG-2-0.6 and SANG-3-0.4. These hydrogels were made by mixing a weighted ratio of 4 wt% Sangelose® solution and either a 2 wt% or 4 wt% Tween® 20 solution, and PBS or water containing GLP-1 RAs (semaglutide, tirzepatide, or liraglutide). The Tween® 20 and aqueous components were mixed using an elbow connector. After mixing, the elbow was replaced with a 21 -gauge needle for injection.

[0357] Table 8 summarizes semaglutide-containing hydrogel compositions and their corresponding ingredients.

[0358] Table 8.Semaglutide (ug / mL) Sangelose® (mg / mL) Tween® 20 (mg / mL) Propylene Glycol (mg / mL) Phenol (mg / mL) Sodium Chloride (mg / mL) Sodium Phosphate (mg / mL) Potassium Phosphate (mg / mL) SANG-1.8-0.5-S1 1800 18 5.50 18.9 7.425 8.79 2.694 0.141 SANG-1.8-0.5-Sla 1800 18 5.50 18.9 7.425 0.00 1.917 0.000 SANG-2-0.6-S2 1800 20 3.30 18.9 7.425 0.00 1.917 0.000 SANG-3-0.4-S3 1800 28.8 2.16 7.56 2.97 8.71 1.536 0.139

[0359] Table 9 summarizes tirzepatide Sangelose® hydrogel formulations and their corresponding ingredients.[0360| Table 9.Tirzepatide Sangelose® Tween® 20 Propylene Glycol Sodium Chloride Sodium Phosphate Potassium Phosphate (ug / mL) (mg / mL)(mg / mL)(mg / mL) (mg / mL) (mg / mL) (mg / mL) SANG-1.8-0.5-T1 1800 18 5.5 0 10.02 0.99 0.14 SANG-1.8-0.5-T2 1800 18 5.5 0 1.23 0.21 0.000 SANG-2-0.6-T3 4500 20 3.30 18.9 1.23 0.21 0.000 |0361] Table 10 summarizes liraglutide Sangelose® hydrogel formulations and their corresponding ingredients.

[0362] Table 10.Liraglutide (ug / mL) Sangelose® (mg / mL) Tween® 20 (mg / mL) Propylene Glycol (mg / mL) Phenol (mg / mL) Sodium Chloride (mg / mL) Sodium Phosphate (mg / mL) Potassium Phosphate (mg / mL) SANG-1.8-0.5-L1 1800 18 5.50 18.9 7.425 8.79 2.694 0.141 SANG-2-0.6-L2 1800 20 3.30 18.9 7.425 0.00 1.917 0.000 SANG-3-0.4-L3 1800 28.8 2.16 7.56 2.97 8.71 1.536 0.139

[0363] Rheological testing was performed at 25 °C using a 20-mm-diameter serrated parallel plate at a 600-pm gap on a stress-controlled TA Instruments DHR-2 rheometer. All experiments were performed at 25 °C. Frequency sweeps were performed from 0.1 to 100 rad s-1 with a constant oscillation strain within the linear viscoelastic regime (1%). Amplitude sweeps were performed at a constant angular frequency of 10 rad-s1from 0.01% to 10000% strain with a gap height of 500 pm. Flow sweeps were performed from low to high stress with steady-state sensing. Steady shear experiments were performed by alternating between a low shear rate (0.1 s⁻¹) and high shear rate (10 s⁻¹) for 60 s each for three full cycles. Shear rate sweep experiments were performed from 10 to 0.001 s⁻¹. Stress controlled yield stress measurements (stress sweeps) were performed from low to high stress with steady-state sensing and 10 points per decade.

[0364] FIGS. 26A-26C are plots illustrating frequency-dependent oscillatory shear sweep rheological characterization of Sangelose® hydrogel formulations without (SANG-2-0.6-no cargo) and with semaglutide cargo (SANG-2-0.6-sema 1.8 mg / mL, corresponding toSANG-2-0.6-S2 in Table 3) (FIG. 26A), without and with tirzepatide cargo (SANG-2-0.6-TZP 4.5 mg / mL, corresponding to SANG-2-0.6-T3 in Table 4) (FIG. 26B), and without and with liraglutide cargo (SANG-2.0-0.6-L 1.8 mg / mL, corresponding to SANG-2-0.6-L2 in Table 5) (FIG. 26C). The results indicate that incorporation of the semaglutide, tirzepatide, and liraglutide cargo did not significantly affect the rheological properties of the hydrogels.Example 12: fa Vitro Release Profile of Semaglutide from Hydrogel Composition

[0365] This example describes an experiment to evaluate the in vitro release of semaglutide from hydrogel compositions discussed in Example 11 and listed in Table 8.

[0366] 100 pL of each hydrogel formulation was loaded into four-inch capillaries and 400 pL of PBS medium was added slowly on top. The surrounding PBS was removed for analysis after 1, 3, 6, 12, 24, and 48 hours and at one week and two weeks after injection into the capillary, and fresh PBS was replaced after each aliquot removal. Semaglutide was quantified by ELISA to determine release kinetics over time.

[0367] FIG. 27A is a plot of the % cumulative in vitro release of semaglutide over the course of one week from Sangelose®-Tween® 20 hydrogel compositions. These results show that all compositions exhibited low levels of burst release and high cargo retention over the one-week period, indicating successful encapsulation of semaglutide in the hydrogels. The SANG-2-0.6-S2 formulation was the most successful at slowing the release of the semaglutide cargo, and thus was used in the in vivo studies described in Example 8 below.Example 13: fa Vitro Release Profile of Liraglutide from Hydrogel Composition

[0368] This example describes an experiment to evaluate the in vitro release of liraglutide from hydrogel compositions discussed in Example 11 and listed in Table 9.[0369J FIG. 27B is a plot of the % cumulative in vitro release of liraglutide over the course of one week from Sangelose®-Tween® 20 hydrogel compositions. These results show that all compositions exhibited low levels of burst release and high cargo retention over the one-week period, indicating successful encapsulation of liraglutide in the hydrogels.Example 14: fa Vivo Delivery of Semaglutide-Loaded Sangelose® Hydrogel Composition

[0370] This example describes an experiment to evaluate in vivo delivery of semaglutide from a Sangelose® hydrogel composition. A single dose of semaglutide-loadedSangelose® hydrogel composition was compared to a daily 20 pg semaglutide bolus injection and a daily PBS bolus injection over a period of six weeks in diabetic rats.

[0371] Animal model. Male Sprague Dawley rats 160-230 g (8-10 weeks, Charles River) were weighed and fasted in the morning 6-8 h prior to treatment with nicotinamide (NA) and streptozotocin (STZ). NA was dissolved in IX PBS and administered intraperitoneally at 110 mg / kg. STZ was diluted to 10 mg / mL in sodium citrate buffer immediately before injection. STZ solution was injected intraperitoneally at 65 mg / kg into each rat. Rats were provided with water containing 10% sucrose for 24 h after injection with STZ. Rat blood glucose (BG) levels were tested for hyperglycemia daily after the STZ treatment via tail vein blood collection using a handheld blood glucose monitor (Bayer Contour Next). Type 2 diabetes (T2D) was defined as having three consecutive BG measurements in the range of 130-200 mg / dL in non-fasted rats.

[0372] Semaglutide delivery and sample collection. Diabetic rats received either a) a single subcutaneous injection of a Sangelose® hydrogel loaded with 1.8 mg / mL semaglutide (SANG-2-0.6-sema 1.86 mg / mL, corresponding to SANG-2-0.6-S2 in Table 8), or b) daily subcutaneous bolus injections of either PBS or 20 pg semaglutide. Glucose measurements and serum collection were taken 5 days before injection of semaglutide and continued over a period of 6 weeks from injection. FIG. 28 is a schematic illustration of the treatment schedule and timing of blood glucose measurements and serum collection for analysis.]0373| Oral glucose tolerance test. An oral glucose tolerance test (OGTT) was conducted in order to group the diabetic rats into separate treatment groups. Blood glucose was measured at -5, 0, 5, 15, 30, 45, 60, and 120 min. Using the area under the curve (AUC), rats with similar glucose tolerance were grouped and then randomized into treatment groups. FIG.29 is a plot of data from an oral glucose tolerance test in rats, showing the area under the curve (AUC) of blood glucose after administration of a semaglutide bolus, semaglutide in a hydrogel composition, or PBS.

[0374] Blood glucose reduction. A single administration of the semaglutide-loaded Sangelose® hydrogel reduced the blood glucose of Type 2-like diabetic male rats over the course of the first 5 days post treatment, compared to daily PBS bolus injections. FIG. 30 is a plot showing a change in blood glucose over 5 days following each treatment group regimen (n = 6).

[0375] Weight reduction. A single administration of the semaglutide-loaded Sangelose® hydrogel reduced the overall weight gain in Type 2-like diabetic male rats over the course of the first 5 days post treatment. FIG. 31 is a plot showing a change in weight over 5 days of each treatment group (n = 6).

[0376] Pharmacokinetic (PK) profile. The semaglutide serum concentration was measured daily by ELISA. FIG. 32 is a plot of semaglutide serum concentration in diabetic rats over the course of the first 5 days post administration of a single semaglutide hydrogel composition and daily bolus injections of 20 pg semaglutide. The hydrogel composition achieved comparable semaglutide serum levels as the daily bolus injections throughout the study period.Example 15: Long-Acting Semaglutide Preparation

[0377] This example describes studies to evaluate sustained-release formulations for semaglutide.

[0378] Current sustained-release formulations for semaglutide, a glucagon-like peptide- 1 (GLP-1) drug, often experience burst release during the initial week, characterized by a rapid release of high drug concentrations. This phenomenon may compromise the safety and efficacy of the sustained-release system for diabetes therapeutics. The present technology enhances sustained-release semaglutide delivery systems by incorporating multivalent salts, which form metal-ion complexes with semaglutide within the hydrogel matrix, thereby stabilizing the drug and reducing burst release. Additionally, lowering the pH increases the hydrophobicity of the multivalent salts, further strengthening their interaction with semaglutide. These combined strategies effectively mitigate the initial rapid release, addressing associated complications, and ensure a safer, more controlled, and sustained release profile, ultimately improving therapeutic efficacy and patient compliance.

[0379] Materials. HPMC (meets USP testing specifications), N, N-diisopropylethylamine (Hunig's base), hexanes, diethyl ether, N-methyl-2-pyrrolidone (NMP), di chloromethane (DCM), lactide (LA), 1-dodecylisocynate, and diazobicylcoundecene (DBU) were purchased from Sigma-Aldrich and used as received. Monomethoxy-PEG (5 kDa) was purchased from Sigma-Aldrich and was dried under vacuum prior to use. Glassware and stir bars were oven-dried at 180 °C. When specified, solvents were degassed by three cycles of freeze, pump, and thaw.

[0380] Preparations ofHPMC-C12. HPMC (1.0 g) was dissolved in NMP (40 mL) by stirring at 80 °C for 1 h. Once the solution reached room temperature (RT), 1-dodecylisocynate (105 mg, 0.5 mmol) and N, N-diisopropylethylamine (catalyst, ~3 drops) were dissolved in NMP (5.0 mL). This solution was added dropwise to the reaction mixture, which was then stirred at RT for 16 h. This solution was then precipitated from acetone, decanted, redissolved in water (~2 wt%), and placed in a dialysis tube for dialysis for 3-4 days. The polymer was lyophilized and reconstituted to a 60 mg ml1solution with sterile PBS.

[0381] Preparation ofPEG-PLA NPs. Monomethoxy -PEG (5 kDa; 0.25 g, 4.1 mmol) and DBU (15 pL, 0.1 mmol; 1.4 mol% relative to LA) were dissolved in anhydrous dichloromethane (1.0 mL). LA (1.0 g, 6.9 mmol) was dissolved in anhydrous DCM (3.0 mL) with mild heating. The LA solution was added rapidly to the PEG / DBU solution and was allowed to stir for 10 min. The reaction mixture was quenched and precipitated by a 1: 1 hexane and ethyl ether solution. The synthesized PEG-PLA was collected and dried under vacuum. Hydrogel permeation chromatography (GPC) was used to verify that the molecular weight and dispersity of polymers meet our quality control (QC) parameters. A 1 mL solution of PEG-PLA in DMSO (50 mg mL1) was added dropwise to 10 mL of water at RT under a high stir rate (600 rpm). NPs were purified by centrifugation over a filter (molecular weight cutoff of 10 kDa; Millipore Amicon Ultra- 15) followed by resuspension in PBS to a final concentration of 200 mg mL1. NPs were characterized by dynamic light scattering (DLS) to find the NP diameter, 37 ± 4 nm.

[0382] PNP Hydrogel Preparation. Hydrogel formulations contained 1 wt% HPMC-C12 and 10 wt% PEG-PLA NPs combined with additional additives listed in Table 1 IB. These hydrogels were made by mixing a weighted ratio of 6 wt% HPMC-C12 polymer solution, 20 wt% NP solution, and PBS or water containing semaglutide. The NP and aqueous components were loaded into one syringe, the HPMC-C12 was loaded into a second syringe and components were mixed using an elbow connector. After mixing, the elbow was replaced with a 21 -gauge needle for injection.

[0383] Sangelose® Hydrogel Preparation. Sangelose® (hydroxypropylmethylcellulose stearoxy ether) formulations included 3 wt% Sangelose® combined with additional additives listed in Table 11 A. These hydrogels were prepared by mixing a 6 wt% Sangelose® solution with either Tris buffer (Tris(hydroxymethyl)aminomethane, pH 7) or acetate buffer (a mixture of acetic acid and sodium acetate, adjusted to pH 4). Certain formulations also incorporated propylene glycol asa tonicity agent. Semaglutide was obtained from Biosynth. The Sangelose® solution and aqueous components were thoroughly mixed using an elbow connector. After mixing, the elbow connector was replaced with a 21 -gauge needle for injection.

[0384] In vivo release and GLP-1 serum concentration analysis. FIG. 33 A is a schematic illustration of the experimental scheme. A volume of 100 pL or 200 pL (for Formulation G) of the formulation was subcutaneously injected into healthy female C57BL / 6 mice. Blood samples were collected from the tail and centrifuged to separate the serum in the first two weeks. A semaglutide-specific competitive ELISA kit was then used to analyze the serum samples.

[0385] Table 11A summarizes Semaglutide Sangelose® hydrogel formulations.[0386| Table 11 A.Formulation Semaglutide (ug / mL) Sangelose® (mg / mL) propylene glycol (mg / mL) carboxymethyl cellulose (mg / mL) zinc chloride (mg / mL) calcium chloride (mg / mL) strontium chloride (mg / mL) pH A San-S-7 1000 30 14 0 0 0 0 7 B San-S-4 1000 30 14 0 0 0 0 4 C San-S-Zn(leq)-7 1000 30 14 0 0.033 0 0 7 D San-S-Zn(1.5eq)-7 1000 30 14 0 0.0497 0 0 7 E San-S-Zn(6eq)-7 1000 30 14 0 0.2 0 0 7 F San-S-Zn(6eq)-4 1000 30 14 0 0.2 0 0 4 G San-0.5S-Zn(6eq)-4 500 30 14 0 0.1 0 0 4 H San-CMC-S-Sr-7 1000 30 14 25 0 0.022 0 7 J San-CMC-S-Ca-7 1000 30 14 25 0 0 0.053 7

[0387] Table 1 IB summarizes Semaglutide PNP hydrogel formulations.

[0388] Table 11B.„ Formu,lat.i. „on Se,mag.... HPMC- PEG- propylene.°luTti.de C12 PLA NPsrglyco.l, albu.mTin., hp-b, C, D x pTHT(ugH / mL) ’ (,mg / .mTL.) (,ug / .mTL.) (.mg / ,mTL.) (mg / mL) ’ (vmg& / mL) ’rX PNP-S 1000 10 100 14 0 0 7Y PNP-S-A 1000 10 100 14 1.5 0 7Z PNP-S-bCD 1000 10 100 14 0 0.35 7

[0389] FIG. 33B is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations A and B. The Sangelose®-semaglutide hydrogel formulations were tested at two different pH values (pH 7 and pH 4) to evaluate their performance.

[0390] FIG. 33C is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations C, D, and E, comparing Sangelose®-semaglutide hydrogel formulations with three different concentrations of zinc chloride additives at neutral pH.[03911 FIG. 33D is a graph illustrating in vivo release profiles showing the GLP-1 serum concentration for formulation E, and F over 2 weeks, comparing Sangelose®-semaglutide hydrogel formulations with 6 molar equivalents of zinc chloride additives at pH 7 and 4.

[0392] FIG. 33E is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations F and G, comparing Sangelose®-semaglutide hydrogel formulations with 6 molar equivalents of zinc chloride additives at pH 4, using a double injection volume while maintaining a constant dosage.

[0393] FIG. 33F is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations H, and J, comparing Sangelose®-CMC-semaglutide hydrogel formulations containing 200 mM additives of calcium chloride and strontium chloride.

[0394] FIG. 33G is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations X, Y, and Z, comparing PNP hydrogel formulations containing albumin or hydroxypropyl-beta-cyclodextrin (hp-bCD).

[0395] FIG. 33H is a graph illustrating in vivo release profiles of GLP-1 serum concentrations over 2 weeks for formulations A and X, comparing Sangelose® and PNP hydrogel formulations.

[0396] Table 12 below lists ratios between maximum concentration (Cmax), steady state concentration (Css, calculated as the average concentration from Day 7 to Day 14), Day 6 concentration (C6), and Day 7 concentration (C7) for the semaglutide hydrogel formulations.

[0397] Table 12. Release profile ratios for semaglutide hydrogel formulations.Formulation Cmax / Css Cmax / C6 Cmax / C7A 406 345B 214 45C 251 22D 297 23E 25F 22G 214 45H 137 60J 443 46X 1432 990Y 4267 2175Z 2415 3212

[0398] These results show that Sangelose®-semaglutide hydrogels formulated with multivalent salts and / or lower pH produced improved release profiles compared to the control formulation A, as evidenced by the lower Cmax / Css, Cmax / C6, or Cmax / C7 ratios. Moreover, PNP hydrogels exhibit a similar release profile as the Sangelose® hydrogels (FIG. 33H), and thus multivalent salts and lower pH formulations are expected to produce similar improvements.Example 16: In Vivo Release of Therapeutic Peptides from Sangelose® Hydrogels

[0399] This example describes in vivo release profiles for therapeutic peptides (e.g., semaglutide) that may be achieved using the Sangelose® hydrogels described herein.|0400] FIG. 34A is a graph illustrating an in vivo release profile for a therapeutic peptide that results from a typical daily / weekly bolus injection regimen (arrows indicate injection timing). As shown in FIG. 34A, the concentration of the therapeutic peptide fluctuates considerably, spiking immediately after injection and falling below the therapeutic window shortly thereafter. Accordingly, the observed C max to Cmin ratio is very high.10401] FIG. 34B is a graph illustrating an in vivo release profile for a therapeutic peptide delivered via a dynamic hydrogel (arrows indicate timing of hydrogel delivery). As shown in FIG. 34B, a consistent release rate is achieved shortly after hydrogel delivery, resulting in a lower C max to Cmin ratio compared to the bolus injection regimen of FIG. 34A.

[0402] FIG. 34C is a graph illustrating an in vivo release profile for a therapeutic peptide delivered via a dynamic hydrogel (arrows indicate timing of hydrogel delivery). Asshown in FIG. 34C, the release rate of the therapeutic peptide declines slowly over time, resulting in a lower C max to Cmin ratio compared to the bolus injection regimen of FIG. 34A.Example 17: Examples of HPMC-C18 Dynamic Hydrogel Depot Formulations

[0403] This example describes hydrogel vehicle formulation to tune micellar crosslink density and bulk mechanical properties.

[0404] Hydrophobically modified cellulose polymers, such as hydroxypropyl methylcellulose bearing pendant stearyl chains (HPMC-C18), form physically cross-linked hydrogels through micelle-mediated association of Cl 8 groups. These micellar junctions act as reversible crosslinks, which offer the shear-thinning and self-healing properties for dynamic hydrogels and are suitable for forming injectable depots for therapeutic peptides. Lipidated peptides possess both a hydrophilic peptide backbone and a hydrophobic alkyl chain. Lipidated peptide cargos exhibit amphiphilic character that enables the lipid moiety interacts with micellar cores to form micelle-like self-assembly while the peptide domain remains solvated in the surrounding aqueous phase. This dual affinity promotes selective association with hydrophobic micellar junctions within the hydrogel network, providing an intrinsic mechanism for depot formation and extended sustained release for therapeutic cargos. Semaglutide is an exemplar lipidated GLP-1 receptor agonist, which contains a saturated C18 chain that enables association with stearyl micelles and other hydrophobic domains within the hydrogel network. This interaction provides an opportunity to engineer depot stability and release kinetics through rational modification of vehicle structure, excipient chemistry, and cargo complexation.[04051 Dynamic Hydrogel Crosslinking. Hydroxypropyl methylcellulose (HPMC) derivatives bearing stearyl (Cl 8) side chains are widely used commercial polymers owing to the simplicity, low cost, and reproducibility of their hydrophobic modification, with established applications in hydrophobic drug solubilization and topical formulations. The grafted Cl 8 groups confer amphiphilicity, enabling the hydrophobic chains to associate into transient nanodomains that function as physical crosslinks in aqueous media. Specifically, the hydrophobic Cl 8 side chains associate to form the micellar cores, while the hydrophilic HPMC backbone remains solvated, forming the corona around these cores. These core-corona assemblies behave like micelle-like crosslinking domains within the hydrogel network. These domains act as physical crosslinks.

[0406] These reversible junctions allow HPMC-C18 to form viscoelastic gels without additional crosslinkers. Hydrophobic small molecules or hydrophobic peptide segments readilypartition into the CIS-associated domains, further strengthening these junctions. Because the associations are non-covalent and dynamically exchanging, the network exhibits pronounced shear-thinning, rapid self-healing, and injectability, and re-forms a stable subcutaneous depot after injection to support sustained release.

[0407] The release profile from HPMC-C18 depots proceeds through three characteristic phases: an initial burst phase, which rapidly reduces local concentration and osmotic stress; an extended diffusion-dominated phase that maintains therapeutically relevant plasma levels; and a late erosion-mediated phase during which the depot softens, contracts, and fully dissipates. To suppress burst release, a stiffer formulation (3 wt%) was selected to promote formation of a more robust depot. This composition was used as the baseline for subsequent studies. Notably, this HPMC-C18 platform reproduces the characteristic viscoelasticity and pharmacokinetic behavior of the PNP system while exhibiting a reduced burst release and providing a substantially simpler and more reproducible formulation process. FIG. 35 A illustrates how lipidated GLP-1 receptor agonists interact with hydrophobically modified HPMC-C18 to assemble into a long-acting subcutaneous depot.

[0408] Table 13 shows the naming scheme for all hydrogel formulations used in Examples 17-21.Table 13Other Injection Sangelose® semaglutideName Pg excipients / Buffer volume (wt%) (pg / mL) (mg / mL) pHNote (pL) methionine (1.5 Tris-HClP3-M 3 900 14 7.4 1000 mg / mL) 25mMmethionine,Tris-HClP3-M-Sr 3 900 14 SrC12 (0.108 7.4 100025mMug / mL)Tris-HClP2-0.6T20 2 1800 14 Tween20 7.4 50025mMdailyDaily 20N / A 40 subcutaneous lx PBS 7.4 500 Pg bolus, Rat10 mMP3 3 100 14 - 7.4 100 phosphate10 mMP3@pH4 3 100 14 - 4 100 phosphateTween80 10 mMP3-2T80 3 100 14 7.4 100(20mg / mL) phosphaterat serum10 mMP3-alb 3 100 14 albumin 7.4 100 phosphate(1.5mg / mL)ZnC12 Tris-HClP3-Zn 3 100 14 7.4 100(0.2mg / mL) 25mMP3- ZnC12 Acetate3 100 14 4 100 Zn@pH4 (0.2mg / mL) bufferSrC12P3-Sr 3 100 14 Tris-HCl 7.4 100(0.12mg / mL) 25mMP3- SrC12 Citric3 50 14 4 200 Sr@pH4 (0.12mg / mL) buffera-tocopherol Tris-HClP3-aT 3 100 14 7.4 100(0.5mg / mL) 25mMa-tocopherol(0.5mg / mL), Tris-HClP3-aT-Sr 3 100 14 7.4 100SrC12 25mM(2.5mg / ml)methionine Tris-HClP3-M 3 100 14 7.4 100(1.5mg / mL) 25mMmethionine(1.5mg / mL), Tris-HClP3-M-Sr 3 100 14 7.4 100SrC12 25mM(0.12mg / mL)CarboxymethylCelluloseTris-HClP-CMC-Sr 3 100 14 (25mg / mL), 7.4 10025mMSrC12(0.022mg / mL)CarboxymethylCelluloseTris-HClP-CMC-Ca 3 100 14 (25mg / mL), 7.4 10025mMCaC12(0.053mg / mL)singleBolus 2 pg 20 subcutaneous PBS 7.4 100bolus, Mice

[0409] Rheological Measurements. Rheological measurements were carried out on a TA Instruments DHR-2 stress-controlled rheometer equipped with a 20-mm serrated parallel plate geometry. Samples were loaded at a 600 pm geometry gap, and all tests were conducted at 21 °C. Oscillatory frequency sweeps were collected over 0.1-100 rad s⁻¹ using a 1% strain amplitude, which falls within the linear viscoelastic range. Strain-dependent behavior was assessed through amplitude sweeps at 10 rad s⁻¹, spanning 0.01% to 10,000% strain. Flow behavior was characterized by stress sweeps run from low to high stress under steady-state sensing conditions from shear rates of 0.1 to 100 s⁻¹. Yield stress was determined under stress-controlled conditions using a logarithmic progression of 10 points per decade.

[0410] FIG. 35B is a graph of an oscillatory frequency sweep of 3 wt% HPMC-C18 (P3) with and without 1 mg / mL semaglutide. FIG. 35C is a graph comparing the plateau modulus (G′o) of P3 and P3-S. FIG. 35D is a graph of the relaxation time (τR) extracted from rheological measurements and FIG. 35F is a graph of the Herschel–Bulkley–fit dynamic yield stress for P3 and P3-S.[04111 Rheological measurements from frequency sweep (FIGS. 35B-35E) show that both P3-noCargo and P3-S behave as solid-like, weakly frequency-dependent gels over the probed range, indicating a robust, physically cross-linked network suitable for subcutaneous depots. However, addition of semaglutide slightly decreases the oscillatory moduli across frequency (FIG. 35B), which is reflected in a lower plateau modulus G′o for P3-S compared with P3-noCargo (FIG. 35C) and a modest reduction in dynamic yield stress (FIG. 35E). These trends suggest that peptide loading partially disrupts or plasticizes the hydrophobic junctions rather than further stiffening them, consistent with semaglutide inserting into micellar domains and loosening packing. The relaxation time τR (FIG. 35D) is longer in P3-S, indicating semaglutide association slows down the network dynamics, making the polymer-particle junctions more persistent. Together, these point to a slightly softer gel upon drug loading, but one that still maintains clear yield behavior and solid-like character.

[0412] Injection Force Measurements. Injection force measurements were conducted using a syringe pump to control the volumetric flow rate through a 1 mL BD syringe fitted with a 40 mm, 21-gauge needle. A calibrated load cell (FUTEK LLB300) mounted to the syringe pump recorded the axial force required for injection. Force-time data were collected using a Lab VIEW acquisition program. Each measurement was continued until the injection force reached a steady-state plateau, after which the pump was stopped.

[0413] FIG. 35F. is a graph of the injection forces for P3-S at 0.5, 1, and 2 mL / min through a 21 -gauge needle, demonstrating practical injectability. Despite the modest softening, P3-S remains readily injectable. As shown in FIG. 35F, the injection forces through a 21 -gauge needle at 0.5-2 mL / min fall within a practical range for subcutaneous administration by hand. Semaglutide-HMPC-C18 association generates a self-assembled depot that maintains sufficient mechanical integrity to localize at the injection site.

[0414] Influence of Surfactant-like Excipients. Semaglutide (trade name Ozempic®) was selected as the therapeutic cargo for sustained delivery from the hydrogel depot materials due to the hydrophobic fatty acid modification and its longer plasma half-life compared to similar GLP-1 RAs (e.g. liraglutide). Drug release from HPMC-C18 hydrogels is regulated not only by stiffness but also by network relaxation dynamics. As such, it was investigated how the surfactant-mediated modulation of micellar crosslinks alters rheology and release behavior. Surfactants such as polysorbate 80 (Tween 80) can soften physically crosslinked hydrogels by inserting into hydrophobic junctions, thereby accelerating crosslink exchange and reducing burst release for non-lipidated macromolecular cargos such as human IgG. FIG. 36A is a schematic illustrating surfactant-mediated micelle stabilization of semaglutide in the presence of Tween-20. However, the addition of Tween 80 to the HPMC-C18 system loaded with semaglutide did not demonstrate this effect.

[0415] Dynamic light scattering (DLS) confirmed that Tween80 increased micelle size relative to semaglutide alone, consistent with surfactant interaction with C18 micellar cores. FIG. 36B shows the DLS size distributions of semaglutide with and without Tween-20, showing formation of larger peptide-surfactant assemblies.

[0416] FIG. 36C illustrates frequency sweeps of P3 hydrogels with semaglutide or Tween-20-semaglutide, demonstrating changes in viscoelastic behavior. Loading semaglutide into P3 slightly decreased the modulus relative to the cargo-free control (FIG. 35B), and this softening was comparable compared to the Tween80-modified formulations (FIG. 36C). This behavior suggests that semaglutide behaves as a weak amphiphile, and this property enables the peptides to insert into Cl 8 micelles but not to the extent that it disrupts the micellar junctions. Semaglutide’ s hydrophobic tail favors micelle incorporation, but the peptide backbone maintains sufficient intermolecular interactions to avoid wholesale disruption of the network. As a result, semaglutide reinforces hydrophobic clustering rather than destabilizing it.

[0417] This comparison demonstrates that HPMC-C18 hydrogels intrinsically accommodate lipidated peptides without the need for additional surfactant. In contrast to IgG and other hydrophilic cargos, where surfactants are required to tune relaxation kinetics, the semaglutide cargo itself provides the necessary hydrophobic interactions to form robust micellar junctions. Consequently, Tween80 is counterproductive for lipidated peptides depots, leading to weaker gels and worsened pharmacokinetic performance. However, it is important to note that such behaviors is different for a non-lipidated active pharmaceutical ingredient (API), such as human IgG. Surfactant addition may be necessary when the cargo is non-lipidated, because hydrophilic proteins do not partition into the C18 micellar cores and therefore do not reinforce the hydrophobic crosslinks.

[0418] The use of HPMC-C18 can be a stand-alone delivery vehicle for lipidated therapeutics. The polymer-micelle network formed by the Cl 8 domains is sufficiently stable to provide sustained release, and lipidated APIs such as semaglutide can act as co-assembling components. This mechanism is likely generalizable to other fatty-acid-modified peptides and proteins, positioning HPMC-C18 as a broadly applicable, formulation-simple platform for long-acting depot delivery.Example 18: Examples of HPMC-C18 Hydrogel Depot Cargo Complexation

[0419] This example describes cargo complexation to modulate hydrophobic interactions and effective diffusion characteristics of HMPC-C18 based hydrogel depots.

[0420] Cargo Size Tunability. Semaglutide is a relatively small molecule that diffuses rapidly through the hydrogel network. As such, strategies that increase its effective hydrodynamic size can modulate release kinetics. Two approaches were evaluated for enlarging the apparent cargo size: pre-binding semaglutide to human serum albumin and conjugating the peptide to multivalent ions. Both methods provide tunable size augmentation prior to depot formation, enabling control over transport within the HPMC-C18 network.[04211 Semaglutide is engineered with a Cl 8 fatty-acid modification that enables high-affinity binding to serum albumin, a key mechanism underlying its extended circulating halflife (FIG. 36D). We hypothesized that pre-binding semaglutide to serum albumin prior to hydrogel formulation would increase the effective cargo size and potentially prolong in vivo retention. FIG. 36E is a graph of DLS size distributions of semaglutide mixed with albumin, indicating formation of higher-molecular- weight complexes and confirming the expected slight increase in hydrodynamic diameter upon rat serum albumin-semaglutide complexation.Unintuitively, this strategy produced unfavorable effects on the hydrogel network that offsets the cargo-size increase. Incorporation of albumin markedly reduced the stiffness of the HPMC-C18 gel and accelerated early-phase release, indicating substantial burst behavior. Albumin is widely used as a model cargo in sustained-release studies, but in this system it acts as a competitive hydrophobic excipient: the protein’s surface patches and fatty-acid-binding pockets interfere with Cl 8 micellar clustering and disrupt hydrophobic junctions within the network. The magnitude of this disruption exceeded that observed with Tween 80, underscoring albumin’s strong ability to perturb the micellar crosslinks. FIG. 36F is a graph of the corresponding frequency sweeps of albumin-loaded P3 hydrogels,

[0422] Multivalent ion complexation increases cargo size. Semaglutide can coordinate with multivalent cations through multiple negatively charged residues and metal-binding motifs. At physiological pH, it carries several anionic groups, and addition of multivalent cations such as Zn2+and Sr2+crosslinks these polyanionic peptides into larger aggregates (FIG.36G). DLS confirms this clustering (FIG. 36H): semaglutide-Zn complexes have an average hydrodynamic diameter of 243.5 nm, whereas semaglutide-Sr complexes average 162.7 nm. Semaglutide-Zn2+ conjugates exhibit a significantly larger hydrodynamic diameter compared to free semaglutide and semaglutide-Sr2+ complexes, while Sr2+ results in only a modest increase in particle size. This difference arises from the stronger coordination affinity of Zn2+ for donor atoms within semaglutide, leading to the formation of compact multimeric ion-peptide aggregates. In contrast, Sr2+, with its larger ionic radius and weaker ligand-binding strength, forms weaker associations, resulting in smaller complex sizes. These findings suggest that Zn2+ induces more extensive peptide-ion crosslinking, which could influence both molecular assembly and sustained-release behavior in hydrogel formulations[04231 Rheological measurements were carried out on a TA Instruments DHR-2 stress-controlled rheometer equipped with a 20-mm serrated parallel plate geometry. Samples were loaded at a 600 pm geometry gap, and all tests were conducted at 21 °C. Oscillatory frequency sweeps were collected over 0.1-100 rad s⁻¹ using a 1% strain amplitude, which falls within the linear viscoelastic range. Strain-dependent behavior was assessed through amplitude sweeps at 10 rad s⁻¹, spanning 0.01% to 10,000% strain. Flow behavior was characterized by stress sweeps run from low to high stress under steady-state sensing conditions from shear rates of 0.1 to 100 s '. Yield stress was determined under stress-controlled conditions using a logarithmic progression of 10 points per decade.

[0424] FIG. 361 is a graph of frequency sweeps of Sr2+-loaded P3 hydrogels showing altered mechanical properties. Both ions stiffen the gel, and Zn- versus Sr-complexed semaglutide show similar frequency-sweep rheology. Taspoglutide, a GLP-1 RA previously evaluated in phase III trials, employed Zn2+complexation to prolong half-life. However, the stronger and more specific coordination of Zn2+with histidine and carboxylates from semaglutide raises the concern that excessively tight complexes may hinder dissociation and release. Because Sr2+has weaker binding energy to semaglutide and forms smaller clusters than Zn2+, it was hypothesized that more labile, charge-mediated clustering would yield improved sustained release.

[0425] Consistent with this, pharmacokinetic studies in C57BL / 6 mice, FIGS. 37 E-37H show that the Sr-formulated HPMC-C18 depot (P3-Sr) exhibits a longer elimination halflife, a comparable release half-life, and an area under the curve (AUC) nearly twice that of the Zn formulation. These data support the idea that weaker metal-peptide binding can produce greater overall systemic exposure than stronger Zn2+coordination.

[0426] FIGS. 37A-37C are graphs of summary of plateau modulus (G'0), relaxation time (T_R), and yield stress, respectively, across formulations including P3-S, P3-S-Alb, and P3-S-Sr.

[0427] FIG. 38A is a schematic of an in vitro assay for semaglutide sustained release in a capillary tube. The in vitro capillary tube release assay serves as an experimental model to evaluate hydrogel release behavior in an enzyme-free, quiescent saline environment (PBS). At each predetermined time point, all PBS buffer from the in vitro tube was collected and analyzed by ELISA to quantify the concentration of released drug in the collected PBS. New PBS is replenished when the sample from the previous timepoint is collected. Hydrogel samples (100 pL per formulation) were dispensed into four-inch glass capillary tubes. A 400 pL layer of phosphate-buffered saline (PBS) was added above each sample to act as the release phase. At predetermined intervals (1, 3, 6, 12, 24, and 48 hours, as well as 1 and 2 weeks), the PBS layer was removed for analysis and replaced with fresh buffer to maintain sink conditions. Semaglutide concentrations in collected fractions were measured using a commercial ELISA kit (BMA Biomedicals, S-1530). Cumulative release curves were generated from triplicate samples (n = 3).

[0428] FIG. 38B shows cumulative release example profiles for P3 and P3-T80 over 7 days, with Korsmeyer-Peppas fits highlighting differences in diffusional behavior.

[0429] Influence of pH on Pharmacokinetics. Commercial semaglutide (Ozempic® and Wegovy®) is formulated around neutral pH (7.4). Adjusting pH induces semaglutide clustering by making semaglutide more hydrophobic, which increases the cargo size and effectively lowers Cmax in the mice pharmacokinetics profile. However, the impact on overall bioavailability remains uncertain.

[0430] The pharmacokinetic profiles of P3 formulated in different buffer systems reveal clear pH-dependent release characteristics. At pH 7.4 (phosphate buffer, no saline), P3 exhibited a shorter release half-life (ti / 2 = 0.4 days) and faster absorption, reaching a tmax of 0.61 days with a Cmax of 9241.3 ng / mL. However, the total drug exposure (AUC = 22,324) was lower, indicating a more rapid initial release followed by faster clearance (elimination ti / 2 = 1.27 days). At pH 4 (citric buffer), P3 demonstrated a longer release half-life (ti / 2 = 1.05 days) and delayed tmax (1.07 days), accompanied by a comparable Cmax (8,735.1 ng / mL) but a higher area under the curve (AUC) (29,602). The elimination half-life remained similar (ti / 2 = 1.17 days), suggesting that the extended exposure was primarily due to slower release rather than altered systemic clearance. The difference in release behavior can be attributed to the effect of pH on semaglutide’ s ionization and hydrophobicity. At lower pH, the peptide becomes more hydrophobic due to protonation of ionizable groups, promoting stronger association with the hydrophobic domains of the HPMC-C18 network. This increased hydrophobic interaction enhances peptide retention within the gel matrix, leading to slower diffusion, reduced burst release, and a more sustained release profile. Acidic buffering (pH 4) improves the formulation’s stability and release control from a pharmacokinetic perspective. However, additional evidence is needed to determine whether bioavailability and therapeutic efficacy can be fully retained in the treatment of type 2 diabetes.

[0431] Pharmacokinetics Profiles in Healthy Mice. FIG. 39A is schematic of the mice pharmacokinetics study design. To avoid variability from uneven type 2 diabetes induction affecting semaglutide uptake, healthy 8-week-old female C57BL / 6 mice were used and administered a single subcutaneous hydrogel dose. Serum samples were collected at 2, 8, 12, 24, 48, and 144 hours post-injection. Pharmacokinetics were fit to a two-compartment model (SC depot and systemic compartment). Elimination half-life, release half-life, Cmax, tmax, and area under the curve (AUC) were used to characterize in vivo release. Elimination half-life reflects the time required for serum semaglutide concentration to drop by half; release half-life reflects the time required for half of the depot-bound semaglutide to be released; Cmax and tmax represent the peak semaglutide serum concentration and time to peak; and area under the curve(AUC) captures total systemic exposure. Together, these parameters provide a comprehensive basis for selecting formulations with the lowest burst release and most sustained delivery.

[0432] FIGS. 39B-39D show semaglutide serum concentration-time curves for P3 and modified P3 formulations, including Tween-80 (P3-T80), albumin prebinding (P3-alb), and Zn2+-complexed formulations (P3-Zn). FIG. 39E is a graph of the elimination half-life (t1 / 2,elim), FIG. 39F is a graph of the release half-life (t1 / 2,rel), FIG. 39G is a graph of Cmax, and FIG. 39H is a graph of the area under the curve (AUC) for all conditions. Together, these metrics highlight how excipient-dependent semaglutide clustering or conjugation modulates burst release and sustained exposure in vivo.[04331 From a translational perspective, pre-complexing semaglutide with recombinant human serum albumin introduces substantial manufacturing complexity, regulatory burden, and cost for GMP production. Taken together, while pre-binding increases apparent cargo size, its destabilizing effect on the hydrogel network and practical challenges make it an unattractive strategy for depot design.Example 19: Examples of Oxidation-Prevention Strategies for HPMC-C18 Dynamic Hydrogel Depots[0434| This example describes oxidation-prevention strategies, including sacrificial antioxidant excipients, to protect sensitive residues and stabilize the peptide during residence in HPMC-C18 hydrogel depots. Semaglutide is vulnerable to oxidative degradation both in vivo and during analytical quantification. Key residues such as His7, Tyr10, and Trp31 are oxidation-prone, and modification at these positions disrupts structural integrity and receptorbinding activity. Tryptophan (Trp31) shows very high susceptibility to oxidation through cleavage or modification of the indole ring, which can alter protein stability and fluorescence properties; Histidine (His7) is highly reactive toward reactive oxygen species (ROS), forming oxo-histidine or imidazolone derivatives that can distort binding-site geometry and impair receptor activation; Tyrosine (Tyr10) undergoes oxidation to form tyrosyl radicals via phenol oxidation, which may influence receptor binding affinity and peptide activity. To mitigate these degradative pathways, sacrificial antioxidants such as L-methionine and a-tocopherol are introduced into the formulation (FIG. 40A). These antioxidants preferentially react with and neutralize reactive oxygen species, thereby preserving the native chemical structure and bioactivity of semaglutide throughout its sustained release period.

[0435] The weaker antioxidant, L-methionine (M), and the stronger antioxidant, a-tocopherol (aT) were evaluated. Their oxidation pathways reflect their relative reactivity: methionine is converted to its sulfoxide and sulfone forms, while a-tocopherol undergoes multi-step oxidative degradation. Importantly, methionine is hydrophilic whereas a-tocopherol is hydrophobic, which affects how each excipient partitions within the depot. Because the P3-Sr formulation showed the most favorable pharmacokinetics, P3-Sr was selected as the base formulation to further optimize formulation based on anti oxidant- API interactions. To isolate burst-release behavior, which is where antioxidant effects are most visible due to their small size and rapid diffusion, a burst release assay was developed to complement the capillary release assay. GLP-1 RA-loaded hydrogels were incubated at 37 °C for 90 min, and released cargo was quantified. This short-timescale assay reveals the initial time point differences that are obscured in long-term capillary release experiments, where diffusion is slow and dominated by quiescent, surface-area-limited, enzyme-free conditions during extended release.

[0436] FIG. 40A is a diagram of the semaglutide structure highlighting oxidation-prone residues (His7, Tyr10, Trp31) and schematic of antioxidant incorporation into the hydrogel depot. L-methionine (M, hydrophilic sacrificial antioxidant) and a-tocopherol (aT, hydrophobic antioxidant) were evaluated. FIG. 40B is a schematic of the capillary burst-release assay used to quantify early-stage release of semaglutide and antioxidants.

[0437] FIG. 40C is a graph of in vitro burst release across formulations showing reduced initial release with methionine-containing depots. To conduct these studies, each hydrogel formulation, 800 μL of PBS was added to 1.5 mL microcentrifuge tubes to serve as the incubation medium. Gel samples were delivered into the tubes using 21 -gauge or comparable needle sizes, and 100 μL of each gel was dispensed to the bottom of the tube. Samples were incubated at 37 °C for 1.5 hours. Following incubation, the PBS supernatant was carefully aspirated and analyzed by semaglutide ELISA.

[0438] FIGS. 40D-40F are graphs of the short-timescale release kinetics for semaglutide and antioxidant-modified formulations.

[0439] FIGS. 40G-40H are graphs of serum pharmacokinetics in healthy mice for P3-M-Sr and P3-aT-Sr, fit with a two-compartment model. The in vivo pharmacokinetics GLP-1 molecules released from subcutaneous gel administrations were quantified using a two-compartment model to obtain the release and elimination half-lives of the drug. Animal studieswere performed with the approval of the Stanford Administrative Panel on Laboratory Animal Care (APLAC-32873) in accordance with NIH guidelines.

[0440] FIGS. 41A-41E are graphs of the extracted PK parameters, including elimination half-life, release half-life, lag time, Cmax, and area under the curve (AUC). These results indicate that antioxidant excipients reduce oxidative degradation, suppress burst release, and enhance sustained semaglutide exposure in vivo.Example 20: Long-term Pharmacokinetics of Semaglutide from HPMC-C18 Hydrogels in T2D Rats

[0441] This example describes the long-term pharmacokinetics of semaglutide release form HMPC-C18 hydrogels when implanted in T2D rats. FIG. 42 evaluates how methionine-containing hydrogels control long-term semaglutide exposure in a T2D rat model. Animal studies were performed with the approval of the Stanford Administrative Panel on Laboratory Animal Care (APLAC-32873) in accordance with NIH guidelines.

[0442] Streptozotocin / Nicotinamide (STZ / NA) Induced Model of Type-2 Diabetes in Rats. Male Sprague Dawley rats (8-10 weeks old, 230-350 g; Charles River) were used for T2D rat studies for evaluation of long-term pharmacokinetics and bioavailability of the GLP- 1 RA hydrogel formulations under Stanford Administrative Panel on Laboratory Animal Care protocol #32873. Type 2 diabetes was induced using a combined nicotinamide (NA) and streptozotocin (STZ) regimen. Animals were fasted for 6-8 hours prior to induction. NA was prepared in sterile 1× PBS and administered intraperitoneally at 110 mg / kg. STZ was freshly dissolved in 10 mg / mL sodium citrate buffer and delivered intraperitoneally at 65 mg / kg. To reduce the risk of hypoglycemia following STZ dosing, rats were provided 10% sucrose solution in place of drinking water for the subsequent 24 hours. Blood glucose was monitored daily via tail vein sampling using a Contour Next handheld glucometer. Animals were classified as diabetic once they displayed at least three consecutive non-fasting glucose measurements between 140 and 290 mg / dL. The overall induction efficiency was approximately 50%.

[0443] In vivo Pharmacokinetics Modeling for IV and SC in T2D Rats. A 24-hour IV PK study was conducted to validate the PK parameters of Semaglutide in rats, and a singlecompartment model was fit to obtain the elimination half-life (clearance rate) of the drugs. The differential equation governing the PK is:d / dt M(t) = -keM(t)

[0444] The IV PK data is fit to this equation. Here, keis the elimination rate constant, from which the elimination half-life is determined as Te=(ln2) / (ke). The SC gel data was also fit to the one-compartment model outlined above to obtain the half-lives of drug elimination from the body.|0445] In vivo Pharmacokinetics and Pharmacodynamics in T2D Rats. For each treatment groups (n = 6-8), baseline blood samples were collected from the tail vein on Day 0. Daily blood glucose levels were monitored using a handheld blood glucose monitor (Bayer Contour Next) for 42 days post-treatment. Blood glucose measurement was immediately followed by blood sampling from the tail vein for serum semaglutide quantification by competitive ELISA, performed daily during the first seven days and twice weekly thereafter. Plasma GLP-1 RA concentrations were determined by ELISA at each time point, and total semaglutide bioavailability was examined at the study endpoint. For the Bolus-R group, sampling was conducted every 24 hours following each injection in the previous study.

[0446] The 42-day study design (FIG. 42 A) allows for the following of both early and late phases of release, something short-window PK studies cannot capture. Because the depot volume was increased to 1 mL, the material persisted for the full study duration, enabling clean resolve of both diffusional and erosion-driven elimination components using the two-mode, two-compartment model in FIG. 42B. This modeling strategy was used to interpret the pharmacokinetics profile from the T2D rat study because the 42-day window, combined with a larger depot volume, permitted the tracking of semaglutide release across the full duration rather than only the first week. This longer profile enables calculation of additional parameters, including the peak-to-trough ratio, which reflects therapeutic stability by comparing Cmax to the steady-state plateau. A lower ratio corresponds to reduced burst release and more sustained delivery.

[0447] Based on the earlier burst-release assays, P3-M-Sr and P3-M were selected for evaluation in streptozotocin-nicotinamide-induced T2D rats. Methionine was prioritized because it consistently reduced burst release while extending sustained release, and its use as a sacrificial antioxidant in injectable depots is well established. The influence of Sr2+on semaglutide bioavailability remained uncertain, warranting direct comparison. Rats received a 1 mL depot, increased from 500 μL used in the pilot study, because supplemental data showed that the smaller depots fully degraded by day 42 and released very little cargo after day 10.Serum semaglutide, body weight, and blood glucose were monitored over 42 days. The glucose-lowering profile aligned with the pharmacokinetics, confirming that the depot-mediated release drives the observed pharmacodynamic response.

[0448] Pharmacokinetics Modeling for T2D Rat PK. The in vivo pharmacokinetics of GLP-1 molecules released from subcutaneous gel administrations were quantified using a twomode, two-compartment model to obtain the release and elimination half-lives of the drug, with the total release happening by a sum of diffusive and erosive mechanisms, and each mode having its characteristic release and elimination constants.

[0449] The serum concentration time curves for P3-M-Sr and P3-M (FIGS. 42C-42D) show the hallmark shape of depot-based release: an initial rise, a relatively flat mid-phase, and a slow, multi-week decay. The Cmax values appear modest relative to bolus dosing, indicating that both depots effectively suppress burst release. P3-M-Sr shows a more gradual decline than P3-M, but both maintain measurable semaglutide levels through day 42.10450] Model-derived half-lives (FIGS. 42E-42F) separate the contributions from diffusional versus erosion-driven loss. The two formulations produce similar diffusional halflives, suggesting that early-phase transport is governed primarily by polymer-peptide interactions rather than Sr2+modulation. In contrast, the erosive half-life differs slightly between the groups, consistent with Sr2+influencing late-stage retention by altering semaglutide multimerization or its interaction with the degrading matrix. Even so, both formulations exhibit erosion half-lives long enough to sustain exposure well beyond what is achievable with direct injection. The gray area was obtained by the 24h pharmacokinetics of a single IV injection of semaglutide in T2D model rat, with Cmax and lowest concentration at the end of 24h.

[0451] Peak-to-Trough Analysis for T2D Rat PK. Peak-to-trough calculations were performed by dividing the maximum observed plasma concentration (Cmax) from the longterm pharmacokinetic profile by the mean peptide concentration measured during the terminal steady-state period (days 11-42). Concentrations within this interval were averaged to obtain a representative trough value, as levels in this phase exhibit minimal day-to-day variation and reflect the sustained-release portion of the curve rather than early burst kinetics. This approach intends to provide a standardized metric to quantify depot stability and the magnitude of concentration decline from peak to late-phase exposure.

[0452] The peak-to-trough analysis (FIG. 42G) provides a simple readout of exposure stability. The ratios remain low for both depots, confirming minimized burst release and a smooth transition into sustained delivery. This stabilization is the primary practical advantage of methionine-containing networks: by blunting oxidation-driven early leakage, the system produces a flatter PK curve without sacrificing total exposure.

[0453] Area under the curve (AUC) measurements over the full 42-day period (FIG.42H) show that both P3-M-Sr and P3-M deliver comparable total drug exposure to the daily 20 pg bolus reference, despite relying on only a single injection. The absence of a marked decrease in AUC for the Sr2+formulation indicates that concerns about reduced bioavailability were not borne out in vivo.

[0454] These data demonstrate that HPMC-C18 and methionine-based depots can sustain long-acting semaglutide delivery with controlled early-phase behavior, low peak-to-trough fluctuation, and multi-week exposure in diabetic animals. The Sr2+-loaded variant behaves similarly, with subtle differences in late-phase kinetics but no loss of total bioavailability. This long-term T2D rat study validates methionine-reinforced hydrogels as a viable platform for stabilizing lipidated incretin therapeutics in chronic disease models in pharmacokinetics perspective.Example 21: Toxicity, Biocompatibility, and Tissue Response of HMPC-C18 Depots in T2D Mice

[0455] This example describes experiments related to assessing the toxicity and biocompatibility of HMPC-C18 depots when implanted in T2D mice.

[0456] Blood chemistry and histology analysis for biocompatibility. Blood chemistry panel was evaluated at Day 47 post injection. On Day 55, a total of sixteen T2D rats were euthanized using carbon dioxide and cardiac puncture for necropsy, including one T2D control rat without hydrogel injection, eight rats that received P3-M-Sr, and seven rats that received P3-M. The pancreas, liver, kidneys, and the skin from the injection site were collected for histological evaluation. Tissues were fixed in 10% neutral -buffered formalin for 72 hours, trimmed and submitted to an external vendor for embedding and slide preparation (HistoTec Inc. Hayward, California). Tissue collection was performed at the Veterinary Service Center, Department of Comparative Medicine at Stanford University School of Medicine. Hematoxylin and eosin (H& E) staining was used to assess overall tissue morphology and local tissue response, while Masson’s tri chrome staining was used to evaluate fibrosis at the injection sites.Histopathological examination was conducted by a veterinary pathologist (WR) blinded to treatment groups. For morphological examination, slides were examined with an Olympus BX53 microscope, Olympus DP27 Digital Camera and the respective cellSens Entry software, version 1.18 (Olympus America Inc. Waltham MA).[04571 For the quantitative evaluation of the islet area, H& E stained slides were digitized with the Olympus VS 120 system and respective VS-ASW-S6 software, version 2.9.2 (Olympus America Inc. Waltham MA) at 40x magnification. The whole-slide images of pancreas were evaluated with the QuPath software, version 0.6.0.17 The whole pancreas area was detected by thresholding (Channel: Green, threshold: 170.0) and irrelevant tissues (e.g. lymph nodes) were excluded manually. Thereafter, islets were annotated manually. Single islet areas and whole organ area measurements were exported to Excel, summed up and islet-to-organ percentages were calculated. Data visualization and statistical analysis was performed using GraphPad Prism, version 10.6.1 (GraphPad Software LLC, Boston, MA).

[0458] Depot Persistence. Following T2D induction, hydrogel injection, and eight weeks of monitoring, the gel depots from 15 rats were excised. Nearly all rats, except one in the P3-M group, retained a detectable hydrogel depot within the subcutis at the Day 55 endpoint. No differences were observed between P3-M-Sr and P3-M in terms of infiltrate composition or fibrosis. The persistence of the retained depot suggests that the residual concentrations observed at the end of the pharmacokinetic study are plausibly due to ongoing release rather than pure ELISA noise.

[0459] Biocompatibility and Tissue Response. To evaluate systemic and local biocompatibility of the sustained-release hydrogels, kidney, pancreas, and liver, and skin at the injection site were examined 55 days post-administration. In all rats, regenerative and occasionally degenerative lesions are detected within the pancreatic islets. Lesions are characterized by mild to moderate cellular atypia (hypertrophy, increased basophilia, increased nuclear size, spindle-like appearance) and mild vacuolation of islet cells.|046()[ Within the pancreas, both treatment groups exhibited mild regenerative islet changes consistent with adaptive remodeling in the diabetic state, without inflammatory cell infiltration. P3-M group exhibited a significantly higher islet area relative to total pancreas area compared to P3-M-Sr (p = 0.0023, Welch’s t-test), suggesting enhanced islet preservation or regeneration, whereas the P3-M-Sr resembled the untreated diabetic control. The increased islet-organ-ratio in P3-M treated rats indicates a greater capacity for islet remodeling relativeto P3-M-Sr. These findings suggest that P3-M hydrogel treatment supports islet maintenance or regeneration in diabetic rats, consistent with regenerative trends reported for GLP-1 based therapies, although specific endocrine cell types cannot be distinguished by routine histology.

[0461] At the subcutaneous injection site, retained hydrogel material was observed in all treated rats except one in P3-M. The hydrogels were surrounded by a well-demarcated layer of foamy macrophages and low numbers of neutrophils and lymphocytes, accompanied by mild peripheral fibroblast proliferation and collagen deposition, which are features consistent with a controlled foreign-body response during gradual material resorption. No necrosis, abscess formation, or tissue destruction was observed in either group, and the severity and character of the local tissue response were comparable between P3-M-Sr and P3-M. The hydrogel was encapsulated by histiocytes and neutrophilic granulocytes with mild fibrosis, indicative of an ongoing but well-regulated degradative process. Overall, depots persisted through Day 55 with only mild inflammation and fibrosis, supporting good local tolerability and an intended multiweek release profile.[04621 Across all biochemical parameters, the results indicate overall metabolic and organ stability following treatment. For HbAlc, only the Bolus group showed a statistically significant reduction, whereas both P3 formulations maintained comparable values between Day 0 and Day 47, indicating stable glycemic control over the study period. A slight rise was observed in P3-M-Sr, while P3-M remained largely within normal ranges, suggesting that strontium addition may modestly influence GLP-1 RA bioavailability, consistent with the higher daily blood glucose trend in P3-M-Sr compared with P3-M after first week post treatment. For AST, an elevation was observed in the Bolus-R group, consistent with mild hepatic enzyme induction, while both P3 groups showed no significant changes, indicating stable hepatic function. ALT remained stable across all treatments, with no evidence of hepatocellular injury. Bilirubin values were within normal limits in all groups, with P3-M-Sr showing a small but statistically significant decrease (p = 0.0082, paired t test) that remained well within the physiological range. For BUN, both P3-M-Sr and P3-M exhibited statistically significant decreases (p = 0.018 and 0.0062, respectively), whereas the Bolus-R group showed consistent but nonsignificant levels, supporting good renal tolerance and absence of nephrotoxicity for all three treatments. Creatinine decreased in all groups; although a subset of Bolus rats dropped slightly below the physiological range, both P3 formulations showed modest, statistically significant reductions that remained within normal limits, potentially reflecting mild hydration effects rather than renal impairment.

[0463] FIGS. 43 A-43G illustrate the pharmacodynamic efficacy, metabolic outcomes, pancreatic remodeling, and local tissue response of long-acting GLP-1 hydrogels in T2D rats. FIG. 43 A is a graph of blood glucose profiles over 6 weeks comparing daily bolus semaglutide, P3-M-Sr, and P3-M depots. Green shading indicates the normoglycemic range. FIG. 43B is a graph of body -weight trajectories showing weight stabilization in depot-treated groups relative to daily bolus. FIG. 43C is a graph of percent weight change at study end. FIG. 43D is a graph of HbAlc before treatment (Day 0) and after 47 days, demonstrating significant HbAlc reduction in daily bolus and P3-M-Sr groups, with stable low levels maintained by P3-M.

[0464] FIG. 43E shows representative whole-pancreas H& E sections from untreated T2D, P3-M-Sr, and P3-M groups. Black boxes denote example regions used for islet quantification (scale bars: 2 mm). FIG. 43F is a graph of quantified islet-to-pancreas area ratios, showing robust islet preservation / regeneration in P3-M. FIG. 43 G shows the local skin response at the injection site (Day 0 and Day 47), excised depot images, and the corresponding H& E and Masson’s tri chrome histology. No adverse tissue reactions or fibrosis were observed in either depot group.Additional Examples (I)

[0465] Additional examples of aspects of the present technology are described below as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the subject technology.

[0466] Clause I-1. A composition for treating a disease or condition, the composition comprising:a dynamic hydrogel comprising a polymer and a plurality of nanoparticles, wherein the polymer is non-covalently crosslinked with the plurality of nanoparticles; andan acylated peptide encapsulated by the dynamic hydrogel.

[0467] Clause I-2. The composition of Clause I-1, wherein the acylated peptide includes a lipophilic substituent.

[0468] Clause I-3. The composition of Clause I-2, wherein the acylated peptide is encapsulated in the dynamic hydrogel via interactions between the lipophilic substituent and the dynamic hydrogel.

[0469] Clause I-4. The composition of Clause I-3, wherein the plurality of nanoparticles have hydrophobic surfaces that interact with the lipophilic substituent of the dynamic hydrogel.

[0470] Clause I-5. The composition of any one of Clauses I-2 to I-4, wherein the lipophilic substituent comprises an acyl group of a fatty acid.[04711 Clause I-6. The composition of Clause I-5, wherein the fatty acid is a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a CIO fatty acid, a C12 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.[0472| Clause I-7. The composition of any one of Clauses I-2 to I-6, further comprising a binding agent that interacts with the lipophilic substituent to physically entrap the acylated peptide in the dynamic hydrogel.|0473] Clause I-8. The composition of Clause I-7, wherein the binding agent comprises albumin.

[0474] Clause I-9. The composition of Clause I-7 or I-8, wherein the binding agent is larger than a mesh size of the dynamic hydrogel.

[0475] Clause I-10. The composition of any one of Clauses I-1 to I-9, wherein the acylated peptide is smaller than a mesh size of the dynamic hydrogel.

[0476] Clause I-11. The composition of any one of Clauses I-1 to I-10, further comprising a dispersing agent that inhibits aggregation of the acylated peptide.

[0477] Clause I-12. The composition of Clause I-11, wherein the dispersing agent comprises one or more of a surfactant or a tonicity agent.

[0478] Clause I-13. The composition of Clause I-12, wherein the dispersing agent comprises the surfactant, and the surfactant comprises one or more of a polysorbate, a sorbitan fatty acid ester, a poloxamer, a polyoxyethylene alkyl ether, an alkyl sulfate, a fatty acid, a fatty acid alcohol, or a phospholipid.

[0479] Clause I-14. The composition of Clause I-12 or I-13, wherein the dispersing agent comprises the tonicity agent, and the tonicity agent comprises one or more of propylene glycol, glycerol, or mannitol.

[0480] Clause 1-15. The composition of any one of Clauses I-12 to I-14, wherein a concentration of the dispersing agent in the composition is within a range from 0.1 mg / mL to 5 mg / mL, 0.5 mg / mL to 1.5 mg / mL, 1 mg / mL to 2 mg / mL, 1 mg / mL to 50 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 16 mg / mL to 26 mg / mL.

[0481] Clause I-16. The composition of any one of Clauses I-1 to I-15, wherein the acylated peptide comprises an incretin mimetic.

[0482] Clause 1-17. The composition of Clause I-16, wherein the incretin mimetic comprises a glucagon-like peptide-1 receptor agonist (GLP-1 RA).

[0483] Clause 1-18. The composition of Clause I-17, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

[0484] Clause 1-19. The composition of any one of Clauses I-16 to I-18, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

[0485] Clause 1-20. The composition of any one of Clauses I-16 to I-18, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

[0486] Clause 1-21. The composition of Clause I-20, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

[0487] Clause 1-22. The composition of any one of Clauses I-1 to I-15, wherein the acylated peptide comprises an analogue of a proglucagon-derived peptide.

[0488] Clause 1-23. The composition of any one of Clauses I-1 to I-15, wherein the acylated peptide comprises an amylin analogue.

[0489] Clause 1-24. The composition of any one of Clauses I-1 to I-15, wherein the acylated peptide comprises an insulin analogue.

[0490] Clause 1-25. The composition of any one of Clauses I-1 to I-24, wherein the composition comprises from 1 mg to 250 mg of the acylated peptide.

[0491] Clause I-26. The composition of any one of Clauses I-1 to I-25, wherein a concentration of the acylated peptide in the composition is within a range from 1 mg / mL to 200 mg / mL.

[0492] Clause 1-27. The composition of any one of Clauses I-1 to I-26, wherein the polymer comprises a hydrophobically-modified polysaccharide.

[0493] Clause 1-28. The composition of Clause I-27, wherein the hydrophobically-modified polysaccharide comprises a hydrophobically-modified cellulose derivative.

[0494] Clause 1-29. The composition of Clause I-28, wherein the hydrophobically-modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

[0495] Clause 1-30. The composition of any one of Clauses I-1 to I-29, wherein the plurality of nanoparticles comprise a plurality of polymeric nanoparticles.

[0496] Clause 1-31. The composition of Clause I-30, wherein the plurality of polymeric nanoparticles are amphiphilic.

[0497] Clause I-32. The composition of Clause I-30 or I-31, wherein the plurality of polymeric nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

[0498] Clause 1-33. The composition of any one of Clauses I-1 to I-32, wherein a concentration of the polymer in the dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.|< 99] Clause 1-34. The composition of any one of Clauses I-1 to I-33, wherein a concentration of the nanoparticles in the dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0500] Clause 1-35. The composition of any one of Clauses I-1 to I-34, wherein the dynamic hydrogel encapsulating the acylated peptide has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the dynamic hydrogel.

[0501] Clause 1-36. The composition of any one of Clauses I-1 to I-35, wherein the dynamic hydrogel encapsulating the acylated peptide has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0502] Clause 1-37. The composition of any one of Clauses I-1 to I-36, wherein the dynamic hydrogel encapsulating the acylated peptide has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s-1.

[0503] Clause 1-38. The composition of any one of Clauses I-1 to I-37, wherein, upon administration to a subject, the composition delivers the acylated peptide to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0504] Clause 1-39. The composition of any one of Clauses I-1 to I-38, wherein, upon administration to a subject, the composition delivers the acylated peptide at a rate to the subject within a range from 0.25 mg / week to 25 mg / week, 0.5 mg / week to 20 mg / week, 0.5 mg / week to 2 mg / week, 0.5 mg / week to 1.5 mg / week, 1 mg / week to 2 mg / week, 2 mg / week to 5 mg / week, 5 mg / week to 15 mg / week, 5 mg / week to 10 mg / week, 10 mg / week to 20 mg / week, 10 mg / week to 15 mg / week, 12 mg / week to 25 mg / week, 12 mg / week to 15 mg / week, 15 mg / week to 25 mg / week, 15 mg / week to 20 mg / week, or 20 mg / week to 25 mg / week.

[0505] Clause 1-40. The composition of any one of Clauses I-1 to I-39, wherein, upon administration to a subject, the composition produces an average concentration of the acylated peptide in serum of the subject within a range from 50 ng / mL to 500 ng / mL, 100 ng / mL to 300 ng / mL, or 150 ng / mL to 250 ng / mL.

[0506] Clause 1-41. The composition of any one of Clauses I-1 to I-40, wherein, upon administration to a subject, the composition produces a Cmaxof the acylated peptide in serum of the subject less than or equal to 2000 ng / mL.

[0507] Clause 1-42. The composition of any one of Clauses I-1 to I-41, wherein the composition is configured for administration via subcutaneous injection.

[0508] Clause I-43. The composition of any one of Clauses I-1 to I-42, wherein the disease or condition comprises diabetes or a diabetes-related condition.

[0509] Clause 1-44. The composition of Clause I-43, wherein the diabetes or diabetes-related condition comprises prediabetes, type I diabetes, type II diabetes, hyperglycemia, or impaired glucose tolerance.

[0510] Clause 1-45. The composition of any one of Clauses I-1 to I-44, wherein the disease or condition comprises one or more of obesity, excessive body weight, an eating disorder, or obstructive sleep apnea.

[0511] Clause 1-46. The composition of any one of Clauses I-1 to I-45, wherein the disease or condition comprises one or more of the following: a cardiovascular disease, a liver disease, a neurological or neurodegenerative disease, an inflammatory disease, a renal disease, a bone disease, a hormonal disease, or a gastrointestinal disease.

[0512] Clause 1-47. A method of treating a disease or condition, the method comprising: administering a composition to a subject, wherein the composition comprises: a dynamic hydrogel comprising a polymer and a plurality of nanoparticles, wherein the polymer is non-covalently crosslinked with the plurality of nanoparticles, and an acylated peptide encapsulated by the dynamic hydrogel.

[0513] Clause I-48. The method of Clause I-47, wherein the acylated peptide includes a lipophilic substituent.

[0514] Clause 1-49. The method of Clause I-48, wherein the acylated peptide is encapsulated in the dynamic hydrogel via interactions between the lipophilic substituent and the dynamic hydrogel.

[0515] Clause 1-50. The method of Clause I-49, wherein the plurality of nanoparticles have hydrophobic surfaces that interact with the lipophilic substituent of the dynamic hydrogel.

[0516] Clause 1-51. The method of any one of Clauses I-48 to I-50, wherein the lipophilic substituent comprises an acyl group of a fatty acid.

[0517] Clause 1-52. The method of Clause I-51, wherein the fatty acid is a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a C10 fatty acid, a C12 fatty acid, a C14 fatty acid, a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.

[0518] Clause 1-53. The method of any one of Clauses I-48 to I-52, further comprising a binding agent that interacts with the lipophilic substituent to physically entrap the acylated peptide in the dynamic hydrogel.

[0519] Clause 1-54. The method of Clause I-53, wherein the binding agent comprises albumin.

[0520] Clause 1-55. The composition of Clause I-53 or I-54, wherein the binding agent is larger than a mesh size of the dynamic hydrogel.

[0521] Clause 1-56. The method of any one of Clauses I-47 to I-55, wherein the acylated peptide is smaller than a mesh size of the dynamic hydrogel.

[0522] Clause 1-57. The method of any one of Clauses I-47 to I-56, further comprising a dispersing agent that inhibits aggregation of the acylated peptide.

[0523] Clause 1-58. The method of Clause I-57, wherein the dispersing agent comprises one or more of a surfactant or a tonicity agent.

[0524] Clause 1-59. The method of Clause I-58, wherein the dispersing agent comprises the surfactant, and the surfactant comprises one or more of a polysorbate, a sorbitan fatty acid ester, a poloxamer, a polyoxyethylene alkyl ether, an alkyl sulfate or a salt thereof, a fatty acid, a fatty acid alcohol.

[0525] Clause 1-60. The method of Clause I-58 or I-59, wherein the dispersing agent comprises the tonicity agent, and the tonicity agent comprises one or more of propylene glycol, glycerol, or mannitol.

[0526] Clause I-61. The method of any one of Clauses I-58 to I-60, wherein a concentration of the dispersing agent in the composition is within a range from 0.1 mg / mL to 5 mg / mL, 0.5 mg / mL to 1.5 mg / mL, 1 mg / mL to 2 mg / mL, 1 mg / mL to 50 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 16 mg / mL to 26 mg / mL.

[0527] Clause 1-62. The method of any one of Clauses I-47 to I-61, wherein the acylated peptide comprises an incretin mimetic.

[0528] Clause 1-63. The method of Clause I-62, wherein the incretin mimetic comprises a glucagon-like peptide-1 receptor agonist (GLP-1 RA).

[0529] Clause 1-64. The method of Clause I-63, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

[0530] Clause 1-65. The method of any one of Clauses I-62 to I-64, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

[0531] Clause 1-66. The method of any one of Clauses I-62 to I-64, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

[0532] Clause I-67. The method of Clause I-66, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

[0533] Clause I-68. The method of any one of Clauses I-47 to I-67, wherein the acylated peptide comprises an analogue of a proglucagon-derived peptide.

[0534] Clause 1-69. The method of any one of Clauses I-47 to I-67, wherein the acylated peptide comprises an amylin analogue.

[0535] Clause 1-70. The method of any one of Clauses I-47-67 or I-69, wherein the acylated peptide comprises an insulin analogue.

[0536] Clause I-71. The method of any one of Clauses I-47 to I-70, wherein the composition comprises from 1 mg to 250 mg of the acylated peptide.

[0537] Clause 1-72. The method of any one of Clauses I-47 to I-71, wherein a concentration of the acylated peptide in the composition is within a range from 1 mg / mL to 200 mg / mL.

[0538] Clause 1-73. The method of any one of Clauses I-47 to I-72, wherein the polymer comprises a hydrophobically-modified polysaccharide.

[0539] Clause 1-74. The method of Clause I-73, wherein the hydrophobically-modified polysaccharide comprises a hydrophobically-modified cellulose derivative.

[0540] Clause I-75. The method of Clause I-74, wherein the hydrophobically-modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

[0541] Clause 1-76. The method of any one of Clauses I-47 to I-75, wherein the plurality of nanoparticles comprise a plurality of polymeric nanoparticles.

[0542] Clause 1-77. The method of Clause I-76, wherein the plurality of polymeric nanoparticles are amphiphilic.

[0543] Clause 1-78. The method of Clause I-76 or I-77, wherein the plurality of polymeric nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

[0544] Clause 1-79. The method of any one of Clauses I-47 to I-78, wherein a concentration of the polymer in the dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0545] Clause 1-80. The method of any one of Clauses I-47 to I-79, wherein a concentration of the nanoparticles in the dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0546] Clause 1-81. The method of any one of Clauses I-47 to I-80, wherein the dynamic hydrogel encapsulating the acylated peptide has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the dynamic hydrogel.

[0547] Clause 1-82. The method of any one of Clauses I-47 to I-81, wherein the dynamic hydrogel encapsulating the acylated peptide has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0548] Clause 1-83. The method of any one of Clauses I-47 to I-82, wherein the dynamic hydrogel encapsulating the acylated peptide has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s-1.

[0549] Clause 1-84. The method of any one of Clauses I-47 to I-83, wherein, upon administration to the subject, the composition delivers the acylated peptide to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0550] Clause 1-85. The method of any one of Clauses I-47 to I-84, wherein, upon administration to the subject, the composition delivers the acylated peptide at a rate to the subject within a range from 0.25 mg / week to 25 mg / week, 0.5 mg / week to 20 mg / week, 0.5 mg / week to 2 mg / week, 0.5 mg / week to 1.5 mg / week, 1 mg / week to 2 mg / week, 2 mg / week to 5 mg / week, 5 mg / week to 15 mg / week, 5 mg / week to 10 mg / week, 10 mg / week to 20 mg / week, 10 mg / week to 15 mg / week, 12 mg / week to 25 mg / week, 12 mg / week to 15mg / week, 15 mg / week to 25 mg / week, 15 mg / week to 20 mg / week, or 20 mg / week to 25 mg / week.

[0551] Clause 1-86. The method of any one of Clauses I-47 to I-85, wherein, upon administration to the subject, the composition produces an average concentration of the acylated peptide in serum of the subject within a range from 50 ng / mL to 500 ng / mL, 100 ng / mL to 300 ng / mL, or 150 ng / mL to 250 ng / mL.

[0552] Clause 1-87. The method of any one of Clauses I-47 to I-86, wherein, upon administration to the subject, the composition produces a Cmaxof the acylated peptide in serum of the subject less than or equal to 2000 ng / mL.

[0553] Clause 1-88. The method of any one of Clauses I-47 to I-87, wherein the composition is administered via subcutaneous injection.

[0554] Clause 1-89. The method of any one of Clauses I-47 to I-88, wherein the disease or condition comprises diabetes or a diabetes-related condition.

[0555] Clause 1-90. The method of Clause I-89, wherein the diabetes or diabetes-related condition comprises prediabetes, type I diabetes, type II diabetes, hyperglycemia, or impaired glucose tolerance.

[0556] Clause I-91. The method of any one of Clauses I-47 to I-90, wherein the disease or condition comprises one or more of obesity, excessive body weight, an eating disorder, or obstructive sleep apnea.

[0557] Clause 1-92. The method of any one of Clauses I-47 to I-91, wherein the disease or condition comprises one or more of the following: a cardiovascular disease, a liver disease, a neurological or neurodegenerative disease, an inflammatory disease, a renal disease, a bone disease, a hormonal disease, or a gastrointestinal disease.

[0558] Clause 1-93. A composition for treating a disease or condition, the composition comprising: a dynamic hydrogel comprising a polymer and a plurality of nanoparticles, wherein the polymer is non-covalently crosslinked with the plurality of nanoparticles; and an incretin mimetic encapsulated by the dynamic hydrogel.

[0559] Clause I-94. The composition of Clause I-93, wherein the incretin mimetic comprises a glucagon-like peptide-1 receptor agonist (GLP-1 RA).

[0560] Clause 1-95. The composition of Clause I-94, wherein the GLP-1 RA comprises exenatide, exenatide-LAR, lixisenatide, liraglutide, semaglutide, albiglutide,dulaglutide, efpeglenatide, ecnoglutide, efinopegdutide, cotadutide, mazdutide, BI 45690, tirzepatide, LY3493269, VK2735, CT-868, AMG133, or retatrutide.

[0561] Clause 1-96. The composition of Clause I-94, wherein the GLP-1 RA comprises liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

[0562] Clause I-97. The composition of any one of Clauses I-94 to I-96, wherein the GLP-1 RA comprises a peptide having a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 1 or SEQ ID NO:2.

[0563] Clause 1-98. The composition of any one of Clauses I-93 to I-97, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

[0564] Clause I-99. The composition of any one of Clauses I-93 to I-97, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

[0565] Clause 1-100. The composition of Clause I-99, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

[0566] Clause I-101. The composition of any one of Clauses I-93 to I-100, wherein the incretin mimetic comprises an acylated peptide.

[0567] Clause 1-102. The composition of Clause I-101, wherein the acylated peptide includes a lipophilic substituent.

[0568] Clause 1-103. The composition of Clause I-102, wherein the acylated peptide is encapsulated in the dynamic hydrogel via interactions between the lipophilic substituent and the dynamic hydrogel.

[0569] Clause 1-104. The composition of Clause I-103, wherein the plurality of nanoparticles have hydrophobic surfaces that interact with the lipophilic substituent of the dynamic hydrogel.

[0570] Clause 1-105. The composition of any one of Clauses I-102 to I-104, wherein the lipophilic substituent comprises an acyl group of a fatty acid.

[0571] Clause 1-106. The composition of Clause I-105, wherein the fatty acid is a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a C10 fatty acid, a C12 fatty acid, a C14 fatty acid,a C15 fatty acid, a C16 fatty acid, a C17 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, a C30 fatty acid, a C32 fatty acid, a C34 fatty acid, a C36 fatty acid, or a C38 fatty acid.

[0572] Clause 1-107. The composition of any one of Clauses I-102 to I-106, further comprising a binding agent that interacts with the lipophilic substituent to physically entrap the acylated peptide in the dynamic hydrogel.

[0573] Clause 1-108. The composition of Clause I-107, wherein the binding agent comprises albumin.

[0574] Clause 1-109. The composition of Clause I-107 or I-108, wherein the binding agent is larger than a mesh size of the dynamic hydrogel.

[0575] Clause 1-110. The composition of any one of Clauses I-101 to I-109, wherein the acylated peptide is smaller than a mesh size of the dynamic hydrogel.

[0576] Clause 1-111. The composition of any one of Clauses I-101 to I-110, further comprising a dispersing agent that inhibits aggregation of the acylated peptide.

[0577] Clause 1-112. The composition of Clause I-111, wherein the dispersing agent comprises one or more of a surfactant or a tonicity agent.

[0578] Clause 1-113. The composition of Clause I-112, wherein the dispersing agent comprises the surfactant, and the surfactant comprises one or more of a polysorbate, a sorbitan fatty acid ester, a poloxamer, a polyoxyethylene alkyl ether, an alkyl sulfate or a salt thereof, a fatty acid, a fatty acid alcohol, or a phospholipid.

[0579] Clause 1-114. The composition of Clause I-112 or I-113, wherein the dispersing agent comprises the tonicity agent, and the tonicity agent comprises one or more of propylene glycol, glycerol, or mannitol.

[0580] Clause 1-115. The composition of any one of Clauses I-112 to I-114, wherein a concentration of the dispersing agent in the composition is within a range from 0.1 mg / mL to 5 mg / mL, 0.5 mg / mL to 1.5 mg / mL, 1 mg / mL to 2 mg / mL, 1 mg / mL to 50 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 16 mg / mL to 26 mg / mL.

[0581] Clause 1-116. The composition of any one of Clauses I-93 to I-100, wherein the incretin mimetic comprises a peptide conjugated to a protein or protein fragment.

[0582] Clause 1-117. The composition of any one of Clauses 1-93 to 1-100, wherein the incretin mimetic comprises a peptide encapsulated in a microparticle.

[0583] Clause 1-118. The composition of any one of Clauses 1-93 to 1-117, wherein the composition comprises from 1 mg to 250 mg of the incretin mimetic.

[0584] Clause 1-119. The composition of any one of Clauses 1-93 to 1-118, wherein a concentration of the incretin mimetic in the composition is within a range from 1 mg / mL to 200 mg / mL.

[0585] Clause 1-120. The composition of any one of Clauses 1-93 to 1-119, wherein the polymer comprises a hydrophobically-modified polysaccharide.

[0586] Clause 1-121. The composition of Clause 1-120, wherein the hydrophobically-modified polysaccharide comprises a hydrophobically-modified cellulose derivative.

[0587] Clause 1-122. The composition of Clause 1-121, wherein the hydrophobically-modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

[0588] Clause 1-123. The composition of any one of Clauses 1-93 to 1-122, wherein the plurality of nanoparticles comprise a plurality of polymeric nanoparticles.

[0589] Clause 1-124. The composition of Clause 1-123, wherein the plurality of polymeric nanoparticles are amphiphilic.

[0590] Clause 1-125. The composition of Clause 1-123 or 1-124, wherein the plurality of polymeric nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

[0591] Clause 1-126. The composition of any one of Clauses 1-93 to 1-125, wherein a concentration of the polymer in the dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt%...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising:a dynamic hydrogel;an incretin mimetic encapsulated by the dynamic hydrogel; andan antioxidant encapsulated by the dynamic hydrogel, wherein the antioxidant is configured to stabilize the incretin mimetic.

2. The composition of claim 1, wherein the dynamic hydrogel is a PNP hydrogel.

3. The composition of claim 2, wherein the PNP hydrogel comprises a polymer and a plurality of nanoparticles, wherein the polymer is non-covalently crosslinked with the plurality of nanoparticles.

4. The composition of claim 3, wherein the plurality of nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

5. The composition of claim 1, wherein dynamic hydrogel is a PXY hydrogel.

6. The composition of claim 5, wherein the PXY hydrogel is octadecyl modified hydroxypropyl methylcellulose (HPMC-C18).

7. The composition of claim 5 or claim 6, wherein a concentration of the PXY hydrogel is at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.75 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%.

8. The composition of any one of claims 1 to 7, further comprising a divalent cation.

9. The composition of any one of claims 1 to 8, wherein the composition has a pH less than or equal to 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2.

10. The composition of any one of claims 1 to 9, wherein when administered to a subject in vivo, the composition produces a Cmax to Csteady-state ratio of the incretin mimetic that is less than or equal to 1000:1, 500:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, or 1:1.

11. The composition of any one of claims 1 to 10, wherein the incretin mimetic comprises a glucagon-like peptide- 1 receptor agonist (GLP-1 RA).

12. The composition of claim 11, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

13. The composition of claim 11 or claim 12, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

14. The composition of claim 11 or claim 12, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

15. The composition of claim 14, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

16. The composition of any one of claims 1 to 15, wherein the antioxidant is a hydrophilic antioxidant.

17. The composition of claim 16, wherein the antioxidant is a weak antioxidant.

18. The composition of claim 17, wherein the antioxidant is L-methionine.

19. The composition of claim 17, wherein the antioxidant is L-histidine.

20. The composition of any one of claims 1 to 15, wherein the antioxidant is a hydrophobic antioxidant.

21. The composition of claim 20, wherein the antioxidant is a strong antioxidant.

22. The composition of claim 21, wherein the antioxidant is a tocopherol.

23. The composition of claim 21, wherein the antioxidant is a-tocopherol.

24. The composition of any one of claims 1 to 15, wherein the antioxidant is acetyl methionine.

25. The composition of claim 24, wherein the antioxidant is N-acetylmethionine.

26. The composition of any one of claims 1-4 and 8 to 25, wherein the dynamic hydrogel comprises a polymer and a plurality of nanoparticles.

27. The composition of claim 26, wherein the polymer comprises a hydrophobically-modified polysaccharide.

28. The composition of claim 27, wherein the hydrophobically-modified polysaccharide comprises a hydrophobically-modified cellulose derivative.

29. The composition of claim 28, wherein the hydrophobically-modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

30. The composition of any one of claims 26 to 29, wherein the plurality of nanoparticles comprise a plurality of polymeric nanoparticles.

31. The composition of claim 30, wherein the plurality of polymeric nanoparticles are amphiphilic.

32. The composition of claim 30 or 31, wherein the plurality of polymeric nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

33. The composition of any one of claims 26 to 32, wherein a concentration of the polymer in the dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

34. The composition of any one of claims 26 to 33, wherein a concentration of the nanoparticles in the dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

35. The composition of any one of claims 1 to 34, wherein the dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the dynamic hydrogel.

36. The composition of any one of claims 1 to 35, wherein the dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25°C.

37. The composition of any one of claims 1 to 36, wherein the dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s-1.

38. The composition of any one of claims 1 to 37, wherein, upon administration to a subject, the composition delivers the incretin mimetic to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

39. A method of treating a disease or condition, the method comprising: administering a composition to a subject, wherein the composition comprises: a dynamic hydrogel; an incretin mimetic encapsulated by the dynamic hydrogel; and an antioxidantencapsulated by the dynamic hydrogel, wherein the antioxidant is configured to stabilize the incretin mimetic.

40. The method of claim 39, wherein the dynamic hydrogel is a PNP hydrogel.

41. The method of claim 40, wherein the PNP hydrogel comprises a polymer and a plurality of nanoparticles, wherein the polymer is non-covalently crosslinked with the plurality of nanoparticles.

42. The method of claim 40 or claim 41, wherein the plurality of nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

43. The method of claim 39, wherein dynamic hydrogel is a PXY hydrogel.

44. The method of claim 43, wherein the PXY hydrogel is octadecyl modified hydroxypropyl methylcellulose (HPMC-C18).

45. The method of claim 43 or claim 44, wherein a concentration of the PXY hydrogel is at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.75 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%.

46. The method of any one of claims 39 to 45, further comprising a divalent cation.

47. The method of any one of claims 39 to 46, wherein the composition has a pH less than or equal to 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2.

48. The method of any one of claims 39 to 47, wherein when administered to a subject in vivo, the composition produces a Cmax to Csteady-state ratio of the incretin mimetic that is less than or equal to 1000:1, 500:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, or 1:1.

49. The method of any one of claims 39 to 48, wherein the incretin mimetic comprises a glucagon-like peptide- 1 receptor agonist (GLP-1 RA).

50. The method of claim 49, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

51. The method of claim 49 or claim 50, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

52. The method of claim 49 or claim 50, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

53. The method of claim 52, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

54. The method of any one of claims 39 to 53, wherein the antioxidant is hydrophilic.

55. The method of claim 51, wherein the antioxidant is a weak antioxidant.

56. The method of claim 52, wherein the antioxidant is L-methionine.

57. The method of claim 52, wherein the antioxidant is L-histidine.

58. The method of any one of claims 39 to 53, wherein the antioxidant is hydrophobic.

59. The method of claim 58, wherein the antioxidant is a strong antioxidant.

60. The method of claim 59, wherein the antioxidant is a tocopherol.

61. The method of claim 60, wherein the antioxidant is a-tocopherol.

62. The method of any one of claims 39 to 61, wherein the antioxidant is acetyl methionine.

63. The method of claim 62, wherein the antioxidant is N-acetylmethionine.

64. The method of any one of claims 39-42 and 46 to 63, wherein the dynamic hydrogel comprises a polymer and a plurality of nanoparticles.

65. The method of claim 64, wherein the polymer comprises a hydrophobically-modified polysaccharide.

66. The method of claim 65, wherein the hydrophobically-modified polysaccharide comprises a hydrophobically-modified cellulose derivative.

67. The method of claim 66, wherein the hydrophobically-modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

68. The method of any one of claims 64 to 67, wherein the plurality of nanoparticles comprise a plurality of polymeric nanoparticles.

69. The method of claim 68, wherein the plurality of polymeric nanoparticles are amphiphilic.

70. The method of claim 68 or 69, wherein the plurality of polymeric nanoparticles are poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

71. The method of any one of claims 64 to 70, wherein a concentration of the polymer in the dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

72. The method of any one of claims 64 to 71, wherein a concentration of the nanoparticles in the dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

73. The method of any one of claims 39 to 72, wherein the dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the dynamic hydrogel.

74. The method of any one of claims 39 to 73, wherein the dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25°C.

75. The method of any one of claims 39 to 74, wherein the dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s-1.

76. The method of any one of claims 39 to 75, wherein, upon administration to a subject, the composition delivers the incretin mimetic to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

77. A composition comprising:a first agent, wherein the first agent is octadecyl modified hydroxypropyl methylcellulose (HPMC-C18), and wherein the first agent forms micellar structures; anda second agent comprising an incretin mimetic, wherein the second agent modulates the micellar structures of the first agent.

78. The composition of claim 77, wherein the incretin mimetic is an acylated peptide.

79. The composition of claim 77, wherein the incretin mimetic is a lipidated peptide.

80. The composition of any one of claims 77 to 79, wherein the incretin mimetic comprises a glucagon-like peptide-1 receptor agonist (GLP-1 RA).

81. The composition of claim 80, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

82. The composition of claim 80 or claim 81, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

83. The composition of claim 80 or claim 81, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

84. The composition of claim 83, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

85. The composition of any one of claims 77 to 84, wherein, upon administration to a subject, the composition delivers the incretin mimetic to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

86. The composition of any one of claims 77 to 85, wherein a concentration of the first agent is at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.75 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%.

87. A method of treating a disease or condition, the method comprising: administering a composition to a subject, wherein the composition comprises: a first agent, wherein the first agent is octadecyl modified hydroxypropyl methylcellulose (HPMC-C18), and wherein the first agent forms micellar structures; and a second agent comprising an incretin mimetic, wherein the second agent modulates the micellar structures of the first agent.

88. The method of claim 87, wherein the incretin mimetic is a lipidated peptide.

89. The method of claim 87, wherein the incretin mimetic is an acylated peptide.

90. The method of claim 88 or claim 89, wherein the incretin mimetic comprises a glucagon-like peptide- 1 receptor agonist (GLP-1 RA).

91. The method of claim 90, wherein the GLP-1 RA comprises one or more of liraglutide, semaglutide, ecnoglutide, cotadutide, mazdutide, tirzepatide, or retatrutide.

92. The method of claim 90 or claim 91, wherein the incretin mimetic binds exclusively to a GLP-1 receptor.

93. The method of claim 90 or claim 91, wherein the incretin mimetic binds to a GLP-1 receptor and at least one additional receptor.

94. The method of claim 93, wherein the at least one additional receptor comprises one or more of a glucagon receptor or a gastric inhibitory peptide (GIP) receptor.

95. The method of any one of claims 87 to 94, wherein, upon administration to a subject, the composition delivers the incretin mimetic to the subject over a treatment period of at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

96. The method of any one of claims 87 to 95, wherein a concentration of the first agent is at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, at least 0.75 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%.

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