Injectable cross-linked chitosan hydrogels

Abstract

The subject matter described herein is directed to cross-linked chitosan hydrogels having desired properties and methods for their preparation and use.

Description

Attorney Docket No.035051 / 618192 INJECTABLE CROSS-LINKED CHITOSAN HYDROGELS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No.63 / 581,448, filed on September 8, 2023, the entire contents of which are incorporated herein by reference. FIELD

[0002] The presently disclosed subject matter relates generally to cross-linked chitosan hydrogels and methods of preparing the hydrogels and their use in therapy as delivery vehicles for active agents. SEQUENCE LISTING

[0003] The Sequence Listing written in file ‘618192SEQLIST.xml’ is 8.4 kilobytes in size, was created September 6, 2024, and is hereby incorporate by reference in its entirety. BACKGROUND

[0004] Localized delivery methods with slow-release kinetics have the potential to avoid systemic exposure and improve the therapeutic index of a co-formulated drug, biologic or other agent. Although direct tumor injection is a common localized delivery approach, intratumoral delivery is extremely difficult, as saline-based solutions are rapidly excluded from the high- pressure tumor environment.

[0005] As described herein, utilizing hydrogels as a delivery medium and local depot can address these shortcomings in the art and more. BRIEF SUMMARY

[0006] In certain embodiments, the subject matter disclosed herein is directed to a hydrogel comprising a cross-linked chitosan, wherein the cross-linked chitosan is prepared by contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0007] In certain embodiments, the subject matter disclosed herein is directed to a hydrogel comprising a cross-linked chitosan, wherein the cross-linked chitosan comprises at least one residue of succinylated chitosan crosslinked to a residue of chitosan. - 1 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0008] In certain embodiments, the subject matter disclosed herein is directed to a lyophilized cross-linked chitosan, wherein the cross-linked chitosan comprises at least one residue of succinylated chitosan crosslinked to a residue of chitosan.

[0009] In certain embodiments, the subject matter disclosed herein is directed to a lyophilized cross-linked chitosan, wherein the cross-linked chitosan is prepared by contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0010] In certain embodiments, the subject matter disclosed herein is directed to a method of delivering a cargo to a subject, comprising administering a cross-linked chitosan hydrogel comprising the cargo as described herein to the subject.

[0011] In certain embodiments, the subject matter disclosed herein is directed to a method of preparing a hydrogel comprising a cross-linked chitosan, the method comprising contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, wherein the chitosan is cross-linked.

[0012] These and other embodiments are described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A-D depicts certain macroscopic properties of the chitosan hydrogel, demonstrating (A / B) injectability, (C) retention within a agar tumor phantom, and (D) self-healing post-injection.

[0014] Figure 2A-B are1H NMR spectra of (A) unmodified chitosan and (B) modified chitosan.

[0015] Figure 3A-H depicts continuous flow viscosity tests and frequency sweeps on unpurified crosslinked chitosan gel. Rheology was performed on the following EDC / NHS to chitosan monomer molar ratios: 1 / 0.1, 1 / 0.25, 1 / 0.5, 1 / 1, 1 / 2, 0.5 / 0.1, 0.5 / 0.25, 0.5 / 0.5, 0.5 / 1. Continuous flow viscosity sweeps of (A) 1 molar and (B) 0.5 molar EDC unpurified chitosan gels, demonstrating a change in viscosity, with (C-D) their corresponding values at 10 Hz. Frequency sweeps of (E) 1 molar and (F) 0.5 molar EDC unpurified gels. demonstrating a change in mechanical properties, with their corresponding storage moduli at 10 rad / s (G-H). EDC molar ratios were varied to demonstrate an increase in viscosity and mechanical properties associated with higher EDC molar ratios. NHS molar ratios were varied to demonstrate an optimal rather than excess ratio of NHS yielded higher crosslinking. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = at least 3 reactions per experimental group. - 2 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0016] Figure 4A-D depicts (A-C) SEM images of gels at 500x and 2000x magnification: (A) 95 / 100_1 / 2_

[0030] chitosan gel; (B) 70 / 100_1 / 2_

[0030] chitosan gel; (C) 95 / 100_1 / 2_

[0060] chitosan gel; and (D) Bar graph of the percentage porosity determined at 1000x. The difference was calculated using one-way ANOVA and Tukey’s HSD post-hoc testing. *P < 0.05. n = at least 3 sample images analyzed per experimental group.

[0017] Figure 5A-L depicts rheological analysis of chitosan hydrogels. (A-C) Amplitude sweep (0.1-500%, 10 Hz) of (A) 1000cp gels, (B) 100cp gels, (C) 10cp gels. (D-F) Frequency sweeps (0.1-100 rad / s, 1%) of (D) 1000cp gels, (E) 100cp gels, (F) 10cp gels. (G-I) Continuous flow viscosity sweeps (0.1-10 Hz) of (G) 1000cp gels, (H) 100cp gels, (I) 10cp gels. (J-L) Cyclic deformation tests (1% and 200% at 10Hz) of (J) 1000cp gel, (K) 100cp gel, and (L) 10cp gel.

[0018] Figure 6 depicts cell viability studies with representative chitosan gels at increasing volumes. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 samples per experimental group.

[0019] Figure 7A-N depicts H&E staining of sections of chitosan gel (A-F), chitosan gel + carboxymethyl chitosan (G-J), chitosan gel + heparin (K-N) at 1x and 10x magnification at days 3, 7, and 62 post-implantation. Mice were injected on the flank subcutaneously with 50uL gel, and the presence and severity of inflammation and longevity of the gel was evaluated over time.

[0020] Figure 8A-C depicts weeklong release of small molecule therapeutics demonstrating a release based on electrostatic charge in 95 / 100_1 / 2_

[0030] chitosan hydrogels.10µg of each fluorophore (rhodamine 6G, rhodamine WT, fluorescein, pyranine) was loaded into 50µL gel and released into 1xPBS. (A) The percentage released was calculated via a log-log standard curve at 370 / 580 ex / em. The percent released at (B) 24 hours and (C) 7 days displayed a statistical difference among the four fluorophores based on their charge at neutral pH. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 samples per experimental group.

[0021] Figure 9A-E depicts weeklong in vitro release of interleukin-12 (IL-12).1µg of IL-12 was loaded into 50µL gel and released into 10% FBS in PBS. (A) The EDC / NHS crosslinker molar ratios were varied (1 / 2 and 0.5 / 1) as were the gel concentrations (30, 60, 90 mg / mL). (B) The inherent chitosan viscosity was evaluated in 95 / 10_1 / 2_

[0060] , 95 / 100_1 / 2_

[0030] , and 95 / 1000_1 / 2_

[0030] gels. (C) The mass of IL-12 was varied in 95 / 100_1 / 2_

[0030] gels demonstrate greater chitosan to protein absorption at lower IL-12 amounts (0.01, 0.1, 1, 10µg). (D) The inclusion of carboxymethyl chitosan at increasing - 3 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 concentrations expedited IL-12 release in 95 / 100_1 / 2_

[0030] gels. (E) The inclusion of heparin (0.2, 2 mg / mL) in 95 / 100_1 / 2_

[0030] gels did not expedite release of IL-12, as compared to 95 / 100_1 / 2_

[0030] control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 samples per experimental group.

[0022] Figure 10A-B depicts weeklong in vitro release of interferon gamma (IFNg) and aCTLA4.1µg of IFNg and 10µg of aCTLA4 were loaded into 50µL 95 / 100_1 / 2_

[0030] and 95 / 100_1 / 2_

[0060] gels and released into 10% FBS in PBS. (A) Cumulative release of IFNg from gels of two concentrations. (B) Cumulative release of aCTLA4 from gels of two concentrations. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 samples per experimental group.

[0023] Figure 11A-C shows that iohexol provided temporary radiopaque character to the hydrogel in vitro and in vivo. (A) Increasing concentrations of iohexol (0, 100, 200 mgI / mL) were loaded into a chitosan hydrogel, and a photograph and x-ray via an AMI HTX imager were obtained in vitro. (B) Weeklong in vitro release of 1ug IL-12 in 50uL 95 / 100_1 / 2_

[0030] gel into 10% FBS in PBS, demonstrating that iohexol does not impact release of a model therapeutic. (C) 200 mgI / mL OMNIPAQUETMwas loaded into 50µL gel, alongside 10µg aCTLA4 and delivered subcutaneously on the right flank of BALB / c mice. Images were taken immediately after injection at Day 0. X-ray images taken 24 hours later did not indicate any iohexol remained localized at the site of injection. n = 3 samples per experimental group for in vitro release. n=5 for in vivo experiments.

[0024] Figure 12A-C depicts aCTLA-4 release in vivo.10µg of aCTLA4 was loaded into 95 / 100_0.5 / 1_

[0060] and 95 / 100_1 / 2_

[0030] + 1 mg / mL CM-CS gels, co-formulated with 200 mgI / mL OMNIPAQUETM.50µL of the therapeutic carrier was delivered subcutaneously on the flanks of mice. (A) Images at 605 / 690 nm (Ex / Em) were taken at days 0, 1, 2, 3, 7, 14, 21, and 28 post-injection. (B) The percentage of the initial radiance were taken and plotted over time. For 95 / 100_1 / 2_

[0060] vs. Saline, & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001. For 95 / 100_1 / 2_

[0030] + CM-CS vs. Saline, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For 95 / 100_1 / 2_

[0060] vs.95 / 100_1 / 2_

[0030] + CM- CS, $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001. (C) The area under the curve of the percent remaining was plotted against time, until 28 days post-treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 5 mice per experimental group.

[0025] Figure 13A-B depicts weeklong in vitro release of interleukin-12 (IL-12) in 70% deacetylated chitosan gels.1µg of IL-12 was loaded into 50µL gel and released into 10% FBS in PBS. (A) The inclusion of carboxymethyl chitosan at 1 mg / ml concentration - 4 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 expedited IL-12 release in 70 / 100_1 / 2_

[0030] gels. (B) The inclusion of heparin (0.2 mg / mL) in 70 / 100_1 / 2_

[0030] gels expedited release of IL-12, as compared to 70 / 100_1 / 2_

[0030] control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 samples per experimental group.

[0026] Figure 14A-C depicts retention testing of XCSgel and saline in gelatin tumor phantoms. (A) Young’s moduli of gelatin tumor phantoms at various gelatin concentrations. (B) Injection of 50µL XCSgel-dye, 100µL XCSgel-dye, 50µL dye in saline, and 100µL dye in saline in 14 w / v% gelatin. (C) Injection of 50µL XCSgel-dye, 100µL XCSgel-dye, 50µL dye in saline, and 100µL dye in saline in 10 w / v% gelatin.

[0027] Figure 15A-D depict XCSgel as a delivery medium localizes therapies following i.t. delivery. (A) Dye in a saline solution was excluded following injection into gelatin tumor phantoms, while (B) dye within XCSgelslowwas retained within the gelatin tumor phantom. (C) ex vivo fluorescence imaging of i.t. delivered IL-12-AF647 within saline, which was not retained within a murine E0771 tumor ex vivo. (D) ex vivo fluorescence imaging of i.t. delivered IL-12-AF647, which was localized within a murine E0771 tumor when delivered in XCSgelslow.

[0028] Figure 16A-T depicts histologic changes via H&E stain noted with i.t. injected XCSgel at different time points demonstrating inflammation that reduced over time. Chemical structures of (A) chitosan and (B) carboxymethyl chitosan. Two gels, (C-K) XCSgelslowand (L-T) XCSgelfast, were evaluated. (C-E) XCSgelslowday one. (C) Low magnification shows tissue track (dashed line) containing gel (arrowhead) from implant procedure with surrounding tumor necrosis (asterix).2x objective. (D) Gel (arrowhead) and tumor interface showing early tumor necrosis (white asterix).40x objective. (E) Gel (arrowhead) and tumor interface with tumor necrosis (white asterix) and marked neutrophilic inflammation (white arrow).40x objective. (F-H) XCSgelslowday three. (F) Low magnification shows persistent tissue track containing gel (arrowhead) with reduced tumor necrosis (asterix).2x objective. (G) Gel (arrowhead) and tumor interface showing tumor necrosis (asterix).40x objective. (H) Gel (arrowhead) with surrounding moderate neutrophilic inflammation (white bracket) and peripheral granulation tissue (black bracket).40x objective. (I-K) XCSgelslowday seven. (I) Low magnification shows tissue track containing gel (arrowhead) from implant procedure with minimal tumor necrosis (asterix).2x objective. (J) Gel (arrowhead) and tumor interface showing a band of cell debris mixed with neutrophils (white asterix) but adjacent tumor necrosis is absent.40x objective. (K) Gel (arrowhead) with surrounding mild neutrophilic inflammation (white - 5 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 bracket) and peripheral fibrosis (black bracket).40x objective. (L-N) XCSgelfastday one. (L) Low magnification shows tissue track containing gel (arrowhead) from implant procedure with surrounding tumor necrosis (asterix).2x objective. (M) Gel (arrowhead) and tumor interface showing early tumor necrosis (asterix).40x objective. (N) Gel (arrowhead) and tumor interface with tumor necrosis asterix) and marked neutrophilic inflammation (white arrow).40x objective. (O-Q) ) XCSgelfastday three (O-Q). (O) Low magnification shows persistent tissue track containing gel (arrowhead) with reduced tumor necrosis (asterix).2x objective. (P) Tumor and tissue track interface showing on- going tumor necrosis (asterix) and less severe neutrophilic inflammation (white arrow). 40x objective. (Q) Tumor periphery with moderate neutrophilic inflammation and peripheral granulation tissue (black bracket).40x objective. (R-T) ) XCSgelfastday seven. (R) Low magnification shows tissue track containing gel (arrowhead) from implant procedure with minimal tumor necrosis (asterix).2x objective. (S) Tumor and tissue track interface showing a band of cell debris (necrosis) mixed with neutrophils (asterix).40x objective. (T) Tumor periphery showing mild neutrophilic inflammation and peripheral fibrosis (black bracket).40x objective.

[0029] Figure 17 depicts in vitro release of cytokines IFNα2 and IL-12 from XCSgelslowover a period of 7 days.

[0030] Figure 18A-I depicts in vivo fluorescence imaging of labeled cytokines in XCSgels demonstrating long-term release. (A-B) Variable cytokine release. AF647- labled cytokines IL-12, IL-15, and IFNα2 were loaded into a different XCSgelslowgels and delivered subcutaneously on the flanks of Balb / c mice. (A) Images at 640 nm / 690 nm (Ex / Em) were taken on days 0, 1, 2, 3, 7, 10, 14, 21, and 28 post-injection. (B) Normalized efficiency values were taken and plotted over time. The significance was determined using one-way ANOVA and Tukey’s HSD posthoc testing. For IL-12-AF647 vs IFNα2-AF647, *P<0.01. n= 5 mice per experimental group. (C-I) Anti-tumor studies with variable cytokines. (C) Orthotopic primary E0771 tumors were established 12 days prior to treatment. (D) Survival and tumor growth curves of (E) untreated, (F) XCSgelslow- IL125μg, (G) XCSgelslow-IL1530μg, (H) XCSgelslow- IFNα210μg, and (I) XCSgelslow- GMCSF5μgwere monitored for 70 days post treatment. For the survival data, statistical significance was calculated using the Log-rank (Mantel–Cox) test. n = 8 mice per experimental group.

[0031] Figure 19A-M depicts in vivo fluorescence imaging of IL-12 and pharmacokinetics of serum IL-12 and IFNγ following delivery of XCSgel-IL12. (A-C) - 6 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 Variable XCSgel release with IL-12-AF657. AF647-labeled IL-12 was loaded into XCSgelfast, XCSgelslow, and saline and delivered subcutaneously on the flanks of Balb / c mice. (A) Images at 640 nm / 690 nm (Ex / Em) were taken on days 0, 1, 2, 3, 7, 10, 14, 17, 21, and 28 post-injection. (B) Normalized efficiency values were taken and plotted over time. The significance was determined using one-way ANOVA and Tukey’s HSD posthoc testing and listed in (C).*P < 0.05,**P < 0.01,****P < 0.0001. n= 5 mice per experimental group. (D-M) Pharmacokinetics and pharmacodynamics of i.t. IL-12 in XCSgels. IL-12 (5 μg) was loaded XCSgelfast, XCSgelslow,, and saline and delivered intratumorally in orthotopic E0771 breast cancer tumors in C57BL / 6 mice. Blood was collected from the facial mandibular vein at 4, 24, 48, and 72 hours post-injection. (D) IL- 12 serum concentrations were plotted over time, with statistical significance plotted at (E) 4 hours, (F) 24 hours, (G) 48 hours, and (H) 72 hours. (I) IFNγ serum concentrations were plotted over time, with statistical significance plotted at (J) 4 hours, (K) 24 hours, (L) 48 hours, and (M) 72 hours. The significance was determined using one-way ANOVA and Tukey’s HSD posthoc testing.*P < 0.05,**P < 0.01,***P < 0.001,****P < 0.0001. n= 5 mice per experimental group.

[0032] Figure 20A-K depicts intratumoral delivery of IL-12 co-formulated in XCSgel eliminating murine triple negative breast cancer tumors. (A-H) IL-12 was delivered within XCSgelsslowto orthotopic E0771 tumors at variable doses (0, 1, 5, 20 μg). (A) Orthotopic E0771 tumors were treated at either 17 or 21 days post tumor implantation with a single dose of IL-12 in XCSgel. (B) Survival and tumor growth curves of (C) XCSgelslow, (D) XCSgelslow-IL121μg, (E) XCSgelslow-IL125μg, (F) XCSgelslow-IL1220μgwere monitored for 60 days post treatment. For the survival data, statistical significance was calculated using the Log-rank (Mantel–Cox) test. (G) Mice whose tumors were eliminated were rechallenged with 5x105E0771 cells on the right flank, and their tumor growth was monitored. Mice that rejected the E0771 rechallenge were rechallenged with 5x105mWnt cells on the left flank. (H-K) IL-12 in XCSgel rejected TNBC mWnt tumors without toxicity. (H) Orthotopic mWnt tumors were treated nine days post tumor implantation with a single dose of XCSgelslow-IL125μg. (I) mWnt tumor growth curves and (J) survival curves up to 85 days post treatment following treatment. (K) Body weights of treated mice, measured daily for seven days following treatment. The significance was determined using the Log-rank (Mantel-Cox) test.***P < 0.001. The number of mice per experimental group are as follows: (A-F) n = 7, (G) n = 5 additional naïve mice for E0771 rechallenge and n = 4 additional naïve mice for mWnt rechallenge, (H-K) n = 5. - 7 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0033] Figure 21A-S shows that XCSgel-IL12 significantly alters the lymphoid cell compartment. Orthotopic primary E0771 tumors were established 12 days prior to treatment with XCSgelslow-IL125μgor XCSgelslow.Seven days after treatment when tumor volumes became palpable, tumors were resected and digested. Multi-spectral flow cytometry was run on tumor samples. (A-C) UMAP visualization of leukocytes populations and phenotypes of (A) untreated tumors, (B) tumors treated with or XCSgelslow,(C) tumors treated with XCSgelslow-IL125μg. (D-X) Frequencies of cell populations and phenotypes. (D) CD4+ T cell population of T cells. For cell frequencies of CD4+ T cells, phenotypes include (E) terminally exhausted (PD-1+Tim3+), (F) proliferating (Ki67+), (G)Th1-like (Ly6C+), (H) effector-like (CD11b+). (I) CD8+ T cell population of T cells. For cell frequencies of CD8+ T cells, phenotypes include (J) terminally exhausted (PD-1+Tim3+), (K) central memory (CD44+CD62L+), (L) activated and proliferating (CD44+Ki67+). (M) NK T cell (CD3+NK1.1+) frequencies of CD45+ leukocytes, (P) NK cell (CD3-NK1.1+) frequencies of CD45+ leukocytes. Cells within the myeloid cell compartment include (O) myeloid derived suppressor cells, (P) macrophages, (Q) Ly6Clomacrophages, (R) granulocytes, and (S) conventional dendritic cells. The significance was determined using one-way ANOVA and Tukey’s HSD posthoc testing.*P < 0.05,**P < 0.01,***P < 0.001,****P < 0.0001. n= 3 mice per experimental group.

[0034] Figure 22A-L shows additional cell populations and phenotype frequencies following XCSgel-IL12 treatment. Orthotopic primary E0771 tumors were established 12 days prior to treatment with XCSgelslow-IL125μg or XCSgelslow. Seven days after treatment when tumor volumes became palpable, tumors were resected and digested. Multi-spectral flow cytometry was run on tumor samples. (A) CD45+ leukocyte frequencies of all viable cells. (B) CD3+T cell frequencies of all viable CD45+leukocytes. (C) FoxP3+regulatory T cell frequencies of CD4+T cells. (D) TCRγδ+T cell frequencies of all CD4+T cells. (E) Overall CD19+B cell frequencies of all viable CD45+leukocytes. (F) B220+CD138- mature B cell, (G) CD138+B220+plasmoblast, and (H) CD138-B220- immature B cell frequencies of all CD19+B cells. (I) Myeloid cell frequencies of all viable CD45+leukocytes. (J) Myeloid and (K) polymorphonuclear MDSC frequencies of all MSDCs. (L) Neutrophil frequencies of all viable CD45+leukocytes. The significance was determined using one-way ANOVA and Tukey’s HSD posthoc testing. *P < 0.05. n= 3 mice per experimental group. - 8 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0035] Figure 23A-L shows intratumoral delivery of IL-12 co-formulated in XCSgel eliminated both primary and untreated abscopal E0771 tumors. (A) Orthotopic primary E0771 tumors were established 16 days prior to treatment, and untreated abscopal tumors were implanted on the flanks of mice 13 days prior to treatment. XCSgel-IL125µgwas delivered i.t. into the orthotopic primary tumors. (B) Survival was monitored for 90 days post-treatment. Statistical significance was calculated using the Log-rank (Mantel–Cox) test. Tumor growth curves of the primary and abscopal tumors for (C) XCSgelslow- IL125µg, (D) XCSgelfast-IL125µg, (E) 5 μg IL-12 in saline i.t, and (F) no treatment were monitored for 80 days post-treatment. The significance was determined using the Log- rank (Mantel-Cox) test.**P < 0.01. n= 5-6 mice per experimental group. DETAILED DESCRIPTION

[0036] Described herein are clinically translatable hydrogel systems comprising the polysaccharide chitosan. Varying certain chitosan hydrogel parameters has resulted in demonstrable tunable release of protein therapeutics, which heretofore are generally clinically limited by their toxicity. Furthermore, disclosed herein are compositions and methods that have addressed current limitations in hydrogel technology for intratumoral delivery, namely biocompatibility, biodegradability, injectability, imageability, and ability to tunably release therapeutics.

[0037] The crosslinking method for the chitosan hydrogel is unique and unexpected. Canonically, EDC / NHS crosslinking is utilized to create zero-order linkers between carboxylic acid and amine groups. In this coupling process, EDC reacts with the carboxylic acid to form an O-acylisourea intermediate. This unstable intermediate can react with an amino group to form an amide bond, the process of which releases an isourea byproduct. However, the O-acylisourea intermediate is susceptible to hydrolysis, and NHS is often added to form a semi-stable NHS ester to react with amines [1]. However, chitosan does not contain carboxylic acid groups for activation via EDC, even though our data demonstrated a clear increase in viscosity dependent on EDC / NHS molar ratios. There has not been a reported hydrogel formation via this type of crosslinking [2]. NMR data on the products of the reactions described herein suggest that chitosan is succinylated following this reaction, and that two small molecules are also present in our chitosan gel preparation. NHS can undergo hydrolysis, one pathway of which contains the intermediate succinamic acid and leads to the formation of succinic acid. Additionally, the succinimidyl group on glycosides has previously been demonstrated to be able to be - 9 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 opened to form a succinyl group on the glycoside [3]. This ring opening may also be occurring on the modified chitosan. From the NMR (Figure 2) and rheology data (Figure 3), chitosan is chemically modified to form succinylated chitosan and covalently crosslinked, with additional small molecule byproducts of hydrolyzed NHS.

[0038] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented herein. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. I. Overview

[0039] Systemically delivered cancer immunotherapeutics lead to adverse events and dose-related toxicities. For example, the systemic delivery of checkpoint inhibitors risks the development of immune-related adverse events (irAEs) in up to 85% of patients [4]. At a maximum tolerated dose, systemically administered immunotherapies, be they checkpoint antibodies or immune-stimulating cytokines, such as interleukin-2 (IL-2) or interferon-alpha (IFN-α), are unlikely to reach optimal concentrations within a tumor

[0074] . Thus, irAEs limit the dose of current immunotherapies, which in turn limit clinical benefit. These dose-limitations also preclude the use of potent immunotherapies in earlier stage cancer patients who are likely to respond more favorably to immune-based interventions

[0096] .

[0040] Localized immunotherapy delivery at the site of the tumor promises to reduce irAEs and increase the local therapeutic concentration. Furthermore, intratumoral delivery has increased in clinical prevalence in the recent decade [5]. Advances in image-guided injections have made practically all solid tumors accessible. Local delivery of - 10 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 immunotherapeutics has been demonstrated to be safe and feasible in clinical trials, yet clinical benefit has been underwhelming with only one approved product. Currently, the oncolytic herpes virus talimogene laherparepvec (T-VEC) is the only therapeutic approved for intratumoral delivery by the Food and Drug Administration (FDA) in patients with melanoma [6]. However, currently 190 recruiting and / or active clinical trials are evaluating drug candidates via the intratumoral route, with five trials (Table 1), having progressed to Phase 3 / 4. Table 1.

[0041] Recently, two Phase 3 clinical trials using intratumoral delivery have published disappointing results [7]. ILLUMINATE-301 (NCT03445533), which evaluated the addition of the intratumoral delivery of the toll like receptor 9 agonist IMO-2125 to systemic ipilimumab in advanced melanoma had an overall response rate (ORR) of 8.8% v.8.6% despite a 22.4% ORR in the ILLUMINATE-204 Phase 1 / 2 trial (NCT02644967) [8]. Additionally, MASTERKEY-265 (NCT02263508), which evaluated the addition of intratumoral T-VEC to systemic pembrolizumab, also failed meet its primary endpoints of improved progression free survival (PFS) or overall survival (OS) [9].

[0042] One explanation for these unfortunate results may be the poor retention of intratumorally injected agents. Dense tumor architectures and high interstitial pressures have been shown to exclude saline-based therapeutics following intratumoral injections

[0010] ,

[0011] . A significant fraction, if not a majority, of intratumorally injected solutions may leak out of the tumor and into low pressure surrounding tissues.

[0043] Hydrogels are viscoelastic solids that resist shear stress and thus have the potential to resist leakage from an injected tumor. Additionally, they present the opportunity for an engineered slow and controlled release of co-formulated immunotherapeutic. Hydrogels are three-dimensional polymeric matrices, encapsulating large amounts of water. They are composed of natural and / or synthetic polymers and are crosslinked via physical and / or - 11 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 chemical bonds. Their properties can be tuned for the variable release of therapeutics, based on their hydrophobic and electrostatic compositions, size of interpenetrating pores, swelling capacity, and rate of degradation in vivo.

[0044] Of the thirty-two hydrogels that have been approved as part of a clinical application applications, only four are used in cancer indications. SPACEOAR® (Augmenix, Inc.), a polyethylene glycol (PEG)-based hydrogel, serves as a spacer to protect vulnerable tissues from x-rays in prostate cancer radiotherapy

[0012] ,

[0013] . Second, BARRIGEL® (Pallete Life Sciences, Inc.) is a recently-FDA-approved hydrogel spacer for prostate cancer radiotherapy, which is composed of the naturally occurring polymer hyaluronic acid

[0014] . Third, TRACEIT® (Augmenix, Inc.) is a PEG gel with iodinated contrast utilized to mark soft tissues for image guided therapy, such as ultrasound, computed tomography, and Magnetic Resonance Imaging, for up to three months post- injection

[0015] ,

[0016] . Fourth, VANTAS® (Endo Pharmaceuticals Inc.) is delivered subcutaneously for the palliative treatment of prostate cancer through sustained delivery of gonadotropin hormone-releasing hormone agonist histrelin acetate

[0017] . However, Endo Pharmaceuticals Inc. discontinued this product on September 21, 2021

[0018] . Though these hydrogels are clinically approved in cancer indications, no hydrogels are currently a component of an FDA-approved intratumoral treatment

[0019] .

[0045] A particular hydrogel which is thermoresponsive, has been shown to potentially deliver a therapeutic the chemotherapeutic mitomycin C for urothelial cancer

[0020] –

[0022] .

[0046] In certain aspects, the subject matter is directed to unique hydrogels that meet the criteria described herein that are beneficial for clinical translation. At the preclinical level, numerous hydrogels are in development for intratumoral delivery of chemo and immunotherapeutics. However, most will not be translated clinically. To translate clinically, a useful hydrogel delivery system must be (1) a shear-thinning, self-healing hydrogel, (2) biocompatible, (3) biodegradable, (4) clinically imageable, and (5) capable of sustained local release of biologically active immunotherapeutics. Ideally, though not a requirement, the hydrogel could be “tuned” to release immunotherapeutics at a desired rate. To our knowledge, no existing hydrogel meets all these requirements. Notably, the ability to be imaged via common imaging modalities is a significantly limiting factor of these preclinical hydrogel delivery systems

[0023] .

[0047] Described herein are injectable cross-linked chitosan hydrogels for the local, tunable release of a cargo, such as an active agent, following injection. Assays have elucidated the chemical and material properties, verified its biocompatibility, - 12 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 demonstrated its slow and tunable release of a variety of therapeutics in vitro. Also described herein are in vivo release studies with a clinically relevant checkpoint inhibitor anti-Cytotoxic T-Lymphocyte Antigen 4 (aCTLA-4). These hydrogels incorporate the necessary engineering design criteria for a clinically translatable, local release platform and are ready for use in a range of localized delivery applications.

[0048] The chitosan hydrogels described herein for the local release of cargo meet the properties for a clinically translatable delivery system. Development of hydrogels for clinical application should meet the needs for translatable application, which are: (1) a shear-thinning, self-healing character, (2) biocompatibility, (3) biodegradability, (4) imageability via clinically relevant modalities, and (5) ability to slowly and tunably release and retain the biologic activity of delivered therapeutics.

[0049] Injectable hydrogels afford a minimally invasive means for therapeutic delivery

[0024] –

[0026] . Due to the high pressures involved with injecting into tumor microenvironment, hydrogel systems that do not require external stimulation (i.e. light, pH, temperature) for gel formation represent the ideal candidates for delivery systems. Such systems must additionally be shear-thinning to be extruded through a syringe and self-healing to re-form post-injection. Described herein are data that macroscopically demonstrate the injectability of a representative hydrogel, which could be retained in a tumor phantom (Figure 1). Additionally, through rheology, the chitosan hydrogel demonstrated shear-thinning character through rotational continuous flow experiments and self-healed following high strain, with slight thixotropic character (Figure 5).

[0050] The hydrogels should additionally demonstrate biocompatibility. Naturally derived polymers demonstrate lower toxicity as compared to synthetic polymers, but have other drawbacks, such as possible pyrogen and pathogen contamination and higher batch to batch source variability

[0027] . For biocompatibility, the starting material is the naturally occurring polysaccharide chitosan, which has demonstrated robust biocompatibility and even demonstrates antimicrobial and antifungal activity

[0028] , but does have limitations. The chitosan must have low endotoxin content. When evaluated in vitro with 3T3 fibroblasts, representative subsets and increasing volumes of the hydrogel did not inhibit cellular viability compared to controls (Figure 6). Furthermore, in vivo data demonstrated a transient inflammatory response that lessened in severity over time, similar to a classical foreign body reaction (Figure 7). Although mild, the inflammatory response indicates that the chitosan hydrogel is ideal for intratumoral delivery. The addition of polysaccharides to reduce the high cationic charge of chitosan gel could alter the severity of inflammation, - 13 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 as was shown with CM-CS, as well as reduced granulation tissue with the incorporation of heparin. Interestingly, the sustained presence of heparin may be detrimental to healing as the CS / heparin gel was the least resolved after seven days.

[0051] In addition to biocompatibility, clinically useful hydrogels as therapeutic delivery systems must also be naturally degraded. In the human body, numerous enzymes can break down chitosan, such as lysozyme

[0029] , colonic bacterial enzyme

[0030] , chitosanase

[0031] , proteases

[0032] , and glucosidase

[0033] , which contribute to chitosan’s degradation in addition to cellular phagocytosis and other inflammatory responses in the foreign body reaction

[0034] –

[0037] . Following a s.c. injection, the volume of chitosan gel diminished over time, with only small amounts of gel being present after 6 weeks post-injection (Figure 7). As a clinical comparison, the TraceIT® polyethylene glycol gel, which is FDA- approved for soft tissue marking in radiation therapy, is stable for about three months and is resolved over a seven month period post-implantation

[0038] –

[0040] .

[0052] Another desirable property for clinically applicable hydrogels is their ability to be imaged via CT or ultrasound, as these are the two most commonly used imaging modalities in intratumoral delivery

[0023] . Clinicians need real-time feedback to ensure that the material was localized to the tumor

[0010] ,

[0023] . Furthermore, the contrast within the hydrogel must be transient and eliminated over time so as not to interfere with subsequent imaging of the tumor. Cross-linked chitosan hydrogels were co-formulated with clinical grade iohexol (OMNIPAQUETM), which provided radiopacity without compromising therapeutic release (Figure 11). Additionally, its imageability via x-ray in vivo was verified when delivered in a 50μL volume with the therapeutic aCTLA-4 in mice (Figure 11). Following one day post-injection, the contrast could not be visualized via x-ray (Figure 11C), indicating its elimination from the body. This elimination further indicated its usefulness in transiently verifying target tumor localization without interfering with future x-ray imaging. Should longer-term imageability be desired, iodine could be covalently conjugated to chitosan, which would persist as long as the gel did. Furthermore, for translation into ultrasound-guided imaging, other contrast such as nanodroplets and microbubbles could be incorporated within the gel

[0041] .

[0053] Also, a clinically useful hydrogel must improve intratumoral delivery through local and slow release of the delivered therapeutic. The cross-linked chitosan hydrogels have demonstrated in vitro the ability to sustain the release of therapeutics via electrostatic charge via small molecule fluorophores (Figure 8). Chitosan is a highly positively charged polymer due to its abundant amine groups, which provide a strong - 14 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 electrostatic force capable of associating with negatively charged particles or pockets of proteins while repelling those highly positively charged. In particular, a dominant force facilitating non-specific, physical association between proteins and polymers are electrostatic forces

[0042] –

[0044] . The pKa of the polymer, pI of the protein, and pH of the solution dictate these forces. The cross-linked chitosan hydrogels have demonstrated tunable release of protein therapeutics based on varied isoelectric point of each therapeutic, with IFNg being released most quickly, followed by aCTLA4, then IL-12, regardless of protein size (Figure 9, 10). Increasing the chitosan gel concentration decreases release for all therapeutics (Figure 9, 10). This phenomenon was hypothesized to be due to an increased overall electrostatic bond as well as decreased interpenetrating pore network size, which increases the path length for a therapeutic to leave the material

[0045] . Additionally, a negatively charged chitosan derivative when incorporated into the hydrogel expedited the release of the most negatively charged therapeutic, IL-12, potentially due to its shielding of positive charge from the therapeutic (Figure 9D). This positive / negative charge association can be leveraged through a variety of means, such as the use of other derivatized chitosans

[0046] as well as negative or zwitterionic components to enhance protein association. For example, one study incorporated Laponite nanoparticle disks, bearing negative surface charge and positive rim charges at neutral pH, to alter protein release

[0047] . Characterizing the chitosan gel has now provided the ability to rationally design therapeutic release based on the desired delivered therapeutic.

[0054] Furthermore, the chitosan-crosslinked hydrogels have demonstrated retaining and slowly releasing the model protein therapeutic, monoclonal antibody aCTLA-4, which was FDA-approved for a variety of cancer indications yet clinically limited by its deleterious systemic toxicity, in vivo. Over 90% of the therapeutic delivered in a saline solution was eliminated after one day post-injection, while over half remained in the fast and slow releasing gels (Figure 12). By the second day following injection, a greater percentage of the therapeutic had left the faster releasing gel (95 / 100_1 / 2_

[0030] + CM-CS) as compared to the slower releasing gel (95 / 100_0.5 / 1_

[0060] ) (Figure 12B), confirming the tunability of the release of therapeutics in vivo. II. Definitions

[0055] The term “chitosan” is used as it is known in the art. Chitosan does not contain a carboxylic acid group and is a linear polysaccharide composed of randomly distributed β- linked D-glucosamine and N-acetyl-D-glucosamine having a monomer structure of: - 15 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192wherein n is an integer that results in a molecular weight of chitosan typically ranging from 5,000 to 2,000,000 daltons. The molecular weight of each chitosan monomer (g / mol / monomer) was calculated according to equation 1: ^^^^ ^^^^^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^= ( ^^^^ ^^^^^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^∗ ^^^^ ^^^^ ^^^^) + ( ^^^^ ^^^^^^^^− ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^∗ (1 − ^^^^ ^^^^ ^^^^)) (1) Where ^^^^ ^^^^^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^is the molecular weight of each glucosamine unit (161.2 g / mol), ^^^^ ^^^^^^^^− ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^is the molecular weight of each N-acetylglucoasmine unit (203.2 g / mol),and ^^^^ ^^^^ ^^^^ is the degree of deacetylation.

[0056] As used herein, the term “succinylated chitosan” refers to a chitosan residue that is covalently linked to a -C(O)-CH2-CH2-COOH, succinyl group.

[0057] As used herein, the term “tunable” and the like refers to the ability to modify the properties of a hydrogel to release cargo at a desired rate or location. The tunability can be controlled by adjusting the MW of the hydrogel, the charge of the hydrogel, or other physical properties or additives purposefully included in the hydrogel.

[0058] As used herein, the term “biocompatible” means a property or characteristic of a hydrogel to disintegrate or break down, over a prolonged period of time, in response to the biological environment in the subject, by one or more physical or chemical degradative processes, for example by enzymatic action, oxidation or reduction, hydrolysis (proteolysis), dissolution by solubilization, and which the break down materials are then absorbed by the body and surrounding tissue, or otherwise dissipated harmlessly.

[0059] As used herein, the term “injectable” refers to a property of a hydrogel to be extruded or dispensed through a lumen, for example, a needle as small as about 27 gauge from a syringe under normal manual pressure and preferably without substantial spiking.

[0060] As used herein, the term “self-healing” refers to the ability of a hydrogel to autonomously recover from physical stress and to repair mechanical damage. Self- healing can involve viscoelastic properties of the hydrogel to allow flow of the hydrogel to close back or revert back to an original state after physical perturbation. - 16 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0061] As used herein, the term “cargo” refers to a substance that is contained within the hydrogel and is released from the hydrogel upon administration of the hydrogel.

[0062] The term “active agent” refers to any chemical compound or molecule, which can be an Active Pharmaceutical Ingredient (API), complex, biologic or composition that has a beneficial biological effect, preferably a therapeutic effect in the treatment of a disease or abnormal physiological condition, and also includes any delivery vehicle that can be incorporated into a hydrogel, such as a liposome or a nanoparticle. The term also encompasses pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term “active agent” is used, it is to be understood that it can include the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, etc. Active agents include, but are not limited to, pharmaceutical drugs, active proteins, antibodies (e.g., monoclonal antibodies including checkpoint inhibitors and immune agonists), antibody drug conjugates, immunomodulators (TLR agonists, STING agonists, glucocorticoids, etc), exosomes, liposomes, nanoparticles, microparticles, etc.

[0063] As used herein, the term “immune-stimulating cytokine” refers to a small protein secreted by cells that induces an anti-tumor immune response. Immune-stimulating cytokines include interferons, interleukins, and growth factors. Non-limiting examples include IFN-α, IFN-β, IFN-γ, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and granulocyte macrophage colony-stimulating factor (GM-CSF). The immune-stimulating cytokines disclosed herein include both endogenous cytokines and recombinant cytokines. The amino acid sequence of the immune-stimulating cytokine may be identical to the naturally occurring sequence or the cytokine may be genetically modified to include one or more mutations such as amino acid substitutions, deletions, or insertions.

[0064] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0065] As used herein, the term “about,” when referring to a measurable value such as an amount of a compound or agent of the current subject matter, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. - 17 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0066] As used herein, conditional language used herein, such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or steps. Thus, such conditional language is not generally intended to imply that features, elements and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or steps are included or are to be performed in any particular embodiment.

[0067] Further definitions may be provided elsewhere herein. III. Compositions

[0068] In certain embodiments, the subject matter described herein is directed to self- healing, aqueous-based cross-linked chitosan hydrogel formed by reacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0069] In certain embodiments of the compositions, the average molecular weight of the chitosan can be from about 5,000 to about 2,000,000 daltons, or from about 10,000 to 500,000 daltons.

[0070] In certain embodiments of the compositions, the cross-linked chitosan hydrogel is prepared from chitosan having a dynamic viscosity of 5-2000 cp, as measured as 1 w / v% solution in 1 v / v% acetic acid.

[0071] In certain embodiments of the compositions, the cross-linked chitosan hydrogel is injectable, biocompatible and / or tunable.

[0072] In certain embodiments of the compositions, the cross-linked chitosan hydrogel is porous. In certain aspects of these embodiments, the cross-linked chitosan hydrogel can comprise an interpenetrating pore network, composed of irregularly shaped and non- uniform pores. In certain aspects of these embodiments, the pore length scales ranged from about 0.5-50μm, or from about 1-20μm, or from about 1-10μm, or from about 1- 5μm.

[0073] In certain embodiments of the compositions, the cross-linked chitosan hydrogel further comprises a cargo. In certain aspects of these embodiments, the cargo is an active agent or contrast agent. In aspects of this embodiment, the active agent is a small molecule pharmaceutical drug or a biologic. - 18 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0074] In certain embodiments, the chitosan hydrogel is a XCSgel. Compositions of XCSgels are described in Examples 1-12.

[0075] In certain embodiments of the compositions, the active agent is an immune- stimulating cytokine. In certain embodiments of the compositions, the immune- stimulating cytokine is IL-12, IL-15, IFNα2, or GM-CSF. In certain embodiments of the compositions, the active agent is IL-12.

[0076] IL-12 is a heterodimer of IL-12A and IL-12B linked through a disulfide bond. In certain embodiments, the IL-12 is human IL-12 (hIL-12). In certain embodiments, the active agent comprises hIL-12A and hIL-12B. In certain embodiments, the hIL-12A comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1. In certain embodiments, the hIL-12B comprises an amino acid sequence having at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:2. In certain embodiments, the active agent comprises a protein comprising an amino acid sequence set forth in SEQ ID NO:1 and a protein comprising an amino acid sequence set forth in SEQ ID NO:2.

[0077] In certain embodiments, the IL-12 is canine IL-12 (cIL-12). In certain embodiments, the active agent comprises cIL-12A and cIL-12B. In certain embodiments, the cIL-12A comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:6. In certain embodiments, the cIL-12B comprises an amino acid sequence having at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:7. In certain embodiments, the active agent comprises a protein comprising an amino acid sequence set forth in SEQ ID NO:6 and a protein comprising an amino acid sequence set forth in SEQ ID NO:7.

[0078] In certain embodiments, the hydrogel comprises a concentration of 0.01 to 0.4, 0.01 to 0.2, or 0.05 to 0.2 µg of IL-12 per µL of hydrogel. In certain embodiments, the hydrogel comprises a concentration of about 0.1 µg of IL-12 per µL of hydrogel.

[0079] In certain embodiments, the active agent is human IL-15 (hIL-15). In certain embodiments, the hIL-15 comprises an amino acid sequence having at least 85%, at least - 19 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:3. In certain embodiments, the active agent comprises a protein comprising an amino acid sequence set forth in SEQ ID NO:3

[0080] In certain embodiments, the active agent is human IFNα2 (hIFNα2). In certain embodiments, the hIFNα2 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:4. In certain embodiments, the active agent comprises a protein comprising an amino acid sequence set forth in SEQ ID NO:4.

[0081] In certain embodiments, the active agent is human GM-CSF (hGM-CSF). In certain embodiments, the hGM-CSF comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:5. In certain embodiments, the active agent comprises a protein comprising an amino acid sequence set forth in SEQ ID NO:5.

[0082] Table A. Amino acid sequences of immune-stimulating cytokines- 20 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192- 21 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0083] In certain embodiments of the compositions, the active agent is a monoclonal antibody. In certain embodiments, the monoclonal antibody is anti-PD-1, anti-PD-L1, anti-LAG3, anti-TIM3, anti-CD40, or anti-41BBL.

[0084] In certain embodiments of the compositions, the cross-linked chitosan hydrogel is combined with an anionic polymer after formation of the hydrogel. The anionic polymer can modify the electrostatic charge of the hydrogel to tune the release of a cargo. In certain aspects of these embodiments, the anionic polymer is selected from the group consisting of carboxymethyl chitosan, hyaluronic acid, sodium alginate and heparin, or combinations thereof.

[0085] In certain embodiments of the compositions, the cross-linked chitosan hydrogel releases cargo at a rate such that about 50% to about 90% of the cargo is released within about 1 day, 1 week or 1 month or longer.

[0086] In certain embodiments of the compositions, the cross-linked chitosan is from about 70 to about 99% deacetylated, or from about 90 to about 99% deacetylated, or is about 95% deacetylated. In certain aspects of these embodiments, the cross-linked chitosan is about 70%, about 75%, about 80%, about 85%, about 90% or about 95% deacetylated.

[0087] In certain embodiments of the compositions, the cross-linked chitosan is cross- linked with from about 0.1:1 to about 2:11-ethyl-3-(3- dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer. In certain embodiments of the compositions, the cross-linked chitosan is cross-linked with from about 0.5:1 to about 2:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer, or about 1:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer.

[0088] In certain embodiments of the compositions, the cross-linked chitosan is cross- linked with from about 1:1 to about 5:1 N-hydroxysuccinimide molar ratio to chitosan monomer. In certain embodiments of the compositions, the cross-linked chitosan is cross- linked with from about 1:1 to about 3:1 N-hydroxysuccinimide molar ratio to chitosan monomer, or about 1:1 N-hydroxysuccinimide molar ratio to chitosan monomer.

[0089] In certain embodiments of the compositions, the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is from about 0.1:10 to about 2:1, or the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is about 0.5:1. - 22 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0090] Molar ratios within the ranges can be adjusted depending on the molecular weight of the chitosan. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0091] In certain embodiments of the compositions, the chitosan is in a neutralized solution. In certain aspects of these embodiments, the pH of the neutralized chitosan solution is from about 5.5 to about 6.2.

[0092] In certain embodiments, the subject matter described herein is directed to a lyophilized cross-linked chitosan, wherein the cross-linked chitosan comprises at least one residue of succinylated chitosan crosslinked to a residue of chitosan.

[0093] In certain embodiments, the subject matter described herein is directed to a cross- linked chitosan prepared by contacting chitosan with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. IV. Methods

[0094] In certain embodiments, the subject matter described herein is directed to a method of delivering a cargo to a subject, comprising administering a cross-linked chitosan hydrogel comprising the cargo as described herein to the subject. In aspects of this embodiment, the delivering is a controlled release of the cargo. In aspects of this embodiment, the delivering is localized.

[0095] In certain embodiments, the subject matter described herein is directed to a method of treating cancer, the method comprising administering to a subject a hydrogel comprising the cargo as described herein. In certain embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancers, lung cancer, kidney cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, penile cancer, gastric cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, bladder cancer, hematologic cancer, endometrial cancer, melanoma, glioblastoma, sarcoma, osteosarcoma, cholangiocarcinoma, neuroblastoma, and glioma. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is triple-negative breast cancer. In certain embodiments, the method of treating cancer comprises administering the hydrogel comprising IL-12. - 23 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0096] In certain embodiments, the subject is a mammal selected from the group consisting of humans, mice, rats, dogs, cats, livestock and horses. Subjects may be of any age, including infant, juvenile, adolescent, adult, and geriatric subjects. In certain embodiments, the subject is human. In certain embodiments, the subject is canine. The amino acid sequence of cytokines as active agents may be selected based on the species of the subject.

[0097] In certain embodiments of the methods, the cargo is an active agent or contrast agent. In aspects of this embodiment, the active agent is a small molecule pharmaceutical drug or a biologic. In aspects of this embodiment, the active agent is an immunomodulator.

[0098] In certain embodiments of the methods, the administering is not systemic. In certain embodiments of the methods, the hydrogel is administered locally. In certain embodiments of the methods, the administering is by injection. In certain embodiments, the method comprises injecting the hydrogel comprising the cargo (e.g., an immune- stimulating cytokine such as IL-12) into a tumor.

[0099] In certain embodiments of the methods, the subject matter described herein is directed to a method of preparing a hydrogel comprising a cross-linked chitosan, the method comprising contacting chitosan with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, wherein the chitosan is cross-linked.

[0100] In certain embodiments of the methods, the cross-linked chitosan is from about 70 to about 99% deacetylated, or from about 90 to about 99% deacetylated, or is about 95% deacetylated. In certain aspects of these embodiments, the cross-linked chitosan is about 70%, about 75%, about 80%, about 85%, about 90% or about 95% deacetylated.

[0101] In certain embodiments of the methods, the cross-linked chitosan is cross-linked with from about 0.5:1 to about 2:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer, or about 1:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer.

[0102] In certain embodiments of the methods, the cross-linked chitosan is cross-linked with from about 1:1 to about 3:1 N-hydroxysuccinimide molar ratio to chitosan monomer, or about 1:1 N-hydroxysuccinimide molar ratio to chitosan monomer.

[0103] In certain embodiments of the methods, the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is from about 0.1:10 to - 24 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 about 2:1, or the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is about 0.5:1.

[0104] In certain embodiments of the methods, the chitosan is in a neutralized solution. In certain aspects of these embodiments, the pH of the neutralized chitosan solution is from about 5.5 to about 6.2, or is about 6.0. In certain aspects of these embodiments, the chitosan solution is neutralized with base, such sodium hydroxide.

[0105] In certain embodiments of the methods, the 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are in a single crosslinking solution further comprising a solvent. In certain aspects of these embodiments, the solvent is selected from the group consisting of water, an aqueous solution, aqueous saline buffer, phosphate buffered saline, phosphate buffer, an organic solvent, DMSO, DMF and NMP or a combination thereof or a combination thereof. In certain aspects of these embodiments, the solvent is a mixture of DMSO and water. In certain aspects of these embodiments, the solvent is a mixture of about 50-80% v / v% DMSO and about 20-50% v / v% water.

[0106] In certain embodiments of the methods, the contacting comprises adding dropwise with stirring a neutralized chitosan solution to a crosslinking mixture comprising 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0107] In certain embodiments of the methods, after contacting, the cross-linked chitosan is dialyzed.

[0108] In certain embodiments of the methods, the methods further comprise lyophilizing or otherwise concentrating the cross-linked chitosan.

[0109] In certain embodiments of the methods, the methods further comprise contacting the lyophilized cross-linked chitosan with water or other aqueous solutions to prepare a hydrogel.

[0110] In one embodiment, the subject matter described herein is directed to a method of preparing a cross-linked chitosan hydrogel, comprising: stirring chitosan in an aqueous acidic solution at room temperature to prepare a chitosan solution, for example, stirring high molecular weight (1000cp) chitosan at 10 mg / ml and medium (100cp) to low (10cp) molecular weight chitosan at 15 mg / mL; neutralizing the chitosan solution to pH 6.0 with a basic aqueous solution, for example, adding 0.5M NaOH dropwise while stirring to a 14 mg / ml final concentration or 9 mg / ml final concentration; - 25 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 stirring the neutralized chitosan solution overnight until it redissolves, for example, at room temperature for about 18-24 hours; centrifuging the redissolved, neutralized chitosan solution to remove particulates, for example, centrifuging at 10,000 rpm for 20 minutes ; preparing a crosslinking solution of EDC and NHS by dissolving ECD and NHS in an aqueous DMSO solution, for example, 75% DMSO and 25% water solution at the desired molar ratio relative to chitosan and desired volume, for example, a volume that is about 35% of final reaction volume; adding the centrifuged neutralized chitosan solution, dropwise, to the crosslinking solution with stirring, for example at a rate of about 50 rpm, at room temperature to prepare a cross-linked chitosan solution; dialyzing the cross-linked chitosan solution, for example, in 7000 Da molecular weight cut-off tubing against 2L of deionized water at room temperature to prepare a dialyzed cross-linked chitosan solution; freeze-drying the dialyzed cross-linked chitosan solution to prepare a lyophilized cross-linked chitosan composition; and, preparing a cross-linked chitosan hydrogel by reconstituting the lyophilized cross- linked chitosan in water to desired concentration, for example a higher molecular weight chitosan at about 10 mg / mL to a lower molecular weight chitosan at 100 mg / mL or higher.

[0111] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0112] Compounds and Reagents—Chitosan was purchased from Heppe Medical Chitosan (Halle, Germany). The following products were used: 95 / 1000 (cat. #24707), 95 / 100 (cat. #24704), 95 / 10 (cat. #24701), 70 / 1000 (cat. #24207), 70 / 100 (cat. #24204), 70 / 10 (cat. #24201). N-Hydroxysuccinimide (NHS) (cat. #804518), deuterium oxide (cat. #151882-10G), deuterium chloride (cat. #543047-10G), rhodamine 6G (cat. #R4127-5G), and fluorescein sodium salt (cat. #46960-25G-F) were purchased from Sigma Aldrich (Burlington, Massachusetts). N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (cat. #10118-238), dimethyl sulfoxide (DMSO) (cat. #BDH1115- 1LP), and pyranine (cat. #H1172-1GM) were purchased from VWR (Radnor, Pennsylvania). Rhodamine WT (cat. # 46971000) was purchased from ThermoFisher - 26 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 Scientific (Waltham, Massachusetts). Hydrochloric acid (cat. # 2033-1L) was purchased from Honeywell International Inc. (Muskegon, Michigan). Recombinant murine interleukin-12 (IL-12) was overexpressed and produced in-house, as previously described

[0048] . Recombinant murine interferon gamma (IFNγ) (cat. # 315-05) was purchased from PeproTech (Cranbury, New Jersey). The checkpoint inhibitor aCTLA4 (cat. #BE0164) was purchased from Bio X Cell (Lebanon, New Hampshire). mIL-12, mIFNγ, and total murine IgG ELISAs (cat. # BMS6004, 88-7314-88, 88-50400-22, respectively) were purchased from Invivogen (San Diego, California). Medical grade imaging contrast agent OMNIPAQUETMInjection 300 mgL / mL was purchased from GE Healthcare (Marlborough, Massachusetts).

[0113] Methods:

[0114] Nuclear Magnetic Resonance—The chemical structure of the modified chitosan was analyzed through nuclear magnetic resonance (NMR). Approximately 5 mg of 95% deacetylated, 5cp chitosan, crosslinked with 1:1 molar ratio EDC to chitosan monomer and 2:1 molar ratio NHS to chitosan monomer concentration, was dissolved in 600 μL 10% / 90% deuterated water / deionized water as the solvent.1H NMR,1H-1H COrrelation SpectroscopY (COSY),1H-13C Heteronuclear Single Quantum Correlation (HSQC), and1H-1H Nuclear Overhauser Effect Spectroscopy (NOESY) NMR were acquired on a Bruker NEO 700 MHz equipped with a 5 mm TCI cryoprobe (H&F-C / N-D).

[0115] Rheological Characterization—Rheological testing was performed on an Anton Paar Physica MCR 301 Rheometer. Briefly, 500 μL of sample was loaded onto the rheometer, and the 25mm diameter parallel plate was lowered to a gap height of 500 μm. To characterize the change in viscosity due to variations in EDC and NHS molar concentrations prior to gel purification, continuous flow experiments were performed for 30 seconds from 1-100 Hz shear rate on unpurified reaction samples. To characterize the mechanical properties of the unpurified reaction samples, frequency sweeps were performed at 1% shear strain (1-10 Hz).

[0116] To characterize the mechanical properties of the gel, oscillatory amplitude sweeps were performed at a frequency of 10 Hz (0.1-500%) and oscillatory frequency sweeps were performed at a strain of 1% (0.1-100 Hz). To demonstrate the shear-thinning behavior of the gel, viscosity measurements were obtained from rotational continuous flow experiments, which were run over a shear-rate of 0.1-10 Hz. To demonstrate the self-healing nature of the gel, cyclic deformation measurements (10 Hz) were performed in five continuous intervals: intervals 1, 3, and 5 being performed at 1% strain for 30 - 27 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 seconds with 30 data points, and intervals 2 and 4 performed at 200% strain for 15 seconds with 30 data points.

[0117] Tumor Agar Phantom—Agar was dissolved at 8 w / v% in 1x phosphate buffered saline with heating and stirring. While the agar was still hot, it was aliquoted into 50 mL conical tubes and allowed to cool prior to storage at 4 °C.

[0118] Scanning Electron Microscopy (SEM) and Analysis—Hydrogels were hydrated overnight in deionized water. CryoSEM analyses were conducted using a JEOL JSM- 7600 FE SEM (JEOL USA, Peabody, MA) outfitted with Alto-2500 (Gatan, Warrendale, PA). Samples were plunge frozen in liquid nitrogen slush, transferred under vacuum to the Alto-2500 preparation chamber, and cryo fractured. The fractured sample was etched for 5 min at -89 °C under 4 x 10-6mbar vacuum to reveal the microstructure. Samples were allowed to cool down to -135 °C. An in-situ cold magnetron coater was used to make a 5 nm thickness Au / Pd coating on etched samples. The SEM images were taken using 5 keV energy at 11 mm working distance under cryo-temperature. Pore size and porosity was determined via ImageJ software.

[0119] 3T3 Cell Culture—Murine NIH / 3T3 fibroblasts originally isolated from a mouse NIH / Swiss embryo were purchased from the American Type Culture Collection (ATCC). They and their subpassages were stored in the vapor phase of liquid nitrogen until use. All cells were visually monitored for obvious contaminants, such as bacterial and fungal growth. They were frequently tested for mycoplasma with a commercially available kit (MYCOALERT PLUS Mycoplasma Detection Kit from Lonza (cat. #LT07-703). Cells were cultured in DMEM media (Corning, cat. #15-013-CV), 1mM penicillin- streptomycin (GenClone, cat. # 25-513), 10% fetal bovine serum (FBS) (GenClone, cat. #25-550), and glutamine (Corning, cat. # 25-005-CI) at 37°C in 5% CO2and 95% humidity.

[0120] In vitro Viability Assay—Gel samples were prepared in deionized water and stored at 4OC overnight.10,0003T3 cells in 100 μL DMEM media were plated in a 96- well plate overnight. The following day, 10 μL or 30 μL of gel was placed on top of the cells.0.1% Tween and deionized water were used as negative and positive controls, respectively. After 24 hours, cell viability was determined via CELLTITER-GLO 3D Viability Assay kit (Promega, cat. # G9681) according to the manufacturer’s instructions. Briefly, the CELLTITER-GLO reagent was warmed to room temperature 30 minutes prior to use.100 μL of the CELLTITER-GLO reagent was added to each of the wells. The plate was shaken on a plate rocker for 10 minutes at room temperature. Subsequently, - 28 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 the plate was placed in the dark for 20 minutes to stabilize the luminescent sample. The luminescence was read, and the media background signal was subtracted from the luminescence values.

[0121] In vivo Biocompatibility and Histology—Gels were prepared overnight.50 μL of gel was injected subcutaneously on the right flank of C57B6 mice (Charles River Laboratories). On days 3 and 7 and 42, mice were euthanized and tissue collected. The skin, gel, and underlying muscle were dissected and fixed in 10% neutral buffered formalin. Sectioning and H&E staining was performed by the Histopathology Laboratory at the NC State College of Veterinary Medicine. Blinded analysis of inflammation and immune cell infiltrates was performed by a board-certified veterinary pathologist.

[0122] Release Studies—50 μL of gel loaded with the appropriate small molecule or therapeutic in deionized water was prepared at least one day prior to release. Subsequently, 1 mL of 1x PBS was loaded on top of the gel for non-protein samples, and 1 mL of 10% FBS in PBS was utilized for protein-samples. For each timepoint, all release media was removed. For the release of biologics, release media was stored at 4OC, and for fluorophore release, samples were kept in the dark at room temperature. Protein concentrations were measured via ELISA. Fluorophores were measured via fluorescence at 370 excitation / 580 emission on a BioTek Cytation 5 microplate reader. Standard curves using samples with no hydrogel were plotted using log-log regression.

[0123] aCTLA-4 Fluorophore Labeling—aCTLA4 monoclonal antibodies were fluorescently labeled using Molecular Probes™ Fluorescent Protein Labeling Kits (Alexa Fluor 660) (ThermoFisher, cat. # A20171). The degree of labeling was calculated to be ~1-2 moles of fluorophore per mole of antibody according to manufacturer’s instructions.

[0124] In vivo X-ray Imaging and Pharmacokinetics—50 μL of gel loaded with 10μg aCTLA-4 and 200 mgI / mL iohexol (OMNIPAQUETM) was injected subcutaneously on the shaved flank of pathogen-free Balb / c mice (Charles River Laboratories). On days 0, 1, 2, 3, 7, 14, 21, and 28 post-injection, fluorescence (Ex / Em 605 / 690 nm) and x-ray images were taken on a Spectral Instruments Imaging Ami HTX optical imaging system.

[0125] Statistical Analysis—A standard two-tailed t test was used to test for significance between two sample groups. Differences between three or more treatment groups were determined using ANOVA with Tukey’s HSD post-hoc testing. The above analyses were conducted using Prism (GraphPad Software Inc., San Diego, CA). Example 1—Preparation of a Hydrogel Comprising Cross-linked Chitosan - 29 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0126] Hydrogel Preparation— Chitosan was dissolved at a concentration of 15 mg / mL (or 10 mg / mL for 1000cp chitosan) in 0.1 M hydrochloric acid overnight at room temperature. The following day, sodium hydroxide was added dropwise to the solution to bring the pH up to 6 and the overall chitosan concentration to 14 mg / mL (or 9 mg / mL for 1000cp chitosan). This solution was stirred overnight until the chitosan re-dissolved. The neutralized chitosan solution was centrifuged at 10,000 rpm for 10 minutes to remove undissolved particulates. In a scintillation vial, EDC and NHS at the appropriate molar ratios to the calculated chitosan monomer were dissolved in 75 v / v% DMSO and 25 v / v% water solution, the total volume of which 35% of the overall reaction volume. The neutralized chitosan solution was dropwise added to the EDC-NHS solution, and the reaction was stirred overnight at room temperature. The following day, the crosslinked chitosan solution was dialyzed in 7000 Da molecular weight cut-off tubing (SpectraPor® RC Tubing, cat. #132660) against 2 liters of deionized water. The dialysate was changed 7 times over 4 days. The solution was extremely hygroscopic and amassed water over four times its original volume. The purified chitosan solution was freeze-dried over a minimum of 3 days and stored at 4 °C until further use. To construct the hydrogel, water was added to the lyophilized crosslinked chitosan at varying concentrations and hydrated overnight. The hydrogel was centrifuged at 10,000xg for at least 3 minutes to remove bubbles. Descriptions of chitosan hydrogels follow the format: %DDA / inherent chitosan viscosity (cp)_EDC / NHS molar ratios[gel concentration (mg / mL)]. Example 2—Cross-linked Chitosan Hydrogel Characterization of Self-healing and Injectability

[0127] Injectable, self-healing chitosan hydrogel resists leakage post-injection—A novel chitosan-based hydrogel was synthesized via EDC / NHS crosslinking (Figure 1A). The chitosan hydrogel (95 / 100_1 / 2_

[0030] ) was loaded with the fluorophore rhodamine WT and extruded through a 23-gauge needle, macroscopically demonstrating its injectability (Figure 1A-B). The hydrogel could be injected through needles as narrow as 27-gauge (data not shown). Additionally, the gel was retained within an agar tumor phantom post- injection (Figure 1Ci), whereas a saline-based solution was rapidly excluded from the dense agar matrix (Figure 1Cii), demonstrating the importance of a local delivery system for intratumoral therapies. Finally, the gel demonstrated self-healing properties, as rhodamine WT and fluorescein from separate gels freely diffused across the interface when the gels were placed in contact and became indistinguishable less than 24 hours after injection (Figure 1D). - 30 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 Example 3—Cross-linked Chitosan Hydrogel Chemical and Rheological Characterization

[0128] Chitosan is modified and crosslinked through EDC / NHS chemistry, as characterized by 1D and 2D NMR and rheology—Chitosan is a naturally occurring, positively charged, linear polysaccharide composed of N-acetyl-d-glucosamine and glucosamine units linked by β(1→4) glycosidic bonds (Fig 2A). Previously groups have suggested that EDC can react with both the hydroxyl

[0049] and amine [2] groups on chitosan, though EDC / NHS crosslinking is canonically used for carboxylic acid to amine coupling. It is unexpected that chitosan can be crosslinked and form a hydrogel using EDC / NHS chemistry.

[0129] From the1H NMR data, there are six distinct new proton peaks at 2.33 (peak a), 2.41 (peak b), 2.48 (peak c), 2.55 (peak d), 3.15 (peak e), and 3.33 (peak f) (Fig 2B) ppm, compared to unmodified chitosan peaks (Fig 1A). Further characterization with 2D1H-13C multiplicity-edited HSQC NMR demonstrated that these six protons belonged to secondary carbons, as indicated by their negative phasing. Chitosan alone 2D1H-13C multiplicity-edited HSQC NMR was generated. On the 2D1H-1H NOESY NMR spectrum, peaks d and e shared a cross peak, meaning they were near each other in space. Furthermore, they also likely belong to the same small molecule, as indicated by their negative phase. Peaks a and f share a cross peak on the1H-1H NOESY NMR spectrum and individually shared a cross peak with a new amide peak (7.98 ppm). To confirm the coupling of the protons from peaks d and e as well as peaks a and f,1H-1H COSY NMR spectrum demonstrated cross peaks between peaks d and e as well as peaks a and f, further suggesting these pairs of protons were coupled. From these data, it is proposed that these pairs of peaks belong to NHS hydrolysis by-product small molecules or other derivative

[0050] . Finally, peaks b and c both exhibit cross peaks to an amide H attached to chitosan in the1H-1H NOESY spectrum, indicating a likely covalent attachment to chitosan. These peaks further yielded a chemical signature similar to succinyl chitosan found in literature

[0051] –

[0054] . This NMR data suggested a covalent modification to chitosan, as well as a presence of hydrolyzed products, which may stabilize the positive charge of chitosan.

[0130] Because chitosan chain covalent crosslinking could not be confirmed via NMR spectroscopy, an increase in polymer molecular weight was explored indirectly via rheological techniques. An increase in the molecular weight of a polymer also correlates with an increase in viscosity, as longer and / or more branched chains entangle more easily and have stronger internal friction and hence flow less readily when a deformation force - 31 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 is applied. Additionally, studies previously utilized viscosity to quantify the degradation of chitosan chains by enzymatic digestion and self-resonating cavitation

[0033] ,

[0055] . Hence, observing an increase in viscosity can serve as a qualitative measurement for covalent chitosan crosslinking.95% deacetylated, 100 cp viscosity chitosan was reacted under the following conditions: (A) 1 EDC:chitosan monomer molar ratio (1 EDC) and 0.1, 0.25, 0.5, 1, and 2 NHS:chitosan monomer molar ratio (0.1, 0.25, 0.5, 1, and 2 NHS, respectively), and (B) 0.5 EDC:chitosan monomer molar ratio (0.5 EDC) and 0.1, 0.25, 0.5, 1 NHS:chitosan monomer molar ratio (0.1, 0.25, 0.5, and 1 NHS, respectively). Rheological experiments were performed with the unpurified reactions one day (22-24 hours) after the reaction was started.

[0131] Rotational continuous flow viscosity tests demonstrated a shear-thinning sample, as indicated by a decrease in viscosity with an increase in shear rate (Fig.3A-B). At a shear rate of 10 Hz, the viscosities of 1 EDC samples were higher than those of 0.5 EDC samples (Fig.3C-D). Furthermore, in both the 1 and 0.5 EDC samples, optimal rather than excess molar ratios of NHS led to an increase in viscosity (Fig.3C-D). In particular 1 EDC / 0.5 NHS and 1 EDC / 1 NHS had to highest viscosity values in the 1 EDC samples, and 0.5 EDC / 0.25 NHS had the highest viscosity value in the 0.5 EDC samples (Fig.3C- D).

[0132] Oscillatory frequency sweeps were used to determine the viscoelastic properties of the reacted chitosan samples. The 1 EDC and 0.25, 0.5, and 1 NHS samples formed viscoelastic solids, as demonstrated by the storage moduli (G’) being higher than the loss moduli (G’’) (Fig.3E). The lower EDC concentration (0.5 EDC) samples remained viscoelastic fluids (G’’ > G’), regardless of NHS concentration (Fig.3F). Additionally, in both the 1 EDC and 0.5 EDC samples, an optimal rather than excess molar ratio of NHS led to higher storage moduli and hence stiffer material, with statistical difference in values at a frequency of 10 rad / s (Fig.3G-H). These data demonstrated that viscosity and solid-like viscoelastic character increase based on EDC / NHS concentrations. Increased EDC concentrations and optimal NHS ratios led to higher viscosity values and storage moduli. Taken together, these rheology findings suggest that EDC / NHS chemistry can increase the length of chitosan chains in a tunable manner. Example 4—Cross-linked Chitosan Hydrogel Pore Size Characterization

[0133] Chitosan hydrogel pore size shrinks as chitosan concentration increases but does not depend on degree of deacetylation—To characterize the impact of reaction conditions on pore size, a range of chitosan hydrogels were prepared and analyzed via cryo-SEM. - 32 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 Chitosan gels displayed an interpenetrating pore network, composed of irregularly shaped and non-uniform pores (Figure 5). For the 30 mg / mL chitosan hydrogels, pore length scales ranged from 5-20μm, irrespective of degree of deacetylation (Figure 4A-B). However, at the 60 mg / mL concentration, pore length scales shrunk to 1-5μm (Figure 4C). The 60 mg / mL chitosan hydrogels displayed lower porosity (73.6 ± 2.4%) compared to the 30 mg / mL hydrogels (77.5±2.4% for 95 / 100_1 / 2_

[0030] , 78.3 ± 2.4% for 70 / 100_1 / 2_

[0030] ), with statistical significance between the low and high concentrations (Figure 4D). Example 5—Cross-linked Chitosan Hydrogel Rheological Characterization

[0134] The chitosan gel affords numerous potential parameters for rheological tunability, particularly the inherent chitosan viscosity, gel concentration, molar ratios of EDC / NHS reactants, and DDA. Rheology studies were performed to understand the impact of inherent chitosan viscosity (10, 100, 1000 cp), hydrogel concentration (30, 45, 60 mg / mL), EDC and NHS crosslinking molar ratios (1 EDC / 2 NHS and 0.5 EDC / 1 NHS molar ratio to chitosan monomer), and DDA (70% and 95%). These studies included oscillatory amplitude sweeps (0.1-500%, at 10 Hz), oscillatory frequency sweeps (0.1-100 rad / s, at 1% amplitude), rotational continuous flow tests (0.1-10 s-1), and cyclic deformation measurements (two cycles of 1% strain for 30 seconds followed by 200% strain for 15 seconds).

[0135] The storage moduli in linear viscoelastic region (LVER) for the chitosan hydrogels, at which strains the storage moduli are not dependent on the deformation force, were used to evaluate the mechanical strength of the different gel iterations

[0056] . Increasing the EDC / NHS ratios in the 1000cp and 100cp chitosan samples created stronger gels (Figure 5A, B). Similarly, increasing the gel concentration also increased strength, as shown in the 100cp (Figure 5B) and 10cp (Figure 5C) samples. Also, the data suggests that higher deacetylated chitosan (95%) produced a stronger gel at lower intrinsic viscosities (10cp) or molecular weights, compared to lower deacetylated chitosan (70%) (Figure 5C), though this effect was not noticeably observed at high viscosities (1000cp) (Figure 5A). Finally, all else equal, increasing the viscosity (1000 vs 100cp) led to greater storage moduli as did increasing chitosan concentration at a fixed viscosity (Figure 5A-C). Across all chitosan viscosities, as gels increased in strength, they became more brittle, as indicated by a shorter LVER (Figure 5A-C).

[0136] A comparison of storage and loss moduli from the frequency sweeps was used to determine the formation of a viscoelastic solid versus viscoelastic liquid for each of the - 33 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 chitosan iterations. Samples with the 1000cp viscosity chitosan at lower crosslinker ratios resulted in a gel at 30 mg / mL (G’>G’’) (Figure 5D). However, with the 100cp viscosity chitosan groups, the lower EDC / NHS (0.5 / 1) ratio did not yield a gel at 30 mg / mL (G’<G’’) (Figure 5E). Increasing the gel concentration led to a critical polymer concentration past which the 100cp viscosity did form a gel (95 / 100_1 / 2_

[0045] and 95 / 100_1 / 2_

[0060] ) (Figure 5E). This phenomenon was additionally observed with the 10cp chitosan samples, as an insufficient concentration of gel resulted in a viscous liquid rather than a viscoelastic solid (G’<G’’) in the 70 / 10_1 / 2_

[0045] but formed at gel in the 70_1 / 2_

[0060] sample (Figure 5F).

[0137] All of the samples displayed shear-thinning behavior, indicated by the negative slope of the continuous flow viscosity vs shear rate curve (Figure 5G-I). Differences in concentration had the largest impact on measured viscosity values at within the same inherent chitosan viscosities, as greater concentrations led to higher viscosity values (Figure 5G-I). Additionally, all of the gels demonstrated self-healing behavior in the continuous strain step tests. Under low shear, they remained a solid (G’>G’’), became a liquid under high shear (G’<G’’), and reverted back to a gel when the high shear was removed (G’>G’’) (Figure 5J-L).

[0138] In all, this rheological data demonstrated the shear-thinning and self-healing properties as well as the rheological tunability of the chitosan hydrogel. In summary, higher chitosan concentrations, higher EDC / NHS concentrations, higher inherent chitosan molecular weights, and higher degrees of deacetylation all resulted in higher storage moduli and hence stronger gels (Figure 5). Example 6—Cross-linked Chitosan Hydrogel Biocompatibility and Non-toxic Characterization

[0139] Chitosan hydrogels are non-toxic and biocompatible—To assess biocompatibility, representative chitosan hydrogels of varying compositions (95 / 100_1 / 2_

[0030] and 70 / 100_1 / 2_

[0030] ) were co-incubated with 3T3 fibroblast cells for 24 hours, at increasing gel volumes (10 μL and 30 μL). Viability data revealed that chitosan hydrogels do not impact cell viability (Figure 6).

[0140] The inflammatory response to chitosan hydrogels was evaluated in vivo. A representative chitosan hydrogel (95 / 100_1 / 2_

[0030] ) was implanted subcutaneously on the flank of C57BL / 6 mice. To understand the impact of altering the charge and chemical composition of the chitosan gel on the degree of inflammation, heparin and carboxymethyl chitosan were incorporated into gels prior to subcutaneous delivery. At - 34 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 days 3 and 7 post-implantation as well as 42 days for the chitosan gel alone sample, the sample and surrounding tissues were collected.

[0141] After both 3 and 7 days post-injection, inflammation was observed in all samples (Table 3, Figure 7). The 95 / 100_1 / 2_

[0030] chitosan hydrogel induced inflammation, ranging from moderate to marked, with neutrophilic and granulomatous inflammation observed, and peripheral necrosis at day 3 post-injection (Figure 7A, D). Inflammation remained moderate to marked at day seven, with mostly neutrophilic inflammation and rare foci of granulomatous inflammation. At this weeklong time point, less gel material was observed, with granulation tissue and fibrosis surrounding the gel (Figure 7B, E). At 42 days post-implantation, moderate inflammation, primarily granulomatous, forming discrete granulomas around the gel material, was observed (Figure 7C, F). The hydrogel material content was further reduced in size at this time point.

[0142] The additional incorporation of carboxymethyl chitosan to alter the charge of the gel led to moderate to marked inflammation, with samples varying from primarily neutrophilic with some foci of granulomatous inflammation to primarily granulomatous with fewer neutrophils at day 3 post-injection (Figure 7G, I). After 7 days post-injection, inflammation was reduced compared to the earlier time point as well as the chitosan gel at this time point, with only minimal to moderate inflammation observed (Figure 7H, J). Inflammation was neutrophilic and lymphoplasmacytic in character. Similar to the chitosan hydrogel, less hydrogel material was observed at this time point, with granulation tissue and fibrosis surrounding the gel.

[0143] Heparin introduced in the gel induced marked inflammation at day 3 post- injection, which was primarily neutrophilic (Figure 7K, M). Peripheral necrosis was also observed. Lymphocytes and macrophages were found at the periphery, in addition to skeletal muscle necrosis. Inflammation had reduced at day 7, primarily being moderate to marked, with mild to moderate peripheral necrosis (Figure 7L, N). The type of inflammation varied across samples, from neutrophilic to granulomatous with fewer neutrophils. However, in these samples at the 7-day time point unlike the chitosan gel alone and chitosan gel with carboxymethyl chitosan, minimal granulation tissue was observed around the gel. Furthermore, this group had the least reduction in the amount of gel over time. Though the nature of the inflammation did transition from neutrophilic to granulomatous, there was considerably less healing (granulation tissue / fibrosis) in this group. - 35 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0144] These data demonstrate that the chitosan hydrogel does elicit inflammation, which reduces over time. Furthermore, less gel material was observed at later time points, suggesting the body naturally degraded the material over time. The addition of carboxymethyl chitosan reduced the severity of inflammation and necrosis at later time points, and less material was observed compared to the other samples. The addition of heparin increased the severity of inflammation and led to less healing compared to the other two groups. It should be noted that none of the injections resulted in overt clinical symptoms, signs of discomfort, or injection site ulcerations in any of the treated mice. Example 7—Cross-linked Chitosan Hydrogel Tunable Release of Small Molecules

[0145] Release of hydrophilic small molecules from chitosan hydrogels is charge dependent—In solution, chitosan’s amine groups (Figure 2) are protonated, which leads to a strong positive charge. Thus, release of molecules loaded into a chitosan hydrogel can depend on charge. Accordingly, four hydrophilic small molecules were chosen based on their molecular properties and charge at neutral pH: rhodamine 6G (cationic, +1), rhodamine WT (anionic, -1), fluorescein (anionic, -2)

[0057] , pyranine (anionic, -3 charge) (Figure 8A). In a release study, after 24 hours, a higher percentage of rhodamine 6G released compared to rhodamine WT (*, p=0.0186), fluorescein (**, p=0.0012), and pyranine (****, p<0.0001) (Figure 8B). After 7 days, the difference between rhodamine 6G and rhodamine WT was no longer observable, but the effect remained for fluorescein (**, p=0.0023 compared to rhodamine 6G; and ***, p = 0.006 compared to rhodamine WT) and pyranine (****, p<0.0001 compared to rhodamine 6G and rhodamine WT) (Figure 8C). Notably, over half of the most negatively charged therapeutic, pyranine, despite its small size, 524.4 Da, was retained within the gel after one week. Example 8—Cross-linked Chitosan Hydrogel Tunable Release of Protein

[0146] Chitosan hydrogels tunably release the protein therapeutic interleukin-12 based on concentration and hydrogel polymer composition—Localized delivery can unlock the potential of strong immunotherapeutics that result in significant toxicity following systemic injections. In particular, immune stimulating cytokines and checkpoint inhibitors that cause serious AEs when delivered systemically may be more effective and less toxic if delivered and maintained intratumorally. In light of interleukin-12 (IL-12)

[0058] –

[0061] having a robust anti-tumor response with limited clinical application due to its high toxicity profile, initial in vitro release studies focused on formulations of IL-12 in a range of chitosan hydrogels. In particular, the influence of hydrogel density, controlled by the concentration of chitosan, and hydrogel charge were explored - 36 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0147] Increasing the concentration or density of chitosan was likely to delay the release of protein therapeutics. Indeed, IL-12 release from 95 / 100_1 / 2_

[0030] led to a sustained release over a week in vitro, with 32.6±1.3% released at 7 days. Increasing the concentration of the gel to 90 mg / mL (95 / 100_0.5 / 1_

[0090] ) slowed down the release, with only 18.7±6.2% (*, p=0.0172) released after a week (Figure 9A). The actual molecular weight of the chitosan strands did not impact release, as no difference in release was detected from 95 / 1000_1 / 2_

[0030] and 95 / 100_1 / 2_

[0030] (Figure 9B). Furthermore, the impact of concentration effect was not seen at very low chitosan molecular weights, as release from 95 / 10_1 / 2_

[0060] followed the same profile as that from 95 / 100_1 / 2_

[0030] (Figure 9B). The concentration of the loaded therapeutic significantly impact release profiles, as a higher percentage of IL-12 was retained in the gel as less IL-12 was loaded into the same 95 / 100_1 / 2_

[0030] gel (Figure 9C).

[0148] As seen in the small molecule release studies, electrostatic interactions also significantly affect release. A significant if not the primary method in which polysaccharides and proteins interact is through electrostatic interactions, which are determined by the pH of the solution and the isoelectric point (pI) of the protein

[0042] –

[0044] . Due to chitosan’s highly positive charge and pKa of its primary amines (~6.5), proteins with an isoelectric point below neutral will electrostatically bond with chitosan to a higher degree than proteins with an isoelectric point above neutral within a neutral solution (pH ~7). IL-12 has an isoelectric point of 6.27 and a charge of -10.9 at physiologic pH, which increase its likelihood to be electrostatically bound to chitosan hydrogels. Altering the electrostatic environment of the hydrogel could expedite the release of IL-12. To alter the charge of the hydrogel without changing the polysaccharide, N-carboxymethyl chitosan (CM-CS) was incorporated into the hydrogel to shield the charge of IL-12 from chitosan. At increasing concentration of CM-CS, IL-12 could be released more quickly from the hydrogel, with 53.7±3.8% release at 7 days with 1 mg / mL CM-CS (***, p=0.0002) (Figure 9D). This effect was also observed for a lower deacetylated chitosan (Figure 13A). Incorporating any negatively charged polymer, though, did not always have this effect. Heparin at 0.2 and 2 mg / mL did not expedite release with 95% deacetylated chitosan gel (Figure 9E), though it did slightly increase release with 70% deacetylated chitosan gel (Figure 13B). Example 9—Cross-linked Chitosan Hydrogel Tunable Release of Protein at Isoelectric Point - 37 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0149] Chitosan hydrogels tunably release protein therapeutic based on protein isoelectric point—To further explore the importance of electrostatic interactions the release of interferon gamma (IFNγ), which has an isoelectric point of 8.43 and charge of +2.4 at physiologic pH, was quantified. After 7 days, all of the loaded IFNγ had been released from 95 / 100_1 / 2_

[0030] (Figure 10A). Because of its fast release profile, efforts were initiated to delay the release through increasing the concentration of the chitosan gel. Doubling the concentration of the chitosan hydrogel significantly delayed release, with 54.5±12.8% released after a week (**, p=0.0070) (Figure 10A).

[0150] Additionally, because monoclonal antibodies are a major class of therapeutics, the release profile of aCTLA-4 monoclonal antibody, an immune checkpoint inhibitor with an isoelectric point between 7.6-7.9 and a molecular weight of ~150 kDa according to the manufacturer. Though larger than IL-12, aCTLA-4 released more quickly than IL-12 after one week (46.6±3.7%) in 95 / 100_1 / 2_

[0030] gel (Figure 10B). Furthermore, similar to IFNγ, doubling the gel concentration could slow down release (33.0±2.4%) (**, p=0.0058) (Figure 10B). These results demonstrate the tunable release of the chitosan hydrogel based on the delivered therapeutic as well as parameters of the hydrogel to tune this release. Example 10—Cross-linked Chitosan Hydrogel Imaging

[0151] An x-ray contrast agent can be incorporated within the gel to impart radiopaque character and does not affect protein release—Fluoroscopy is a commonly used clinical imaging modality to visualize interventional injections. Because chitosan is not radiopaque, a contrast agent, iohexol was co-formulated into the chitosan hydrogel to provide x-ray contrast. Increasing concentrations of iodine within the hydrogel could be imaged via x-ray in vitro (Figure 11A). Importantly, iohexol incorporation into the hydrogel did not impact the release of the model therapeutic IL-12 (Figure 11B). When 200 mgI / mL iohexol was incorporated into a chitosan hydrogel, the gel could be imaged in vivo when injected subcutaneously on the flanks of mice (Figure 11C). Iohexol was eliminated quickly after being delivered, as none was observed following one day after injection, though the gel was still visibly present (Figure 11C). Example 11—Cross-linked Chitosan In-Vivo Slow Release of Antibody

[0152] The hydrogel slowly releases a proof-of-concept antibody aCTLA4 in vivo—The in vivo release kinetics of fluorescence-labeled antibody, aCTLA4, from chitosan hydrogels were documented via non-invasive imaging

[0060] . Briefly, Alexa Fluor 660 (AF660) labeled aCTLA-4 was loaded into chitosan hydrogels composed of - 38 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 95 / 100_1 / 2_

[0030] with 1 mg / mL CM-CS (“fast releasing gel”) or 95 / 100_0.5 / 1_

[0060] (“slow releasing gel”) and injected subcutaneously on the flanks of Balb / c mice. At days 0, 1, 2, 3, 7, 14, 21, and 28 post-injection, fluorescence images of the remaining therapeutic were acquired (Figure 12A).

[0153] After twenty-four hours following injection, the slow releasing gel and the fast releasing gel were indistinguishable with 49.6±2.5% and 47.2±9.1% released. However, 93.0±4.0% of the injected aCTLA-4 had dissipated from the injection site in the control saline group (****, p<0.0001 for both groups). By three days post-injection, no aCTLA-4 was detected in any of the mice in the saline-control group. In contrast, aCTLA-4 was observed in both hydrogel groups at day 28, demonstrating its utility for extended release of antibody therapeutics. Additionally, after two days post-injection, the slow releasing gel retained more aCTLA-4 than the fast releasing gel, with statistical significance (**, p=0.0084) (Figure 12B). Overall exposure, as evidenced by the area under the curve of the normalized radiance values, were statistically higher in the gel groups (Figure 12C). These data demonstrate the ability to retain and slowly release protein therapeutics, including antibodies, and suggest that the hydrogel formulation can be tuned for faster or slower release. Example 12 - Injectable chitosan hydrogel localizes and tunably releases immunotherapeutics intratumorally

[0154] A chitosan-based hydrogel, XCSgel, was investigated as a localized intratumoral delivery platform in the context of murine models of orthotopic triple-negative breast cancer (TNBC). The ability of XCSgel to resist expulsion following i.t. injection was characterized in vitro and ex vivo. Tissue responses to XCSgel delivered i.t. in an orthotopic TNBC murine model were characterized via histological analyses. In vivo retention and sustained release of three cytokines in XCSgel were demonstrated with fluorescence imaging. The antitumor efficacies of diverse antitumor cytokines were compared. Based on differences in efficacy, additional antitumor and pharmacokinetics studies of XCSgel when co-formulated with the cytokine IL-12 (XCSgel-IL12) were performed in multiple TNBC models. Changes in the tumor-immune microenvironment (TIME) following i.t. immunotherapy were evaluated with multi-spectral flow cytometry. Finally, XCSgel-IL12-induced systemic antitumor immunity was assessed via rechallenge studies and analyses of abscopal tumor control. Materials and Methods

[0155] Compounds, Reagents, and Animals - 39 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0156] All compounds and reagents are listed in Table 2. Recombinant murine interleukin-12 (IL-12) was overexpressed and purified in-house, as previously described

[0062] . Seven- to 10-week-old female C57BL / 6NCr and Balb / cAnNCr mice were purchased from Charles River Laboratories. Animal use complied with the Public Health Service Policy on Human Care and Use of Laboratory Animals. All experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee at North Carolina State University (protocols 19-795, 22-414, and 23-126).

[0157] Table 2. Reagents and resources used, listed along with their source and identifying information.- 40 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0158] XCSgel Preparation

[0159] XCSgel was prepared following Example 1, as previously described

[0063] . XCSgelslowrefers to 95% deacetylated chitosan, at a molecular weight range 100-250kDa, crosslinked with 1:2 EDC:NHS molar ratio, at a concentration of 45 mg / mL. XCSgelfastrefers to 70% deacetylated chitosan, at a molecular weight range 100-250kDa, crosslinked with 1:2 EDC:NHS molar ratio, at a concentration of 30mg / mL, co- formulated with 3mg / mL carboxymethyl-chitosan (CM-CS). All XCSgels were prepared one day prior to delivery and stored overnight at 4°C.

[0160] Cell Growth and Maintenance - 41 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0161] E0771 cell lines were purchased from the American Type Culture Collection. The mWnt cell line was a gift from Dr. Stephen D. Hursting

[0064] . The cells and their subpassages were stored in the vapor phase of liquid nitrogen until use. All cells were visually monitored for obvious contaminants, such as bacterial and fungal growth. They were frequently tested for mycoplasma with a commercially available kit (MycoAlert® PLUS Mycoplasma Detection Kit). E0771 cells were cultured in DMEM media, 1 mM penicillin-streptomycin, 10% fetal bovine serum (FBS), glutamine, and 20mM HEPES at 37°C in 5% CO2 and 95% humidity. mWnt cells were cultured in DMEM media, 1mM penicillin-streptomycin, 10% FBS, glutamine, 5mM HEPES, 0.1% 2-mercaptoethanol at 37°C in 5% CO2 and 95% humidity.

[0162] Gelatin Tumor Phantoms

[0163] Gelatin was dissolved in 100mL boiling deionized water with stirring. The solution solidified at room temperature overnight in dome-shaped silicone molds for tumor retention visualization and in cylindrical silicone molds for compression testing. For retention visualization, 50 or 100μL of food dye or 50 or 100μL of XCSgelslowcontaining blue food dye was injected into a gelatin tumor phantom using a 26g needle.

[0164] Compression Testing

[0165] Gelatin tumor phantoms were compressed using an Instron 5944 single-column mechanical testing system (Instron; Norwood, Massachusetts, USA) at a ramp rate of 0.20mm / s with a 500N load cell. Force (N) and displacement (mm) were recorded. Stress was calculated as force / cross-sectional area. Strain was calculated as displacement / initial length. Cross-sectional area and initial length were measured with digital calipers. The Young’s modulus was calculated as the slope of the stress-strain curve in the initial linear region before the inflection point.

[0166] In vitro Cytokine Release

[0167] 50μL of XCSgelslowloaded with IFNα2 and IL-12 were prepared one day prior to release. Cytokines were released in 1mL of 10% FBS in 1xPBS. For each time point, all release media was removed to simulate sink conditions and stored at 4°C to prevent degradation. Protein concentrations were measured via ELISA.

[0168] Cytokine Fluorophore Labeling

[0169] Cytokines IL-15 and IFNα2 were fluorescently labeled using Alexa Fluor™ 647 Microscale Protein Labeling Kit. IL-12 was labeled using Alexa Fluor™ 647 Protein Labeling Kit. The degrees of labeling, moles of fluorophore per mole of cytokine, were - 42 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 calculated to be 1.4 (IL-12), 0.6 (IL-15), 1.6 (IFNα2), according to manufacturer’s instructions.

[0170] Tumor Cytokine Localization and Tissue Clearing

[0171] 5x105 E0771 cells were implanted in the 4th mammary fat pad of C57BL / 6 mice and allowed to grow until 199±47mm3.50μL XCSgelslowco-formulated with 5μg IL-12 labeled with AF647 (IL-12-AF647) or 5μg IL-12-AF647 in 50μL saline were delivered i.t. in E0771 tumors. Following delivery, tumors were excised and fixed in 1xPBS / 4% paraformaldehyde at 4°C overnight. Tissues were cleared using the iDISCO clearing protocol (https: / / idisco.info / ). Briefly, tumors were dehydrated with increasing concentrations (20-100%) of methanol at room temperature. Samples were then shaken with 66% dichloromethane / 33% methanol at room temperature for three hours, followed by two fifteen-minute washes in 100% dichloromethane to remove excess methanol. Tissues were finally placed in dibenzyl ether for imaging. Cleared tumors were imaged using an LaVision UltraMicroscope II equipped with zoom body optics and an Olympus MVPLAPO 2X / 0.5 NA lens with a corrected dipping cap. Zoom was set to 0.80x, resulting in a pixel size of 3.75µm. Z stacks were taken with 5µm spacing. The sample was illuminated with 3 sheets from one side (2 of them angled, to minimize shadowing), with the horizontal focus in the center of the field of a view, and a sheet NA setting of 0.016, which results in a 16.9µm sheet thickness. Fluorescence was collected using two channels: 488nm laser excitation and emission collection through a Chroma ET525 / 50 filter, for autofluorescence, and 647nm laser excitation and collection through a Chroma ET690 / 50 filter, for staining. Images were analyzed using IMARIS software version 10.1.1.

[0172] In vivo Immunogenicity and Histology

[0173] 5x105E0771 cells were implanted in the left 4th mammary fat pad of C57BL / 6 mice. When E0771 tumors reached 154±41mm3, mice were randomized into two groups, and 50μL of XCSgelslowor XCSgelfastwas injected i.t.. On days 1, 3, and 7, mice were euthanized, and tissue collected. The tumor, gel, and surrounding tissue were dissected and fixed in 10% neutral buffered formalin. Tissue processing – including paraffin- embedding, sectioning, and staining – was performed by the Histopathology Laboratory at the NC State College of Veterinary Medicine. Blinded analysis of tissue reactions and immune cell infiltrates was performed by a board-certified veterinary pathologist (D.M.M.) on routine hematoxylin and eosin-stained slides.

[0174] In vivo Fluorescence Imaging - 43 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0175] For the variable cytokine release study, 50μL of XCSgelslowloaded with 73.5nmol of AF647-labeled cytokine (IL-12, IL-15, or IFNα2) was injected subcutaneously on the shaved flank of female Balb / c mice. For the variable XCSgel with IL-12 study, 50μL of XCSgelslowor XCSgelfastloaded with 5μg (73.5nmol) of IL-12-AF647 was injected subcutaneously on the shaved flank of female Balb / c mice, with a saline medium as a control. Fluorescence (Ex / Em 640 / 690 nm) images were taken on a Spectral Instruments Imaging Ami HTX (Tucson, Arizona, USA) optical imaging system. Background values were subtracted, and the data were then normalized to the maximum efficiency in the experiment

[0065] .

[0176] Pharmacokinetics / Pharmacodynamics

[0177] 5x105E0771 cells were implanted in the left 4th mammary fat pad of C57BL / 6 mice. Fourteen days after tumor implantation (tumor volumes 159±101 mm3), mice were randomized into three treatment groups.50μL of XCSgelslowco-formulated with 5μg IL- 12 (XCSgelslow-IL-125μg) or XCSgelfast-IL-125μgwere delivered i.t., with a saline medium as a control. Separate cohorts of mice were bled one day prior to treatment and 4-, 24-, 48-, and 72-hours following treatment. Following each bleed, mice received 100-200μL injection of Hanks' Balanced Salt Solution i.p. to restore lost liquid volume. Sera were collected and stored at -20°C until use. Serum IL-12 and IFNγ concentrations were determined via ELISA.

[0178] Antitumor Studies

[0179] For the E0771 single orthotopic tumor study with variable cytokines, 5x105E0771 cells were implanted in the left 4th mammary fat pad of C57BL / 6 mice. When tumors reached 182±72mm3, (day 12 post implantation), mice were treated i.t. with 50μL of XCSgelslow-IL125μg, XCSgelslow-IL1530μg, XCSgelslow-IFNα210μg, XCSgelslow-GMCSF5μg, or left untreated. IL-15, IFNα2, and GM-CSF doses were determined using the maximum delivered dose effective in immune competent mice in preclinical studies, and the IL-12 dose was determined via prior work in our lab [66-68]. For the E0771 single orthotopic tumor study, 5x105E0771 cells were implanted in the left 4th mammary fat pad of C57BL / 6 mice. When tumors reached 125±57mm3, (day 17-21 post implantation), mice were treated i.t. with 50μL of XCSgelslow-IL12, XCSgelslow-IL121μg, XCSgelslow-IL125μg, or XCSgelslow-IL1220μg. Cured mice were rechallenged with 5x105E0771 cells on the opposite flank at least 100 days following initial treatment. Mice that rejected the E0771 rechallenge were rechallenged with 5x105mWnt cells on the left flank 95 days following the E0771 rechallenge. For the mWnt single orthotopic tumor study, 5x105mWnt cells - 44 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 were implanted in the left 4th mammary fat pad of C57BL / 6 mice. When tumors reached 114±63mm3, (day 9 post implantation), mice were treated i.t. with 50μL of XCSgelslow- IL125μgor were left untreated. For the abscopal E0771 tumor study, 5x105E0771 cells were implanted in the left 4th mammary fat pad of C57BL / 6 mice. To establish abscopal tumors, 5x105E0771 cells were implanted subcutaneously on the right flank three days after the primary tumor was implanted. When primary tumors reached 165±76 mm3(16 days post implantation), 50μL of XCSgelslow-IL125μgor XCSgelfast-IL125μgwere delivered i.t., with XCSgelslowand no treatment as controls. Tumor volumes were measured every 2-3 days and calculated using the formula: V = (w × w × l) / 2, where V is tumor volume, w is tumor width and l is tumor length. Mice were euthanized when total tumor burden exceeded 2000mm3for single tumors and 3000mm3for two tumors, when tumors became ulcerated, or when mice became moribund.

[0180] Spectral Flow Cytometry

[0181] Tissue samples were minced, incubated with Type IV collagenase and DNase I at 37°C for 30min with shaking at 250RPM, and then passed through 70-micron filters. Cell suspensions were then resuspended in phosphate buffered saline (PBS) with 1:800 Live / Dead Blue and incubated at room temperature for 10 minutes, protected from light in a 96-well V-bottom plate. Antibodies for extracellular antigens were prepared in HorizonTM Brilliant staining buffer at a 2x dilution and added directly to the cell suspensions (Table 3). Cells were stained for 30min at 4°C and protected from light. Stained cells were washed with PBS containing 2% fetal calf serum. Cells were then fixed and permeabilized, and intracellular antigens were stained using the eBioscience FoxP3 / Transcription factor staining kit. Samples were run on a 5-laser Cytek Aurora following daily quality control experiments using SpectroFlow QC beads. Data were analyzed with FlowJo software (TreeStar, Version 10.9.0) and OMIQ (Dotmatics).

[0182] Table 3. Antibodies used for multi-spectral flow cytometry.- 45 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192- 46 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0183] Statistical Analysis

[0184] A standard two-tailed t test was used to test for significance between two sample groups. Differences between three or more treatment groups were determined using ANOVA with Tukey’s HSD post-hoc testing. The above analyses were conducted using Prism (GraphPad Software Inc., San Diego, California, USA). Results

[0185] XCSgel effectively localizes cytokine immunotherapy in vivo when delivered intratumorally

[0186] Local retention of i.t. injections is limited by dense tumor architectures as well as high interstitial fluid and injection pressures, which exclude low viscosity injectates. To visualize the leakage of injectate, we developed transparent gelatin tumor phantoms that were mechanically similar to solid tumors. Compression testing of different concentrations of gelatin tumor phantoms revealed a Young’s modulus range of 9.8- 41.9kPa (Figure 14A). These data mapped onto the range of tumors’ Young’s Modulus, which for breast cancer, for example, range between 10-43 kPa

[0069] .14 and 10w / v% gelatin had Young’s moduli of 41.9±0.8 and 26.0±1.2 kPa, respectively. Blue dye in a saline solution injected into a gelatin tumor phantom offered a qualitative representation of poor retention, as most of the injectate returned via the needle track. (Figure 15A; Figure 14B-C). When co-formulated with shear-thinning, self-healing XCSgel, the blue dye was completely retained within the gelatin tumor phantom (Figure 15B; Figure 14B- C). These results were confirmed in vivo in the E0771 TNBC model. Fluorescently- labeled IL-12-AF647 in saline did not remain within the tumor when delivered in vivo and post-tissue clearing (Figure 15C), whereas IL-12-AF647 in XCSgel remained localized within the tumor when delivered following the same delivery and processing conditions (Figure 15D). These data demonstrate that XCSgel overcomes a major barrier, i.e. poor intratumoral retention, to the success of i.t. immunotherapy.

[0187] XCSgel alone induces a transient, tunable inflammatory response within injected tumors

[0188] Any foreign material delivered within the body will induce inflammation. The intensity and duration of which will vary based on the injected tissue, the host immune response, and the material delivered. In general, naturally derived materials, such as chitosan, elicit less inflammation compared to their synthetic counterparts. Knowledge of the type of immune response induced by a biomaterial can facilitate rational combinations of immunotherapies to leverage this response. To this end, the i.t. delivery of different - 47 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 compositions of XCSgel were delivered i.t. in the E0771 TNBC model. Two compositions, called XCSgelslowand XCSgelfastdue their relative release kinetics, were chosen. XCSgelslow, had a high degree of deacetylation (DDA) (95%) and therefore a higher positive charge by virtue of its higher density of primary amine groups (Figure 16A). The second, XCSgelfast, had a reduced negative charge due to its 70% deacetylation and was co-formulated with a more negatively charged derivative of chitosan, CM-CS (Figure 16B). XCSgelfastreleased protein therapeutics more quickly in vitro with reduced inflammatory responses in vivo compared to XCSgelslow. Higher inflammatory responses to positively charged versus negatively charged polymers have previously been reported (21–23).

[0189] Mice bearing E0771 tumors were treated i.t. with either gel, and timepoints were taken at day 1, 3, and 7 post-injection (Table 4). One day post-injection, XCSgelslow induced moderate i.t. inflammation, with 42±18% tumor necrosis (Figure 16C-E). Inflammation was neutrophilic, with polymorphonuclear leukocytes (PMNs) found around the gel and scattered lymphoplasmacytic (LP) inflammation around the periphery (Figure 16E). On day three, inflammation remained moderate, with 35±9% tumor necrosis (Figure 16F-H). Inflammation was neutrophilic and eosinophilic, with granulation tissue surrounding the gel and PMN inflammation. The granulation contained scattered LP inflammation (Figure 16H). One week post-injection, the severity of the inflammation had decreased to mild, with reduced necrosis within the tumor (Figure 16I- K). Inflammation remained both neutrophilic and eosinophilic, though it had noticeably decreased around the gel (Figure 16K). The presence of granulation tissue was increased at this timepoint with some progressing towards mature fibrous tissue.

[0190] Table 4. Histologic changes noted with intratumorally injected XCSgel at different time points.- 48 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192injection into the E0771 tumor. The gel induced tumor necrosis (57±6%), and inflammation was primarily neutrophilic (Figure 16L-N). On the third day post-injection, inflammation was moderate, with 28±6% tumor necrosis (Figure 16O-Q). Inflammation was both neutrophilic and eosinophilic (Figure 16P-Q). Gel was not visualized at this timepoint, but granulation tissue was observed as well as PMN infiltration in necrotic tumor regions. Seven days post-injection, inflammation was noticeably reduced to mild or minimal (Figure 16R-T). Tumor necrosis was noted at 30±9%. XCSgel was likely fully resolved as it could not be visualized, and PMN infiltration was observed in areas of tumor necrosis (Figure 16T).

[0192] It is important to note that the tumor necrosis induced by XCSgel alone was limited to the region directly in contact with the gel and did not result in tumor control. Over time, XCSgels were resolved following i.t. injections in a similar manner as subcutaneous (s.c.) injections in Example 6.

[0193] XCSgel releases cytokine therapeutics irrespective of charge or size

[0194] To explore release kinetics in vivo, three clinically relevant antitumor cytokines, IL-12

[0070] , IL-15

[0071] , and IFNα2

[0072] , were chosen based on their distinct sizes, charges at neutral pH, and isoelectric points. These parameters are listed as follows: IL-12 (68kDa, -10.9 at pH 7.4, pI~6.3), IL-15 (13kDa, -10.7 at pH 7.4, pI~4.5), and IFNα2 (19kDa, +1.8 at pH 7.4, pI~7.9)

[0073] . The release profile of IFNα2 and IL-12 in vitro demonstrated significantly different release kinetics with the smaller, positively charged IFNα2 releasing much more quicky (Figure 17). For in vivo studies, each cytokine was labeled with AF467 and delivered s.c. in XCSgelslow. All three cytokines displayed similar slow-release profiles and were imageable up to 28 days post-injection, with statistical significance only between IL-12 and IFNα2 at day 21 (Figure 18A-B). Unlike the in vitro data of Examples 7-9, no difference between the cytokine release rates was observed. Without being bound by theory, other parameters, such as the complex environment or the inflammatory tissue response in vivo, may overcome the importance of electrostatic interactions.

[0195] XCSgel-IL12 outperforms other commonly used antitumor cytokines co- formulated in XCSgel - 49 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0196] To compare antitumor activities, a range of antitumor cytokines (IL-12, IL-15, IFNα2, GM-CSF) commonly pursued in clinical studies [74-76] were co-formulated in XCSgelslowand injected i.t. in an orthotopic E0771 primary tumor (Figure18C). XCSgelslow-IL12 induced complete tumor rejection in 5 of 8 treated mice (Figure 18D). Tumor elimination was durable for at least 70 days (Figure 18F). A statistically significant slight initial tumor growth delay was noted in XCSgel-IFNα2, but this effect waned over time (Figure 18H). There were no discernible antitumor effects among untreated, XCSgelslow-IL15, XCSgelslow-IFNα2, and XCSgelslow-GMCSF cohorts (Figure 18E,G-I). Due to XCSgelslow-IL12’s superior performance compared to the other treatments, the remaining studies focused on localization of IL-12.

[0197] Different compositions of XCSgel release cytokine IL-12 at different rates

[0198] As XCSgelslowdid not release different cytokines at distinct rates, the composition of XCSgel was engineered to determine if a single cytokine could be released at variable rates. Based on IL-12’s demonstrated antitumor activity (Figure 18F), IL-12 was chosen as a model cytokine therapeutic. IL-12-AF647 was co-formulated with both XCSgelslowand XCSgelfastand injected subcutaneously on the flanks of mice in vivo, with IL-12 delivered in saline as a control (Figure 19A). After 24 hours post-injection IL-12 delivered in saline was nearly undetectable, with high levels of cytokine remaining in both XCSgels (Figure 19A-C). XCSgelfastretained IL-12 for about 2 weeks post-injection (Figure 18B). XCSgelslowcontinued to release IL-12 over 3 to 4 weeks, with detectable levels up to day 28 (Figure 18A-C). Beginning at timepoint day 3, statistical differences in IL-12 release were observed between the two gels, which was again observed at days 10, 17, and 21 post-injection (Figure 18C). These results demonstrate that XCSgel can be tuned to manipulate the rate of delivery in vivo.

[0199] XCSgel reduces systemic IL-12 exposure

[0200] To determine if intratumoral delivery and retention of a potent cytokine, IL-12, within XCSgel mitigates systemic exposure, the pharmacokinetics of IL-12 were evaluated. XCSgelslow-IL12 and XCSgelfast-IL12 were injected i.t. in an E0771 TNBC orthotopic tumor. IL-12 alone served as a control. At timepoints 4, 24, 48, and 72 hours, mice were bled, and their sera collected for circulating cytokine concentrations of both IL-12 and IFNγ, a downstream cytokine produced by activated T and NK cells in response to IL-12

[0077] .

[0201] IL-12 alone caused a spike in serum IL-12 levels 4 hours post-i.t. injection, which then diminished significantly 24-hours post-i.t. injection with barely detectable levels at - 50 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 48-hours and undetectable levels at 72-hours (Figure 19D). At 4 hours post-injection, XCSgelfast-IL12 treatment resulted in significantly lower serum IL-12 levels compared to unformulated IL-12 (Figure 19E). However, at 24-, 48-, and 72-hours, mice treated with i.t. XCSgelfast-IL12 had higher serum IL-12 concentrations than unformulated IL-12 although concentrations were quite low – less than 1ng / ml (Figure 19F-H). XCSgelslow- IL12 treatment resulted in the lowest levels of circulating IL-124 hours post-injection (Figure 19E). At 24, 48, and 72-hours post-injection, XCSgelslow-IL12 treatments maintained very low but measurable levels of IL-12 (Figure 19F-H). Taken together, these data demonstrated that co-formulation in XCSgel can reduce systemic exposure to IL-12 and that XCSgel can be modified to control systemic exposure. It is important to note that XCSgel markedly reduced but did not completely eliminate systemic exposure to IL-12.

[0202] Given that IL-12-induced toxicity is largely attributed to IFNγ

[0078] , systemic circulation of this cytokine was also tracked. Circulating IFNγ levels peaked at 24 hours post injection for all IL-12 treatment groups (Figure 19I). IFNγ levels were reduced at 48 hours and still further reduced at 72 hours, indicating a resolution to baseline (Figure 19I). There were no statistical differences among treatment groups at the 4-, 24-, and 48-hour timepoints (Figure 19J-L) and only XCSgelslowmaintained a slightly elevated IFNγ concentration at 72-hours (Figure 19M).

[0203] XCSgel-IL12 eliminates primary orthotopic TNBC tumors and induces protective immunity

[0204] The antitumor activity of i.t. XCSgelslow-IL12 at a range of IL-12 doses, 0, 1, 5, or 20µg, was evaluated in orthotopic E0771 tumors (Figure 20A). XCSgel-IL121µgtreatment delayed tumor growth and extended survival compared to the XCSgel alone group (Figure 20B-D), however, none of the treated mice survived beyond 40 days. XCSgel- IL125µgtreatment eliminated nearly all (6 / 7) tumors (Figure 20E). After complete regression, none of the treated tumors recurred, and mice remained tumor-free for 60 days (Figure 20B). Interestingly, increasing the dose of IL-12 in XCSgel to 20 µg reduced antitumor activity, with only 3 / 7 tumors completely regressing (Figure 20E). Cured mice from all groups were rechallenged in the opposite flank with E0771 cells and remained tumor-free, demonstrating protective immunity (Figure 20G). Following the E0771 rechallenge, mice were then challenged with the mWnt TNBC model. As expected, mWnt tumors grew in all mice, demonstrating immune specificity (Figure 20G). - 51 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0205] To evaluate function in another, more difficult to treat tumor model, XCSgelslow- IL125µgwas delivered i.t. in immunologically ‘cold’ mWnt orthotopic tumors (Figure 20I)

[0064] .1 / 5 mice experienced complete tumor rejection, with survival measured up to 85 days (Figure 20J). Body weight measurements were taken to assess potential systemic toxicities. however, no mouse experienced significant weight loss after XCSgelslow- IL125µgtreatment (Figure 20K).

[0206] XCSgel-localized IL-12 reshapes the T cell landscape within the TIME

[0207] To determine how XCSgel-IL12 alters the TIME, tumors were resected seven days after treatment when tumor volume differences became obvious. As illustrated in the UMAP plots in Figure 21A-C, high dimensional spectral flow cytometry revealed untreated and XCSgel-treated tumors displayed similar immune compositions, but XCSgelslow-IL125µgdramatically altered the composition of the TIME, particularly T cell populations (Figure 21A-C). Cell markers are listed in Table 5.

[0208] Table 5. Markers used in flow cytometry to identify cell populations, with their respective abbreviations.- 52 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192

[0209] The overall percentages of CD4+T cells decreased from 63% to 41% of all T cells with XCSgelslow-IL125µgtreatment (Figure 21D). Conversely, the frequency of CD8+T cells increased from 25% to 44% with treatment (Figure 21I). More striking differences were found within these T cell lineages. The frequency of proliferating CD4+T cells increased 2-fold, while the frequency of terminally exhausted CD4+T cells decreased by 2.4-fold with XCSgelslow-IL125µgtreatment (Figure 21E-F). A striking 9.4-fold increase in Ly6C+Th1-like CD4+cells was detected as well as a 2.1-fold increase in effector-like CD11b+CD4+cells (Figure 21G-H). The frequency of terminally exhausted CD8+T cells decreased by 3-fold while central memory-like CD8+T cells, presumably containing favorable features of T cell stemness, increased by 4.2-fold (Figure 21J,K). Notably, the - 53 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 frequency of activated, proliferating CD8+T cells increased by 3.2-fold with XCSgelslow- IL125µgtreatment (Figure 21L). No differences were observed in the overall frequency of CD45+leukocytes, T cells, regulatory T cells, and TCR γδ T cells (Figure 22A-D). In contrast to beneficial changes in anti-tumor T cell populations, XCSgelslow-IL125µgdecreased the frequency of NK T cells and NK cells by 1.6- and 2.3-fold, respectively (Figure 21M,N). Last, no dramatic differences in B cell populations were observed (Figure 22E-H).

[0210] While the overall frequency of myeloid cells did not change with treatment, we detected changes in the composition of myeloid populations in the TIME (Figure 22I). Myeloid derived suppressor cells (MDCSs) decreased 2.4-fold after XCSgelslow-IL125µgtreatment, but the mononuclear (M-MDSCs) and polymorphonuclear (PMN-MDSCs) constituents exhibited no treatment-related changes (Figure 21O, Figure 22J,K). Overall macrophage frequencies remained relatively stable; however, we detected a 3-fold decrease in the frequency of immunosuppressive Ly6Clo macrophages, which have previously been shown to be pro-tumorigenic, in the XCSgel-IL12 group (Figure 21P,Q)

[0079] . XCSgelslow-IL125µgalso increased granulocytes from 0.04 to 0.08% and conventional dendritic cells (cDCs) over 2-fold from 5.2 to 11.4% (Figure 21R,S). No differences in neutrophils were noted (Figure 22L).

[0211] XCSgel-IL12 eliminates both primary orthotopic and abscopal flank TNBC tumors

[0212] A primary argument in favor of the use of systemic immunotherapies is for the treatment of disseminated disease. However, locally and rationally delivered immunotherapies can not only eliminate primary / treated tumors but also induce an adaptive immune response capable of eliminating untreated, distant tumors. To determine if XCSgel-IL125µgcould eliminate abscopal tumors, a bilateral E0771 tumor model was developed in which the implantation of primary and abscopal tumors was staggered by three days. Sixteen days after primary tumor implantation, when abscopal tumors were palpable, XCSgelslow-IL125µgand XCSgelfast-IL125µgwas injected i.t. in the primary tumor (Figure 23A). Controls included an untreated group and IL-12 alone. XCSgelslow- IL125µgled to long-term cures, with both primary and abscopal tumor elimination (Figure 23B-C). XCSgelfast-IL125µgtreatment led to tumor rejection in 3 / 6 primary tumors, and only one mouse was cured of both the primary and untreated abscopal tumor (Figure 23D). There were no tumor regressions in either the IL-12 alone (Figure 23E) or untreated groups (Figure 23F). Comparisons of survival data between either XCSgel-IL12 - 54 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 group and the untreated cohort revealed statistically significant differences (Figure 23B). These data demonstrate that both injected and anenestic solid tumors can be eliminated with a single injection of XCSgel-IL125µg.

[0213] In summary, XCSgel resisted tumor leakage and slowly released three model cytokines. XCSgel could be tuned for faster or slower release of embedded therapeutics. XCSgel-IL12 outperformed XCSgel formulations with other commonly used antitumor cytokines. A single injection of XCSgel-IL12 eliminated 86% E0771 and 20% mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated 67% untreated abscopal E0771 tumors. XCSgel- IL12 induced profound changes to the TIME, including a 3-fold reduction in the frequency of exhausted CD8+T cells and a 3.2-fold increase in activated, proliferating CD8+T cells. Discussion

[0214] I.t. immunotherapies aim to reduce irAEs while enhancing delivery to the tumor microenvironment. However, in the absence of a delivery system, i.t. injections are likely to rapidly leak from solid tumors, thus failing to maximize delivery to the tumor microenvironment. I.t. injections of low viscosity solutions are not likely to be retained locally, as we have modeled in vitro and in vivo (Figure 15). To improve local retention, we developed XCSgel, an injectable, biodegradable, and clinically imageable hydrogel capable of coformulation and release of any number of immunobiologics. In stark contrast to saline-based injections, XCSgel effectively localizes its cargo within the tumors for sustained release (Figure 15).

[0215] XCSgel by itself induced a robust inflammatory response that was reduced over seven days after i.t. injection (Table 4, Figure 16). The immune response was primarily neutrophilic at first, with eosinophils appearing 3 and 7 days after injection. Of particular note, XCSgel injected within a tumor induced a different reaction than XCSgel administered subcutaneously on the flanks of mice, which had both neutrophilic and granulomatous but not eosinophilic character. Location of implanted foreign materials have previously been demonstrated to impact the immune response

[0080] . For example, fat necrosis often induces a more granulomatous response, which was seen with the subcutaneous injections. In comparing different XCSgel prototypes, the gel with the higher concentration, higher positive charge, and less hydrophobicity (XCSgelslow), was still present one week following injection, whereas the lower concentration, lower positive charge, and more hydrophobic, XCSgelfastwas not found one week following - 55 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 injection and induced less inflammation at later timepoints (Table 4). This faster resolution of 70% deacetylated chitosan demonstrated that higher acetylation of chitosan led to faster degradation in vivo

[0081] . Additionally, the incorporation of carboxylic acid groups via CM-CS may have also reduced inflammation by the addition of carboxylic acid groups reducing the foreign body reaction

[0082] . Both XCSgels induced tumor necrosis, which is typical of cationic polymers [83-84], and this necrosis was incapable of controlling the tumor (Figure 20C) and was reduced over time (Figure 16).

[0216] The immune reaction to XCSgel may have enhanced IL-12 activity and overall antitumor efficacy. Neutrophils produce IFNγ in response to IL-12

[0085] . Thus, the brisk XCSgel-induced neutrophilic infiltration may have enhanced the activity of co- formulated IL-12 in antitumor studies. In addition, the necrosis induced by XCSgel may provide a source of tumor antigen for the induction of an in situ vaccine response. Finally, DDA may have impacted antitumor efficacy. For example, a recent study delivering a highly deacetylated polymer derived from chitin demonstrated that a higher DDA led to a more robust antitumor response

[0086] . Mechanistically, higher DDA led to higher induced mitochondrial stress, cGAS-STING activation, and IFNAR-dependent dendritic cell maturation

[0087] . Taken together, XCSgel likely pairs well with potent pro-inflammatory immunotherapeutics, such as IL-12.

[0217] Our in vitro release studies demonstrated charge-dependent release of macromolecules from XCSgel, supporting the impact of charge on protein and polysaccharide interactions [63, 88-90]. To evaluate the impact of this charge-based release in vivo, three different cytokines with varying isoelectric points were formulated in XCSgel and delivered subcutaneously. Surprisingly, no difference in the release among the cytokines could be observed, though differences were observed in vitro (Figure 18A- B, Figure 14). Without being bound by theory, the complex, proteinaceous in vivo environment may disrupt or shield any charge effects or that immune cells recruited by XCSgel-induced tissue responses may alter cytokine availability.

[0218] Additional in vivo imaging studies demonstrated that the release of a cytokine could be manipulated by changing the composition of XCSgel. Specifically, XCSgelfastand XCSgelslowwere shown capable of maintaining measurable IL-12 in a tumor for 2 and 4 weeks, respectively (Figure 19A-B).

[0219] In addition to increasing localization of IL-12, XCSgel significantly reduced systemic exposure to IL-12 (Figure 19D-H). That XCSgel did not completely eliminate IL-12 dissemination may be a feature, as some amount of systemic IL-12, as long as it - 56 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 does not induce toxicity, may be beneficial. Serum IFNγ levels were in the low ng / ml range and remained largely unaffected, except at 72-hours post-treatment (Figure 19I-M), indicating that the relationship between IL-12 and IFNγ may not be proportional. Even though IL-12 related toxicity is attributed to IFNγ, the low levels measured did not result in observable clinical symptoms or significant body weight changes (Figure 20K). Advantageously, the enhanced local retention of IL-12 allows for reduced treatment frequency and likely a reduced number of clinic visits. Notably, all antitumor experiments described herein were performed with a single injection, and not multiple IL-12 treatments

[0091] .

[0220] Another finding was the loss of antitumor activity when the dose of co-formulated IL-12 was increased from 5µg to 20µg (Figure 20E,F). These data indicate that the optimal biological dose of XCSgel-IL12 may not be the same as the maximum tolerated dose

[0092] .

[0221] Immunoprofiling studies revealed that XCSgelslow-IL125μginduced profound changes in the TIME that likely contributed to the robust antitumor efficacy. Of note, XCSgelslow-IL125μgincreased the frequency of activated and proliferating CD8+T cells and reduced terminally exhausted CD4+and CD8+T cells, while reducing suppressive MDSCs (Figure 21). In comparison, seven days after a single i.t. injection of IL-12 in saline shows limited antitumor activity [93,94]. Without being bound by theory, it may be the persistent Th1 signaling due to the sustained presence of IL-12 that drove alterations in the TIME.

[0222] In the context of TNBC, local and distant recurrences cause the greatest number of BC-related deaths

[0095] . Rechallenge studies described herein demonstrated that XCSgel- IL12 induced antitumor immunity that rejected live tumor rechallenges in a tumor- specific manner (Figure 20) as well as anenestic tumors (Figure 23). To evaluate the effectiveness of IL-12 co-formulated with XCSgel, an abscopal tumor on the opposite side of the primary tumor was implanted. Following treatment of only the primary tumor, 4 / 6 mice in XCSgelslow-IL125μgeliminated both tumors, with long-term survival (Figure 23B-C). Interestingly, XCSgelslow-IL125μgeliminated both treated and untreated tumors in 4 / 6 mice whereas XCSgelfast-IL125μgonly eliminated both tumors in 1 / 5 mice (Figure 23D). This draws attention to the effect of release timing and / or delivery composition.

[0223] In conclusion, XCSgel is a promising localized delivery platform well-suited to enhance the retention and antitumor activity of potent immunotherapeutics. A single injection of XCSgel-IL12 can eliminate both primary and abscopal solid tumors, - 57 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 indicating that systemic immunotherapy may not be required for systemic control of cancer. A delivery system such as XCSgel, is likely to improve intratumoral therapeutic exposure and consequently, antitumor efficacy while mitigating toxicity.

[0224] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0225] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practicing the subject matter described herein. The present disclosure is in no way limited to just the methods and materials described.

[0226] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs.

[0227] Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. It is understood that embodiments described herein include “consisting of” and / or “consisting essentially of” embodiments.

[0228] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these small ranges which may independently be included in the smaller rangers is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0229] Many modifications and other embodiments set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. - 58 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 BIBLIOGRAPHY [1] S. K. Vashist, “Comparison of 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Based Strategies to Crosslink Antibodies on Amine-Functionalized Platforms for Immunodiagnostic Applications,” Diagnostics, vol.2, no.3, pp.23–33, 2012, doi: 10.3390 / diagnostics2030023. [2] F. Costa, S. Maia, J. Gomes, P. Gomes, and M. C. L. Martins, “Characterization of hLF1-11 immobilization onto chitosan ultrathin films, and its effects on antimicrobial activity,” Acta Biomater, vol.10, no.8, pp.3513–3521, 2014, doi: 10.1016 / j.actbio.2014.02.028. [3] M. Andersson and S. Oscarson, “Synthesis of O-glycopyranosyl-N- hydroxysuccinimides of glucose and lactose and their opening by nucleophiles into prespacer glycosides,” Glycoconj J, vol.9, no.3, pp.122–125, 1992, doi: 10.1007 / BF00780758. [4] J. W. Hommes, R. J. Verheijden, K. P. M. Suijkerbuijk, and D. Hamann, “Biomarkers of Checkpoint Inhibitor Induced Immune-Related Adverse Events— A Comprehensive Review,” Front Oncol, vol.10, no. February, pp.1–16, 2021, doi: 10.3389 / fonc.2020.585311. [5] A. Marabelle et al., “Starting the fight in the tumor: Expert recommendations for the development of human intratumoral immunotherapy (HIT-IT),” Annals of Oncology, vol.29, no.11, pp.2163–2174, 2018, doi: 10.1093 / annonc / mdy423. [6] R. H. I. Andtbacka et al., “Patterns of Clinical Response with Talimogene Laherparepvec (T-VEC) in Patients with Melanoma Treated in the OPTiM Phase III Clinical Trial,” Ann Surg Oncol, vol.23, no.13, pp.4169–4177, 2016, doi: 10.1245 / s10434-016-5286-0. [7] M. Ghosn et al., “Intratumoral Immunotherapy: Is It Ready for Prime Time?,” Curr Oncol Rep, no.0123456789, 2023, doi: 10.1007 / s11912-023-01422-4. [8] “Idera Pharmaceuticals Announces Results From ILLUMINATE-301 Trial of Tilsotolimod + Ipilimumab in anti-PD-1 Refractory Advanced Melanoma,” 2021. https: / / ir.iderapharma.com / news-releases / news-release-details / idera- pharmaceuticals-announces-results-illuminate-301-trial (accessed May 09, 2023). [9] A. Ribas et al., “1037O MASTERKEY-265: A phase III, randomized, placebo (Pbo)-controlled study of talimogene laherparepvec (T) plus pembrolizumab (P) for unresectable stage IIIB–IVM1c melanoma (MEL),” Annals of Oncology, vol. 32, pp. S868–S869, Sep.2021, doi: 10.1016 / j.annonc.2021.08.1422.

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Claims

Attorney Docket No.035051 / 618192 What is claimed is:

1. A hydrogel comprising a cross-linked chitosan, wherein the cross-linked chitosan is crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

2. The hydrogel of claim 1, further comprising a cargo.

3. The hydrogel of claim 2, wherein the cargo is an active agent or contrast agent.

4. The hydrogel of claim 3, wherein the active agent is a small molecule pharmaceutical drug, a biologic or an immunomodulator.

5. The hydrogel of claim 4, wherein the active agent is an immune-stimulating cytokine or a monoclonal antibody.

6. The hydrogel of claim 5, wherein the immune-stimulating cytokine is IL-12, IL- 15, IFNα2, or GM-CSF; and the monoclonal antibody is anti-PD-1, anti-PD-L1, anti- LAG3, anti-TIM3, anti-CD40, or anti-41BBL.

7. The hydrogel of claim 6, wherein the active agent is IL-12.

8. The hydrogel of any one of claims 2-7, wherein release of the cargo from the hydrogel is tunable.

9. The hydrogel of any one of claims 1-8, wherein the hydrogel is porous.

10. The hydrogel of any one of claims 1-9, wherein the hydrogel is biocompatible.

11. The hydrogel of any one of claims 1-10, wherein the hydrogel is injectable.

12. The hydrogel of any one of claims 1-11, wherein the cross-linked chitosan is from about 70 to about 99% deacetylated.

13. The hydrogel of claim 12, wherein the cross-linked chitosan is from about 90 to about 99% deacetylated.

14. The hydrogel of claim 13, wherein the cross-linked chitosan is about 95% deacetylated. - 66 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 15. The hydrogel of any one of claims 2-14, wherein about 50% to about 99% of the cargo is released within about 1 month.

16. The hydrogel of claim 15, wherein about 50% to about 99% of the cargo is released within about 1 week.

17. The hydrogel of claim 16, wherein about 50% to about 99% of the cargo is released within about 1 day.

18. The hydrogel of any one of claims 15-17, wherein about 60% to about 90% of the cargo is released.

19. The hydrogel of any one of claims 15-18, wherein about 70% to about 90% of the cargo is released.

20. The hydrogel of any one of claims 15-19, wherein the release is sustained via electrostatic charge.

21. The hydrogel of any one of claims 1-20, wherein the hydrogel further comprises an anionic polymer.

22. The hydrogel of claim 21, wherein the anionic polymer is a polymer that modifies the electrostatic charge of the hydrogel to tune the release of a cargo.

23. The hydrogel of claim 22, wherein the anionic polymer is selected from the group consisting of carboxymethyl chitosan, hyaluronic acid, sodium alginate and heparin, or combinations thereof.

24. The hydrogel of any one of claims 1-23, wherein the hydrogel is self-healing.

25. The hydrogel of any one of claims 1-24, wherein the hydrogel is imageable.

26. The hydrogel of any one of claims 1-25, wherein the cross-linked chitosan is cross-linked with from about 0.1:1 to about 2:11-ethyl-3-(3- dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer.

27. The hydrogel of claim 26, wherein the cross-linked chitosan is cross-linked with about 1:1 or 0.5:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer. - 67 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 28. The hydrogel of any one of claims 1-27, wherein the cross-linked chitosan is cross-linked with from about 0.1:1 to about 3:1 N-hydroxysuccinimide molar ratio to chitosan monomer.

29. The hydrogel of claim 28, wherein the cross-linked chitosan is cross-linked with about 1:1 or 2:1 N-hydroxysuccinimide molar ratio to chitosan monomer.

30. The hydrogel of any one of claims 1-29, wherein the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is from about 0.1:10 to about 2:

1.

31. The hydrogel of claim 30, wherein the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is about 0.5:

1.

32. The hydrogel of any one of claims 1-31, wherein the chitosan is in a neutralized solution.

33. The hydrogel of any one of claims 1-32, wherein the 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are in a single crosslinking solution further comprising a solvent.

34. The hydrogel of claim 33, wherein the solvent is selected from the group consisting of water, an aqueous solution, aqueous saline buffer, phosphate buffered saline, phosphate buffer, an organic solvent, DMSO, DMF and NMP or a combination thereof.

35. The hydrogel of claim 34, wherein the solvent is a mixture of DMSO and water.

36. A method of delivering a cargo to a subject, comprising administering a hydrogel of claim 2 to the subject.

37. The method of claim 36, wherein the delivering is a controlled release of the cargo.

38. The method of claim 37, wherein the cargo is an active agent or contrast agent.

39. The method of claim 38, wherein the active agent is a small molecule pharmaceutical drug, a biologic or an immunomodulator. - 68 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 40. The method of claim 39, wherein the active agent is an immune-stimulating cytokine or a monoclonal antibody.

41. The method of claim 40, wherein the immune-stimulating cytokine is IL-12, IL- 15, IFNα2, or GM-CSF; and the monoclonal antibody is anti-PD-1, anti-PD-L1, anti- LAG3, anti-TIM3, anti-CD40, or anti-41BBL.

42. The method of claim 41, wherein the active agent is IL-12.

43. The method of any one of claims 36-42, wherein the administering is not systemic.

44. The method of any one of claims 36-43, wherein the administering is by injection.

45. A method of preparing a hydrogel comprising a cross-linked chitosan, the method comprising contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, wherein the chitosan is cross-linked.

46. The method of claim 45, wherein the cross-linked chitosan is from about 70 to about 99% deacetylated.

47. The method of claim 46, wherein the cross-linked chitosan is from about 90 to about 99% deacetylated.

48. The method of claim 47, wherein the cross-linked chitosan is about 95% deacetylated.

49. The method of any one of claims 45-48, wherein the cross-linked chitosan is cross-linked with from about 0.1:1 to about 2:11-ethyl-3-(3- dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer.

50. The method of claim 49, wherein the cross-linked chitosan is cross-linked with about 1:11-ethyl-3-(3-dimethylaminopropyl)carbodiimide molar ratio to chitosan monomer.

51. The method of any one of claims 45-50, wherein the cross-linked chitosan is cross-linked with from about 0.1:1 to about 3:1 N-hydroxysuccinimide molar ratio to chitosan monomer. - 69 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 52. The method of claim 51, wherein the cross-linked chitosan is cross-linked with about 1:1 N-hydroxysuccinimide molar ratio to chitosan monomer.

53. The method of any one of claims 45-52, wherein the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is from about 0.1:10 to about 2:

1.

54. The method of claim 53, wherein the molar ratio of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is about 0.5:

1.

55. The method of any one of claims 45-54, wherein the chitosan is in a neutralized solution.

56. The method of any one of claims 45-55, wherein the 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are in a single crosslinking solution further comprising a solvent.

57. The method of claim 56, wherein the solvent is selected from the group consisting of water, an aqueous solution, aqueous saline buffer, phosphate buffered saline, phosphate buffer, an organic solvent, DMSO, DMF and NMP or a combination thereof.

58. The method of claim 57, wherein the solvent is a mixture of DMSO and water.

59. The method of claim 45, wherein the contacting comprises adding dropwise with stirring a neutralized chitosan solution to a crosslinking mixture comprising 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

60. The method of claim 45 or 59, wherein the pH of the neutralized chitosan solution is from about 5.5 to about 6.

2.

61. The method of any one of claims 45-60, further comprising after contacting, dialyzing the cross-linked chitosan.

62. The method of any one of claims 45-61, further comprising lyophilizing the cross- linked chitosan.

63. The method of claim 62, contacting the lyophilized cross-linked chitosan with water or an aqueous solution to prepare a hydrogel. - 70 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 64. The method of claim 57, wherein the aqueous solution comprises a cargo.

65. The method of claim 64, wherein the cargo is an immune-stimulating cytokine or a monoclonal antibody.

66. The method of claim 65, wherein the immune-stimulating cytokine is IL-12, IL- 15, IFNα2, or GM-CSF; and the monoclonal antibody is anti-PD-1, anti-PD-L1, anti- LAG3, anti-TIM3, anti-CD40, or anti-41BBL.

67. The method of claim 66, wherein the active agent is IL-12.

68. The method of any one of claims 45-67, further comprising contacting the hydrogel with an anionic polymer.

69. A lyophilized cross-linked chitosan, wherein the cross-linked chitosan comprises at least one residue of succinylated chitosan crosslinked to a residue of chitosan.

70. A lyophilized cross-linked chitosan, wherein the cross-linked chitosan is prepared by contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

71. A cross-linked chitosan, wherein the cross-linked chitosan is prepared by contacting chitosan with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

72. A hydrogel comprising a cross-linked chitosan, wherein the cross-linked chitosan comprises at least one residue of succinylated chitosan crosslinked to a residue of chitosan.

73. The hydrogel of claim 72, further comprising a cargo.

74. The hydrogel of claim 73, wherein the cargo is an active agent or contrast agent.

75. The hydrogel of claim 74, wherein the active agent is a small molecule drug, a biologic or immunomodulator.

76. The hydrogel of any one of claims 72-75, wherein release of the cargo from the hydrogel is tunable. - 71 - LEGAL02 / 44832132v1Attorney Docket No.035051 / 618192 77. A method of treating cancer, the method comprising administering to a subject the hydrogel of any one of claims 2-35 and 72-76.

78. The method of claim 77, wherein the hydrogel is administered locally.

79. The method of claim 77 or 78, wherein the method comprises injecting the hydrogel into a tumor.

80. The method of any one of claims 77-79, wherein the cancer is selected from the group consisting of breast cancer, head and neck cancers, lung cancer, kidney cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, penile cancer, gastric cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, bladder cancer, hematologic cancer, endometrial cancer, melanoma, glioblastoma, sarcoma, osteosarcoma, cholangiocarcinoma, neuroblastoma, and glioma.

81. The method of claim 80, wherein the cancer is breast cancer.

82. The method of claim 81, wherein the cancer is triple-negative breast cancer. - 72 - LEGAL02 / 44832132v1

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