**3. Results and Discussion**

According to our previously reported method [37], mPEG-PELG was prepared via the ROP of ELG-NCA using mPEG-NH<sup>2</sup> as the macroinitiator. The chemical structure and molecule weight of the obtained block copolymer were identified by <sup>1</sup>H NMR and GPC, respectively (Figure S1). The degree of polymerization (DP) of mPEG-PELG was estimated as approximately 18 by comparing the integration of the peak at 0.82 ppm ascribed to the pendant methyl group of ELG residues with that at 3.09 ppm assigned to the terminal methyl group of mPEG in the <sup>1</sup>H NMR spectrum. Additionally, the monomodal distribution of the resulting copolymer in the GPC trace confirmed the successful synthesis of the diblock copolymer. The *M*<sup>n</sup> was determined as 4100 Da with a PDI of 1.2 according to GPC data.

It was found that the mPEG-PELG aqueous solutions showed a sol–gel phase transition with increasing temperature, which was dependent on the polymer concentration. To investigate the gelation temperature range of the mPEG-PELG aqueous solutions, the thermo-induced sol–gel phase transitions of the polymer solutions were tested. As shown in Figure 1A, the mPEG-PELG solutions in PBS with polymer concentrations of 4~8 wt% exhibited sol–gel phase transitions with the increase in temperature, and the gelation temperature declined from 38 ◦C to 24 ◦C as the polymer concentration increased from 4 wt% to 8 wt% (Figures 1B and S2). The thermo-induced gelation of the mPEG-PELG solutions may be owing to the cooperative effects of partial dehydration of mPEG blocks and the maintained β-sheet conformation at elevated temperatures (Figure S3) [38,39]. The partial dehydration of mPEG segments may result in enhanced chain entanglement and aggregation of the mPEG-PELG micelles in aqueous solution, and the β-sheet conformation of the polypeptide blocks facilitates the formation of intermolecular hydrogen bonding. These effects promote the formation of a physical crosslinking network and hydrogel.

Considering the suitable gelation temperature (~30 ◦C), the 6 wt% mPEG-PELG solution in PBS was chosen for fabrication of the injectable hydrogel. Based on the rheological test, the 6 wt% mPEG-PELG solution showed an abrupt increase in the storage modulus (G0 ) with increasing temperature over 20 ◦C, indicating the hydrogel formation (Figure 1C). Moreover, the mixing of Dox and a model antibody, IgG, with the mPEG-PELG solution showed no obvious influence on the variations of G0 and G" with the temperature increase, indicating that the Dox and antibody encapsulation did not markedly affect the thermoinduced gelation behavior of the polypeptide solution. Additionally, the freeze-dried hydrogel sample indicated a porous structure in the SEM image (Figure 1D), which may facilitate the encapsulation and transportation of the drugs and bioactive agents [27,28]. Considering the suitable gelation temperature (~30 °C), the 6 wt% mPEG-PELG solution in PBS was chosen for fabrication of the injectable hydrogel. Based on the rheological test, the 6 wt% mPEG-PELG solution showed an abrupt increase in the storage modulus (G′) with increasing temperature over 20 °C, indicating the hydrogel formation (Figure 1C). Moreover, the mixing of Dox and a model antibody, IgG, with the mPEG-PELG solution showed no obvious influence on the variations of G′ and G″ with the temperature increase, indicating that the Dox and antibody encapsulation did not markedly affect the thermoinduced gelation behavior of the polypeptide solution. Additionally, the freeze-dried hydrogel sample indicated a porous structure in the SEM image (Figure 1D), which may facilitate the encapsulation and transportation of the drugs and bioactive agents [27,28].

dehydration of mPEG segments may result in enhanced chain entanglement and aggregation of the mPEG-PELG micelles in aqueous solution, and the β-sheet conformation of the polypeptide blocks facilitates the formation of intermolecular hydrogen bonding. These effects promote the formation of a physical crosslinking network and hydrogel.

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**Figure 1.** (**A**) Temperature-dependent sol–gel phase diagram of mPEG-PELG hydrogel. (**B**) Photographs of 6 wt% mPEG-PELG solution in PBS at 0 °C and the hydrogel formation at 37 °C. (**C**) Temperature-dependent variations in the storage modulus (G′) and loss modulus (G″) of the 6 wt% blank mPEG-PELG hydrogel or Dox/IgG-loaded hydrogel (Dox: 1 mg/mL; IgG: 1 mg/mL). (**D**) SEM image of lyophilized mPEG-PELG hydrogel (6 wt%). Scale bar = 20 μm. **Figure 1.** (**A**) Temperature-dependent sol–gel phase diagram of mPEG-PELG hydrogel. (**B**) Photographs of 6 wt% mPEG-PELG solution in PBS at 0 ◦C and the hydrogel formation at 37 ◦C. (**C**) Temperature-dependent variations in the storage modulus (G0 ) and loss modulus (G00) of the 6 wt% blank mPEG-PELG hydrogel or Dox/IgG-loaded hydrogel (Dox: 1 mg/mL; IgG: 1 mg/mL). (**D**) SEM image of lyophilized mPEG-PELG hydrogel (6 wt%). Scale bar = 20 µm.

The degradation of hydrogels plays an important role in affecting the release behaviors of encapsulated agents. The degradation behavior of the mPEG-PELG hydrogels was evaluated in vitro and in vivo, respectively. As shown in Figure 2A, the hydrogel showed ~30% mass loss in 15 days in PBS, which may be mainly due to the surface erosion of the hydrogel. When proteinase K was added, the hydrogel degradation was obviously accelerated with ~60% mass loss within 15 days. This is likely due to the enzymatic cleavage of the polypeptide segments in addition to the hydrogel erosion [40,41]. Moreover, after The degradation of hydrogels plays an important role in affecting the release behaviors of encapsulated agents. The degradation behavior of the mPEG-PELG hydrogels was evaluated in vitro and in vivo, respectively. As shown in Figure 2A, the hydrogel showed ~30% mass loss in 15 days in PBS, which may be mainly due to the surface erosion of the hydrogel. When proteinase K was added, the hydrogel degradation was obviously accelerated with ~60% mass loss within 15 days. This is likely due to the enzymatic cleavage of the polypeptide segments in addition to the hydrogel erosion [40,41]. Moreover, after subcutaneous injection into rats, the 6 wt% hydrogels degraded continuously in the subcutaneous layer of rats and completely disappeared within 5 weeks in vivo (Figure S4). It is known that there are different enzymes, such as cathepsin B, cathepsin C and elastase, in the subcutaneous layer of mammals [42]. Therefore, the degradation of the hydrogel in the subcutaneous layer of rats may be accelerated by enzymatic hydrolysis in vivo. These

results indicated that the hydrogels underwent continuous degradation in vitro and in vivo.

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**Figure 2.** (**A**) In vitro degradation behaviors of 6 wt% hydrogel in PBS or PBS containing proteinase K (5 U/mL) (*n* = 3). (**B**) Accumulative Dox release profiles from 6 wt% hydrogels in PBS or PBS containing proteinase K (5 U/mL) (*n* = 3). (**C**) Accumulative IgG release from 6 wt% hydrogel in PBS (*n* = 3). (**D**) Fluorescence intensity of CRT expressed on B16F10 cells after different treatments. **Figure 2.** (**A**) In vitro degradation behaviors of 6 wt% hydrogel in PBS or PBS containing proteinase K (5 U/mL) (*n* = 3). (**B**) Accumulative Dox release profiles from 6 wt% hydrogels in PBS or PBS containing proteinase K (5 U/mL) (*n* = 3). (**C**) Accumulative IgG release from 6 wt% hydrogel in PBS (*n* = 3). (**D**) Fluorescence intensity of CRT expressed on B16F10 cells after different treatments.

Calreticulin (CRT) is a typical marker on cells undergoing immunogenic cell death (ICD), which can promote antigen processing and presentation for adaptive immune response [47]. To reveal the ability of the drug-loaded hydrogel to induce ICD of tumor cells, the CRT expressions on the B16F10 melanoma cells after different treatments were tested (Figure 2D). It was found that incubation of tumor cells with either free Dox or Dox-loaded hydrogel caused enhanced CRT expression of B16F10 cells, indicating the occurrence of ICD in tumor cells. To investigate the combination effects of Dox-mediated ICD of tumor cells and ICB therapy, Dox, aCTLA-4 and aPD-1 were co-loaded into the mPEG-PELG hydrogel to construct a depot for sustained antitumor chemo-immunotherapy. Different drug-containing systems were then peritumorally injected into C57BL/6 mice bearing B16F10 melanoma for evaluating the antitumor efficacy in vivo. The tumor-bearing mice were assigned into seven groups at random including PBS group, aCTLA-4 + aPD-1 group, (aCTLA-4 + aPD-1)@gel group, Dox solution group, Dox@gel group, Dox + aCTLA-4 + aPD-1 mixed solution group, and (Dox + aCTLA-4 + aPD-1)@gel group. It was observed that all the formulations containing Dox at a dose of 4 mg/kg exhibited an inhibition effect on tumor growth (Figure 3B). Moreover, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel exhibited significantly improved tumor suppression efficiency compared to the formulations (hydrogels or solutions) containing either Dox or antibodies. Additionally, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel led to a significantly extended animal survival time compared to sin-Further, the release behaviors of chemotherapeutic drug and antibodies from the mPEG-PELG hydrogel were measured in vitro using Dox and IgG as the model drug and antibody, respectively. It was found that the Dox-loaded hydrogel showed a burst Dox release in the first 2 days, but exhibited more continuous and slower Dox release rates during the subsequent 13 days (Figure 2B). The two-stage release profile of Dox may be due to the rapid diffusion of Dox near the hydrogel surface at the first stage, and subsequent combined mechanisms of retarded Dox diffusion and slow hydrogel degradation [35]. In contrast, the Dox release was obviously accelerated with the addition of proteinase K, likely attributed to increased hydrogel disintegration in the presence of enzyme. Additionally, the release of IgG from the hydrogel was slower than Dox. This may be due to the slower diffusion rate of the antibody from the hydrogel than the small molecule (Figure 2C). The in vitro drug release data in the first 6 days were fitted to different kinetic models, including the zero-order model, first-order model, Korsmeyer–Peppas model and Higuchi model [43–45] (Figure S5). Through comparing the R-squared value, the Dox release profile from the hydrogel in PBS showed a relatively better degree of fitting to the Korsmeyer– Peppas model. It is noteworthy that the R-squared value was reduced for Dox release in the presence of proteinase K. Additionally, the IgG release from the hydrogel did not fit well with these kinetic models, which may be attributed to the fact that the IgG release behavior was influenced by complicated interactions between the protein and hydrogel network [46]. Therefore, the drug release tests in vitro suggested that the drug-loaded

gle or multiple antitumor agent solutions, or hydrogels loaded with Dox or antibodies (Figure 3D). The results suggested that the sustained co-delivery of Dox, aCTLA-4 and

aPD-1 using the hydrogel showed the best antitumor efficacy in vivo.

hydrogels exhibited continuous release profiles of both Dox and antibodies, which could be adjusted by the hydrogel degradation.

Calreticulin (CRT) is a typical marker on cells undergoing immunogenic cell death (ICD), which can promote antigen processing and presentation for adaptive immune response [47]. To reveal the ability of the drug-loaded hydrogel to induce ICD of tumor cells, the CRT expressions on the B16F10 melanoma cells after different treatments were tested (Figure 2D). It was found that incubation of tumor cells with either free Dox or Dox-loaded hydrogel caused enhanced CRT expression of B16F10 cells, indicating the occurrence of ICD in tumor cells.

To investigate the combination effects of Dox-mediated ICD of tumor cells and ICB therapy, Dox, aCTLA-4 and aPD-1 were co-loaded into the mPEG-PELG hydrogel to construct a depot for sustained antitumor chemo-immunotherapy. Different drug-containing systems were then peritumorally injected into C57BL/6 mice bearing B16F10 melanoma for evaluating the antitumor efficacy in vivo. The tumor-bearing mice were assigned into seven groups at random including PBS group, aCTLA-4 + aPD-1 group, (aCTLA-4 + aPD-1)@gel group, Dox solution group, Dox@gel group, Dox + aCTLA-4 + aPD-1 mixed solution group, and (Dox + aCTLA-4 + aPD-1)@gel group. It was observed that all the formulations containing Dox at a dose of 4 mg/kg exhibited an inhibition effect on tumor growth (Figure 3B). Moreover, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel exhibited significantly improved tumor suppression efficiency compared to the formulations (hydrogels or solutions) containing either Dox or antibodies. Additionally, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel led to a significantly extended animal survival time compared to single or multiple antitumor agent solutions, or hydrogels loaded with Dox or antibodies (Figure 3D). The results suggested that the sustained co-delivery of Dox, aCTLA-4 and aPD-1 using the hydrogel showed the best antitumor efficacy in vivo. *Pharmaceutics* **2023**, *15*, x FOR PEER REVIEW 10 of 17

**Figure 3.** (**A**) Schematic illustration of localized treatment of melanoma-bearing mice with peritumoral injection of drug-containing systems. (**B**) Tumor volume curves during therapy (*n* = 5). (**C**) Changes of the body weight of mice during therapy (*n* = 5). (**D**) Survival periods of various experiment groups (*n* = 7). (\* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001) **Figure 3.** (**A**) Schematic illustration of localized treatment of melanoma-bearing mice with peritumoral injection of drug-containing systems. (**B**) Tumor volume curves during therapy (*n* = 5). (**C**) Changes of the body weight of mice during therapy (*n* = 5). (**D**) Survival periods of various experiment groups (*n* = 7). (\* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001).

Additionally, it was found that all the local treatments resulted in no obvious effect on the body weight in the mice (Figure 3C), indicating a reduced systemic side effect. After treatment for 14 days, the main organs of the mice, such as the heart, liver, spleen, lung

logical changes were found in any of the groups. This confirmed that the treatments with localized injection of drug-containing formulations showed no obvious toxic side effects to normal organs. Compared to systemic administration, the local and sustained release of Dox and ICB antibodies at tumor sites can markedly reduce the blood drug concentration and drug distribution in normal tissues [29]. Thus, low systemic toxicity was ob-

served for the local treatments.

Additionally, it was found that all the local treatments resulted in no obvious effect on the body weight in the mice (Figure 3C), indicating a reduced systemic side effect. After treatment for 14 days, the main organs of the mice, such as the heart, liver, spleen, lung and kidney, were taken and stained with H&E staining. Observation and pathological analysis were carried out under a microscope. As shown in Figure 4, no obvious pathological changes were found in any of the groups. This confirmed that the treatments with localized injection of drug-containing formulations showed no obvious toxic side effects to normal organs. Compared to systemic administration, the local and sustained release of Dox and ICB antibodies at tumor sites can markedly reduce the blood drug concentration and drug distribution in normal tissues [29]. Thus, low systemic toxicity was observed for the local treatments. *Pharmaceutics* **2023**, *15*, x FOR PEER REVIEW 11 of 17

**Figure 4.** H&E staining images of the main organs of mice, including the heart, liver, spleen, lung and kidney, after 14 days of treatment with various therapeutic methods. Scale bar = 50 μm. **Figure 4.** H&E staining images of the main organs of mice, including the heart, liver, spleen, lung and kidney, after 14 days of treatment with various therapeutic methods. Scale bar = 50 µm.

1 co-loaded hydrogel treatment reduced the ratio of immunosuppressive regulatory T cells (Tregs) in CD4+ cells in tumors (Figure 5D). This indicated that the continuous, simultaneous release of Dox, aCTLA-4 and aPD-1 promoted the generation and tumor accumulation of tumor-killing CD8+ T cells, and inhibited the accumulation of Tregs at tumor

To analyze the immune response of the mice following treatments, typical immune cells and pro-inflammatory cytokines were evaluated. It was observed that the ratios of

sites.

To analyze the immune response of the mice following treatments, typical immune cells and pro-inflammatory cytokines were evaluated. It was observed that the ratios of CD8<sup>+</sup> T cells in the spleen, lymph nodes and tumors were enhanced after the therapy with Dox/aCTLA-4/aPD-1 co-loaded hydrogel in Figure 5A–C. In addition, Dox/aCTLA-4/aPD-1 co-loaded hydrogel treatment reduced the ratio of immunosuppressive regulatory T cells (Tregs) in CD4<sup>+</sup> cells in tumors (Figure 5D). This indicated that the continuous, simultaneous release of Dox, aCTLA-4 and aPD-1 promoted the generation and tumor accumulation of tumor-killing CD8<sup>+</sup> T cells, and inhibited the accumulation of Tregs at tumor sites. tions on T cells, NK cells and NK-T cells [50]. IL-2 plays a role in promoting the proliferation of lymphocytes including T cells and NK cells. TNF-α is able to exert antitumor effects by direct showing cytotoxicity on tumor cells and inducing an antitumor immune response. IFN-γ is a crucial cytokine for cell-mediated immunity, which plays key roles in stimulating antigen presentation, cytokine secretion, as well as the activation of macrophages, NK cells and neutrophils. Overall, the localized co-delivery of Dox, aCTLA-4 and aPD-1 from the hydrogel depot resulted in a significantly enhanced antitumor immune response, which may contribute to the increased tumor inhibition efficacy in vivo.

Moreover, the pro-inflammatory cytokines, including IL-2, TNF-α and IFN-γ, in the blood were examined. It was observed that the concentrations of pro-inflammatory cytokines were significantly enhanced after the treatment with the hydrogel loading Dox, aCTLA-4 and aPD-1 (Figure 6). IL-2 is a cytokine that undertakes immunoregulatory func-

It has been established that CTLA-4 acts on immunosuppressive functions in several aspects [7,48]. First, it inhibits antigen presentation from DCs to T cells through competitively binding CD80/CD86. Second, CTLA-4 is responsive for the functions of Tregs. Thus, the blockade of CTLA-4 by the sustained release of aCTLA-4 near the tumor site promoted the presentation of TAAs, which was generated by Dox-mediated ICD of tumor cells [22], resulting in enhanced T cell activation (Scheme 1). Moreover, the ratio of Tregs at the tumor site was also reduced. In addition, PD-1, another immune checkpoint receptor usually expressed on activated T cells, also acts as a "brake" during the tumor recognition and killing, through binding to its ligand (PD-L1) expressed on tumor cells, resulting in the immune escape of tumor cells [49]. Therefore, the inhibition of the PD-1/PD-L1 pathway by a localized release of aPD-1 near the tumor site led to enhanced antigen recogni-

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tion and tumor killing by CD8+ cytotoxic T lymphocytes (CTLs).

**Figure 5.** Immune cell analysis results of spleens, lymph nodes and tumors of the melanoma-bearing mice model after receiving various treatments, obtained by flow cytometry. (**A**) Ratio of CD8+:CD3+ **Figure 5.** Immune cell analysis results of spleens, lymph nodes and tumors of the melanoma-bearing mice model after receiving various treatments, obtained by flow cytometry. (**A**) Ratio of CD8<sup>+</sup> :CD3<sup>+</sup> in spleen (*n* = 5). (**B**) Ratio of CD8<sup>+</sup> :CD3<sup>+</sup> in lymph (*n* = 5). (**C**) Ratio of CD8<sup>+</sup> :CD3<sup>+</sup> in the tumor (*n* = 5). (**D**) Ratio of Treg:CD4<sup>+</sup> in the tumor (*n* = 3). (\* *p* < 0.05 and \*\*\* *p* < 0.001).

It has been established that CTLA-4 acts on immunosuppressive functions in several aspects [7,48]. First, it inhibits antigen presentation from DCs to T cells through competitively binding CD80/CD86. Second, CTLA-4 is responsive for the functions of Tregs. Thus, the blockade of CTLA-4 by the sustained release of aCTLA-4 near the tumor site promoted the presentation of TAAs, which was generated by Dox-mediated ICD of tumor cells [22], resulting in enhanced T cell activation (Scheme 1). Moreover, the ratio of Tregs at the tumor site was also reduced. In addition, PD-1, another immune checkpoint receptor usually expressed on activated T cells, also acts as a "brake" during the tumor recognition and killing, through binding to its ligand (PD-L1) expressed on tumor cells, resulting in the immune escape of tumor cells [49]. Therefore, the inhibition of the PD-1/PD-L1 pathway by a localized release of aPD-1 near the tumor site led to enhanced antigen recognition and tumor killing by CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs).

Moreover, the pro-inflammatory cytokines, including IL-2, TNF-α and IFN-γ, in the blood were examined. It was observed that the concentrations of pro-inflammatory cytokines were significantly enhanced after the treatment with the hydrogel loading Dox, aCTLA-4 and aPD-1 (Figure 6). IL-2 is a cytokine that undertakes immunoregulatory functions on T cells, NK cells and NK-T cells [50]. IL-2 plays a role in promoting the proliferation of lymphocytes including T cells and NK cells. TNF-α is able to exert antitumor effects by direct showing cytotoxicity on tumor cells and inducing an antitumor immune response. IFN-γ is a crucial cytokine for cell-mediated immunity, which plays key roles in stimulating antigen presentation, cytokine secretion, as well as the activation of macrophages, NK cells and neutrophils. Overall, the localized co-delivery of Dox, aCTLA-4 and aPD-1 from the hydrogel depot resulted in a significantly enhanced antitumor immune response, which may contribute to the increased tumor inhibition efficacy in vivo. *Pharmaceutics* **2023**, *15*, x FOR PEER REVIEW 13 of 17 in spleen (*n* = 5). (**B**) Ratio of CD8+:CD3+ in lymph (*n* = 5). (**C**) Ratio of CD8+:CD3+ in the tumor (*n* = 5). (**D**) Ratio of Treg:CD4+ in the tumor (*n* = 3). (\* *p* < 0.05 and \*\*\* *p* < 0.001)

**Figure 6.** Pro-inflammatory cytokine concentration in the serum of various groups after treatment for 14 days. (**A**) The concentration of IL-2 (*n* = 5). (**B**) The concentration of TNF-α (*n* = 5). (**C**) The concentration of IFN-γ (*n* = 5). (\* *p* < 0.05 and \*\*\* *p* < 0.001) **Figure 6.** Pro-inflammatory cytokine concentration in the serum of various groups after treatment for 14 days. (**A**) The concentration of IL-2 (*n* = 5). (**B**) The concentration of TNF-α (*n* = 5). (**C**) The concentration of IFN-γ (*n* = 5). (\* *p* < 0.05 and \*\*\* *p* < 0.001).

To further evaluate the ability of the Dox/aCTLA-4/aPD-1 co-loaded hydrogel to inhibit post-operative tumor reoccurrence, a tumor resection model was established using melanoma-bearing C57BL/6 mice. As shown in Figure 7, after ~90% of the tumor was resected, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel was injected into the surgical site, with PBS or the mixed solution of the multiple agents as the controls. It was observed that the remaining tumors regrew rapidly after the tumor resection surgery with additional PBS treatment (Figure 7B). The local injection of Dox/aCTLA-4/aPD-1 mixed solution at the surgery site resulted in the effective inhibition of tumor reoccurrence. Notably, the local treatment of the Dox/aCTLA-4/aPD-1 co-loaded hydrogel showed a significantly To further evaluate the ability of the Dox/aCTLA-4/aPD-1 co-loaded hydrogel to inhibit post-operative tumor reoccurrence, a tumor resection model was established using melanoma-bearing C57BL/6 mice. As shown in Figure 7, after ~90% of the tumor was resected, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel was injected into the surgical site, with PBS or the mixed solution of the multiple agents as the controls. It was observed that the remaining tumors regrew rapidly after the tumor resection surgery with additional PBS treatment (Figure 7B). The local injection of Dox/aCTLA-4/aPD-1 mixed solution at the surgery site resulted in the effective inhibition of tumor reoccurrence. Notably, the local treatment of the Dox/aCTLA-4/aPD-1 co-loaded hydrogel showed a significantly stronger

stronger inhibition effect on the tumor reoccurrence, compared to either PBS or Dox/aCTLA-4/aPD-1 mixed solution. Moreover, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel therapy led to a significantly extended survival time compared to other treatments

4/aPD-1 co-loaded hydrogel may be attributed to the sustained and prolonged release manners of the chemotherapy drug and ICB antibodies from the hydrogel depot [29,30]. In addition, the immune cells in the spleens, lymph nodes and tumors of the treated mice were analyzed at day 12 post-surgery. It was observed that the Dox/aCTLA-4/aPD-1 co-loaded hydrogel treatment enhanced the ratio of CD8+ T cells in the spleen and lymph

inhibition effect on the tumor reoccurrence, compared to either PBS or Dox/aCTLA-4/aPD-1 mixed solution. Moreover, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel therapy led to a significantly extended survival time compared to other treatments (Figure 7D). The higher efficiency in tumor reoccurrence inhibition of the Dox/aCTLA-4/aPD-1 co-loaded hydrogel may be attributed to the sustained and prolonged release manners of the chemotherapy drug and ICB antibodies from the hydrogel depot [29,30]. ment of B16F10 melanoma-bearing mice and post-operative mice models, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel showed a significantly increased antitumor immune response, enhanced tumor inhibition efficacy and prolonged animal survival time. Additionally, it was demonstrated that the local co-delivery of the ICB antibodies and chemotherapeutics caused no obvious systemic side effects, compared to the risk of severe irAEs by systemic administration of ICB antibodies [5].

node, and caused a decrease in the ratio of Treg cells in the tumor (Figure S6). Thus, the results suggested that the Dox/aCTLA-4/aPD-1 co-loaded hydrogel can effectively inhibit post-operative tumor reoccurrence and extend animal survival time, through strengthen-

In recent studies, hydrogel-based local delivery systems have been investigated for the co-delivery of chemotherapeutics and single ICB antibody, aPD-1 or anti-PD-L1, as a strategy for local chemo-immunotherapy [31–34,36]. It has been found that the chemotherapy-mediated ICD of tumor cells could promote the antitumor immune response when combining with ICB blockade. Moreover, some agents for modulating the immunosuppressive tumor micro-environment, e.g., indoleamine-(2,3)-dioxygenase (IDO) inhibitors, were further incorporated into the hydrogel depots for improving the antitumor efficacy [35,51]. In this study, a new formulation of a hydrogel depot co-loaded with dual ICB antibodies, aCTLA-4 and aPD-1, and a chemotherapeutics was developed for topical antitumor chemo-immunotherapy. The sustained release of aCTLA-4 at the tumor site can strengthen the antigen presentation of DCs to T cells, and the simultaneous delivery of aPD-1 is able to enhance the subsequent tumor recognition and killing. Based on the treat-

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ing the antitumor immune response.

**Figure 7.** (**A**) Schematic illustration of post-surgical treatment by the injection of drug-containing systems to the surgical site after ~90% tumor resection. (**B**) Tumor volume curves during treatment (*n* = 12). (**C**) Changes of the body weight of mice during treatment (*n* = 12). (**D**) Survival periods of various groups after treatment (*n* = 7). (\* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001).

In addition, the immune cells in the spleens, lymph nodes and tumors of the treated mice were analyzed at day 12 post-surgery. It was observed that the Dox/aCTLA-4/aPD-1 co-loaded hydrogel treatment enhanced the ratio of CD8<sup>+</sup> T cells in the spleen and lymph node, and caused a decrease in the ratio of Treg cells in the tumor (Figure S6). Thus, the results suggested that the Dox/aCTLA-4/aPD-1 co-loaded hydrogel can effectively inhibit post-operative tumor reoccurrence and extend animal survival time, through strengthening the antitumor immune response.

In recent studies, hydrogel-based local delivery systems have been investigated for the co-delivery of chemotherapeutics and single ICB antibody, aPD-1 or anti-PD-L1, as a strategy for local chemo-immunotherapy [31–34,36]. It has been found that the chemotherapymediated ICD of tumor cells could promote the antitumor immune response when combin-

ing with ICB blockade. Moreover, some agents for modulating the immunosuppressive tumor micro-environment, e.g., indoleamine-(2,3)-dioxygenase (IDO) inhibitors, were further incorporated into the hydrogel depots for improving the antitumor efficacy [35,51]. In this study, a new formulation of a hydrogel depot co-loaded with dual ICB antibodies, aCTLA-4 and aPD-1, and a chemotherapeutics was developed for topical antitumor chemo-immunotherapy. The sustained release of aCTLA-4 at the tumor site can strengthen the antigen presentation of DCs to T cells, and the simultaneous delivery of aPD-1 is able to enhance the subsequent tumor recognition and killing. Based on the treatment of B16F10 melanoma-bearing mice and post-operative mice models, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel showed a significantly increased antitumor immune response, enhanced tumor inhibition efficacy and prolonged animal survival time. Additionally, it was demonstrated that the local co-delivery of the ICB antibodies and chemotherapeutics caused no obvious systemic side effects, compared to the risk of severe irAEs by systemic administration of ICB antibodies [5].
