**4. Conclusions**

In the present study, an antitumor chemo-immunotherapy system was prepared by the incorporation of Dox, aCTLA-4 and aPD-1 into thermosensitive mPEG-PELG hydrogel. The polypeptide hydrogel exhibited enzyme-accelerated degradation behavior in vitro for over 15 days, and gradually degraded in the subcutaneous layer of mice. The sustained release of Dox or IgG model antibody from the hydrogel persisted for over 12 days in vitro, which can be further accelerated by the addition of proteinase K. A combination antitumor strategy was developed by simultaneous, sustained delivery of Dox and the dual ICB antibodies through peritumoral injection of the multiple-agent co-loaded hydrogel into mice bearing B16F10 melanoma. The treatment with Dox/aCTLA-4/aPD-1 co-loaded hydrogel demonstrated markedly enhanced tumor inhibition efficacy, significantly extended animal survival time and strengthened antitumor immune response, compared to the mixed drug solutions or hydrogel containing Dox or antibodies. Moreover, the localized treatments with the hydrogel-based formulations showed no obvious systemic side effects. Additionally, the treatment with Dox/aCTLA-4/aPD-1 co-loaded hydrogel following tumor resection surgery achieved significantly increased efficiency in inhibiting tumor reoccurrence and extending animal survival time. In summary, the hydrogels encapsulating chemotherapeutics and dual ICB antibodies can serve as a promising candidate as depots for antitumor combination therapy and the prevention of post-operative tumor reoccurrence.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15020428/s1. Scheme S1. Synthesis route for mPEG-PELG. Figure S1. (A) <sup>1</sup>H NMR spectrum of mPEG-PELG in CF3COOD. (B) GPC data of mPEG-PELG. Figure S2. Photographs of the mPEG-PELG (4–10 wt%) at different temperatures. The gelation temperatures and syneresis temperatures were marked specially. Figure S3. The ellipticity of polymer aqueous solution (0.05 wt%) with increasing temperature from 10 to 60 ◦C. Figure S4. Photographs of the hydrogels after injection into the subcutaneous layer of rats at different time points. Figure S5. Fitting of the in vitro drug release data to kinetic models of zero-order model (A), first-order model (B), Korsmeyer-Peppas model (C) and Higuchi model (D). (a) Dox release from Dox-loaded hydrogel in PBS; (b) Dox release from Dox-loaded hydrogel in PBS containing proteinase K; (c) IgG release from IgG-loaded hydrogel in PBS [43–45]. Figure S6. Immune cell analysis data of spleens, lymph nodes and tumors of the post-operative mice model after receiving various treatments, obtained by flow cytometry. (A) The ratio of CD8<sup>+</sup> :CD3<sup>+</sup> in spleen (*n* = 5). (B) The ratio of CD8<sup>+</sup> :CD3<sup>+</sup> in lymph nodes (*n* = 5). (C) The ratio of Tregs:CD4<sup>+</sup> in tumor (*n* = 5).

**Author Contributions:** Conceptualization, Z.C., X.C. (Xuesi Chen) and C.H.; Methodology, Z.C., Y.R., J.D., X.C. (Xuesi Chen), X.C. (Xueliang Cheng) and C.H.; Validation, C.H.; Investigation, Z.C. and Y.R.; Data curation, Z.C. and Y.R.; Writing—original draft preparation, Z.C. and Y.R.; Writing—review and editing, Y.R., J.D., X.C. (Xuesi Chen), X.C. (Xueliang Cheng) and C.H.; Supervision, X.C. (Xuesi Chen) and C.H.; Project administration, X.C. (Xuesi Chen) and C.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** We thank the National Natural Science Foundation of China (No. 51973218, 51833010, 52173147, 52203202) and the Scientific and Technological Development Projects of Jilin Province (20210204136YY) for financial support.

**Institutional Review Board Statement:** All animal procedures were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Changchun Institute of Applied Chemistry, CAS (No. 2020-010).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data supporting the study is available from the authors on request.

**Conflicts of Interest:** The authors declare no conflict of interest.
