Crucial Impact of Residue Chirality on the Gelation Process and Biodegradability of Thermoresponsive Polypeptide Hydrogels.

Thermosensitive polypeptide hydrogels have gained considerable attention in potential biomedical applications, of which the polymer structure may be tuned by residue chirality. In this study, polypeptide-based block copolymers with different chiralities were synthesized by ring-opening polymerization of γ-ethyl-l-glutamate N-carboxyanhydride and/or γ-ethyl-d-glutamate N-carboxyanhydride using amino-terminated monomethoxy poly(ethylene glycol) as a macroinitiator. All mPEG-polypeptide copolymers underwent sol-gel transition with an increase in temperature. The block copolymers with mixed enantiomeric residues of γ-ethyl-l-glutamate (ELG) and γ-ethyl-d-glutamate (EDG) in the polypeptide blocks exhibited lower critical gelation concentrations and lower critical gelation temperatures compared with those composed of pure ELG or EDG residues. We established that the difference in gelation properties between the copolymers was derived from the distinction of the secondary structures. We further demonstrated the influence of polypeptide chirality on the degradability and biocompatibility of hydrogels in vivo. Our findings provide insights into the design of hydrogels having tailored secondary conformation, gelation property, and biodegradability.

Rapidly Thermoreversible and Biodegradable Polypeptide Hydrogels with Sol-Gel-Sol Transition Dependent on Subtle Manipulation of Side Groups.

Thermoreversible hydrogels are attractive materials for biomedical applications, but their applications are still limited by nonbiodegradability and/or slow temperature-dependent gel-to-sol transition rates. In this research, we prepared a range of amphiphilic diblock, triblock, and four-armed star block copolymers composed of poly(ethylene glycol) (PEG) and poly(γ-(2-(2-ethoxyethoxy)ethyl)-l-glutamate) (P(EEO2LG)) segments, which can form rapidly thermoreversible hydrogels at physiological temperature. Intriguingly, the obtained hydrogels can transform from gel to sol within 10-70 s in response to the temperature decrease from 37 to 0 °C. The thermosensitive sol-gel-sol transitions are markedly faster than previously reported thermoreversible PEG-poly(l-glutamate) derivative hydrogels with subtle differences in the side groups and a widely studied poly(d,l-lactide-co-glycolide)-b-PEG-b-poly(d,l-lactide-co-glycolide) (PLGA-PEG-PLGA) hydrogel that required a much longer time of 40∼150 min. Further investigation of the relationship between the hydrogel property and polymer structure is performed, and the self-assembly mechanisms of different copolymers are proposed. Cytotoxicity assays and subcutaneous degradation experiments reveal that the PEG/P(EEO2LG) block copolymers are biocompatible and biodegradable. The polypeptide hydrogel can therefore be used as a three-dimensional platform for facile cell culture and collection by regulating the temperature.

pH- and Amylase-Responsive Carboxymethyl Starch/Poly(2-isobutyl-acrylic acid) Hybrid Microgels as Effective Enteric Carriers for Oral Insulin Delivery.

Oral delivery of insulin has the potential to revolutionize diabetes care since it is regarded as a noninvasive therapeutic approach without the side effects caused by frequent subcutaneous injection. However, the insulin delivery efficiency through oral route was still limited, likely due to the chemical, enzymatic and absorption barriers. In this study, a novel type of pH- and amylase-responsive microgels as an insulin drug carrier for oral administration was developed to improve the drug delivery efficiency. The microgels were prepared via aqueous dispersion copolymerization of acrylate- grafted-carboxymethyl starch (CMS- g-AA) and 2-isobutyl-acrylic acid ( iBAA). The resulting hybrid microgels with the P iBAA contents of 13.6-45.3 wt% exhibited sharp pH-sensitivity, which was revealed by the changes in particle size of the microgels and the transmittance of the microgel aqueous solution. The accelerated decomposition of the CMS-containing microgels in response to amylase was demonstrated by chromogenic reaction and morphology change. Insulin was loaded into the microgels by swelling-diffusion method, and the insulin release from the insulin-loaded microgels in vitro was found to be triggered by pH change and addition of amylase, which was highly dependent on the microgel component. Cytotoxicity assay was performed to show the good biocompatibility of the microgels. In addition, the tests of cellular uptake by Caco-2 cells and transmembrane transport through the Caco-2 cell monolayers were carried out to confirm the intestinal absorption ability of the insulin-loaded microgels. Finally, the oral administration of insulin-loaded microgels to STZ-induced diabetic rats led to a continuous decline in the fasting blood glucose level within 2 to 4 h, and the hypoglycemic effect maintained over 6 h in vivo. The relative pharmacological availability of the insulin-loaded microgels was enhanced 23-38 times compared to free-form insulin solution through oral route. Therefore, the novel starch-based microgels may have potential as an efficient platform for oral insulin delivery.

Injectable Enzymatically Cross-linked Hydrogels with Light-Controlled Degradation Profile.

An advanced hydrogel that features facile formation and injectability as well as light-controlled degradation profile is reported here. By modifying 4-arm poly(ethylene glycol) (4-arm PEG) with 2-nitrobenzyl (NB) and phenol, the 4-arm PEG precursor solutions could form enzymatically cross-linked hydrogels in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2 O2 ). The gelation time, mechanical strength, and porous structure could be simply tuned by the concentration of HRP and H2 O2 . Moreover, the hydrogels underwent controlled degradation under UV light irradiation via photo-cleavage reaction of the NB ester bond. The hydrogels exhibited negligible cytotoxicity toward mouse fibroblast L929 cells in vitro and can be manipulated through injection in vivo.

Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable Bioactivity and Controllable Cell Adhesion.

Injectable hydrogels have been widely investigated for applications in biomedical fields, for instance, as biomimetic scaffolds mimicking the extracellular matrix (ECM). In addition to as scaffolds for mechanical support and transferring of nutrients, the dynamic bioactivity of ECM is another critical factor that affects cell behavior. In this work, a novel injectable poly(l-glutamic acid)-based hydrogel decorated with RGD was fabricated. The presentation of RGD significantly enhanced the cell-matrix interaction and promoted cell adhesion and proliferation. Moreover, the cell-adhesive RGD was conjugated to the network via a disulfide bond, so that the density of RGD and the bioactivity of hydrogel can be well controlled by tuning the RGD content through treating with glutathione. As a result, the cell behaviors on the hydrogel can be tuned on demand. The injectable hydrogel with controllable bioactivity may provide an interesting strategy to develop a scaffold mimicking ECM that can regulate cell adhesion dynamically.

Injectable Thermosensitive Polypeptide-Based CDDP-Complexed Hydrogel for Improving Localized Antitumor Efficacy.

In this study, a type of novel thermosensitive polypeptide-based hydrogel with tunable gelation behavior through changing the content of carboxyl groups was developed for the purpose of improving the cisplatin (CDDP) release behavior and enhancing the localized antitumor efficiency. The introduction of carboxyl groups in methoxy-poly(ethylene glycol)-b-(poly(γ-ethyl-l-glutamate-co-l-glutamic acid) (mPEG-b-P(ELG-co-LG)) not only led to adjustable mechanical properties of the hydrogel but also significantly reduced the burst release of the drug through the complexation between the carboxyl groups of polypeptide and CDDP. Furthermore, both the good biocompatibility and the biodegradable properties of mPEG-b-P(ELG-co-LG) hydrogel were observed in vivo. Interestingly, the CDDP-complexed mPEG-b-P(ELG-co-LG) hydrogel exhibited significantly enhanced antitumor efficacy in vivo compared to the mPEG-b-PELG hydrogel loaded with CDDP without complexation, although a lower cytotoxicity and IC50 of the CDDP-complexed hydrogel was observed in vitro. Overall, the new type of injectable CDDP-complexed hydrogel may serve as an efficient platform for sustained CDDP delivery in localized tumor therapy.

Injectable Polypeptide Hydrogels with Tunable Microenvironment for 3D Spreading and Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells.

Bone-marrow-derived mesenchymal stem cells (BMSCs) possess vast potential for tissue engineering and regenerative medicine. In this study, an injectable hydrogel comprising poly(l-glutamic acid)-graft-tyramine (PLG-g-TA) with tunable microenvironment was developed via enzyme-catalyzed cross-linking and used as an artificial extracellular matrix (ECM) to explore the behaviors of BMSCs during three-dimensional (3D) culture. It was found that the mechanical property, porous structure as well as degradation process of the hydrogels could be tuned by changing the copolymer concentration. The PLG-g-TA hydrogels showed good cytocompatibility in vitro. After being subcutaneously injected into the back of rats, the hydrogels degraded gradually within 8 weeks and exhibited good biocompatibility in vivo. BMSCs were then encapsulated in the polypeptide-based hydrogels with different copolymer concentration to investigate the influence of 3D matrix microenvironment on stem cell behaviors. It is intriguing to note that the BMSCs within the 2% hydrogel showed a well-spread morphology after 24 h and a higher proliferation rate during 7 days of culture, in contrast to a rounded morphology and lower proliferation rate of BMSCs in the 4% hydrogel. Furthermore, the hydrogels with different microenvironment also regulated the matrix biosynthesis and the gene expression of BMSCs. After incubation in the 2% hydrogel for 4 weeks, the BMSCs produced more type II collagen and expressed higher amounts of chondrogenic markers, compared to the cells in the 4% hydrogel. Therefore, the PLG-g-TA hydrogels with tunable microenvironment may serve as an efficient 3D platform for guiding the lineage specification of BMSCs.

N-Isopropylacrylamide Modified Polyethylenimines as Effective siRNA Carriers for Cancer Therapy.

N-isopropylacrylamide modified PEI (PEN) was synthesized via Michael addition and was developed as an efficient siRNA delivery system both in vitro and in vivo. PEN showed significant enhanced cytocompatibility compared with commercial PEI-25k. The complexation of PEN with siRNA was studied by gel retardation, particle size and zeta potential measurement. The in vitro transfection ability of PEN was measured by qRT-PCR assay, and achieved obviously enhanced gene silencing efficiency compared with PEI-25k. The confocal imaging and flow cytometric analysis further validated its excellent intracellular trafficking ability. For antitumor treatment experiment, PEN mediated siVEGF showed obviously therapeutic effects for the treatment of CT26 tumor. Therefore, the present study demonstrated a useful strategy for constructing efficient siRNA delivery vehicles for antitumor therapy.

Metallo-supramolecular nanogels for intracellular pH-responsive drug release.

A simple process is developed to fabricate metallo-supramolecular nanogels (MSNs) by the metallo-supramolecular-coordinated interaction between histidine and iron-meso-tetraphenylporphin. MSNs are composed of histidine-modified dextran (DH) and iron-meso-tetraphenylporphin (Fe-Por) and exhibit excellent biocompatibility and stability. MSNs show pH responsiveness in the intracellular mildly acidic environment, which has great potential for acid-triggered drug release delivery. In vitro drug release profiles demonstrate that the pH-dependent disassembly of MSNs to histidine and Por results in a quicker release rate of loaded-DOX at pH 5.3, while at pH 7.4 MSNs could hinder the release of loaded-DOX due to the enhanced stability of MSNs.

Designing Hydrogels for Immunomodulation in Cancer Therapy and Regenerative Medicine.

The immune system not only acts as a defense against pathogen and cancer cells, but also plays an important role in homeostasis and tissue regeneration. Targeting immune systems is a promising strategy for efficient cancer treatment and regenerative medicine. Current systemic immunomodulation therapies are usually associated with low persistence time, poor targeting to action sites, and severe side effects. Due to their extracellular matrix-mimetic nature, tunable properties and diverse bioactivities, hydrogels are intriguing platforms to locally deliver immunomodulatory agents and cells, as well as provide an immunomodulatory microenvironment to recruit, activate, and expand host immune cells. In this review, the design considerations, including polymer backbones, crosslinking mechanisms, physicochemical nature, and immunomodulation-related components, of the hydrogel platforms, are focused on. The immunomodulatory effects and therapeutic outcomes in cancer therapy and tissue regeneration of different hydrogel systems are emphasized, including hydrogel depots for delivery of immunomodulatory agents, hydrogel scaffolds for cell delivery, and immunomodulatory hydrogels depending on the intrinsic properties of materials. Finally, the remained challenges in current systems and future development of immunomodulatory hydrogels are discussed.

Stimuli-Responsive Hydrogel Adhesives for Wound Closure and Tissue Regeneration.

Sutures and staplers, as gold standards for clinical wound closure, usually cause secondary tissue injury and require professional technicians and equipment. The noninvasive hydrogel adhesives are used in various biomedical applications, such as wound closure, tissue sealing, and tissue regeneration, due to their remarkable properties. Recently-developed hydrogel adhesives, especially stimuli-responsive hydrogels, have shown great potential owing to their advantages in regulating their performance and functions according to the wound situations or external conditions, thus allowing the wounds to heal gradually. However, comprehensive summary on stimuli-responsive hydrogels as tissue adhesives is rarely reported to date. This review focuses on the advances in the design of various stimuli-responsive hydrogel adhesives over the past decade, including the systems responsive to pH, temperature, photo, and enzymes. Their potential biomedical applications, such as skin closure, cardiovascular and liver hemostasis, and gastrointestinal sealing, are emphasized. Meanwhile, the challenges and future development of stimuli-responsive hydrogel adhesives are discussed. This review aims to provide meaningful insights for the further design of next-generation of hydrogel adhesives for wound closure and tissue regeneration.

ROS-responsive thermosensitive polypeptide hydrogels for localized drug delivery and improved tumor chemoimmunotherapy.

In this study, we developed a ROS-responsive thermosensitive poly(ethylene glycol)-polypeptide hydrogel loaded with a chemotherapeutic drug, doxorubicin (Dox), an antiviral imidazoquinoline, resiquimod (R848), and antibody targeting programmed cell death protein 1 (aPD-1) for local chemoimmunotherapy. The hydrogel demonstrated controllable degradation and sustained drug release behavior according to the concentration of ROS in vitro. Following intratumoral injection into mice bearing B16F10 melanoma, the Dox/R848/aPD-1 co-loaded hydrogel effectively inhibited tumor growth, prolonged animal survival time and promoted anti-tumor immune responses with low systemic toxicity. In the postoperative model, the Dox/R848/aPD-1 co-loaded hydrogel exhibited enhanced tumor recurrence prevention and long-term immune memory effects. Thus, the hydrogel-based local chemoimmunotherapy system demonstrates potential for effective anti-tumor treatment and suppression of tumor recurrence.

Injectable, Adhesive Albumin Nanoparticle-Incorporated Hydrogel for Sustained Localized Drug Delivery and Efficient Tumor Treatment.

Injectable hydrogels are receiving increasing attention as local depots for sustained anticancer drug delivery. However, most current hydrogel-based carriers lack tissue-adhesive ability, a property that is important for the immobilization of drug-loaded systems at tumor sites to increase local drug concentration. In this study, we developed a paclitaxel (PTX)-loaded injectable hydrogel with firm tissue adhesion for localized tumor therapy. PTX-loaded bovine serum albumin (BSA) nanoparticles (PTX@BN) were prepared, and the drug-loaded hydrogel was then fabricated by cross-linking PTX@BN with o-phthalaldehyde (OPA)-terminated 4-armed poly(ethylene glycol) (4aPEG-OPA) via a condensation reaction between OPA and the amines in BSA. The hydrogel showed firm adhesion to various organs and tumor tissues ex vivo due to the condensation reaction of unreacted OPA groups and amines in the tissues. The PTX-loaded nanocomposite hydrogels sustained PTX release over 30 days following the Korsmeyer-Peppas model and exhibited notable inhibition activities against mouse C26 colon and 4T1 breast cancer cells in vitro. Following peritumoral injection into mice with C26 or 4T1 tumors, the PTX@BN-loaded hydrogel significantly enhanced the antitumor efficacy and prolonged animal survival time compared to free PTX solutions with low systemic toxicity. Therefore, the adhesive, PTX-loaded nanocomposite hydrogels have the potential for efficient localized tumor therapy.

Spatiotemporal consistency analysis of cerebral small vessel disease: an rs-fMRI study.

Introduction: Cerebral small vessel disease (SVD) affects older adults, but traditional approaches have limited the understanding of the neural mechanisms of SVD. This study aimed to explore the effects of SVD on brain regions and its association with cognitive decline using the four-dimensional (spatiotemporal) consistency of local neural activity (FOCA) method. Methods: Magnetic resonance imaging data from 42 patients with SVD and 38 healthy controls (HCs) were analyzed using the FOCA values. A two-sample t test was performed to compare the differences in FOCA values in the brain between the HCs and SVD groups. Pearson correlation analysis was conducted to analyze the association of various brain regions with SVD scores. Results: The results revealed that the FOCA values in the right frontal_inf_oper, right temporal_pole_sup, and default mode network decreased, whereas those in the temporal_inf, hippocampus, basal ganglia, and cerebellum increased, in patients with SVD. Most of these varying brain regions were negatively correlated with SVD scores. Discussion: This study suggested that the FOCA approach might have the potential to provide useful insights into the understanding of the neurophysiologic mechanisms of patients with SVD.

A low-swelling hydrogel as a multirole sealant for efficient dural defect sealing and prevention of postoperative adhesion.

Dural defects and subsequent complications, including cerebrospinal fluid (CSF) leakage, are common in both spine surgery and neurosurgery, and existing clinical treatments are still unsatisfactory. In this study, a tissue-adhesive and low-swelling hydrogel sealant comprising gelatin and o-phthalaldehyde (OPA)-terminated 4-armed poly(ethylene glycol) (4aPEG-OPA) is developed via the OPA/amine condensation reaction. The hydrogel shows an adhesive strength of 79.9 ± 12.0 kPa on porcine casing and a burst pressure of 208.0 ± 38.0 cmH2O. The hydrogel exhibits a low swelling ratio at physiological conditions, avoiding nerve compression in the limited spinal and intracranial spaces. In rat and rabbit models of lumbar and cerebral dural defects, the 4aPEG-OPA/gelatin hydrogel achieves excellent performance in dural defect sealing and preventing CSF leakage. Moreover, local inflammation, epidural fibrosis and postoperative adhesion in the defect areas are markedly reduced. Thus, these findings establish the strong potential of the hydrogel sealant for the effective watertight closure of dural defects.

An antibacterial, multifunctional nanogel for efficient treatment of neutrophilic asthma.

Asthma is a chronic and heterogeneous disease affecting the lungs and respiratory tract. In particular, the neutrophil subtype of asthma was described as persistent, more severe, and corticosteroid-resistant. Growing evidence suggested that nontypeable Haemophilus influenzae (NTHi) infection contributes to the development of neutrophilic asthma, exacerbating clinical symptoms and increasing the associated medical burden. In this work, arginine-grafted chitosan (CS-Arg) was ionically cross-linked with tris(2-carboxyethyl) phosphine (TCEP), and a highly-efficient antimicrobial agent, poly-ε-L-Lysine (ε-PLL), was incorporated to prepare ε-PLL/CS-Arg/TCEP (ECAT) composite nanogels. The results showed that ECAT nanogels exhibited highly effective inhibition against the proliferation of NTHi, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In addition, ECAT nanogels could effectively inhibit the formation of mucins aggregates in vitro, suggesting that the nanogel might have the potential to destroy mucin in respiratory disease. Furthermore, in the ovalbumin (OVA)/NTHi-induced Balb/c mice model of neutrophilic asthma, the number of neutrophils in the alveolar lavage fluid and the percentage of inflammatory cells in the blood were effectively reduced by exposure to tower nebulized administration of ECAT nanogels, and reversing airway hyperresponsiveness (AHR) and reducing inflammation in neutrophilic asthma mice. In conclusion, the construction of ECAT nanogels was a feasible anti-infective and anti-inflammatory therapeutic strategy, which demonstrated strong potential in the clinical treatment of neutrophilic asthma.

Biodegradable stereocomplex micelles based on dextran-block-polylactide as efficient drug deliveries.

Biodegradable stereocomplex micelles (SCMs) based on amphiphilic dextran-block-polylactide (Dex-b-PLA) were designed and used for efficient intracellular drug deliveries. The Dex-b-PLA copolymers were successfully synthesized by click reaction. The structures of the resultant copolymers were verified by (1)H NMR and FT-IR spectra. The formation of stable micelles through self-assembly driven by the stereocomplexation between enantiomeric l- and d-PLA blocks was characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), and fluorescence techniques. It was interesting to observe that the SCMs showed lower critical micelle concentration values (CMCs) because of the stereocomplex interaction between PLLA and PDLA. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis provided information on the thermal and crystal properties of the copolymers and SCMs. The improved stability of SCMs should be attractive for intracellular drug delivery. Thus, a model anticancer drug doxorubicin (DOX) was loaded into micelles, and the in vitro drug release in was also studied. The release kinetics of DOX showed DOX-loaded SCMs exhibited slower DOX release. Confocal laser scanning microscopy (CLSM) and flow cytometry studies also showed that the DOX-loaded SCMs exhibited a slower drug release behavior. Meanwhile, the MTT assay demonstrated that DOX-loaded SCMs show lower cellular proliferation inhibition against HepG2. In sum, the micelles through self-assembly driven by stereocomplex interaction would have great potential to be used as stable delivery vehicles for pharmaceutical and biomedical applications.

Versatile biofunctionalization of polypeptide-based thermosensitive hydrogels via click chemistry.

In this study, we report thermosensitive hydrogels based on poly(ethylene glycol)-block-poly(γ-propargyl-l-glutamate) (PEG-PPLG). (13)C NMR spectra, DLS, and circular dichroism spectra were employed to study the mechanism of the sol-gel phase transition. Mouse fibroblast L929 cells were encapsulated and cultured within the hydrogel matrices, and the encapsulated cells were shown to be highly viable in the gel matrices, suggesting that the hydrogels have excellent cytocompatibilities. The mass loss of the hydrogels in vitro was accelerated by the presence of proteinase K compared to the control group. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ∼21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. The presence of alkynyl side groups in the PEG-PPLG copolymers allowed convenient further functionalization with azide-modified bioactive molecules, such as biotin and galactose. The biofunctionalized PEG-polypeptide block copolymers showed sol-gel phase transitions similar to the parent copolymers. Interestingly, the incorporation of galactose groups into the hydrogels was found to improve cell adhesion, likely due to the adsorption of fibronectin (FN) in cell-extracellular matrix (ECM). Because bioactive materials have shown unique advantages in biomedical applications, especially tissue engineering and regenerative medicine applications, we believe our novel functionalizable thermosensitive hydrogels have potential to serve as a versatile platform for the development of new biofunctional materials, for example, bioadhesive and bioresponsive hydrogels.

pH- and Temperature-responsive Hydrogels Based on Tertiary Amine-modified Polypeptides for Stimuli-responsive Drug Delivery.

pH- and temperature-responsive hydrogels have attracted considerable attention due to their responsiveness to dual physiologically-relevant stimuli. In this study, we developed stimuli-responsive hydrogels based on monomethoxy poly(ethylene glycol) (mPEG)-polypeptide block copolymers containing various tertiary amine pendants (EEP-TAs). The EEP-TAs were synthesized via ring-opening copolymerization of α-amino acid N-carboxyanhydrides, and further modified post-polymerization with click chemistry. The EEP-TAs exhibited an α-helix-to-ß-sheet transition when the pH was increased from 4.0 to 7.4. At elevated polymer concentrations, aqueous solutions of the EEP-TAs underwent thermo-induced sol-gel phase transitions, which were dependent on the pH. The hydrogels almost fully degraded within 3 weeks in the subcutaneous layer of mice and exhibited good histocompatibility in vivo. Additionally, doxorubicin (DOX)-loaded hydrogels exhibited pH-responsive drug release profiles in vitro, which were composed of rapid release at acidic pH and more sustained release at neutral pH. Thus, these polypeptide hydrogels hold potential as depots for environment-responsive delivery of therapeutic agents.

Injectable Polypeptide Hydrogel Depots Containing Dual Immune Checkpoint Inhibitors and Doxorubicin for Improved Tumor Immunotherapy and Post-Surgical Tumor Treatment.

In this work, we developed a strategy for local chemo-immunotherapy through simultaneous incorporation of dual immune checkpoint blockade (ICB) antibodies, anti-cytotoxic T-lymphocyte-associated protein 4 (aCTLA-4) and anti-programmed cell death protein 1 (aPD-1), and a chemotherapy drug, doxorubicin (Dox), into a thermo-gelling polypeptide hydrogel. The hydrogel encapsulating Dox or IgG model antibody showed sustained release profiles for more than 12 days in vitro, and the drug release and hydrogel degradation were accelerated in the presence of enzymes. In comparison to free drug solutions or hydrogels containing Dox or antibodies only, the Dox/aCTLA-4/aPD-1 co-loaded hydrogel achieved improved tumor suppression efficiency, strengthened antitumor immune response, and prolonged animal survival time after peritumoral injection into mice bearing B16F10 melanoma. Additionally, after injection of Dox/aCTLA-4/aPD-1 co-loaded hydrogel into the surgical site following tumor resection, a significantly enhanced inhibition on tumor reoccurrence was demonstrated. Thus, the polypeptide hydrogel-based chemo-immunotherapy strategy has potential in anti-tumor therapy and the prevention of tumor reoccurrence.