Immunotherapeutic agents mainly include antigens, cytokines, adjuvants, checkpoint inhibitors, and immune cells, which can be individually loaded and transported by the hydrogel (Figure 2). Ovalbumin (OVA) can provoke robust OVA-specific immunity and has a good immune effect (Lee S.-J. et al., 2019; Habibi et al., 2020). According to the study, researchers devised a DNA hydrogel with auxiliary activity (Umeki et al., 2015). By conjugation of ethylenediamine (ED) and cationized OVA, complexes with hexapod structure DNA (ED-OVA) were developed with a slower release of OVA compared to unmodified OVA and induced higher IFN-γ production. Cytosine-guanine (CpG) dinucleotides is a positive immune adjuvant, and the integration of ED-OVA and immune-stimulating DNA injection hydrogels containing it is conducive to effective binding of DCs, thus improving the efficiency of antigen presentation.

Cytokines are a powerful weapon in immunotherapy, however, their half-life in patients is quite short. To overcome this major obstacle, Shimizu et al. loaded IL-12 into the cholesterol-containing pullulan (CHP) hydrogel to ensure its slow release of cytokines (Shimizu et al., 2008). A mouse model of CSA1M fibrosarcoma was established, and the hydrogel was injected subcutaneously into mice, which was found to cause a persisted increase in the concentration of IL-12 in serum. In addition, interleukin-15 (IL-15) is also a vital cytokine indispensable and has vast potential as tumor immunotherapy (Robinson and Schluns, 2017). Santi et al. prepared hydrogel microspheres (MS), which were connected to IL-15, to maintain IL-15 in vivo for a long time, namely ultra-long-acting IL-15 (Hangasky et al., 2022). It was found that the half-life of cytokines released from the library was prolonged, showing moderate tumor growth inhibition and effective and lasting anti-tumor activity.

Hydrogels load and transport immune checkpoint inhibitors to induce immune checkpoint blockade, which is a reliable immune tactic for the therapy of tumors. Chen and Liu’s group developed an injectable hydrogel based on betamethasone phosphate (BetP) for topical delivery of aPD-L1 (Chen et al., 2020). The study found that this steroid-based carrier-free system could reprogram the tumor microenvironment (TME) into anti-tumor TME, and it will continuously release aPD-L1 to combinational enhance the immunity system. The results showed that the gel administration of aPD-L1 greatly extended the retention time in the tumor. Gu’s group has developed a library of heat-sensitive and reactive oxygen species (ROS)-sensitive peptide hydrogels for sustained delivery of aPD-L1 and dextro-1-methyltryptophan (D-1MT), and modulates ROS concentration in the TME, facilitating the release of immunotherapeutic drugs that enhance efficacy against melanoma in vivo (Yu et al., 2018b). In addition, Yang’s group developed injectable supramolecular hydrogels for the topical delivery of DPPA-1 peptides and doxorubicin (DOX). DPPA-1 peptides can block the PD-1/PD-L1 pathway to enhance the T cell-mediated immune response (Liu et al., 2021). Similarly, Shi et al.’s study demonstrated that aPD-L1 and Dox combined hydrogel therapy with B16F10 melanoma models significantly restrained cancer progression and extended animal survival (Shi et al., 2021).

Dendritic cells (DCs) can activate the key APCs of immature T cells, which are effective initiators of the immune response (Banchereau et al., 2000; Steinman and Banchereau, 2007; Gardner et al., 2020). Treatment based on autologous dendritic cell injection has become a well-studied strategy (Bol et al., 2016; Santos and Butterfield, 2018; Bol et al., 2019; Perez and De Palma, 2019). Irvine et al. prepared an injectable hydrogel by combining alginate microsphere with DCs. The purpose was to achieve long-term storage of dendritic cells at a determined location, capture cytokines secreted by dendritic cells, and start the natural T cell immune response (Hori et al., 2008). The DCs vaccine is a promising and powerful immunotherapy for cancer, but it has drawbacks that cannot be ignored, the most critical one is that the transplanted DC dies, or is lost to a non-cancerous site, and rarely returns to the lymph nodes (Palucka and Banchereau, 2013; Abraham and Mitchell, 2016; Cornel et al., 2018), thus causing the T cell response is not strong enough. In recent years, there are still many studies on hydrogel-based dendritic cell vaccines. For example, Wang’s study showed that the hydrogel containing DC, aPD-1, and tumor antigen caused a robust immune response, showing excellent antitumor immunotherapeutic effect (Yang et al., 2018). Recently, Zheng and his colleagues devised a Sendai virus (SeV) based hydrogel vaccine (SHV), which can effectively inhibit the occurrence of melanoma (Zheng et al., 2021). In addition, to overcome the limitations of rapid spread and short half-life of therapeutic drugs at the tumor site (Chang et al., 2011; Liau et al., 2018; Goradel et al., 2021), Yun et al. used gelatin hydrogels to co-deliver onco-lytic adenoviruses AD. (Oh et al., 2017) This study discovered that, compared with single cure, the effect of this method on tumor growth was significantly increased. For hydrogels containing immune cells, some key questions should be considered to facilitate their potential applications. For example, these hydrogels require good biodegradability to achieve DC release.

The biological activity of a single immunotherapeutic drug is limited, so this strategy is not ideal for tumor treatment. Therefore, scientists have considered delivering multiple immunotherapeutic agents at the same time.

Recently, the PCL-PEG-PCL thermos-sensitive hydrogel encapsulated GM-CSF and OVA NPs to improve antigen absorption efficacy (Sun et al., 2018). It could trigger an effective immune response. In another study, Chen et al. co-encapsulated nano-hydroxyapatite (nHA) and GM-CSF into a biocompatible heat-sensitive hydrogel (Chen et al., 2022). Interestingly, the biological activity of nHA and GM-CSF can not only be very kept in hydrogel, but the addition of nHA can also weaken the erupt emancipate of GM-CSF, which is conducive to the continuously release of GM-CSF and realize enhanced and extended anti-melanoma immunity.

In another study, Wang et al. developed a PEG-b-poly (L-alanine) hydrogel, in which the tumor cell lysate, GM-CSF, and a dual immune checkpoint inhibitor (anti-CTLA-4/PD-1 antibodies) were easily loaded into (Song et al., 2019). It disclosed that the continuous release of cancer antigen and GM-CSF continuously recruited and stimulated DCs, and provoked robust T cell response. Immune checkpoint therapy boosted T cell response, and also upregulated the creation of IgG and cytokine secretion. Importantly, hydrogel combination therapy has a better immunotherapeutic effect on melanoma and 4T-1 tumor than using the vaccine alone or blocking immune checkpoints alone.

Overall, the co-delivery of hydrogel-based multiple immune therapeutics generally results in higher efficacy in treating tumors than a single drug (Blidner et al., 2020; Ramos-Casals et al., 2020). In addition, the design of such intelligently delivered composite hydrogels is often complex, which is a test for researchers.

Polymer-based hydrogels can be loaded with multiple chemotherapy drugs for cancer at the same time, and the efficacy can be improved through drug combinations. Melittin is a very potent anti-cancer agent, however, hemolysis is the main limitation of its application (Rady et al., 2017; Wang et al., 2022). To overcome this limitation, Yang et al. devised a peptide hydrogel encapsulated with DOX and Melittin-RADA32 (Jin et al., 2018). It can stimulate DCs, specifically consume M2 TAMs and recruit activated NK cells from tumors to further protect cells from residual tumors. It has significant anti-tumor effects on subcutaneous and metastatic tumors in vivo.

DOX is a potent anti-tumor cytotoxic chemotherapy drug and a representative ICD inducer that can lead to apoptosis of cancer cells (Hu et al., 2021). There has been a lot of research on the effective treatment of cancer using DOX as a chemotherapy drug loaded into hydrogels along with immunotherapeutic agents. Recently, Lv et al. devised an injectable hydrogel for the programmed delivery of DOX and cytosine-phosphate-guanine (CpG) nanoparticles guided by dual fluorescence imaging (Dong et al., 2020). CpG as an immune adjuvant can successfully stimulate an antitumor immune response by interacting with Toll-like receptor 9 (TLR9), and according to previous studies, the amalgamation of DOX and CpG is one of the most useful companions of chemotherapeutic immunotherapy (Kumagai et al., 2008; Wang Z. et al., 2017; Yu et al., 2017; Xia et al., 2018; Kheirolomoom et al., 2019). CpG self-crosslinked nanoparticles from hydrogels ensure long-term immunostimulant effects of dual continuous delivery systems. In addition to inducing apoptosis of tumor cells by doxorubicin, this hydrogel actively modulated the tumor microenvironment, which will construct a more favorable treatment response.

Imiquimod (R837) is also an immune adjuvant that activates TLRs and nuclear factor-kappa B (NF-κB) (Schon and Schon, 2007). Previously, Zhang et al. prepared a novel near-infrared (NIR) in situ resistant tumor vaccine by combining hyaluronic acid-modified polydopamine nanoparticles (HA-PDA NPs) with immunoadjuvants R837 and DOX into a thermosensitive hydrogel (Zhang et al., 2021). Due to the long retention time at the tumor site, after a single injection of near-infrared radiation, the NPs are endowed with multiple photothermal ablation characteristics. What’s more, this pattern enables it to stimulate DC maturation and trigger a robust anti-tumor immune response. Due to membrane permeability, nanoparticles can successfully enter the cancer tissues, generate tumor-associated antigens in situ, induce DC maturation and secrete related cytokines in vitro. Recently, Zhang et al. devised an injectable hydrogel loading DOX and R837 for the synergistic therapy of melanoma (Li J. et al., 2022). It efficiently inhibits melanoma growth and metastasis in vivo by DOX-based ICD, and R837-based immune responses are secreted by DC maturation, M1 type macrophage activation, TNF-α, and IFN-γ. This shows that this continuous release system can provide effective cancer treatment strategies, showing the potential of precise targeting tumor therapy.

Injectable hydrogels can be utilized to load and release chemotherapy agents and cytokines for chemoimmunotherapeutic. Chen et al. prepared an in situ forming hydrogel for the joint delivery of IL-15 and cisplatin (Wu et al., 2017). This peptide-based hydrogel can reduce systemic toxicity as a topical drug delivery vehicle. It can mediate the S-phase cell cycle block and increase CD8⁺ T cells. In another study in the same group, DOX-IL-2-IFN-γ co-loaded hydrogel was used for the chemical immunotherapy of melanoma. The antitumor effect was enhanced by expanding the proportion of apoptosis and G2/S phage cycle block (Lv et al., 2018).

In addition to the above stimulants, other immunomodulators also can be combined with hydrogels for amplified tumor immunotherapy. For example, In the article by Luan et al., the L-norvaline-based immunomodulatory gel is reported, which can effectively block the arginase 1 (ARG1) pathway (Ren et al., 2021). ARG1 can impair the synthesis of the T cell receptor (TCR) chain, contributing to reactive inactivated T cells (Bronte and Zanovello, 2005; Rodriguez et al., 2007; Geiger et al., 2016). The reactive cleavage of the hydrogel peptide bond ensures the controlled release of L-norvaline, thereby efficiently blocking the ARG1 pathway. DOX was further introduced to the hydrogel to trigger the ICD. It was found that this strategy showed a strong immunotherapeutic effect. Recently, Zhang et al. developed biomimetic nanobubbles loading DOX (DOX@LINV) for combined immunochemotherapy by blending synthetic liposomes with tumor-derived nanovesicles (TNVs) (Hu et al., 2021). Overall, DOX@LINV was found to enhance the penetration of effector immune cells and improve the immune suppressive TME.

Injectable hydrogels are widely employed in the combined delivery of chemotherapy agents and immune checkpoint inhibitors for combined chemoimmunotherapy. The immune checkpoint inhibitors provoke a combined antitumor immune response by intervening with suppressive T cell signaling (Jenkins et al., 2018).

In cancer immunotherapy, it significantly enhances treatment response for immune checkpoint inhibitors to aim at the CTLA-4 and PD-1/PD-L1 axes (Littman, 2015; Klevorn and Teague, 2016; Robert, 2020; Naimi et al., 2022). For instance, Gu and others obtained ROS-responsive hydrogels by crosslinking polyvinyl alcohol (PVA) for co-delivery of chemotherapy gemcitabine and aPD-L1 (Wang C. et al., 2018). The designed hydrogels can be used to load and release therapeutic drugs when implanted at the tumor site because ROS is very abundant. This study showed that this type of gel scaffold promoted immunogenic tumor phenotyping and enhanced the anti-tumor response by local release of aPD-L1. In another study, a dual bioresponsive gel depot for co-deliver aPD1 and Zebularine is developed (Ruan et al., 2019). The results showed that this synergetic therapeutic improved tumor cells’ immunogenicity.

Additionally, Yang et al. fabricated an injectable thermosensitive hydrogel loading DOX and PD-L1 agonist peptide (^DPPA-1) (HDU hydrogels) (Liu et al., 2021). The HDU hydrogel steadily emitted encapsulated DOX and ^DPPA-1 to stimulate an antitumor immune response. ^DPPA-1 is based on the local blockade of PD-1/PD-L1 pathway, thereby enhancing the immune response mediated by T cells and reducing toxicity (Figure 3). In another study, Cui’s group established a prodrug hydrogel for locally delivering ICBs to further boost antitumor immunity (Wang et al., 2020). Their research results show that this hydrogel can be used as a continuous response emancipation library in TME, inducing powerful PD-1 to block the immune response.

Chemotherapy combined with injectable hydrogels containing immune checkpoint inhibitors is more effective than hydrogels loading a single immune checkpoint inhibitor (Table 2). Despite this, most synthetic hydrogels with complex structures should be carefully evaluated for long-term toxicity. In addition, the load ratio of agents should be elevated to achieve the maximum therapeutic benefit (Xie et al., 2021).

PTT is a hopeful approach that employs ingredients with extraordinary photothermal conversion efficiency to transform illumination energy into heat (Huang et al., 2006; Yang et al., 2010; Huang P. et al., 2019). The PTT-induced hyperthermic effect defeats cancer cells by damaging cell membranes and generating DNA injury, thereby causing cell necrosis, cell apoptosis as well ICD (Hildebrandt et al., 2002; Li Y. et al., 2020; Gao et al., 2020). By filling it and immunotherapeutic agents into hydrogel carriers, PTT combination immunotherapy can be achieved.

PTT has been reported to promote the discharge of the protein antigens from tumor by inducing immunogenic cell death, which exposes large amounts of autoantigens after PTT treatment, thereby enhancing a robust immune response (Ng et al., 2018; Wang et al., 2019). Wang et al. presented a heat-sensitive nanogels loading MnO₂ NPs to activate anti-tumor immune responses (Fan et al., 2021). In particular, the catechol group is introduced into the structure of the heat-sensitive nanogel, and its strong adhesion gives it the capability to capture the antigens produced by PTT. After intertumoral injection of hydrogel, it continuously releases antigens to realize improved and extended immune stimulation, inducing both strong cytotoxicity and ICD. It indicated that the use of PTT based on injection of adhesive hydrogel resulted in complete ablation of the solid tumor and efficient inhibition of distal and regenerated cancer cells.

The combination of photothermal effect and immune adjuvant will result in a more effective immunotherapeutic effect, there has been a lot of research on PTT-combination immunotherapy based on hydrogels loaded with immunoadjuvants (Guo et al., 2014). Nishikawa’s group devised a compound immune-stimulatory hydrogel consisting of hexapod DNA with CpG sequence and Au NPs (Yata et al., 2017). Laser irradiation l leads to hexapods release, which effectively stimulates the stimulation of macrophages and DCs, and stimulates the emancipation of pro-inflammatory cytokines.

In another study using CpG as an immune adjuvant, Lv and coworkers proposed a conjugated hydrogel with CpG self-crosslinked NPs and IR820 for combined PTT and immunotherapeutic (Dong et al., 2019). It can advance continuous release by blocking rapid degradation of CpG, with CpG-mediated immunostimulatory effects of TLR-9 activation. In the B16 melanoma mouse model, after injection of CpG NPs/IR820 hydrogel and irradiation treatment, DC maturation and CD8⁺ T cell activation were moderately enhanced, and the anti-tumor immune effect was enhanced. More specifically, auto-fluorescent CpG NPs/IR820 hydrogels do not need additional fluorescence and are used for image-guided synergistic PTT and immunotherapeutic using a dual fluorescence imaging technique. It provides a platform full of possibilities and hopes for precision cancer diagnosis and therapy. Furthermore, Wei et al. designed a local injectable platform to achieve combined PTT and immunotherapy, which was formed by the photothermal agent Indocyanine Green (ICG), the TLR-7/8 agonist (R848), and the TLR-9 agonist CPG ODN (Jia et al., 2020). NIR triggered the thermally responsive hydrogel PLEL to release the immune components CPG ODN and R848, which, together with tumor-associated antigens, induce effective and sustained anti-tumor immunity as carcinoma in situ vaccines for postoperative immunotherapy.

Apart from immune adjuvants, strategies for immune checkpoint blocking can also be introduced into injectable hydrogels for photothermal immunotherapeutics. As Sun et al. proposed an all-in-one and fully controlled combination strategy, NIR photothermal agents IR820 and aPD-L1 were loaded into a lipid gel library with excellent thermally reversible gel-sol phase transition properties to achieve native symbiotic PTT-assisted immunotherapeutic (Huang L. et al., 2019). This study shows that symbiotic mild photothermal-assisted immunotherapy is an operational and potential approach for curing “cold” tumors. Other than that Li et al. proposed a personalized cancer vaccine (PVAX) for postoperative immunotherapeutics, by loading tumor cells with a BRD4 inhibitor and ICG into the hydrogel-based matrix (Wang T. et al., 2018). PVAX can advance the growth of DCs, and trigger tumor penetration of cytotoxic T lymphocytes.

PTT can also be used in combination with chemotherapy for cancer immunotherapy. For instance, Liu’s group developed a Ag₂S QD/DOX/Bestatin@PC₁₀ARGD peptide gel for breast cancer treatment as a lasting-release material (Hou et al., 2020). The experiments were carried out and it presented that the laser-irradiated hydrogel could stimulate anti-tumor immunity, relieve tumor pressure, and inhibit primary tumor and lung metastasis. At the same time, the safer low-temperature several laser irradiation approaches showed more effective tumor-killing performance. In summary, the strategy of combining PTT with other therapies based on injectable hydrogels effectively improves the effect of cancer treatment and is a promising and potential option.

Lately, PDT has fascinated widespread notice owing to its little toxicity, great spatiotemporal selectivity, minimally invasive, and considerable therapeutic effect (Zhang and Li, 2018; Shu et al., 2021). It can not only exactly destroy cancer cells, but also initiate ICD, providing anti-tumor immunity. Regrettably, the inherent nature and complex limitations of TMEs drastically reduce the efficacy of PDT (Ji et al., 2022). Emerging smart polymer hydrogels have been developed, loaded with immunotherapeutic agents through the combined action of PDT and immunotherapy, cleverly modulating the pharmacokinetics of drugs and TME, thus improving the anti-tumor efficacy.

Oxygen is essential for PDT to perform therapeutic roles, but the hypoxic TME present in most solid tumors will significantly restrain the efficiency of PDT (Yang et al., 2017; Min et al., 2021). To relieve hypoxic TME, Liu et al. used PEGDA as a polymer matrix to construct a light-triggered gelation procedure encompassing catalase modified by the photosensitizer (Meng et al., 2019). First, the photosensitizer Chlorin e6 (Ce6), which is extensively used in clinical, is coupled with catalase (CAT), and then the resulting coupling is mixed with the polymer matrix PEGDA and R837-loaded PLGA nanoparticles (RPNPs) as immune adjuvants. The retained CAT in the hydrogel reverses immunosuppressive TME by decomposing endogenous H₂O₂ of tumors to produce O₂ leading to sustained remission of tumor hypoxia. Subsequently, tumor cell fragments after PDT-triggered ICDs can pretend TAAs and elicit a strong anti-tumor immune response alongside with RPNPs. In addition, this hybrid hydrogel can offer the chance to continually stimulate the immune response by triggering PDT through several times of lighting, which contribute to an extensively improved immune response. Studies have also found that further combination with a CTLA-4 checkpoint blockade could inhibit the evolution of distant metastatic cancers.

Sun’s team developed a continuous luminescent immune hydrogel by introducing a persistent luminescent material (PLM) and immunoadjuvant R837 into alginate-Ca²⁺ to convert solids into gels (Shu et al., 2021). PLM is a unique property that can stimulate continuous and repetitive PDT (Lécuyer et al., 2016; Wang J. et al., 2017; Wu et al., 2020; Wang et al., 2021). PLM, R837 and alginate-Ca²⁺ solutions were mixed to synthesize a rechargeable immune hydrogel (PRA) with a homogeneous structure. The designed PRA has great biocompatibility and excellent injectability, and can be effortlessly inserted into the tumor site, which the continuous luminescence efficiency of PLM can reach 100%. The experiments have exposed that DCs are activated, resulting in increased emission of TNF-α and IL-6, and a significant amplification of the anti-cancer immune response. It showed that the reliable anti-tumor immune effect is obtained by hydrogel-based PDT synergistic immunotherapy.

Recently, Li and others reported the use of NIR dual-response precursor hydrogels for synergistic immunotherapy for cancer (Ding et al., 2022). This prodrug hydrogel is formed by calcium-induced iron oxide (Fe₃O₄) nanoparticles modified with the photosensitizer protoporphyrin IX (PpIX) and aPD-L1 prodrug nanoparticles crosslinked through ROS-responsive linkers. This hydrogel enabled the on-demand discharge of aPD-L1 during photoactivation. This type of prodrug hydrogel could stimulate ICD by mediating photokinetic and chemokinetic therapy and expand the efficiency of aPD-L1-mediated immune checkpoint blockade. Together, this study provided a double-response hydrogel strategy for precise tumor immunotherapy.

Sometimes, two combinations of therapies still do not meet the therapeutic effect, so researchers try to use multiple treatments to enhance the efficiency and achieve the purpose of treatment. For example, Cao and Liu et al. devised and formulated a hydrogel based on DOX, polypeptide PC₁₀A, and MoS₂ nanosheet (Figure 5). (Jin et al., 2020) Firstly, positively charged DOX and negatively charged PC₁₀A are filled on the exterior of MoS₂ nanosheets by electrostatic adsorption, and then hybrid PC₁₀A/DOX/MoS₂ NPs are ‘dissolved’ in PC₁₀A hydrogel to form an injectable hydrogel. The experiments showed that it has excellent photothermal efficiency. MoS₂ nanosheets are materials with both PTT and PDT effects. It was found that the amount of DC, CD4⁺ T cells, and CD8⁺ T cells increased, and the combination of these treatments greatly increased the number of tumor cell apoptosis. In conclusion, this study showed that the combination therapy meaningfully enhanced the impact of tumor inhibition and inhibited the development of primary tumors and remote metastasis.

In summary, phototherapy-activated ICDs can stimulate immune responses, hydrogel-mediated combination phototherapeutic and immunotherapeutic generally provide better efficiency than hydrogel-mediated immunotherapeutic (Table 4). Compared to hydrogel-mediated combination chemoimmunotherapy, this approach likewise showed excellent controllability in space and time.