Short Review on Advances in Hydrogel-Based Drug Delivery Strategies for Cancer Immunotherapy

Abstract

Cancer immunotherapy has become the new paradigm of cancer treatment. The introduction and discovery of various therapeutic agents have also accelerated the application of immunotherapy in clinical trials. However, despite the significant potency and demonstrated advantages of cancer immunotherapy, its clinical application to patients faces several safety and efficacy issues, including autoimmune reactions, cytokine release syndrome, and vascular leak syndrome-related issues. In addressing these problems, biomaterials traditionally used for tissue engineering and drug delivery are attracting attention. Among them, hydrogels can be easily injected into tumors with drugs, and they can minimize side effects by retaining immune therapeutics at the tumor site for a long time. This article reviews the status of functional hydrogels for effective cancer immunotherapy. First, we describe the basic mechanisms of cancer immunotherapy and the advantages of using hydrogels to apply these mechanisms. Next, we summarize recent advances in the development of functional hydrogels designed to locally release various immunotherapeutic agents, including cytokines, cancer immune vaccines, immune checkpoint inhibitors, and chimeric antigen receptor-T cells. Finally, we briefly discuss the current problems and possible prospects of hydrogels for effective cancer immunotherapy.

Introduction

Cancer is a global health problem that continues to be the leading cause of death worldwide [1, 2]. Over the past century, various therapeutic approaches have been developed to treat cancer, many of which have already shown positive effects in clinical use [3]. However, traditional approaches—including chemotherapy and radiotherapy—have limitations such as their severe side effects, the high risk of cancer recurrence, and their limited therapeutic effects, emphasizing the need for more promising treatments [4].

Cancer immunotherapy has emerged as a revolutionary approach in oncology for several reasons. Unlike traditional therapies that directly kill tumor cells, immunotherapy harnesses the inherent immune systems of individuals to attack the tumor cells [4, 5]. This allows immunotherapy to prevent metastasis and/or tumor recurrence effectively. The first agents marketed for cancer immunotherapy were interferon-α (IFN-α)-derived cytokines, approved by the US Food and Drug Administration (FDA) in 1986 for the treatment of hairy cell leukemia [6, 7]. In 2010, Sipuleucel-T, an agent for dendritic cell-based immunotherapy, was approved as the first successful therapeutic cancer vaccine for prostate cancer [8].

More recently, the second wave of intense research activity has emerged following the introduction of immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR)-T cells to immunotherapy. In 2011, cytotoxic T lymphocyte antigen 4 (CTLA-4) targeting the monoclonal antibody (mAb), ipilimumab, was approved for metastatic melanomas [9]. Since then, many other ICIs have been approved for their clinical efficacy (Table 1). Targeted programmed cell death 1 (PD-1), its ligand PD-L1, and CAR-T cells are examples of immunotherapy agents that continue to show unprecedented benefits in clinical use [13–19].

Table 1.

Approved immunotherapies for cancer treatment

Classification	Name	Description	Major Administration Route	Applications	References
Cytokines	Roferon–A	Recombinant interferon alpha-2a (IFNα2a)	IM1 or SC2	AIDS-related Kaposi’s Sarcoma, hairy cell leukemia, and chronic myelogenous leukemia	[6, 10]
	Intron A	Recombinant interferon alpha-2b (IFNα2b)	IM, SC, or IV3	AIDS-related Kaposi’s Sarcoma, hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma
	Imiquimod	Immune response enhancer that increases IFNγ, tumor necrosis factor, and interleukin-2 (IL-12) production	Topical	Basal cell carcinoma	[11]
	Aldesleukin	Recombinant IL-2	SC or IV	Renal cell carcinoma and metastatic melanoma	[12]
Cancer vaccines	Sipuleucel–T	APCs4 activated with PAPGM-CSF	IV	Asymptomatic or minimally symptomatic metastatic castrateresistant prostate cancer	[8]
Immune checkpoint inhibitors	Ipilimumab	CTLA-4 mAb		Metastatic melanoma	[9, 16]
	Nivolumab	PD-1 mAb		Refractory melanoma, renal cell carcinoma, advanced non-small cell lung cancer, Hodgkin lymphoma, head and neck cancer, and bladder and urinary tract cancer	[13, 14, 16]
	Pembrolizumab
	Atezolizumab	PD-L1 mAb		Bladder and urinary tract cancer and non-small cell lung cancer
	Avelumab			Bladder and urinary tract cancer and merkel cell carcinoma	[16]
	Durvalumab			Bladder and urinary tract cancer
CAR-T cells	Tisagenlecleucel	CD19-specific CAR-T cells		Non-Hodgkin lymphoma and Bcell lymphoblastic leukemia	[122]
	Axicabtagene ciloleucel			Large B cell lymphoma	[123]

¹IM: intramuscular, ²SC: subcutaneous, ³IV: intravenous, ⁴APCs: antigen-presenting cells

Although immunotherapy opened a new chapter of promising cancer therapies, it also encountered various drawbacks. Safety and limited efficacy remain two significant limitations that immunotherapy must overcome to exhibit the full potency anticipated by researchers. There have been various reports that over-activated immune responses during immunotherapy may induce fatal side effects, such as autoimmune reactions, vascular leak syndrome (VLS), or cytokine release syndrome (CRS) [20, 21]. As for efficacy, it has been found that not all patients are responsive to immunotherapy agents, making the therapy only applicable to a selective group of patients [22, 23]. Excluding glioblastomas that express EGFRvIII, most solid tumors also inhibit effective chimeric antigen receptor-T (CAR-T) cell therapies owing to the complex tumor microenvironment (TME) [24, 25]. The introduction of various TME-mimicking platforms, including organoids, enables various in vitro research on drug-TME interactions [26]. Nevertheless, new delivery strategies are desperately needed to compensate for the limitations of tumor immunotherapy.

Hydrogels are biomaterials that are being studied as novel delivery strategies to overcome the limitations associated with current immunotherapy. Various hydrogel parameters, such as low toxicity, high loading efficacy, stimuli responsiveness, and in situ gel-forming ability, have been repeatedly verified, making hydrogels promising carriers of biological agents [27, 28]. Owing to their highly tunable physicochemical properties, hydrogels provide a stable release platform for molecules, cells, and labile therapeutic agents of various sizes [27]. These advantageous characteristics make the use of hydrogels a potent delivery strategy that could profoundly leverage the targeting of cancer/immune cells, enhance the associated immune responses, and reduce the risk of systemic toxicity and adverse side effects of immunotherapy. The rapid advancement and development of novel hydrogels suggest regular overviews focusing on their most current status in immunotherapy. Herein, we present an original review on the current status of functional hydrogels in the delivery of various immunotherapeutic agents. In particular, we categorize the hydrogels by their cargo counterparts, including cytokines, cancer immune vaccines, ICIs, and CAR-T cells, to effectively emphasize the hydrogels and the significance of each immunotherapeutic agent.

Mechanism of the Cancer Immunotherapy

This review does not address only the chemical significance of individual hydrogels, but it primarily provides an overview regarding the significance of potent immunotherapeutic hydrogel platforms as a whole. First, we describe the basic mechanisms of cancer immunotherapy and the advantages of incorporating hydrogels in these mechanisms. Next, we summarize the recent advances in developing functional hydrogels designed to locally release various immunotherapeutic agents, namely cytokines, cancer immune vaccines, ICIs, and CAR-T cells. Finally, we briefly discuss the current problems and possible application prospects of hydrogels in effective cancer immunotherapy.

Figure 1 illustrates the cancer-immunity cycle that occurs during the presence of cancer. When cancer cells undergo necrosis or apoptosis, tumor antigens are released and then captured by antigen-presenting cells (APCs). When these antigen-bearing APCs prime immature T cells found in the lymph node, the immature T cells develop into tumor-specific cytotoxic T cells. The specificity of these T cells allows them to infiltrate the tumor environment and interact with tumor cells via T cell receptors and major histocompatibility complexes. This interaction drives effector T cells to kill cancers by inducing apoptosis. This causes an additional release of tumor antigens, subsequently forming a positive feedback loop of immune-cell-mediated anticancer reactions. Cancer immunotherapy, in essence, is an approach that aims to intervene in the cancer-immunity cycle using external immunostimulatory agents, various signaling molecules, or immune cells [29].

Fig. 1.

Cancer-immunity cycle. Cancer cells that undergo necrosis or apoptosis release tumor antigens subsequently captured by APCs. When these antigen-bearing APCs prime immature T cells located in the lymph node, the immature T cells develop into tumor-specific cytotoxic T cells. The T cells infiltrate the TME and kill cancer cells by inducing apoptosis. This causes an additional release of tumor antigens, subsequently forming a positive feedback loop of immune cell-mediated anticancer reactions (Figure

adapted from Ref [4]; Open Access. Modified by Hee Seung Seo)

Conventional immunotherapies simply focused on facilitating the cancer-immunity cycle to kill cancer cells. However, such methods faced many obstacles limiting the efficacy of anticancer immune responses. The primary problem was the presence of immune-related signaling molecules and mechanisms that promote tumor growth and survival. M1-polarized macrophages are pro-inflammatory cells capable of killing tumor cells [30]. However, these macrophages are repolarized into M2 macrophages in the presence of IL-10, TGF-β, and sphingosine-1-phosphate (S1P), which are immunosuppressive molecules released from dead tumor cells [31]; these molecules also release monocyte chemoattractant protein-1 (MCP-1) and bombesin (BN) that promote monocyte infiltration into the TME [32–34]. Monocytes attracted into the TME differentiate into tumor-associated macrophages that enable tumors to avoid immune surveillance [32–34].

Immune checkpoints are good examples of how cancer cells form immunosuppressive environments. Tumor cells exhibit immune-suppressive molecules (i.e., PD-L1 and PD-L2) that—when bound to receptors on T cells (i.e., PD-1 and CTLA-4)—deactivate the cytotoxic activity of T cells [35–38]. These immunosuppressive mechanisms associated with the TME stress the importance of new approaches that can overcome the limitations of current immunotherapy. Of the many platforms devised to address these limitations, hydrogels have been highlighted as a potentially powerful platform for future immunotherapies.

Hydrogels for Cancer Immunotherapy

The agents most commonly used in cancer immunotherapy include cytokines, anticancer vaccines, checkpoint inhibitors, and CAR-T cells. A common characteristic of these agents is that they affect immune cells regardless of their location or the presence of tumor cells. Systemic administration of such agents—especially checkpoint inhibitors—commonly results in various immune-related adverse events (irADs) [39–42]. Local administration is an approach that significantly lowers the risk of irADs while enforcing the specificity and effectiveness of agents toward the desired level of anticancer immunity [43].

Hydrogels, owing to their high water content, resemble the physical properties of soft tissue. The highly porous net construction of hydrogels forms an extracellular matrix (ECM)-like structure that provides a stable repository for immunotherapy agents without damaging their biological activity. The first attempt in utilizing hydrogels in immunotherapy was to deliver dendritic cells in an injectable hydrogel matrix in 2008 [44]. This research provided evidence that hydrogels were indeed potent carriers that could induce systemic immunity through local administration. Hydrogels have since become a popular area of research in the field of immunotherapy, leading to the discovery and fabrication of various immunotherapy-related hydrogels.

More recently, hydrogels in immunotherapy have been developed to a level that allows precise control over the release of various biological agents [27]. Many hydrogels now have injectable sol–gel characteristics owing to the various advantages of in situ gelation [45]. The following sections will address various hydrogels developed to deliver immunotherapy agents, including cytokines, anticancer vaccines, checkpoint inhibitors, and CAR-T cells.

Delivery of Cytokines (Interferons, Interleukins, and GM-CSF)

Interferons, interleukins, and granulocyte–macrophage colony-stimulating factor (GM-CSF) are three cytokines commonly used in immunotherapy [20]. Unlike ICIs, cytokines directly stimulate immune cells to perform specific actions and/or production.

Interferons, which are signaling proteins released by host and immune cells during microbial infection, are capable of inducing the maturation and activation of other immune cells such as macrophages, dendritic cells (DCs), natural killer (NK) cells, and lymphocytes [46–49]. Immune cells activated by these interferons can inhibit tumor angiogenesis, making interferons highly potent agents [47, 50]. Interleukins are signaling proteins known to modulate T cells' development and activity [51–54]. Thus, GM-CSF has multiple roles in our immune responses, with the following two significant roles: (1) enhancing T cell survival and action by regulating cell homeostasis and (2) supporting the expression of tumor-specific antigens in DCs [55].

Unfortunately, cytokines have very short in vivo half-lives requiring high, frequent dosages to maintain significant therapeutic effects [20]. Such harsh administration patterns pose high risks of inducing adverse side effects, including CRS or VLS [20]. Another risk associated with cytokine therapy is that cytokines may induce apoptosis of activated T cells and promote survival of regulatory T cells (T_regs) to form auto-immunity against healthy tissues [21]. To minimize these risk factors associated with high dosages and frequent administration of cytokines, hydrogels can be actively utilized as effective local delivery platforms.

Recent research has reported the success of local delivery systems combining thermosensitive hydrogels with cytokines and therapeutic agents. Thermosensitive hydrogels effectively release bioactive agents that are sensitive to temperature. Unlike conventional hydrogels that must be implanted surgically, hydrogels with temperature-sensitive properties enable local injection, avoiding the first-pass metabolism effects of the liver and gut wall that absorb the drug before it reaches systemic circulation [56]. Temperature-driven hydrogels undergoing sol–gel transitions show outstanding biocompatibility and are highly suitable for injection as they do not require the use of various potently toxic denaturing crosslinkers [56]. Flowing-state encapsulation allows uniform dispersion of the therapeutic agent across the hydrogel, whereas rapid sol–gel conversion at body temperature prevents the initial explosive release of the therapeutic agents, thus allowing controlled release profiles in vivo [56].

In one study, injectable thermosensitive polypeptide hydrogels of poly(ethylene glycol)/poly(γ-ethyl-L-glutamate) (PEG/PELG) block copolymers were used to co-deliver doxorubicin (DOX) with IL-2 and IFN-γ for the local treatment of melanomas. This local co-delivery strategy showed significant melanoma suppression in B16F10 xenograft mouse models without inducing any systemic side effects. Analysis of the mouse models post-injection showed enhanced apoptosis of tumor cells and proliferation of both CD3⁺/CD4⁺ T cells and CD3⁺/CD8⁺ T cells, indicating the presence of an enhanced anti-tumor immunity [57]. Another study also used PEG/PELG to synthesize an injectable heat-responsive hydrogel system (Fig. 2A). This hydrogel was used to co-deliver the cytokine IL-15 and drug cisplatin for the treatment of melanomas. Once injected into a black mouse, the IL-15 and cisplatin (CDDP) loaded within the thermosensitive hydrogel were able to enhance anti-tumor immunity by systematically regulating CD8⁺ T cell and NK cell activity (Fig. 2B, C) [58].

Fig. 2.

A Mechanism for synergistic anti-tumor effects following co-administration of IL-15 and CDDP released from mPEG-b-PELG heat-responsive hydrogels. B Effect of combination IL-15/CDDP therapy on anti-tumor immune responses in vivo. Percentages of NK-and CD8 + T cells. C in vivo tumor cell-specific fluorescence images of representative mice from each treatment group (Reprinted with permission from Ref. [57];

Copyright 2017 Elsevier B.V. All rights reserved)

Chitosan hydrogels have long been used as effective drug delivery platforms for various cargos, including cytokines [59–61]. In 2009, a hydrophobic chitosan-based thermosensitive hydrogel was used to co-deliver GM-CSF with various cancer drugs, such as DOX, CDDP, or cyclophosphamide (CTX). The GM-CSF-drug conjugates were locally administered to induce activation of tumor-antigen-specific CD8⁺ T cells. CTX showed great synergy with GM-CSF in inducing robust anti-tumor immunity, demonstrating the successful use of chitosan hydrogels in chemo-immunotherapy [61]. More recently, a sulfated chitosan hydrogel was coated onto an implantable gelatin scaffold to create a reservoir for cells as well as various growth-related cytokines (Fig. 3A–C). The 2-N, 6-O-sulfated chitosan coated on the scaffold showed the great capturing ability of vascular endothelial growth factor (VEGF) in vivo (Fig. 3D) [62].

Fig. 3.

A Scheme of sulfated chitosan-coated scaffolds captures VEGF in situ. B Photos of the gelatin sponge, 6-O-SCS-coated scaffold, and 2-N,6-O-SCS-coated scaffold, respectively (from left to right); the minimum division value of the ruler is 1 mm. C Microstructure of (1) the 6-O-SCS hydrogel and (2) the 2-N,6-O-SCS hydrogel coated gelatin sponge. Gelatin fibers were wrapped with (3) the 6-O-SCS hydrogel and (4) the 2-N,6-O-SCS hydrogel. D Immunohistochemical staining of VEGF on the sections of implantations. (1) 6-O-SCS hydrogel scaffold + ; (2) 6-O-SCS hydrogel scaffold; (3) 2-N,6-O-SCS hydrogel scaffold + ; (4) 2-N,6-O-SCS hydrogel scaffold; (5) gelatin sponge. VEGF was dyed to be brown, and the scaffolds appear fibrous; the scale bar equals 100 μm (

Reproduced from Ref. [62]; with permission from The Royal Society of Chemistry)

Apart from the three aforementioned major cytokines, researchers have also found immunotherapeutic potential in agonists related to specific intracellular mechanisms of immune cell activation when delivered via various functional hydrogels. For example, agonists of toll-like receptor 7/8 (TLR7/TLR8) stimulate APCs to improve anti-tumor immunity, whereas agonists of stimulators of interferon genes (STING) cause the release of inflammatory cytokines [63, 64]. One of the studies reported great immunotherapeutic success of a biodegradable hyaluronic acid-based hydrogel loaded with R848 and STING-RR, each an agonist of TLR7/8 and STING. This hyaluronic acid-based hydrogel allowed the sustained release of the agonists, which increased the level of anti-tumor immune cells and cytokines, ultimately exhibiting considerably better therapeutic results compared to intraperitoneal, intravenous, or local injection of the same agonists [65].

Another method employed by Wang et al. was a drug-based supramolecular hydrogel system developed for the local delivery of STING-activating cyclic dinucleotides (CDNs). A tumor-penetrating peptide was chemically conjugated to the anticancer drug camptothecin (CPT) to form supramolecular nanotubes that could form a gel in situ. The CDNs released from the gel activated various immune cells (NK cells, macrophages, T cells) within the TME, whereas the gel itself simultaneously performed chemotherapy via CPT. This chemo-immunotherapy platform significantly increased mouse survival in various tumor models [66].

Delivery of Cancer Immune Vaccines

Cancer immune vaccines aim to promote the host’s immune system to attack cancer cells by reducing the host’s immune tolerance against cancer antigens. Cancer vaccines have utilized various composites of cancer antigen and immunostimulatory adjuvants to achieve this goal [67]. The application of strong immunogenic epitopes against specific tumor antigens induces effective CD8⁺ T cell-mediated immunity against cancer cells [68]. In addition to strong epitopes, diverse agents have been actively researched to develop compelling vaccine compositions. The common focus in cancer vaccine research includes peptides, DNA, mRNA, cancer cell lysates, recombinant virus, and engineered immune cells [69].

DNA and nucleotides have been recently highlighted as a novel delivery platform for bioactive agents such as chemo drugs, photosensitizers, and antisense oligonucleotides [70]. Using this approach, a group of researchers created a DNA cytosine‐phosphate‐guanine (CpG)-based photothermal hydrogel that demonstrated considerable achievement in photothermal therapy-mediated in situ vaccination and delivery of dual adjuvants to DCs (Fig. 4). In this research, rolling circle amplification (RCA) was used to synthesize the CpG sequence-containing DNA hydrogel, which was used to encapsulate and release bis-(3′-5′)-cyclic dimeric guanosine monophosphate, an agonist of STING (Fig. 4A). The hydrogels injected into CT26 colon cancer models successfully activated DCs by producing tumor antigens and remodeled the TME with increased CD8⁺ T cell and decreased T_regs (Fig. 4B–E). When cancer cells were re-administrated to the mice after treatment, the mice inoculated with this hydrogel vaccine showed no sign of cancer recurrence, indicating the successful development of active anti-tumor immunity [71].

Fig. 4.

A Proposed action mechanism of DNA CpG-based photothermal hydrogel. B-E In vivo T cell infiltration and cytokine production in CT26 colon cancer. B Infiltration of CD8 + T cells, C population of T_reg cells, D granzyme B-expressing T cells, and E IFN-γ-expressing T cells (***p < 0.001). F Schematic of injectable hydrogel (GLP Gel). This GLP-RO Gel (R for R848 and O for mOVA) continuously releases nanovaccines to trigger an immune response for cancer immunotherapy. G-I in vivo prevention of metastasis. G Flow cytometry analysis of CD8 + IFN-γ + T cells in splenocytes from mice with various treatments, H ELISA analysis of TNF-α, I Tumor gross images (Reprinted with permission from Ref. [71, 75];

Copyright 2021 Elsevier and 2021 American Chemical Society, respectively)

RNA cancer vaccines traditionally treat cancer by translating tumor-associated antigens from APCs to produce tumor-specific CD4⁺ and/or CD8⁺ T cells [72, 73]. Unfortunately, the low stability and cell absorption of RNAs in vivo limits its full potential as a cancer immune vaccine [74]. These limitations emphasize the need for a new platform that protects RNAs from degradation and allows their delivery to specific immune cells. To address these problems, an injectable hydrogel platform was synthesized from graphene oxide (GO) and linear polyethyleneimine (LPEI) that greatly enhanced the stability and delivery of mRNA cargo to the lymph node (Fig. 4F). This GO-LPEI hydrogel encapsulated with mRNA (mOVA) and R848 demonstrated excellent efficacy inducing high levels of tumor antigen-specific CD8⁺ T cells and tumor antigen-specific antibodies (Fig. 4G–H). Antibodies created via the inoculation of the GO-LPEI hydrogel vaccine limited further metastasis or the formation of secondary tumors (Fig. 4I) [75].

Cancer vaccines that rely on a replication-defective or competent virus to deliver immunotherapy agents are powerful approaches toward treating malignant tumors [76, 77]. Unique viruses like oncolytic viruses can directly lyse tumor cells while also stimulating the formation of anticancer immunity [78]. The adenovirus encoding vector Flagrp170 (Adc-Flagrp170) is a chimeric molecule that induces inflammatory cytokines (e.g., TNF-α, IL-6 IL-12p70, IFN-γ) in the TME, reconstructing the immune surveillance system of the host [79]. Recently, cell infiltration and duration of Flagrp170 were enhanced using an injectable supramolecular hydrogel (SH) made of α-cyclodextrin and 4-arm PEG. Because in situ viral immunotherapy does not require the intensive infection of cancer cells, prolonged local retention of the viral vector via SH was enough to up-regulate tumor-reactive T cells significantly. SH-mediated release of Flagrp170 allowed efficient delivery of the tumor antigens to target APCs, showing increased therapeutic efficacy in murine melanoma models [80].

Other immunotherapeutic vaccines that do not use viral vectors rely instead on vaccine packages comprising three or more independent agents. GM-CSF is a cytokine frequently used as an adjuvant in multi-conjugate vaccines owing to its role in stimulating and activating various immune cells [81–83]. These composite vaccines often require unique platforms that allow stable encapsulation of multiple distinct agents. Song et al. applied an injectable PEG-β-poly(L-alanine) hydrogel for the co-delivery of an immune vaccine (tumor cell lysates, GM-CSF) and ICIs (anti-CTLA-4/PD-1 antibody). Compared to vaccines or ICIs administered independently, the co-delivery of the two agents showed much greater T cell activity, hence greater therapeutic efficacy. When administered in melanomas and the 4T1 mouse model, the hydrogel induced tumor growth suppression and decreased the level of T_regs in the spleen and inguinal draining lymph nodes. This demonstrated that the dual functionality elicited by the co-delivery of the vaccine and ICI stimulated T cell activity and weakened the immunosuppressive environment of the TME [84].

More recently, a similar hydrogel vaccine system used the aforementioned co-delivery method to induce synergistic therapeutic effects. In this research, GM-CSF, CpG-ODN, TLR9, and tumor cell lysates were encapsulated within the thermosensitive PDLLA-PEG-PDLLA copolymer hydrogel (Fig. 5A). The adjuvants locally released from this hydrogel allowed prolonged maturation and activation of DCs and sustained an elevated level of tumor necrosis factors (Fig. 5B–D). The hydrogel vaccine demonstrated great therapeutic and vaccination results in B16F10 and C26 mouse models, validating its therapeutic potency against other various tumors (Fig. 5E–F) [85].

Fig. 5.

A Scheme of in vivo immune regulation mediated by the thermosensitive PDLLA-PEG-PDLLA copolymer hydrogel vaccine. B, C Representative flow cytometry plots analyzing activated bone-marrow-derived cells in various immunized groups. D Concentration of TNF-α in the supernatant of various groups. Statistical differences were calculated using a student’s t-test (B, C, and D) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). E, F Hydrogel vaccine conferred therapeutic protection against melanoma and C26 tumors. E Percentage survival of B16F10 tumor-bearing mice (n = 12). F Survival percentage of C26 tumor-bearing mice. G Schematic of photothermal hyaluronic acid-pluronic F-127 (HP) hydrogel. H, I Anti-tumor recurrence effect of NIR-triggered cancer immunotherapy in 4T1-luc and B16F10-luc tumor model. H Percentage survival of 4T1-luc tumor-bearing mice (n = 10), I Percentage survival of B16F10 tumor-bearing mice (n = 7). J-M Photothermal vaccine induced a robust anti-tumor immune response. J Representative flow cytometry plots of different groups of T cells in the residual tumors from different groups, K Number of CD8 + T cells per field of tumor after treatment, L Serum IFN-γ levels and M serum TNF-α levels in mice after treatment (n = 4); mean ± s.e.m. (#1) PBS, (#2) CCNV, (#3) a-PD1, (#4) Gel-BPQD-CCNVs, (#5) Gel-BPQD-CCNVs + NIR, (#6) Gel-BPQD-CCNVs + NIR + a-PD1. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Tukey post hoc tests (J, K, L, and M) (Reprinted with permission from Ref. [85, 86];

Copyright 2020 Elsevier Ltd. and 2021 American Chemical Society, respectively)

Some unique hydrogel vaccines exhibit dual functionality by taking advantage of the tunable thermoresponsive properties of hydrogels. Photothermal hyaluronic acid-pluronic F-127 (HP) hydrogels allow stable encapsulation of GM-CSF, lipopolysaccharide (LPS), and black phosphorus quantum dot nanovesicles (BPQD-CCNVs) (Fig. 5G). These hydrogels not only recruit and activate T cells and DCs by releasing GM-CSF and LPS, but also act as a heat source. Stimulating HP hydrogels with near-infrared radiation (NIR) generates local heat that accelerates the infiltration of recruited T cells into the TME (Fig. 5J–M). Hypodermic injection of HP hydrogels into 4T1-luc and B16F19-luc models greatly enhanced mice survival rates compared to the control groups (Fig. 5H–I) [86].

The ultrasound-responsive self-healing nanocomposite hydrogel system is another immune vaccine delivery platform that effectively uses the hydrogel’s thermoresponsive property. This hydrogel relies on external stimuli to release its cargo; this allows greater control over the gel’s release profile. In this study, the immunostimulatory antigen ovalbumin and adjuvant imiquimod (R837) were encapsulated into a precursor solution containing oligo (ethylene glycol) methacrylate. When stimulated with ultrasound, the hydrogel transformed into a sol formulation, explosively releasing its vaccine cargo. Interestingly, the gel exhibited self-healing characteristics during the absence of ultrasound. The immunotherapeutic vaccine released from this hydrogel induced significant levels of repeated anti-tumor T cell responses [87].

Delivery of ICIs

Immune checkpoints are inherent shielding mechanisms that maintain proper immune reactions and protect healthy tissue from off-target immune attacks [88]. PD-1/PD-L1 and CTLA-4 are examples of immune checkpoints that are most frequently addressed in immunotherapy [16, 89]. CTLA-4 is a co-inhibitory receptor molecule that controls the activity of T cells. When CTLA-4 binds to specific ligands (CD80 or CD86) on T cells, T cells lose their functionality, leaving tumor cells to develop without immune intervention [90]. Inhibiting CTLA-4 via antibodies before T cell binding allows T cells to function fully in the TME [90]. PD-1 is an immune checkpoint expressed on T cells during inflammation that allows abnormal cells to detect [91]. Unfortunately, tumor cells can bypass T cell surveillance by expressing PD-L1, a conjugate inhibitor molecule of PD-1; PD-L1 inhibits the ability of a T cell to detect abnormal cells [15]. Monoclonal antibodies that target these checkpoints can reinforce T cell surveillance or inhibit the interactions mentioned above to prevent the proliferation of undetected tumor cells [92].

The FDA has already approved many ICIs, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, and durvalumab clinical use [16]. Although ICIs show improved survival and therapeutic efficacy compared to traditional chemotherapy [93], limitations still exist. First, systemic administration of ICIs may induce severe adverse effects across various organs [94–96]. Second, ICIs—similar to immunotherapy in general—apply to only a limited population [97, 98]. Finally, different TMEs show different inhibition mechanisms that limit the universal use of approved ICIs [99]. There is no doubt that ICIs are currently the most potent immunotherapy method available. The heightened interest in ICIs has driven researchers to overcome the limitations of ICIs via various delivery platforms, including hydrogels.

A shear-thinning injectable nanofiber hydrogel based on betamethasone phosphate was developed for the local delivery of anti-PD-L1 (Fig. 6). Betamethasone phosphate is a clinical steroid drug frequently used for the treatment of arthritis and asthma. Surprisingly, intermolecular hydrogen bonds and hydrophobic interactions between steroid molecules, as well as coordination between phosphate groups and calcium ions, enable this drug to take the form of a nanofiber hydrogel (Fig. 6A). This nanofiber hydrogel loaded with anti-PD-L1 can promote efficient T cell infiltration into a tumor and decrease the proportion of immunosuppressive cells, hence reprograming the immunosuppressive TME into an antitumoral TME (Fig. 6C–D). Moreover, this hydrogel also acts as an adequate reservoir for the controlled and sustained release of α-PD-L1, promoting a T cell-mediated immune response to cancer cell attacks and significantly inhibiting local tumor growth (Fig. 6B) [100].

Fig. 6.

A Schematic shows the formation of nanofiber hydrogel. B Tumor growth kinetics in different groups. C, D Systemic immune responses induced by local injection of αPDL1@BetP-Gel. C Percentages of TILs (CD3 +), of CD8 + TILs (CD3 + CD8 +), and Gz-B positive CD8 + TILs (CD3 + CD8 + Gz-B +), respectively (from left to right). D Percentages of regulatory T cells (CD3 + CD4 + FOXP3 + , T_regs) (Reprinted with permission from Ref. [100];

Copyright 2020 American Chemical Society)

Nanofiber hydrogels formed by the cross-linking of filamentous assemblies via physical interaction exhibit repairable sol–gel transformation, allowing injection into the TME with minimal invasion [101–103]. Due to this characteristic, various hydrogelators have been investigated to design effective self-delivery nanofiber hydrogels [104, 105]. A drug-based supramolecular hydrogelator was recently used for the local co-delivery of anti-PD-1 antibody and CPT [106]. The amphiphilic prodrug used in this SH, diCPT-PLGLAGiRGD, includes iRDG, a peptide known to facilitate tumor penetration anticancer agents [107]. In an aqueous environment, the diCPT-PLGLAGiRGD hydrogel self-assembles into supramolecular nanotubes, forming a reservoir for the sustained release of CPT and anti-PD-1. Once localized in the TME, the cargos stimulate the production of type 1 interferons and chemokine CXCL10. This further induces the expression of immune-stimulating phenotypes in the TME and increases the level of effector T cells, leading to 100% tumor regression in GL-261 and CT26 cancer models [106].

Reactive oxygen species (ROS) are integral signaling agents produced within the TME [108]. These agents participate in various biological processes, induce cell apoptosis, control PD-1 expression, regulate T cell activity, and promote tumor development [109]. Early studies emphasized the relationship between ROS and the immunosuppressive TME, stimulating many studies focusing on creating a ROS-free environment in the TME [110, 111]. Subsequently, a unique strategy using ROS to deliver various ICI antibodies was developed [112–114].

The ROS-reactive gel is not only a reservoir to regulate the delivery of therapeutic agents, but a scavenger of ROS, further enhancing immunogenic phenotypes of the TME. This approach can treat low-immunogenic tumors, which have been known to be insensitive to ICIs. In one study, gemcitabine and anti-PD-L1 were locally released via a ROS-degradable hydrogel in B16F10 and 4T1 tumor models to achieve successful chemo-immunotherapy (Fig. 7A). Apart from the anti-tumor responses enhanced by local gemcitabine and anti-PD-L1, the ROS hydrogel itself also stimulated systemic immune responses and the formation of memory T cells, which prevented additional tumor recurrence (Fig. 7B–C) [110]. In another study, different ROS-responsive hydrogels were used to encapsulate anti-PD-L1 and dextro‐1‐methyl tryptophan, an inhibitor indoleamine-2,3-dioxygenase (IDO) (Fig. 7D). Composed of P(Me-D-1MT)-PEG-P(Me-D-1MT) triblock copolymers, this thermos-gelling ROS-responsive hydrogel not only stimulated immune response by sustaining local release of therapeutic agents but also decreased the level of ROS within the tumor (Fig. 7E–I) [115].

Fig. 7.

A Schematic of combination chemo-immunotherapy using a ROS-degradable hydrogel scaffold to deliver GEM and aPDL1 into the TME. B GEM@Gel implantation for eliciting immunogenic tumor phenotypes. Representative flow cytometric analysis of T cell infiltration within the tumor and corresponding quantification results. UnTx, untreated. CD45 + , CD8 + and CD4 + , respectively (from left to right). C ROS-degradable hydrogel for T cell memory. Corresponding quantification of CD4 and CD8 central memory T cells (TCM) in splenocytes, respectively (from left to right). D Schematics of the injectable thermogelling ROS-responsive hydrogel for tumor microenvironmental regulation and immunotherapy. E H₂O₂‐triggered (10.0 × 10⁻³ m) IgG release behavior in PBS (pH = 7.4). F Proportion of tumor‐infiltrating CD45 + T cells. G Proportion of tumor‐infiltrating CD8 + T cells. H Intratumoral H₂O₂ intensity test after treated with/without 8.0 wt% hydrogel in 48 h. I Proportion of intratumoral H₂O₂ intensity according to H. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. **p < 0.01 (From Ref. [110, 115]; reprinted with permission from AAAS and 2018 WILEY–VCH Verlag GmbH & Co. KGaA, Weinheim, respectively)

Other hydrogels recently used in ICI delivery include the bare pluronic F-127 hydrogel and DNA polyaptamer hydrogels [116, 117]. Bare pluronic F-127 was used as a thermosensitive platform for the delivery of anti-CTLA-4 and anti-PD-1 antibodies. Despite the relative simplicity of the hydrogel synthesized in this study, the hydrogel extended the half-life of delivered antibodies by ~ 20 fold. This result minimized the need for repeated injections as the antibody-loaded hydrogel demonstrated strong anti-tumor immunity in mouse breast cancer models [116]. A DNA polyaptamer hydrogel is a unique delivery platform that relies on aptamers to block immune checkpoints instead of antibodies. In this study, the aptamer hydrogel was designed to be precisely cut in the presence of Cas9/sgRNA. The hydrogel was synthesized using the RCA method, the RCA products containing repeats of the aptamer and the sgRNA being cross-linked via internal sites. Even though Cas9/sgRNA is not usually present in the TME, the co-injection of Cas9/sgRNA with the hydrogel initiated the controlled release of PD-1 aptamers, promoting tumor-specific immune responses [117].

Delivery of CAR-T Cells

Immunotherapy using CAR-T cells has become widespread following its success in various clinical applications. T cells isolated from a patient are transferred into a test tube to express a receptor against a specific antigen and re-administered to the patient as CAR-T cells [118]. After injection, the CAR-T cells can uniquely identify and kill tumor cells [119]. These cells can remain active for ten years after injection, making it a more beneficial single-dose therapy than other cancer therapies [120, 121]. There are currently two CD19-targeting CAR-T cells clinically approved by the FDA: tisagenlecleucel for acute lymphocytic leukemia and diffuse large B cell lymphoma (DLBCL) axicabtagene ciloleucel for DLBCL [122, 123]. The clinical success of CD19-targeting CAR-T cells has prompted the further application of CAR-T cells to various disease-related antigens [118, 124, 125].

However, CAR-T cells also suffer serious drawbacks. A significant problem is a phenomenon known as “on-target, off-tumor” activity [126]. Even though CD19 is an antigen commonly expressed on B cell leukemia and lymphoma, CD19-targeting CAR-T cells may also affect normal immature B cells, causing B cell aplasia [126]. Other side effects may include CRS or neural poisoning [127, 128]. The highly time-consuming and costly process associated with CAR-T cell development is also a practical limitation that needs to be overcome [129]. However, the most critical problem of current CAR-T cell therapy is that CAR-T cells show low efficacy against most solid tumors except for glioblastomas that express the protein EGFRvIII [24, 25, 130]. Novel administration strategies and delivery platforms have been studied to increase the efficacy of CAR-T cells against solid tumors.

One approach to avoid the “on-target, off-tumor” effect and increase tumor infiltration of CAR-T cells is to directly inject the cells into the solid tumor [131]. Unfortunately, CAR-T cells need a continuous supply of oxygen and a specific immune niche, making an immunosuppressive and hypoxic TME unsuitable for CAR-T cells [132–134]. To enhance CAR-T cell survival and proliferation within the TME, Luo et al. developed an injectable hydrogel-encapsulated porous immune-microchip system (i-G/MC) that reduced the hypoxic environment and built an immune niche suitable to CAR-T cells (Fig. 8A) [135]. Once injected into the tumor, the hydrogel layer surrounding the microchip degrades rapidly to deliver HEMOXCell, marine extracellular hemoglobin that can function as an oxygen carrier and a depot [136]. Sustained release of oxygen into the TME via HEMOXCell causes high oxygen tension within the TME, facilitating the survival of infiltrating immune cells [135]. Following the degradation of the hydrogel is the release of CAR-T cells and IL-15, a potent cytokine that promotes the proliferation and memory of T cells into the tumor stroma (Fig. 8B–D) [137, 138]. The i-G/MC system improved significantly CAR-T cell survival and immunotherapeutic efficacy in solid tumors via the co-delivery of HEMOXCell and IL-15 (Fig. 8C–F) [135].

Fig. 8.

A Schematic of the i-G/MC with the capability of enhancing CAR-T cell survival and persistence by improving hypoxia in the TME and building an immune niche. B Proliferation of T cells cultured in various MCs under hypoxic conditions. C IL-2 and D IFN-γ release from CAR-T cells detected by ELISA after coculture with Meso + SKOV-3 cells under hypoxic conditions. E, F Anti-tumor efficacy of CAR-T cells delivered intratumorally by the i-G/MC in vivo. E average tumor growth, F survival curves of various treatment groups (Reprinted with permission from Ref. [135];

Copyright 2020 American Chemical Society)

A recent study successfully treated retinoblastoma in mouse models using an injectable chitosan-PEG hydrogel as a platform for CAR-T cell delivery [139]. Because retinoblastoma cells overexpress ganglioside GD2 [140], CAR-T cell therapy has been actively researched. In addition, the biocompatible and biodegradable characteristics of chitosan make it an effective carrier for biological agents [141, 142]. Previous studies on chitosan-PEG hydrogels have already shown that the gel’s low viscosity makes it an effective platform for cell delivery [141, 143]. In one study, GD2-specific CAR-T cells were co-encapsulated with IL-15 in a chitosan hydrogel and were locally injected to the tumor site. The co-delivered IL-15 actively prompted CAR-T cell activity while being locally restricted by the chitosan hydrogel. Local injection of this immunotherapy system in a mouse yielded noteworthy therapeutic effects; the CAR-T cells were able to successfully eliminate retinoblastoma cells without impairing mouse vision [139].

CAR-T cells are not the only cells delivered as therapeutic agents in immunotherapy. Other cells such as DC or normal T cells have also been encapsulated within various hydrogels for enhanced immunotherapy [144, 145]. Although the abovementioned various cell-based immunotherapies are successful, there is still a clinical problem associated with rapid mass production of patient-specific T cells [118]. A recent study addressed this problem by developing an external hydrogel depot resembling the lymph node for T cell expansion and differentiation [146]. To truly mimic the lymph node, three-dimension poly(ethylene) glycol hydrogel was mixed with heparin for its structure and mechanical characteristics, then loaded with CCL21 to promote T cell proliferation [146]. CCL21 is a cytokine native to the lymph node and is known to enhance CD4 + and CD8 + T cell activity by binding to the chemokine receptor CCR7 of naïve T cell and APCs [147]. Isolated T cells incubated within this hydrogel showed significant ex vivo activation and proliferation, making it a powerful platform for rapid immunotherapeutic CAR-T cell production [146].

Conclusion

The continually rising interest in functional polymers has introduced various novel delivery platforms, particularly in cancer immunotherapy. In this review, we have specifically explored the status of hydrogels in the administration of various immunotherapeutic agents. The low toxicity, biocompatibility, and biodegradability of hydrogels make this material an ideal subject for biomedical applications. Nevertheless, to make full use of its potential, hydrogels for immunotherapy must also exhibit high drug-loading capacities and flexible responses to multiple stimuli. Most of the agents used in immunotherapy have low solubility, low stability, and need sustained delivery, making hydrogels even more appealing.

These unique advantages of hydrogel-based systems have inspired many researchers to develop local sustained release platforms for immunotherapeutic agents and overcome the many drawbacks of current immunotherapy methods, including systemic toxicity and limited efficacy. However, several critical considerations must be resolved before transferring these experimental results to clinical applications. First, the pharmacokinetics of therapeutic agents are relatively easily observed and explained in experimental environments, whereas biological interactions in vivo, especially in humans, make real-life analysis extremely difficult. Moreover, even though the short-term biocompatibility of hydrogels may be evaluated with animal models, animal models do not assure identical organ biocompatibility in humans [148]. Novel hydrogels must be carefully analyzed under appropriate experimental conditions before clinical application.

The demand for patient-specific immunotherapies is continually increasing following the recent improvements in cancer immunotherapy. Such an increase in demand by patients may indicate the increasing acceptance and approval of immunotherapy as a novel cancer therapy. Nevertheless, this also emphasizes the need to control the characteristics of therapeutic agents more precisely. Thus, a much more comprehensive investigation into the precise mechanisms and dynamics of cargo release from alternate hydrogels is needed to drive hydrogel-based delivery platforms toward clinical application. Nevertheless, current research in hydrogel and immunotherapy indicates the clinical success of highly controlled hydrogel-based therapies soon.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

There are no animal experiments involved in this work.