Recent Advances in Nanomodulators for Augmenting Cancer Immunotherapy in Cold Tumors: Insights from Drug Delivery to Drug‐Free Strategies

Immunotherapy has significantly improved cancer treatment, yet the immunosuppressive tumor microenvironment (TME) remains a substantial impediment to therapeutic efficacy. Nanomodulators have emerged as promising tools to address immunosuppressive factors within the TME, enhancing clinical interventions such as immunotherapy, chemotherapy, and radiotherapy while minimizing associated safety risks with immune modulators. In this review, recent advancements are spotlighted in TME‐targeted nanomodulators from drug delivery to drug‐free concepts. First, nanomodulators designed to synergize with various immunomodulatory agents, including gene tools (mRNA, siRNA, miRNA, plasmid DNA, and CRISPER system), cytokines, immune agonists, and inhibitors are analyzed. Subsequently, recently developed drug‐free nanomodulators designed to modulate the physicochemical and biological properties in the microenvironment of solid tumors are succinctly presented. Finally, integrative perspectives on the future development and challenges of nanomodulators in assisting cancer immunotherapy are offered as conclusions.


Introduction
Immunotherapy has emerged as a paramount choice in the battle against solid tumor growth, metastasis, and recurrence by diligently monitoring and removing elusive tumor cells that are resistant to conventional treatments. [1]onetheless, the efficacy of cancer immunotherapy has benefited only a minority of patients (<20%), primarily attributed to the profoundly immunosuppressive tumor microenvironment (TME). [2]The TME predominantly comprises tumor cells, stromal cells, tumor-infiltrating immune cells, and the extracellular physiological milieu. [3]The tumor stromal microenvironment comprises vascular networks and tumor-associated fibroblasts (TAFs) and furnishes an ideal milieu for tumor angiogenesis via producing vascular endothelial growth factor (VEGF) and the orchestration of treatment resistance (involves antigen mutation and the high expression of immune checkpoints). [4]The extracellular matrix (ECM), metabolic acidosis, hypoxia, and reactive oxygen species (ROS) collectively intensify the exhaustion and self-renewal ability loss of effector T cells and promote the expansion of regulatory T cells (Tregs) and the repolarization of tumor-associated macrophages (TAMs) from an antitumoral phenotype (M1) to a pro-tumoral phenotype (M2). [5]Meanwhile, cytokines and enzymes are released by pro-tumoral immune cells, such as IL-10 and TGF- from Tregs, [6] TGF-, IL-10, and arginase 1 from pro-tumoral phenotype TAMs and myeloid-derived supressor cells (MDSCs), [7] and indoleamine 2,3-dioxygenase (IDO) and IL-10 secreted by dendritic cells (DCs). [8]These species further dampen the tumor cytotoxicity of effector T cells and natural killer (NK) cells, and consequently foster the formation of an immunosuppressive network.5d,9] To counteract tumor immune tolerance induced by the TME, a variety of immunologic strategies including immune checkpoint blockade (ICB), personalized vaccine, and adoptive chimeric antigen receptor (CAR) T cell therapy have emerged, but their clinical efficacy remains suboptimal, primarily attributed to the absence of a systematic approach for activating the TME. [10]Alternatively, cancer nanomedicines serve as potent novel nanomodulators, which are TME-tailored formulations, leading to enhancement of the host anti-tumor immunity via reversion of the immunosuppressive TME. [11]A nanomodulator typically comprises three essential components, i.e., a nanoplatform, surface ligands, and immunomodulatory cargoes delivered (e.g., cytokines, agonists, inhibitors, genes, proteins, etc.).In previous studies, the pivotal aspect for achieving efficient activation of the TME through nanomodulators has consistently been the meticulous selection of specific surface ligands and potent immune agonists.By proficiently delivering emerging immunomodulatory drugs including gene tools (e.g., siRNA, mRNA, miRNA, plasmid DNA, and the clustered regularly interspaced short palindromic repeats (CRISPR) system), immune stimulatory molecules (e.g., cytokines, agonists), antigenic proteins and small molecule drugs into solid tumors and specifically regulating the activation, inactivation, or deletion of resident cells (e.g., TAFs, tumor cells, T cells, DCs, TAMs, and MDSCs), these nanomodulators effectively converts the TME from a cold to a hot state. [12]Besides, the inhibition of ECM formation and enhanced degradation of the ECM network through nanomedicine also promote the penetration of nanomedicines into solid tumors, relieve chemotherapeutic resistance, normalize the tumor vessels and improve the infiltration of immune cells. [13]Undoubtedly, these nanomodulators targeting the TME exhibit significant potential for converting a cold tumor into a state that enhances immunotherapy efficacy, but the challenges such as unelucidated toxicities and nano-bio-interactions, transparency in reproducibility, and complexity (expense) remain critical issues and continue to pose critical obstacles to their clinical translation. [14] is worth noting that nanoparticles (NPs) have been regarded solely as a drug delivery platform, but mounting evidence has substantiated that the intrinsic physicochemical properties of the materials empower themselves to directly influence the extracellular and intracellular physiological environment, thus enhancing the therapeutic efficacy of immunotherapy. [15]For instance, alkaline NPs for tumor excess acid neutralization, [16] oxygenproducible NPs for ameliorating tumor hypoxia, [17] and nutritional metal cation-doped NPs for relieving the nutritional element deficiency [18] prominently inhibit the tumor cells' proliferation and rescue exhausted T cells.In contrast to nanomodulators (nanomedicines), the maturation of nanotechnology has made the large-scale production of uniform NPs no longer a significant challenge.Henceforth, drug-free nanomodulators have well-defined crystal structures and simple compositions and are free from intricate modifications or delivered drugs, capable of directly or indirectly regulating immune cell functions and modulating the immunosuppressive TME, which will undoubtedly evolve into a pivotal facet of cancer immunotherapy.
In this review, we comprehensively summarize the recent advancements of nanomodulators for activating cold tumor tissues from the insight of drug delivery to drug-free strategies, rendering a better understanding of the design of TME-targeting nanomodulators for augmenting cancer immunotherapy (Figure 1).We first summarize recent progress in developing nanomodulators that are loaded with emerging gene-/protein-/cytokine-/antibody-/agonist-/small moleculebased immunomodulators.Subsequently, we review the most recently developed drug-free nanomodulators for the reversal of immunosuppressive TME by utilizing their physicochemical characteristics.We conclude with our integrative perspectives on the remaining challenges and the development of TME-targeted nanomodulators for efficient cancer immunotherapy.

Nanomodulators Deliver Immunomodulators to Reverse Cold TME
Significant strides have been made in the field of immunomodulatory tools recently, aiming at achieving systemic activation of cold tumors.Notably, the gene toolbox including RNA, plasmid DNA (pDNA), and the CRISPR system has enhanced immune recognition by selectively modifying receptor expression on tumor or immune cells.Other novel immunomodulators such as cytokines and immune agonists/inhibitors have shown promise by broadly activating immune cells residing within the tumor tissues.Compared with the free immunomodulators, nanomodulators carrying immunomodulators can be surface modified to possess in vivo colloidal stability and the enhanced permeability and retention (EPR) effect, enabling efficient delivery of immunomodulators into the TME and the targeted cells and specifically regulating the function of tumor and tumor-resident immune cells. [19]In this section, we comprehensively overview the recent advancements in nanomodulators for cold tumor activation to enhance tumor immunotherapy.

Gene Toolbox
Gene tools such as RNA, pDNA, and the CRISPER system have played vital roles in regulating the expression of targeted genes in cells.mRNA is a template for temporarily regulating the expression of targeted genes. [20]siRNA is able to temporarily downregulate gene expression. [21]miRNA binds to target mRNA and interferes with its translational inhibition or degradation. [22]ifferently, pDNA encoding genetic information exhibits superior physiological stability and long-term modulation of specific gene expression. [23]The CRISPR-associated protein 9 (CRISPR-Cas9) system is composed of Cas9 nuclease and single guide RNA (sgRNA) and is designed to specifically cleave a target sequence. [24]Based on the functions of gene tools, the recently developed gene-related nanomodulators are presented below.

mRNA
mRNA functions as the template of protein expression in the cytoplasm, averting the need for nuclear localization and the risk of genomic integration and mutagenesis.As mRNA carriers, nanomodulators can regulate the intracellular signaling pathway to reprogram the immune TME for effective cancer immunotherapy. [25]The loss of tumor suppressor gene functions p53 not only promotes malignant tumor cell proliferation and treatment resistance, [26] but also restricts the transcriptional regulation of genes encoding for cytokines essential for the activation of NK cells and M1 TAMs. [27]Xiao et al. uti-lized lipid-polymer hybrid NPs to deliver CXCR4-targeted p53encoding synthetic mRNA to restore the p53 expression of tumor cells, which tremendously delayed the growth of p53-null hepatocellular carcinoma (HCC) by triggering cell cycle arrest and apoptosis. [28]Similarly, another work also showed that a redox-responsive NP loaded with p53 mRNA could sensitize p53-deficient HCC cells to achieve mTOR signaling pathway inhibition. [29]Simultaneous targeting of tumor-p53 and mTOR signaling pathways yielded significant tumor growth regressing in animal models of HCC and non-small cell lung cancer (NSCLC).Notably, no detectable innate immune response was observed in mice following p53-mRNA NP treatment even after a 24-h period, which underscores the biosafety and feasibility of the current therapeutic approach.Besides, the loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) not only decreases T cell infiltration but also promotes the accumulation of MDSCs and Tregs within the TME. [30]in et al. designed a methoxy poly(ethylene glycol)-poly(lacticco-glycolic acid) (mPEG-PLGA) copolymer NP loaded with synthetic PTEN mRNA to increase PTEN expression in tumor cells and reversed the immunosuppressive TME. [31]The in vitro studies demonstrated an increased expression of PTEN in Ptenmutated melanoma cells and Pten-null prostate cancer cells significantly induced autophagy and triggered cell death-associated immune activation.The in vivo results further showed that the nanomodulator notably remolded the immunosuppressive TME by augmenting cytotoxic T cell infiltration, elevating proinflammatory cytokines content, and decreasing the proportion of MD-SCs and Tregs.
Moreover, signaling dysfunction in the TME-infiltrating T cells also limits the antitumor immunity for cancer immunotherapy.Li et al. utilized phospholipid (PL) and glycolipid (GL) biomimetic NPs for delivering the OX40 mRNA to increase the expression of OX40 (crucially involved in T-cell proliferation, cytokine secretion, and the stimulation of antitumor immune responses) in tumor-resident T cells, which efficiently elevated the therapeutic response rate of ICB such as anti-OX40 antibody (Figure 2a). [32]n this study, the authors showed that the increased OX40 expression in T cells significantly elevated the response rate of anti-OX40 antibody to 50-60% in A20 and B16F10 tumor models, promoted the infiltration of CD8 + and CD4 + T cells into tumor tissues, and decreased the number of immunosuppressive Tregs.In addition, Wang et al. constructed a lipid NP encapsulated with mRNA encoding a single-domain antibody to specifically bind and neutralize CCL (C-C motif chemokine ligand)2 and CCL5 (BisCCL2/5i), thereby polarizing TAMs toward the M1 phenotype. [33]Zhang et al. leveraged cationic poly(-amino ester) (PbAE) polymer NPs to deliver mRNA encoding interferon regulatory factor 5 (IRF5) and IKK (a kinase that phosphorylates and activates IRF5) to increase M1 phenotype TAMs in the TME, reverse the immunosuppressive response and augment tumor regression in ovarian cancer, melanoma and glioblastoma mouse models. [34]1.2.siRNA siRNA offers an alternative to antibodies as it can down-regulate intracellular expression of target genes based on only a short sequence, which is generally cheap and easy to manufacture.However, the lack of efficient vehicles for its transport to tumor cells or immune cells hinders its clinical translation.Nanomedicines provide a potent strategy to deliver siRNA to enhance cancer immunotherapy.[35] Guo et al. designed a nucleic acid nanogel assembled from DNA backbone, pheophorbide A (PPA) photosensitizers, and programmed death-ligand 1 (PD-L1) siRNA for synergistic cancer photo-immunotherapy. [36] When the nanogel was captured by tumor cells, the PPA photosensitizer-mediated photodynamic treatment induced remarkable immunogenic cell death (ICD) while the released siRNA quickly down-regulated the expression of PD-L1 to produce a potent antitumor immune response, which inhibited both primary and distal tumors.Zhang et al. utilized the cationic moieties of poly(amidoamine) (PA-MAM) to deliver the PD-L1 siRNA to tumor cells to decrease the PD-L1 expression.[37] The PAMAM/siRNA nanocomplex was released in tumor tissues through the tumor-acidity-sensitive linkage, which enhanced the uptake by tumor cells.As a result, PA-MAM/siRNA exhibited 75.4% tumor inhibition and the lowest proportion of Treg infiltration in the CT26 tumor model.Furthermore, Walters et al. constructed stable nucleic acid-lipid NPs co-loaded with PD-L1 siRNA and OX40L mRNA for modulating the TME.[38] The in vivo data showed that the reduction of PD-L1-mediated immune suppression and the augment of OX40L expression together facilitated the activation and proliferation of T cells, thus successfully reversing the TME into an immunos-timulatory state and achieving a comparable high tumor suppression efficiency (80%).In addition, Yan et al. designed selfadaptive platelet pharmacyte-contained polycationic nanocomplexes which were assembled from Rab27 (a key gene for regulating the secretion of exosomes) siRNA and cytotoxic T lymphocyte (CTL)-responsive anti-PD-L1 (aPDL1) nanogels.[39] The silence of Rab27a relieved the immunosuppressive TME via decreasing the tumor-derived exosomes, increasing the infiltration of cytotoxic T cells, and triggering the release of aPDL1, thus inducing strong antitumor immunity and memory effect against syngeneic murine melanoma.
Besides, the "self" signals expressed by tumor cells can also be down-regulated by siRNA-based nanomodulators.Zhang et al. designed PLGA-based NPs to deliver CD47 siRNA and mitoxantrone hydrochloride (MTO•2HCl) to cancer cells (Figure 2b). [40]he CD47 siRNA suppressed the "self" signal while the MTO induced the calreticulin (CRT) exposure to provide "eat me" signal, thereby increasing the phagocytosis of tumor cells by macrophages for antigen presentation and initiating potent immune responses against melanoma and colon tumor.Moreover, Li et al. developed an M2 TAM-targeting nanomicelle to codeliver the PI3K- inhibitor NVP-BEZ 235 and CSF-1R-siRNA for TAM reprogramming. [41]PI3K- blockade and downregulation of CSF-1R significantly elevated intratumor M1 TAM level and downregulated the infiltration of MDSCs.This, in turn, remodeled the TME and activated systematic antitumor immune responses in pancreatic tumors.In another work, Xiao et al. used acid-responsive micellar nanodrug with M2 TAM-targeting peptides to co-deliver the IKK siRNA and STAT6 inhibitor AS1517499.Specific M2-to-M1 repolarization and suppression of tumor growth and metastasis were also been observed in the 4T1 tumor. [42]1.3.miRNA miRNAs play a central role in mRNA translation inhibition or degradation via binding target mRNA, which can regulate cell proliferation, differentiation, and survival.The miRNA dysregulation, or genetic and epigenetic alterations, induces global miRNA depletion in tumor cells, which often leads to oncogenesis.[43] Nanomodulators loading with miRNA to modulate immunosuppressive TME for cancer immunotherapy have thus recently been explored.Zhang et al. assembled doxorubicin (DOX), adenosine triphosphate (ATP), and copper ions (Cu 2+ ) with miR-448 (IDO inhibitor) to generate coordination polymer NPs, the surface of which were further modified with polydopamine (PDA) for photothermal therapy.[44] The downregulation of IDO by miR-448 resulted in a considerably higher level of mature DCs and infiltrated CD4 + and CD8 + T cells but a lower level of Tregs in TME, thereby achieving remarkable inhibition of primary and distant tumor growth in 4T1 tumor-bearing mice.In another study, miR-200c, which can effectively inhibit the PD-L1 expression and sensitize tumor cells to pro-apoptotic signals, was co-delivered into tumor cells with the chemical drug DOX by PLGA-PEG NPs.The DOX/miR-200c NPs induced conspicuous inhibition of PD-L1 expression and ICD in cancer cells, resulting in a high level of DCs maturation and effector T cell response in solid tumors.[45]  Additionally, miRNA has also been used to regulate the repolarization of TAMs.Gao et al. designed a virus-mimicking membrane-coated nucleic acid nanogel (Vir-Gel) with a surface modified with M2pep and HA peptides to increase its blood circulation and promote the TAM uptake of miR155 (Figure 2b).[46] The in vivo immunological data showed that the proportion of M1 phenotype TAMs in glioma was significantly increased due to miR155 polarization of TAMs into pro-inflammatory phenotype through potentiating NF-B activity.[47] As a result, a potent antitumor immunity was initiated and the survival of glioma-bearing mice was greatly prolonged.Moreover, Yang et al. utilized layered double hydroxide (LDH) NPs to load miR155 (LDH@miR155) and treat a TC-1 tumor via intro-tumoral administration.[48] The in vivo studies showed that LDH@miR155 markedly inhibited the content of phosphorylated STAT3 and ERK1/2 and activated the NF-B signal pathway for TAM polarization.Consequently, LDH@miR155-treated mice exhibited diminished infiltration of M2 TAMs and MDSCs and an increase in effector T cells, ultimately leading to the effective suppression of tumor growth.In addition, nanomodulators such as polyethyleneimine-modified dendritic mesoporous silica NPs loaded with microRNA-125a [49] and CD44 modified hyaluronic acid-poly(ethylenimine) (HA-PEI)-based NPs loaded with miR125b [50] were also observed to effectively inhibit the growth of TC-1 and NSCLC tumor in vivo, respectively.

pDNA
Synthetic pDNA encodes genetic information, which offers to deliver exogenous cargoes into living cells by vehicles.Compared with RNA, pDNA has a much higher physiological stability, contains more regulatory elements for regulating specific gene expressions, and induces long-term protein expression, which could be applied in diverse scenarios. [23]19a] Owing to the alterable gene information in pDNA, the delivery of pDNA to specific cells could make the cells restore functions for tumor treatment.Kang et al. recently presented a facile approach to program CAR-M1 TAMs through in vivo injection of mannose-conjugated polyethylenimine (MPEI) carrying CARinterferon (IFN)- pDNA (MPEI/pCAR-IFN-). [51]The expression of CAR in M1 TAMs facilitated direct phagocytosis of cancer cells, leading to potent antitumor effects.Simultaneously, the secretion of IFN- induced a pro-inflammatory TME by polarizing TAMs from the pro-tumoral M2 to the anti-tumoral M1.This enhancement in the activation of cytotoxic T lymphocytes (CTLs) and NK cells subsequently reduced the Treg population, prompted the additional secretion of pro-inflammatory cytokines including TNF- and IFN-, and suppressed the expression of IL-4 and IL-10 within the Neuro-2a tumor.Moreover, Qiu et al. designed an esterase-responsive polymer to enhance the transfection efficacy of IL-12 pDNA in the tumor cells and macrophages, which increased the M1/M2 ratio of TAMs and activated an antitumoral immune response to prolong the mouse survival in three mouse tumor models. [52]Huang et al. used a tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) carrying pDNA encoding IL-2, which was syner-gized with PD-L1 siRNA to promote the proliferation and activation of CD8 + T cells for enhanced cancer immunotherapy. [53]u et al. utilized a lipid nanocomplex to co-deliver CCL-19encoded pDNA and PD-L1 inhibitor into the tumor cells, which enhanced the expression of CCL-19 to induce T-cells proliferation, DC maturation, and TAM repolarization, significantly inhibiting colon cancer and melanoma growth. [54]pDNA encoding a multifunctional inflammatory cytokine LIGHT was loaded into calcium phosphate liposome (CaP). [55]With antifibrotic phosphates-modified -mangostin, this nano-sapper reshaped the TME by decreasing abnormal fibroblasts and collagen deposition, normalizing tumor vessels, recruiting and activating the CTLs and constructing intra-tumoral tertiary lymphoid, thereby remarkably promoting the efficacy of the ICB.
Interestingly, pDNA encoding specific enzymes has also been applied for normalizing the physicochemical properties of the TME such as hypoxia, which severely impedes photodynamic therapy (PDT) efficacy and immune cells' antitumor immunity. [56]Huang et al. designed a pH-sensitive polymetric micelle incorporating pDNA encoding catalase gene (pDNA-cat) and photosensitizer Ce6 to generate oxygen in the TME to relieve tumor hypoxia and amplify tumor PDT. [57]he expressed catalase catalyzed H 2 O 2 to O 2 in the TME while the PDT-responsive Ce6 induced ICD by generating cytotoxic ROS, which subsequently promoted DCs' maturation and augmented CTL's infiltration in the TME.As a result, this nanocomplex elicited strong antitumor immunity to inhibit tumor progress and recurrence in 4T1 tumor-bearing mice.
Furthermore, pDNA is also designed to encode short hairpin RNA (shRNA) to produce RNAi for gene knockdown.Feng et al. developed a poly(amino acid) NP to co-deliver pDNA encoding shRNA of VEGF-A (shVEGF-A) and shRNA of PD-L1 (pshPD-L1) against murine melanoma. [58]The combinatory silence of PD-L1 and VEGF-A gene sensitized tumor cells to the ICB, normalized tumor vessels, and down-regulated exhaustion molecules such as programmed cell death protein-1 (PD-1), cytotoxic T lymphocytes assocaited protein-4, T cell immunoglobulin-and mucin-domain-containing 3, and lymphocyte-activation gene 3 of CD8 + T cells in TME.Another work conducted by Wu et al. constructed an immune cocktail therapy to achieve multiple boosts of the cancer-immunity cycle. [59]The nanomodulator specifically delivered DOX and pDNA-expressed PD-L1 shRNA and Spam1 shRNA to the tumor area, which synergistically promoted the infiltration of CD4 + and CD8 + T cells and decrease of Tregs and M2 TAMs, achieving a 97.3% tumor shrinkage in B16F10 tumor mice models.In addition, Liu et al. engineered a liposome to encapsulate a plasmid encoding short hairpin RNA (shRNA) that targets LncRNA plasmacytoma variant translocation 1, and a chemotherapeutic agent Oxaliplatin (OX), which was then cloaked with a colorectal cancer (CRC) cell membrane and embedded within a bio-scaffold composed of hyaluronic acid and alginate. [60]This complex induced ICD by releasing OX and ameliorating granulocytic myeloid-derived suppressor cell-mediated immunosuppression, which significantly restrained perioperative CRC local recurrence by 97.8% with simultaneous distant metastasis reduction by 70.8%.

CRISPR/Cas9
CRISPR-Cas9 system is composed of Cas9 nuclease and sgRNA.24b] In this context, nanomodulators carrying the CRISPR-Cas system target and penetrate the tumor issue and increase the endocytosis effect, with the potential to overcome physiological barriers such as ECM and stroma.These systems can significantly improve gene editing efficacy and enhance cancer immunotherapy.Tang et al. designed a supramolecular cationic gold nanorod to deliver CRISPR/Cas9 plasmid for genomic disruption of PD-L1 and to conduct photothermal therapy (PTT) based on the second near-infrared-window (NIR-II) light (Figure 3a). [61]By virtue of the heat-inducible promoter (HSP), Cas9 transcriptional activation was initiated at 42 °C, a temperature also conducive to triggering tumor ICD.The genomic disruption of PD-L1 facilitated the maturation of DCs, decreased the level of Tregs and increased the infiltration of CD4 + and CD8 + T cells, increased the level of pro-inflammatory cytokines including INF-, IL-2, and TNF- in TME, thereby effectively inhibiting primary tumor growth and lung metastasis and forming durable immune memory responses to prevent tumor recurrence.Moreover, Yang et al. synthesized a programmable unlocking nano-matryoshka-CRISPR system (PUN) to knockdown PD-L1 and protein tyrosine phosphatase N2 (PTPN2). [62]Gene editing efficacy with 21.6% and 23.3% indel rate at the PD-L1 and PTPN2 locus, respectively, was observed.The knockdown of PD-L1 and deletion of PTPN2 spurred potent T-cell immune responses while amplifying the adaptive immune responses, significantly attenuating the immunosurveillance evasion and exhibiting potent antitumor efficiency and long-term immune memory against murine melanoma.
Interestingly, Zou et al. prepared a glutathione-sensitive polymer shell modified with angiopep-2 peptide and degradable cross-linker to encapsulate the single Cas9/sgRNA complex for polo-like kinase 1 (PLK1) editing to reduce the off-target mutations and avoid genome integration. [63]This nanocomplex (≈30 nm) with nearly neutral surface charges protected the Cas9/sgRNA complex from degradation by ribonuclease (RNase) and promoted its blood stability and circulation lifetime.The small size and angiopep-2 peptide functionalization promoted blood-brain barrier (BBB) penetration and glioblastoma-specific targeting.The disulfide-cross-linker was cleaved in situ by excessive intracellular glutathione (GSH) to release the Cas9/sgRNA complex in tumor cells.This strategy showed up to 38.1% of PLK1 gene editing efficiency with less than 0.5% of off-target in high-risk tissues, which extended the median survival (68 days versus 24 days with blank sgRNA treatment).In addition, Fan et al. designed a hierarchical RNA nano-cocoon containing programmable RNA nanosponges (RNSs) and DOX. [64]The Cas13a/crRNA RNP was anchored on RNSs for its activity suppression.The acidic endo/lysosomal microenvironment induced the decomposition of this complex and released Cas13a/crRNA RNP to specifically recognize and cleave the EGFR variant III (EGFRvIII) mRNA.The combined EGFRvIII mRNA silence and DOX significantly inhibited glioblastoma cancer cells in vitro and in vivo.24a] Zhang et al. developed a multiplexed dendrimer lipid NP (LNP) to co-deliver Cas9 mRNA, sgRNA, and focal adhesion kinase (FAK) siRNA (Figure 3b). [65]This study elucidates that targeted silencing of FAK induced solid tumor penetration and increased tumor cell endocytosis by altering contractility and tumor cell membranes, resulting in more than 10-fold enhancement of gene editing.Knockdown of the PD-L1 observably activated the antitumoral effect of CTLs, thereby inhibiting potential metastasis and tumor growth in a mouse metastasis model of ovarian cancer and prolonging the survival in an aggressive MYCdriven HCC model, which is featured with fibrosis and ECM barriers.

Immunomodulatory Cytokine
Cytokines have pivotal roles in regulating immunity, making them attractive as therapeutics for reversing the immunosuppressive TME.Representatively, interleukins (ILs) are closely linked to the activation of immune cells, [66] chemokines play a vital role in recruiting immune cells into the TME via ligandreceptor interactions, [67] the granulocyte-macrophage colonystimulating factor (GM-CSF) is the most widely used colonystimulating factor to recruit and stimulate the maturation of DCs, [68] and other cytokines such as IFN- and TNF play immune-regulatory function in tumor progression. [69]An appropriate concentration of cytokines is critical, as low doses of interleukins prove ineffective due to enzymatic degradation, while high concentrations of interleukins are frequently associated with systemic toxicity. [70]This section will introduce the utilization of nanomodulators for localized or targeted delivery of cytokines to the TME for immunomodulation.

IL
Representatively, IL-21 serves as a pivotal regulatory cytokine that fosters the proliferation, differentiation, maturation, and longevity of NK cells. [71]Meng et al. constructed a bio-orthogonal nanocarrier (N3-NK-NP) by conjugating IL-21 NPs on the surface of NK cells, then the NK cells and tumor cells in the TME were modified with complementary bio-orthogonal groups (azide (N3)/bicyclo [6.1.0]nonyne separately via nondestructive metabolic glycoengineering (Figure 4a). [72]Compared to the control group, this bio-orthogonal strategy increased the infiltration, recognition, and migration of NK cells into deeper solid tumors by nearly 4-fold.Meanwhile, IL-21 ILNPs hitchhiking on the surface of NK cells released IL-21 in situ at a low dosage (5 μg kg −1 ) to continuously foster NK cell proliferation and activation, recruit and activate innate immune cells such as macrophages and inherent NK cells into TME, thereby significantly inhibiting the progression of solid tumors.Similarly, Luo et al. conjugated the IL-12-loaded human serum albumin (HSA) NPs (INS-CAR T) onto the surface of CAR-T cells via bio-orthogonal chemistry (Figure 4b). [73]Owing to the elevated level of free thiols observed  on activated T cells in comparison to naïve T cells, IL-12 was selectively released from the INS-CAR T biohybrids in response to increased thiols during the activation of tumor antigen-specific CAR-T cells, subsequently recruiting and expanding CAR-T cells to boost CAR-T antitumor efficacy, reshape the immunosuppressive TME and eliminate the solid tumor tissues.Ou et al. devised a Treg cell-based targeted drug delivery system, on the surface of which pH-sensitive liposomes loaded with IL-2, a PD-L1 antibody, and imiquimod were conjugated. [74]Liposome-anchored Treg cells migrated and infiltrated in the TME under the chemoeffect, and then IL-2, PD-L1 antibodies and imiquimod were acidresponsively released in the TME to stimulate maturation and activation of DCs and CTLs, thus dramatically inhibiting both primary tumor and lung metastasis.

Chemokine
The use of chemokines for an elevated infiltration of various immune cells can systematically reshape the immunosup-pressive TME.Chen et al. used acid-responsive NPs to deliver CCL25 and CD47 siRNA for enhanced immunotherapy based on the deficiency of CCL25 expression in human or murine triple-negative breast cancers (Figure 4c). [75]The nanodelivery system promoted the specific release of CCL25 and CD47 siRNA, causing additional infiltration of CCR9 + CD8 + T cells and the blockade of the CD47/SIRP signaling of tumor cells.The nano-system not only inhibited tumor growth and metastasis with improved T-cell immune responses, but also synergistically facilitated ICB therapy in 4T1 tumors.Xiong et al. combined lymphotactin (XCL-1)-loaded sodium alginate with macrophage cell membrane-modified DOX-loaded PLGA NPs to modulate the immunosuppressive TME (Figure 4d). [76]he biomimetic nanocarriers effectively prolonged the circulation time and enhanced the accumulation of DOX in tumor tissues, which induced high expression of CRT and HMGB1.Meanwhile, intratumoral injection of XCL-1-loaded sodium alginate recruited a large quantity of XCR-1 + DCs to migrate into the TME for cross-presentation of TAA to CTLs.As a result, highly activated CTLs infiltrated in the TME and secreted high levels of cytotoxic cytokines, promoting strong antitumoral immunity.
In addition, to realize the slow-release delivery of CCL21, Poelaert et al. loaded CCL21 (elevate the infiltration of immune cells and inhibit tumor growth) into an alginate-based nanoformulation for the treatment of neuroblastoma. [77]Through intra-tumoral injection, nano-formulated CCL21 significantly increased the proportion of DCs, CD4 + and CD8 + T cells, NK cells, NKT cells, and M1 phenotype macrophages in tumor tissues, eliminating the tumor tissues and protecting 33% of the tumor model mice from the tumor rechallenge.In addition, CXCL10, the ligand of CXCR3, also attracts a series of immune cells such as DCs, macrophages, T helper cells (Th cells), CTLs, and NK cells. [78]Zhao et al. encapsulated CXCL10 in PLGA NPs that were then anchored onto the surface of erythrocytes via non-covalent conjunction. [79]Due to the colocalization of intravenously administered erythrocytes with metastatic tumors in the lung, CXCL10 was in situ released at the lung metastatic sites and notably increased the infiltration of effector immune cells such as DCs, NK cells, central memory, and effector memory CD8 cells.

Colony Stimulating Factor
GM-CSF is the most used colony-stimulating factor to recruit and stimulate the maturation of DCs. [68]Ke et al. designed bifunctional fusion membrane NPs (FM-NPs) consisting of autologous tumor cell membranes and bacterium membrane extracts. [80]M-NPs and GM-CSF were then encapsulated in an injectable alginate hydrogel, in which FM-NPs could induce the maturation of recruited DCs with tumor antigens.Furthermore, lowdose bacterium extracts in FM-NPs could also enhance the maturation and proliferation of DCs.Their study showed that the hydrogel not only facilitated DC maturation in tumor-draining lymph nodes (TDLNs) but also increased sufficient effector memory T cells into the TME.Combined with immune checkpoint inhibitor aPD-1, the hydrogel sufficiently regressed tumor growth and completely suppressed the recurrence after tumor resection in mice and human-derived gastric cancer organoids.
In addition to in situ recruiting DCs to TDLNs, the GM-CSF enrichment of DCs into the solid tumors also enables in situ vaccination and activates the suppressive TME.For instance, peritumoral injection of macroporous alginate gels loaded with GM-CSF could recruit intratumor DCs to present endogenous tumor antigens. [81]In this study, CpG oligonucleotide and doxorubicin-iRGD were conjugated on the gel to enhance the ICD of tumor cells, induce the infiltration of CTLs, and promote the repolarization of TAMs into the M1 phenotype.As expected, such macroporous alginate gels significantly delayed 4T1 tumor growth and reduced metastasis with increased DCs, CTLs, and M1 TAMs in solid tumors, thereby effectively preventing tumor recurrence and tumor metastasis upon re-challenge.In another similar work, Ramos et al. used injectable alginate cryo-gels loaded with GM-CSF and CpG oligonucleotide for recruiting DCs and infiltrating CTLs.Spermine-modified acetalated dextran NPs were loaded with the p53 activator Nutlin-3a for induction of ICD and then combined with this gel.As a result, peritumoral injection of this gel formulation inhibited tumor growth and prevented tumor recurrence. [82]

Other Cytokines
69b,83] Sufficient expression of IFN from delivered mRNA substantially polarized TAMs into antitumoral phenotype, induced potent antitumor immunity, and suppressed tumor growth.To harmonize the negative effect of IFN, CERSmediated IFN mRNA delivery was synergized with PD-L1 antibody therapy, which extremely prevented malignant metastasis in the lung.69a] Angelo et al. constructed isoAsp-Gly-Arg (isoDGR)-coated gold nanospheres bearing TNF to treat fibrosarcoma. [84]This formulation not only showed significant therapeutic activity in the murine fibrosarcoma model, but also increased Dox penetration in tumors after injection.In combination with IL12, IsoDGR-coated gold nanospheres served as a versatile platform for the delivery of multiple cytokines.Due to the double-edge effects of IFN and TNF, more efforts need to be made for further use in TME modulation.

Receptor Agonist and Inhibitor
Immune agonists and inhibitors also play a pivotal role in modulating the function of both tumor and immune cells.Representatively, immune agonists associated with stimulators of interferon genes (STING) [85] and toll-like receptors (TLRs) [86] have been shown to effectively activate the host's innate immune systems.Meanwhile, immune inhibitors that target to PI3K [87] and IDO [88] can also reshape the immunosuppressive TME by activating TAMs and CTLs while decreasing suppressive MD-SCs and Tregs.This section summarizes the recently developed nanomodulators that deliver these agonists or inhibitors.

Agonist
STING agonists have greatly elevated the efficacy of immunotherapy.Dane et al. designed discoid lipid nanodiscs (LNDs) to deliver STING-activating cyclic dinucleotides (CDNs) into solid tumors via self-assembly of PEGylated lipids (PEG, polyethylene glycol). [89]The LNDs effectively activated the cyclic GMP-AMP synthase (cGAS)-STING signaling pathway and significantly induced the antigen uptake by DCs and more effective CD8 T-cell priming.Li et al. conjugated biocompatible branched cationic biopolymers loaded with cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) onto APC-targeting microbubbles (MBs) via ultrasound (US)-guided release (Figure 5a). [90]Upon exposure to the US, the targeted MBs bounded to the cell surface and were oscillated to create transient pores in the cell membrane for delivery of cGMAP into the cytoplasm.Spermine-modified dextran (SpeDex) and anti-CD11b antibody (aCD11b) were conjugated onto the surface of MBs to promote the cGAMP loading efficiency and APC targeting ability.As a result, this complex remarkedly activated both cGAS-STING and downstream proinflammatory pathways to induce type I IFN responses and antigen-specific CTL activation, thereby inhibiting both primary tumors and metastasis.To avoid the metabolic instability of cGAMP, Wang et al. encapsulated SR717 (a non-nucleotide as a cGAMP mimetic) into human heavy-chain ferritin (HFn) NPs which possess the intrinsic BBB crossing ability. [91]After an intravenous injection of this nanocomplex, SR717 was durably accumulated in the glioma TME, which significantly elevated expression of STING signaling-related proteins, upregulated the level of proinflammatory cytokines, and recruited more CD8 + T cells, NK cells and DCs.In addition, emerging research has indicated that metal ions, such as Mn 2+ , also function to augment type I IFN responses via stimulating the STING pathway.Sun et al. co-assembled CDNs, Mn 2+ , phospholipid-(histidine) 11 (DOPE-H11), and a PEG-lipid layer into a coordination polymer (CMP). [92]The addition of Mn 2+ to cGAMP notably increased type I IFN (IFN-I) production in murine bone marrow-derived dendritic cells (BMDCs) and THP1 cells in a dose-dependent manner.After intra-tumoral administration of CMP loaded with cyclic di-AMP (CDA) and Mn 2+ (CMP CDA ), robust CD8 + T cell-mediated immune responses were reflected by the elevated level of IFN-, TNF-, CXCL10, and CCL5, which led to a 78% regression of the CT26 tumor.Intravenous administration of CMP CDA also induced potent anti-tumor effects with expanded memory CD8 + T cell subsets, repolarized M1 TAMs, and decreased MD-SCs.In comparison, CMP CDA showed superior anti-tumor therapeutic effects and elevated animal survival in multiple difficultto-treat murine tumor models to other STING-activating formulations.
Besides, intra-tumoral immunotherapy using TLR agonists is a promising modality for the treatment of solid tumors, which avoids the systematic adverse side effects. [93]For example, Bahmani et al. designed platelet membrane-coated PLA NPs loaded with TLR agonist resiquimod (R848) (PNP-R848) to induce antitumor responses. [94]Following intra-tumoral injection, R848 in PNP-R848 at lower doses notably enhanced DC activation, and resulted in an elevated level of CTLs within TME and effector memory T cells in the TDLNs.As expected, this treatment eradicated the established tumors and provided long-term immunity against tumor re-challenges in a mouse breast cancer model.For intravenous injection, the rapid diffusion of TLR agonists to non-target tissues often causes systemic off-target cytotoxicity, a challenge for the in vivo application. [95]To address this challenge, Manna et al. developed pathogen-like nano-assemblies, which were covalently linked with TLR agonists to augment CD8 and NK cell-mediated antitumor immune response (Figure 5b). [96]nspired by immune recognition of pathogens, TLR agonist heterodimer containing cell-surface-active peptide-based TLR 2/6 agonist and small-molecule TLR 7/8 agonist was assembled with sugar polymers into NPs.The TLRa heterodimer was designed to amplify the immune responses.Intravenous administration of this NP significantly augmented the percentage of tumorinfiltrating CD8 + CTLs and NK cells, as compared with the control group in a mouse melanoma model.As expected, limited off-target toxicity was verified in the blood cytokine analysis.Macrophage polarization induced by TLR agonists has been synergized with ICD caused by PDT to modulate the immunosuppressive TME by Chen et al. [97] In this study, photosensitizer pyropheophorbide-a (Pyro) and TLR agonist R848 were assembled into uniform nanosized particles (PyroR) by intermolecular hydrophobic interactions ROS and ICD generated by PDTpromoted maturation of DCs and activated CTLs while R848 polarized macrophages from M2 to M1 phenotype to remodel the cold TME.After intravenous administration of PyroR, the combination strategy greatly restricted the growth of primary, distant, and metastatic tumors, indicating a superior synergetic therapeutic efficacy in comparison with single modular therapy.

Inhibitor
Inhibition of PI3K has been demonstrated to effectively activate TME by Ding et al. [98] In this study, PI3K inhibitor IPI-549 and photosensitizer chlorin e6 (Ce6) were encapsulated into a liposome (LIC) to treat colon cancer.IPI-549 inhibited PI3K in MDSCs, promoting MDSC apoptosis and relieving the immunosuppressive activity of CTLs.LIC treatment significantly inhibited tumor growth and facilitated DC maturation and CTLs infiltration while decreasing the level of Tregs, MDSCs, and M2like TAMs in a mouse colon cancer model.Shen et al. developed a biohydrogel scaffold encapsulated with the PI3K inhibitor IPI549 and immunostimulatory chemotherapy drug OX for post-ablative cancer therapy. [99]After an in situ treatment, this immunostimulant hydrogel sustainably released OX and IPI549 to induce ICD and rescue PI3K blocking-dependent immunosuppressive effect.In another study, Song et al. designed an albumin NP containing PI3K inhibitor IPI-549 and PTX (Nano-PI) to co-modulate the immunosuppressive microenvironment in both lymph nodes and the tumor tissue (Figure 5c). [100]Benefiting from the excellent colloidal stability, Nano-PI not only promoted the delivery of chemo-immunotherapeutic drugs into both the tumor tissue and lymph nodes but also enhanced the drug internalization by macrophages there.The combination of PI-549 and PTX facilitated M2-to-M1 macrophage repolarization in tumors and lymph nodes in tumor-bearing mice.Nano-PI synergized with aPD-1 notably showed prolonged tumor remission and inhibition of lung metastasis, attributed to significantly re-shaping the immunosuppressive TME via polarizing TAMs from M2 to M1 phenotype, increasing the level of CD4 + and CD8 + T cells, B cells, and DCs, while decreasing Tregs and preventing T cell exhaustion in two breast cancer mouse models.
IDO-blockade has greatly elevated the efficacy of immunotherapy.Li et al. prepared a tri-functional immunostimulatory supramolecular containing IDO inhibitor indoximod (IND). [101]he successful inhibition of the IDO-mediated immunosuppressive pathway effectively activated T cell immune responses, remarkedly promoted the infiltration of CTLs and expression of perforin while decreasing the level of Tregs, leading to 80.8% tumor suppression and inhibited metastasis to the lung in a breast cancer mouse model.Moreover, He et al. designed a semiconducting polymer nanomodulator with IDO inhibitor 1-MT (SPN T ), which was conjugated to the side chain of the polymer backbone via an apoptosis-sensitive linker, to reinforce photodynamic-evoked immunotherapeutic outcomes (Figure 5d). [102]Upon near-infrared photoirradiation, SPN T induced rich ROS and ICD with more HMGB1, ATP, and CRT expressed, triggering the specific release of the IDO inhibitor 1-MT in tumor cells.The subsequent immunogenic apoptosis and IDO inhibition facilitated the maturation of DCs, infiltration of CTLs and fewer Tregs in the TME, effective inhibition of primary and distant tumor growth, and prevention of lung metastasis in 4T1 tumor-bearing mice.In addition, Ding et al. developed an intelligent nanomedicine, in which the IDO inhibitor NLG919 was encapsulated in the nanoscale MOF NPs, and small gold NPs loading a chlorambucil-based prodrug were anchored on the surface of MOF NPs to precisely activate chemoimmunotherapy in the TME while avoiding systemic toxicity. [103]A higher intracellular level of phosphate caused the MOF structure to collapse and the release of the IDO inhibitor.The in vivo activation of chemotherapy not only induced apoptosis of tumor cells but also triggered ICD while the IDO inhibition caused a sharp decrease of the Kyn/Trp ratio, significantly reversing the immunosuppressive TME.

Summary
Various types of immunomodulators are more efficiently delivered to the TME via nanoplatforms than free immunomodulators, attributed to the EPR effect.Once within the TME, these immunomodulators are released from nanomodualtors intercellularly or intracellularly.They either directly combine with the target genes or proteins to inhibit the functions or regulate the expression of targeted genes in tumor or immune cells, showing great promise in reversing the TME from immunosuppression to immunostimulation.However, immunomodulator-associated off-target systemic toxicity is still a challenge, [14a,104] which may be overcome by drug-free nanomodulators, as summarized in the following section.

Drug-Free Nanomodulators for Combating Immunosuppressive TME
Recently, many studies have explored the intrinsic immunomodulatory properties of nanomaterials as a drug-free approach to counteracting immunosuppressive TME.The concept of drugfree nanomodulators draws inspiration from the field of drugfree therapeutics, which utilizes the inherent physicochemical and biological properties of nanomaterials rather than combining them with traditional therapeutic agents to directly modulate the TME.Although this is still an emerging research direction, these biocompatible drug-free nanomodulators are expected to play an important role in reducing off-target cytotoxicity, simplifying the production process, and promoting clinical translation.In this section, the recent progress of drug-free nanomodulators for stimulating the immunosuppressive TME is introduced below according to their targets in the TME.

Reversal of Extracellular Microenvironment
The abnormal extracellular microenvironment such as excess acid, hypoxia, and structurally incomplete blood vessels are key factors that can accelerate the heterogeneity and progression of solid tumors. [105]Herein, we summarize recent advances in drug-free nanomodulators, revealing the complex interactions between nanomaterials and the extracellular tumor microenvironment, aiming to develop some guidelines for more efficient nanomodulator designs. [106]ntiacid treatment is feasible to reverse the TME from immunosuppressed to immune-activated state. [107]As demonstrated in previous clinical studies, strategical administration of bicarbonate (NaHCO 3 ) through the tumor-feeding arteries has been shown to increase the pH level in extracellular and intracellular regions of hepatocellular carcinoma (HCC), thereby initiating a cascade of molecular events that ultimately lead to cancer apoptosis and enhance the efficacy of chemotherapy. [108]However, the use of bicarbonate solution to treat solid tumors requires extremely high surgical skills.Instead, Zhang et al. utilized weakly alkaline LDH NPs for simultaneous TME acidity neutralization and tumor cell autophagy blockade for neoadjuvant tumor immunotherapy (Figure 6a). [109]Compared with NaHCO 3 , Na + /H + exchanger inhibitor cariporide, and vacuolar ATPase inhibitor omeprazole, [110] peritumoral injection of drugfree LDH NPs formed local LDH depot for persistent presence at the injection location for at least one week, during which the tumor pH was raised from 6.6 and maintained close to 7.0.Further investigations revealed that the weakly alkaline LDH NPs easily escaped from endosomes of tumor cells and participated in the autophagy pathway, thereby suppressing the acidification of autophagolysosomes to damage the autophagic flux and triggering tumor apoptosis.Peritumoral treatment of LDH significantly promoted the level of antitumor M1 TAMs and cytotoxic T cells in TME while decreasing the concentration of pro-tumor immune cells including M2 TAMs, regulatory T cells, and MDSCs, which collectively inhibited the growth of mice melanoma and colon cancer in both early and advanced stages.Meanwhile, LDH NPs also captured the tumor antigens released from dying tumor cells for initiating in situ tumor vaccination, which induced personalized antigen-specific antitumor T cells to further eliminate tumor cells.Furthermore, peritumoral injection of LDH NPs recruited more antigen-specific T cells into the TME and significantly improved the immune efficacy of the vaccine against solid tumors in combination with cancer therapeutic vaccines.Such a drug-free LDH immunomodulator shows promising potential for clinical translation for solid tumor immunotherapy.Similarly, lipid/alkalescent bicarbonate NP recently prepared by Ding et al. may serve as an alternative tool to neutralize the acidic TME and enhance cancer immunotherapy. [111]In this study, the simple and drug-free inorganic sodium bicarbonate NPs were synthesized via a fast microemulsion.Once the NPs reached TME, they neutralized the extracellular acid to regulate the lactic acid metabolism.Meanwhile, NPs internalized by tumor cells released a high amount of Na + to induce a surge in intracellular osmolarity, thereby activating the pyroptosis pathway, releasing DAMPs and antigens, and improving immune responses.The treatment of alkalescent bicarbonate NPs successfully inhibited primary/distal tumor growth and tumor metastasis.
Apart from neutralizing the extracellular acid, nanomodulators that supply O 2 in the TME also promote the reversal of immunosuppressive TME.Recently, Sun et al. first designed a nanozyme for simultaneous TME hypoxia alleviation and lactate depletion for tumor growth inhibition and antitumor immunity activation through a cascade reaction chain. [112]In this case, the nanozyme was constructed by co-loading partially cross-linked catalase (pcCAT) and lactate oxidase (pcLOX) onto the surface of peroxidized LDH NPs (MgO 2 formed on the surface of LDH).Once the nanozyme reached the TME, the acid-responsive hydrolysis of the surface MgO 2 resulted in the generation of H 2 O 2 , which was subsequently catalyzed into O 2 by pcCAT to relieve the TME hypoxia.Simultaneously, the lactate produced by tumor cells was oxidized into H 2 O 2 and pyruvic acid by pcLOX in parallel or tandem.As a result, the removal of excess lactate acid and relief of hypoxia efficiently activated the tumor-resident immune cells and induced cancer cell apoptosis, significantly inhibiting primary tumor growth (>95%) and suppressing distant tumor growth.Interestingly, in complete contrast, Huang et al. constructed an implantable self-charging battery, which consisted of a biocompatible polyimide electrode and zinc electrode to persistently consume oxygen and maintain a long-term (14 days) hypoxic environment, leading to 100% prevention of tumor formation. [113]During this process, the zinc electrode revolved in the acidic TME and released zinc ions (Zn 2+ ), which have been demonstrated to activate immune cells. [114]imilarly, regulation of the structurally incomplete blood vessels is also a promising avenue for activating TME. [115]Sung et al. designed a drug-free NO-delivery system based on the tumor vessel normalization ability of nitric oxide (NO) (Figure 6b). [116]erein, NO was encapsulated in a biodegradable PLGA polymer (NanoNO) for controlled release, and the PEG was modified on the polymer's surface for prolonged circulation time.Intravenous injection of this formulation resulted in a high accumulation efficacy and steady release of NO in HCC.Interestingly, a high dose (HD) of NanoNO resulted in the eradication of tumor blood vessels, thereby exacerbating tumor hypoxia and limiting nutrient delivery to the tumor, which caused moderate tumor killing.In contrast, a low dose (LD) of NanoNO efficiently promoted angiogenesis and vessel formation, normalizing the vessel network in the TME, and pumping more O 2 into the solid tumor to relieve hypoxia.Moreover, the LD of NanoNO also downregulated the PD-L1 expression in tumor cells, promoted the polarization of TAMs into M1-like antitumoral phenotype, and increased CD4 + and CD8 + T cells in HCC.Benefiting from the normalization of tumor vessels and activation of the TME, the anti-cancer efficacy of a cancer therapeutic vaccine was significantly improved in orthotopic HCC models.

Activation of Tumor-Resident Immune Cells
15b] For example, nutritional metal ions such as Mn 2+ and Mg 2+ have been revealed with the ability to effectively activate tumorresident immune cells to enhance cancer metalloimmunotherapy, which facilitates the rapid development of metal-based drug-free nanomodulators. [117]Besides, the particle size, surface charge, and shape-dependence of nanomodulators also affect the trafficking, phenotype, and cytokine secretion of tumorassociated immune cells. [118]t has been found that nutritional Mn 2+ and Zn 2+ can bind to the DNA that is released from the damaged mitochondria to induce cGAS-STING signaling pathway activation in tumor cells, consequently triggering tumor ICD and eliciting potent antitumor immunity.For instance, Liu et al. recently used an ultra-pH-sensitive polymer with a hydrophilic block of PEG and a hydrophobic block of poly(ethylpropylaminoethyl methacrylate) to encapsulate Mn 2 O 3 NPs for TME modulation. [119]Such nanomodulators dissolved rapidly in the acidic TME to release Mn 2+ , which subsequently activated the STING cascade activation to augment the maturation of DCs and activation of NK cells and specific T cells.Moreover, Cen et al. reported a ZnS@BSA (bovine serum albumin) nanocluster obtained through a selfassembly approach. [120]Zn 2+ was quickly released from the nanocluster in the acidic TME to significantly enhance tumor cGAS/STING signals.At the same time, intracellular Zn 2+ induced the production of ROS, which was amplified by H 2 S gas released from ZnS@BSA via specifically inhibiting catalase enzyme activity in tumor cells.As a result, the ZnS@BSA nanoclusters effectively promoted intratumor infiltration of CD8 T cells and the cross-presentation of DCs, tremendously augmenting cancer metalloimmunotherapy efficacy against HCC.Furthermore, Zhang et al. fabricated a Zn 2+ doped LDH (Zn-LDH)based immunomodulatory adjuvant, which synergistically modulates the immunosuppressive TME and elicits robust antitumor immune response without integrating any cytotoxic agents or immunomodulatory agonists (Figure 7a). [121]The weakly alkaline Zn-LDH NPs not only neutralized tumor excess acid and disrupted tumor lysosomes to block autophagy and trigger tumor cell death, but also simultaneously supplemented Zn 2+ to activate the cGAS-STING signaling pathway for ICD and robust antitumor immunity.The authors demonstrated that the increased release of DAMPs, caused by the ICD (including HMGB1, ATP, and CRT), decreased the expression of "don't eat me" signals including PD-L1 and CD47 on tumor cells.Peritumoral injection of Zn-LDH reversed the immunosuppressive TME through increasing M1-TAMs, NK cells, DCs, and CTLs, significantly inhibiting growth and metastasis of murine melanoma and breast cancer.
Similarly, Mg 2+ released from the nanomodulator is beneficial to activate NK cells and T cells by associating the surface NKG2D receptor. [122]Recently, Zhang et al. developed a weakly alkaline nano-aluminum (NanoAlum) adjuvant from the commercial Imject Alum. [123]Their results showed that peritumoral injection of NanoAlum also efficiently neutralized the acidic TME.Notably, the dissolution of NanoAlum in the TME released plenty of Mg 2+ , which greatly promoted the recruitment and filtration of CD8 + T cells into the solid tumor, and further activated T cells via increasing the surface NKG2D expression.Recently, Li et al. reported a postsurgical flex-patch containing a Mg 2+ Fe 3+ -based LDH for postsurgical adjuvant cancer therapy. [124]After surgical removal of the primary tumor, the LDH flex-patch was implanted and found to boost CTL activation and cytotoxic killing via metal cation (Mg 2+ and Fe 3+ )-mediated activation.In addition, the mitochondrial Ca 2+ overload-mediated pyroptosis pathway or the ICD pathway has been proven to effectively inhibit tumor proliferation. [125]Zheng et al. designed biodegradable Ca 2+ nanomodulators (CaNMs) for cancer immunotherapy through mitochondrial Ca 2+ overload-triggered pyroptosis. [126]CaNMs were composed of CaCO 3 and curcumin (CUR), in which CUR enhanced the Ca 2+ concentration by promoting Ca 2+ release and interfering with the efflux of Ca 2+ , causing mitochondrial Ca 2+ overload and resulting in an increase of intracellular ROS and cytochrome C. The cytochrome C further activated caspase-3 to cleave GSDME and induce pyroptosis. [127]CaNMs-mediated pyroptosis releases large amounts of inflammatory molecules and cell contents to efficiently activate the host immune responses for cancer immunotherapy.In vivo CaNMs injection promoted the maturation of DCs and activation of CTLs, leading to an effective tumor and lung metastasis control.Some drug-free nanomodulators directly target TAMs or DCs based on metal iron ions for enhanced cancer immunotherapy.For instance, Sang et al. prepared an iron nanotrap to polarize pro-tumor M2 to anti-tumor M1 phenotype. [128]Iron was absorbed on S dots (termed SP) with the help of coordination bond formation with oxygen atoms on the surface.The internalization into lysosomes and intracellular H 2 O 2 in TAMs promoted the release of iron from SP while TAMs-targeted peptides were modified on SP for TAM targeting.Such a nanotrap was ultrasmall and penetrated deeply into the solid tumor and achieved durable stimulation toward TAMs.Iron nanotrap treatment significantly boosted the production of NO, TNF-, and CD86, in comparison with the other groups, thereby exerting higher cytotoxicity to 4T1 cells.The in vivo treatment showed a higher content of M1 macrophages, CD8 T cells, and CD4 T cells in the tumor tissue and lymphoid organs including the thymus and spleen, causing a notable suppression of tumor growth.Another work reported modulation of DCs by calcium ions-mediated autophagy boosting.An et al. designed a versatile calcium ion "nanogenerator" to boost chemotherapy-induced immune responses (Figure 7b). [129]his formulation was composed of CaCO 3 NPs and OVA antigen, which not only attenuated tumor excess acidity for maintaining the DC viability, but also disarmed the autophagy inhibition of DCs by increasing the intracellular calcium level.CaCO 3 NPs mediated Ca 2+ overloading induced the DAMPs release from tumor cells, which also promoted DC maturation.After MTX-mediated chemotherapy, intravenous injection of this formulation remarkedly reshaped the immunosuppressive TME and inhibited the growth of both primary and distant colorectal tumors in two bilateral syngeneic mouse models.To inhibit fibroblast activation, Zhou et al. reported enzyme-activated peptide FR17, which could in situ perform a "flame-retarding blanket" mission to extinguish the "fire" of the immunosuppressive TME. [130]The MMP2-activatable self-assembled branched peptide FR17 consists of a self-assembled peptide domain Phe-Phe-Lys-Tyr and a pentapeptide called TP5 with perfect hydrophilic and immunomodulatory properties.After subcutaneous administration of FR17, the peptide nano-blanket could effectively impede the activation of fibroblast, leading to the reconstruction of stromal and vessel, which further inhibited MDSC recruitment and metastatic cascades.These results demonstrated FR17 treatment significantly inhibited pulmonary pre-metastatic niche formation and postoperative metastasis occurrence.
An emerging method for modulating circulatory innate immune cells is the intravenous injection of drug-free nanomodulators. [131]Zhang et al. found that intravenous administration of cargo-free poly (DL-lactide-co-glycolide) (PLG) NPs targeted circulating immune cells and improved the therapeutic efficacy of ICB. [132]The in vitro study showed a decrease in MCP-1 expression by 5-fold and an increment in TNF- content by more than 2-fold upon uptake by innate immune cells.After intravenous injection, PLG NPs were internalized by MDSCs and monocytes, thereby descending MDSCs in the lung.In combination with immune checkpoint inhibitor aPD-1, such drug-free PLG NPs significantly hindered tumor growth and prolonged the survival of tumor-bearing mice.Gene analysis revealed that the inflammatory myeloid cell pathways were inhibited in the lung and boosted in the spleen and tumor.Upregulated extrinsic apoptotic pathways also occurred in the primary tumor, which reshaped the immunosuppressive TME and reprogrammed immune cell responses against tumor cells.Although the biological interaction mechanism between NPs and immune cells has not been fully elucidated but probably involves multiple signaling pathways, drug-free NPs that target circulating innate immune cells have the potential to serve as novel adjuvant strategies for cancer treatment. [133]

Stimulus-Responsive Induction of Tumor ICD
Different from the ICD effect induced by the inherent physicochemical and biological properties of nanomaterials, external stimulus-responsive drug-free nanomodulators provide another venue for modulating immunosuppressive TME by inducing tumor ICD.For example, PDT, as a local non-invasive or minimally invasive treatment, is most widely used for solid tumor ablation, which can generate ROS to trigger tumor cells ICD under light irradiation. [134]However, conventional photosensitizers (PSs) are restrained in effective cancer treatment, including nonspecific tumor targeting and biodistributions, insufficient activation in the hypoxia TME, and restricted light penetration to deep tumor tissues, immensely impeding its potential therapeutic efficacy. [135]Drug-free nanomodulators synergize novel PSs to not only overcome the challenges for high tumor treatment efficacy but also minimize off-target toxicity.
PDT-induced ICD offers numerous tumor antigens to form efficient in situ vaccination. [136]To enable the controllability and lessen the systemic toxicity of neoadjuvant for broad applications, Wang et al. loaded Ce6 with a hypoxia-responsive amphiphilic dendrimer NP to form a light-activable immunological adjuvant (LIA) (Figure 8a). [137]When near-infrared (NIR) light was adopted, PDT-mediated ROS generation elicited tumor cells ICD and tumor antigens release.Simultaneously, the hypoxia TME caused the rapid reduction of 2-nitroimidazole groups of the dendrimer into 2-aminoimidazole.In vitro experiments verified that LIA effectively killed cancer cells upon NIR light irradiated and induced maturation of BMDCs.Multiple gene analyses of LIAtreated BMDCs showed that immune response-associated signaling pathways were upregulated.After intravenous injection, high tumor accumulation of LIA reduced systemic toxicity, and significantly increased mature DCs in LNs and a higher proportion of CTLs in the spleen were observed.Moreover, LIA treatment suppressed primary and abscopal tumor growth with more infiltrated CTLs and decreased Tregs in tumor tissues, efficiently inhibited tumor metastasis, and induced long-term antigenspecific memory effects.Moreover, Li et al. designed a PTT/PDT combined nanohybrid amplifier (FeOOH@STA/Cu-LDH) by integrating ROS producer (FeOOH nanodots), ROS booster (Cucontaining LDH for photothermal therapy) and heat shock protein inhibitor (STA). [138]Upon laser irradiation, the local production of ROS by FeOOH nanodots was dramatically amplified by the photothermal effect of Cu-LDH (maintained at a fever-type temperature of 40-42 °C), thus efficiently inducing tumor ICD.In the 4T1 breast cancer mouse model, only FeOOH@STA/Cu-LDH successfully eradicated the primary tumor inhibited distal tumor growth and induced a potent cytotoxic T lymphocyte immune response.On this basis, Sun et al. further constructed a defective Cu-LDH NP loaded with indocyanine green (ICG) for PTT/PDT/chemodynamic therapy (PTT/PDT/CDT) without the integration of any anticancer drugs. [139]In this case, a synergy between the surface structurally created defects of Cu-LDH and the interlayer ICG molecules significantly augmented photothermal transduction and singlet-oxygen generation upon laser irradiation.Meanwhile, the defective Cu-LDH/ICG exhibited Fentonlike catalysis to produce hydroxyl radicals in the presence of H 2 O 2 .Such efficacy was ultra-strengthened by Cu(II) reduction with glutathione and temperature elevation.In the 4T1 breast cancer model, defective Cu-LDH/ICG significantly suppressed tumor growth with single PTT/PDT/CDT combinatory treatment under a very mild laser (0.23 W cm −2 for 5 min) and at a very low dosage of Cu and ICG (2.5 and 0.5 mg kg −1 ).
Another issue that limits the host immune system activation by PDT is the lack of deep tissue-excitable photosensitive agents and appropriate strategies. [140]To solve this obstacle, Mao et al. coupled an aggregation-induced emission (AIE) photosensitizer TPEBTPy with upconversion NPs (AUNPs) for the activation of PDT in deep tissue. [141]Different from traditional photosensitizers, the AIE character enables TPEBTPy to generate strong fluorescence and ROS in the aggregated state.Meanwhile, upconversion NPs with emission matching the absorption of TPEBTPy were selected as the NIR antennae for improved light penetration.Compared with commonly used photosensitizer Ce6, AUNPs induced more CRT, HMGB1, and HSP70 in tumor cells following NIR treatment, and matured DCs and enhanced antigen cross-presentation upon low-power NIR irradiation.Intra-tumoral injection of this formulation showed the TAA release from damaged tumor cells in the TME and enhanced TAA uptake by DCs and macrophages under high-power NIR irradiation.AUNPs treatment not only efficiently eradicated the B16F10 tumor by PDT, but also established systematic anti-tumor immune responses and a long-term immune memory when it was synergized with ICB, which significantly reshaped the immunosuppressive TME.
Analogous to PDT, photocatalytic therapy could depend on photosensitizers under NIR excitation for energy collection.Dif-ferent from PDT, photocatalytic therapy could generate high oxidative holes for GSH consumption in tumor cells, which are independent of oxygen molecules. [142]For example, Zhao et al. proposed a catalysis-based drug-free therapeutic strategy, in which a Z-scheme SnS 1.68 -WO 2.41 nanocatalyst was synthesized to generate oxidative holes and hydrogen molecules under NIR irradiation. [143]WO 2.41 nanodots possessed strong NIR adsorption and a high oxidative potential for GSH oxidation owning to the oxygen defect-induced localized surface plasmon resonance effect.The sulfur-deficient structure of SnS 1.68 , instead, was responsible for NIR-photocatalytic hydrogen generation.After nanocatalyst treatment, NIR-photocatalytic GSH oxidation induced increased intra-tumoral ROS levels, breakdown of cellular anti-oxidation defense system (ADS), and damage of tumor cell DNAs, leading to tumor cell apoptosis.Furthermore, the 4T1 and HeLa tumors were effectively inhibited and the TME was completely remolded by hole/hydrogen therapy with regressed tumor vasculature and decreased pro-tumoral TAMs.In vivo biosafety evaluation demonstrated the nanocatalyst possessed good biocompatibility and showed no significant toxicity to major organs.
In addition to PDT, emerging sonodynamic immunotherapy (SDT) and electroimmunotherapy have also been applied for modulating the immunosuppressive TME and amplifying cancer immunotherapy.For instance, Xu et al. recently designed ultrasound-responsive LaCoO 3 (LCO) lanthanide-based nanocrystals with multiple nanozyme abilities for triggering cytotoxic ROS and releasing pyroptosis-inducer La 3+ (Figure 8b). [144]he peroxidase-and oxidase-mimetic activities enabled LCO nanocrystals with excellent ROS production ability in the acidic TME, while the catalase-and glutathione peroxidase-like activities enabled LCO nanozyme for tumor hypoxia relief, antioxidant system destruction, and tumor cells sensitization to ROS.The external ultrasound stimulus further accelerated the enzymatic kinetic rate.Notably, La 3+ released from LCO in lysosomes robustly destroyed the lysosomal membrane to induce canonical pyroptotic cell death, which amplified the immunotherapeutic effects to restrain lung cancer growth and metastasis.Similarly, Sun et al. designed an Mn-LDH-based defect-rich nanoplatform as a sono-chemo sensitizer, which enabled ultrasound to efficiently induce ROS generation for augmented sono/chemo-dynamic therapy. [145]In this case, the Mn-LDH nanomodulator responded to external ultrasound to relieve tumor hypoxia and enhance singlet oxygen production by catalyzing H 2 O 2 into O 2 .During this process, Mn 2+ was released from Mn-LDH to amplify immune activation and remold the immunosuppressive TME, thereby effectively regressing both primary and distant tumor growth.In another study, Wu et al. designed a multifunctional sonosensitizer based on MgCaFe-LDH that amplified the SDT and showed excellent capacity to activate T cells and enhance their tumor infiltration. [146]The introduction of Ca 2+ regulated the energy band of MgCaFe-LDH and promoted the separation of electronhole pairs, thereby enhancing sonodynamic performance.Meanwhile, the high valence of Fe 3+ depletes GSH and generates Fe 2+mediated Fenton catalysis to induce oxidative stress.Moreover, the bioactive Ca 2+ and Mg 2+ released from MgCaFe-LDH collaboratively activated CD8 + T cells and repolarized M1 TAMs, formed the inflammatory immune network in the TME to prolong the anti-tumor effect and suppressed metastasis.Furthermore, Yang et al. developed a Mg-based galvanic cell (MgG) by decorating platinum on the surface of Mg rods, which generated H 2 continuously in an aqueous environment. [147]When implanted into tumor tissue, MgG accelerated the water etching of Mg to generate H 2 molecules in the tumor tissue, inducing mitochondrial dysfunction and intracellular redox homeostasis destruction.The produced Mg(OH) 2 could neutralize the acidic tumor environment, leading to increased intratumor CD8 + T cells and decreased MDSCs.The in vivo data showed that MgG treatment not only inhibited 4T1 and CT26 tumors but also exhibited significant therapeutic efficacy in patient-derived xenografts models.

Conclusion and Perspectives
While significant progress has been made in cancer immunotherapy, the formidable challenge posed by the immunosuppressive TME has impeded its broader clinical application. [148]In this comprehensive review, we have overviewed recent advances in emerging nanomodulators that help reverse immunosuppressive TME and enhance cancer immunotherapy (Table 1).Our exploration begins with drug delivery and further extends to embrace the innovative concept of drug-free modalities.Leveraging the EPR effect alongside active targeting has substantially bolstered the localized immunotherapeutic efficacy of nanomodulators and simultaneously reduced off-target toxicities. [149]Some nanoparticles are not included in the category of nanomodulators if they possess an immunoregulatory effect and induce the apoptosis of tumor cells, but have no TME modulation effect.Consequently, nanomodulators are referred to as functioning as highly efficient drug delivery systems, providing immense potential for the precise targeting of tumor or immune cells, facilitating the delivery of gene tools and proteinic antigens for up-/down-regulating specific gene expression, being immune agonists and inhibitors to modulate cellular immune responses, leading to enhancement of the host antitumor immunity and reversion of the immunosuppressive TME.Beyond their role as drug delivery systems, the intrinsic physicochemical attributes of nanomaterials empower themselves to directly interact with the TME at the extracellular and intracellular levels, engage immune cells to regulate their anti-tumor immune functions and respond to external stimuli for tumor eradication and immune modulation. [150]The concept of drug-free therapeutics has recently been defined as a "therapeutic methodology without the use of traditional toxic drugs and the consumption of therapeutic agents during treatment".Drug-free nanomodulators are designed to target the immunosuppressive TME without involving any therapeutic agents, but solely relying on the character of nanomaterials to realize the modulation of the TME.Through reversing the immunosuppressive TME, these drug-free nanomodulators potentiate cancer immunotherapy by facilitating the infiltration and augmenting the killing efficiency of tumor-inhibitory immune cells.
In light of the swift progress in nanotechnology and the evolution of nanomaterial structure and composition, it is imperative to recognize that the scope of nanomaterial applications extends far beyond conventional drug delivery, even though this undeniably remains as their paramount function.Thus far, cancer nanomedicines and nanomodulators have exhibited substantial potential for enhancing the precision and effectiveness of cancer therapy.Their high surface-to-volume ratio enables enhanced interactions with biological interfaces.This unique feature facilitates a significantly heightened drug loading capacity, allowing for more efficient delivery systems.Moreover, versatile surface modification and active targeting of tumors demonstrate promising potential for precise and personalized therapy,  minimizing damage to healthy tissues.However, formidable challenges, including issues related to on-target and off-target drug toxicity, scalability in manufacturing, long-term safety assessments, and clinical efficacy, persist in the journey from laboratory development to clinical implementation.Consequently, a growing inclination is observed toward the utilization of drug-free nanomodulators, particularly nanomaterials characterized by simple compositions and well-defined structures, in the realm of immunomodulation for cancer therapy.These well-established nanomaterials can be prepared with mature and highly controllable synthesis techniques, ensuring their reproducible preparation at a large scale.Similar to pharmaceutical compounds, these nanomaterials can be assessed in preclinical settings as independent entities, streamlining the evaluation process compared to traditional drug-loaded nanomedicines.
In clinical practice, drug-free nanomodulators possess unique physicochemical attributes, simple fabrication, and nonmodified surfaces, which lead to a low cost and high stability in clinical translation. [155]However, they may face challenges in their capacity to sufficiently provoke an immune response for the eradication of solid tumors, primarily due to the intrinsic complexity of the human immune system.Meanwhile, their intrinsic composition requires further exploration during the preparation process and an understanding of the specific impacts of inorganic/organic components on immune modulation.Furthermore, it is crucial to delve more deeply into the biological effects of various drug-free nanomodulators, utilizing omics technologies to explore their interactions with important species within the tumor tissues.This comprehensive research approach aims to expand our understanding of drug-free nanomodulators in promoting immunity, fostering their further applications and development in disease treatments and immune regulation.Initially, the concept of nanomodulators was coined with the goal of alleviating the immunosuppressive TME to a certain extent and regulating the progression of solid tumors.Consequently, drug-free nanomodulators function as neoadjuvant therapeutic tools, with the goal of controlling tumor progression, making surgical resection more amenable, and/or serving as valuable complements to enhance the efficacy of conventional therapies such as chemotherapy, radiotherapy, immune checkpoint blockade, and adoptive cell therapy.Furthermore, through the synergistic integration of nanomodulators with various biomedical materials such as flexible devices and 3D printing scaffolds, there is anticipation for the emergence of implantable immunomodulatory medical devices (e.g., hemostatic gauze and wound-filling stents), aiming at preventing postoperative tumor recurrence.
In summary, harnessing the intrinsic physicochemical properties of biocompatible nanomaterials in alignment with the unique pathological milieu of tumor tissues represents a straightforward approach to the development of emerging TMEtargeting nanomodulators.Preclinical investigations have illuminated the potential of nanomodulators as valuable complements to immunotherapy strategies.Nevertheless, several technical challenges persist on the path from laboratory to clinical implementation, encompassing the need for sufficient TME modulation capacity to amplify the efficacy of existing clinical approaches, a comprehensive understanding of the functional mechanisms and potential toxicity associated with nanomaterials, and the establishment of standardized physicochemical properties for engineering purposes.Nonetheless, we firmly believe that drug-free nanomodulators offer immense potential to advance cancer neoadjuvant immunotherapy.

Figure 1 .
Figure 1.The development of nanomodulator for activating cold tumor tissues to enhance cancer immunotherapy: from drug delivery toward drug-free concepts.

Figure 4 .
Figure 4. Immunomodulatory cytokine-based nanomodulators.a) Responsive release of IL-21 NPs to activate NK cells for solid tumor immunotherapy.Reproduced with permission from Ref. [72] Copyright 2022 Wiley-VCH.b) IL-12 nanoformulation conjugated to CAR-T cell to achieve robust antitumor immunity.Reproduced with permission from Ref. [73] Copyright 2022 Elsevier.c) Intra-tumoral injection of XCL-1-loaded sodium alginate combined with membranes-coated DOX-loaded PLGA NPs for enhanced tumor immunotherapy.Reproduced with permission from Ref. [76] Copyright 2021 Elsevier.d) Tumor acidity-responsive NP delivering CCL25 and CD47 siRNA to recruit CCR9 + T cells for breast cancer treatment.Reproduced with permission from Ref. [75] Copyright 2020 AAAS.

Figure 5 .
Figure 5. Receptor agonist/inhibitor-based nanomodulators.a) Ultrasound-guided nanocomplex loaded with cGAMP for targeted immunotherapy of cancer.Reproduced with permission from Ref. [90] Copyright 2022 Springer Nature.b) Pathogen-like nanoassemblies covalently link TLR agonists to improve CD8 and NK cell-mediated antitumor immunity.Reproduced with permission from Ref. [96] Copyright 2020 American Chemical Society.c) Albumin NP loading IPI-549 and PTX in combination with -PD1 to remodel TME in breast cancer.Reproduced with permission from Ref. [100] Copyright 2022 AAAS.d) Apoptosis induced by near-infrared photoirradiation triggered M-Trp mediated IDO inhibition for photodynamic cancer immunotherapy.Reproduced with permission from Ref. [102] Copyright 2022 Wiley-VCH.

Figure 6 .
Figure 6.Drug-free nanomodulators for normalizing tumor extracellular microenvironment.a) LDH NPs neutralized the excess acid and blocked autophagy of tumor cells for neoadjuvant cancer immunotherapy.Reproduced with permission from Ref. [109] Copyright 2022 American Chemical Society.b) Efficient delivery of NO by PLGA NPs to reprogram tumor vasculature and immune microenvironment.Reproduced with permission from Ref. [116] Copyright 2019 Springer Nature.

Figure 7 .
Figure 7. Drug-free nanomodulators for tumor-associated immune cell stimulation.a) Peritumoral injection of Zn-LDH elicited robust and safe antitumor immunity for cancer metalloimmunotherapy against solid tumors.Reproduced with permission from Ref. [121] Copyright 2022 Wiley-VCH.b) Honeycomb CaCO 3 NPs generated Ca 2+ to disrupt the autophagy inhibition condition in DCs and attenuated acidity in TME for enhanced chemoimmunotherapy.Reproduced with permission from Ref. [129] Copyright 2020 American Chemical Society.

Figure 8 .
Figure 8. Stimulus-responsive nanomodulators for inducing tumor ICD and activating TME.a) LIA served as a light-activable immunological adjuvant to form in situ vaccination.Reproduced with permission from Ref. [137] Copyright 2021 Springer Nature.b) LaCoO 3 nanocrystals with multienzyme characteristics were rationally designed to trigger the generation of ROS and the release of lanthanum ions, which ultimately include the pyroptosis of lung cancer cells.Reproduced with permission from Ref. [144] Copyright 2023 Wiley-VCH.
Zhang received his Ph.D. degree from the Institute of Process Engineering Chinese Academy of Sciences in 2020.Then he served as a research assistant professor at Zhejiang University, subsequently attaining the position of associate professor.Since 2023, he joined Aarhus University as a Marie Sklodowska-Curie Actions Research Fellow.His research focuses on the development of 2D nano-aluminum adjuvants for cancer immunotherapy and neurodegenerative disease therapy.Currently, he serves as editor for the Journal of Nanobiotechnology and young editorial board member for the Asian Journal of Pharmaceutical Science, Exploration, Microstructures, Biomaterials Translational and Neural Regeneration Research.Ruitian Liu received his Ph.D. degree from Shandong University in 1998, then he was promoted to associate professor in 1999.He performed his postdoctoral research at Arizona State University from 2002 to 2005.Then he worked at Tsinghua University as an associate professor from 2005 to 2012.He has been a senior group leader and professor at the Institute of Process Engineering, Chinese Academy of Sciences since 2012.His research focuses on discovering and developing original biotechnological drugs and biosimilar medicines such as vaccines, peptides, recombinant proteins, and genetic engineering antibodies for the treatment of major diseases and infectious diseases.Yingbo Jia received his Ph.D. degree from the Institute of Process Engineering, University of Chinese Academy of Sciences in 2023.He is currently a Postdoctoral Research Fellow at Dana Farber Cancer Institute, Harvard Medical School.His research focuses on synthetic biology and nanobiotechnologybased cancer immunotherapy and immune cell engineering.Zhi Ping Xu received his Ph.D. from the National University of Singapore, then he performed his postdoctoral research at the University of North Texas and the University of Queensland.He worked at The University of Queensland as an associate professor and professor from 2007 to 2022.Currently, he is a senior group leader and professor at the Institute of Biomedical Health Technology and Engineering, and the Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, as well as at the Australian Institute for Bioengineering and Nanotechnology, the University of Queensland.His research focuses on control preparation of layered double hydroxide nanomaterials, calcium phosphate nanoparticles, nanoemulsions, quantum dots, chemosensors, and nanosensors, and their applications in biomedicine and agriculture.