Cell Selective STING‐Activating Polysaccharide Immunomodulators for Cancer Therapy

ABSTRACT

Activation of the stimulator of interferon genes (STING) signaling pathway represents a robust strategy to reverse tumor immunosuppressive microenvironment (TIME) for cancer therapy. However, selective STING activation and its quantitative comparison across heterogeneous cell populations remain a tremendous challenge. Herein, we engineered a type of selective STING‐activating polysaccharide immunomodulators (SSAPIs) with quantitative STING activation efficiency across tumor cell, macrophage, and dendritic cell (DC). Dextran as an immune cell targeting nanocarrier was employed to improve drug delivery to macrophage and DC, and to avoid the impact of macromolecular self‐assembly on drug release kinetics. The STING agonist (DMXAA) was conjugated to dextran via defined linkers to control the selectivity of STING activation in different cell populations. In vitro experiments quantitively revealed the enhanced STING activation of the ester linker SSAPI (DESX) in macrophage, while the disulfide linker SSAPI (DSSX) prompted STING activation across tumor cell and immune cell. In B16F10 and CT26 tumor‐bearing mice models, DSSX exhibited much superior antitumor efficacy with six out of eight complete tumor remission by inducing broad immune responses across diverse cell populations to reprogram TIME. Collectively, this work highlights the significance of activating the STING signaling pathway across cell populations in solid tumor for cancer immunotherapy.

A type of cell selective STING‐activating polysaccharide immunomodulator (SSAPIs) for cancer therapy.

1. Introduction

Immunotherapy via immune checkpoint inhibitors or agonists has become a promising strategy for cancer therapy, which has drawn great attention in material science, medicinal chemistry, cell, and molecular biology [1, 2, 3]. However, the efficacy for immunotherapy is severely limited by tumor immunosuppressive microenvironment (TIME), which is a well‐established feature across multiple cancer types and categories [4]. Strategies for reversing TIME via innovative nanomedicines have been well‐documented over past several years, including the delivery of programmed death‐1 (PD‐1)/programmed death ligand 1 (PD‐L1) inhibitors [5], cluster of differentiation 47 (CD47)/signal regulatory protein alpha (SIRPα) inhibitors [6], toll‐like receptor (TLR) agonists [7], and stimulator of interferon genes (STING) agonists [8]. Nanotherapeutics inducing immunogenic cell death (ICD) also play an important role in reprogramming TIME via certain chemotherapeutic drugs [9, 10], or photodynamic and photothermal therapy [11]. Among them, the STING signaling pathway stands out as a powerful innate immune activation approach, which has been a prominent therapeutic target for tumor immunotherapy.

A variety of drug delivery systems (DDSs) have been engineered to improve the activation efficiency of STING agonists for cancer immunotherapy [12]. The most common methodology is to load these agonists into amphiphilic nanoparticles or carriers to improve tumor delivery. For example, a pH‐responsive DDS was fabricated by encapsulating 2’,3’‐cyclic‐GMP‐AMP (cGAMP) to increase the delivery efficiency to endoplasmic reticulum (ER), which achieved a 33.9‐fold amplification of STING sensibilization [13]. Another pH‐responsive polymer was employed to encapsulate cGAMP to enhance the delivery to type 1 dendritic cells, revealing the specific subtype of immune cells for STING activation [14]. A sandwich‐like scaffold was 3D‐printed as a drug depot for STING agonist delivery, which realized strong immune cell stimulation to reverse TIME [15]. Another extensively utilized strategy is to covalently conjugate STING agonists to form macromolecular prodrugs for advanced DDSs. For example, a cathepsin‐sensitive linker was employed to conjugate cytosolic cyclic dinucleotide (CDN) to a synthetic poly(β‐amino ester) to generate nanoparticles, which expanded the therapeutic window in multiple murine tumor models [16]. A glutathione (GSH)‐responsive polymeric STING proagonist was developed to combine with sono‐irradiation for enhanced tumor immunotherapy [17]. These STING agonist delivery systems exploited different kinds of morphologies, including supramolecular nanoparticles [18], PEG‐lipid nanodiscs [19], polymeric nano‐immunomodulator [20], to boost tumor accumulation and penetration, and exhibited synergistic effect in combination with other cancer therapy strategies [21, 22, 23]. The engineering of multifunctional and smart DDSs has achieved great progress on the stability, bioavailability, tumor accumulation, and targeting of these agonists for STING signaling activation.

The heterogeneity of cell populations in solid tumors frequently brings challenges to efficient antitumor therapy. It has been reported that activation of macrophage STING pathway can reprogram its cell functions to enhance the phagocytosis capability and increase the secretion of proinflammatory cytokines and chemokines to alleviate TIME [24, 25]. Meanwhile, stimulation of dendritic cell STING pathway promotes its maturation to present tumor‐specific antigens to activate T and NK cells [26, 27]. Moreover, provoking tumor cell STING signaling leads to the production of type I interferons (IFNs) to recruit circulating immune cells, reverse TIME and inhibit the reactivation of dormant metastasis [28, 29]. However, the main perspective emphasizes on the activation of dendritic and myeloid cell STING signaling to initiate potent antitumor innate immune responses [14, 30]. The impact on selective activation of STING signaling in tumor cell, macrophage, and dendritic cell is unclear. Challenges remain on how to precisely tune and quantify the selectivity of STING activation among tumor cell, macrophage, and dendritic cell.

To address this challenge, we put forward the concept of selective activation of the STING signaling pathway across tumor cell, macrophage, and dendritic cell via a type of selective STING‐activating polysaccharide immunomodulators (SSAPIs) (Figure 1). Dextran (Dex) was employed as the drug carrier because of its good water solubility and targeting ability to macrophage and dendritic cells, which avoided the impact of macromolecular self‐assembly on drug release kinetics [31, 32, 33]. The targeting to macrophage and dendritic cells compensated for the big difference of cell numbers between macrophage, dendritic cell, and tumor cell. The STING‐activating selectivity was designed via chemical linkers with defined release kinetics, and the activation efficacy of the SSAPIs was carefully investigated in macrophage, dendritic cell, and tumor cell for quantitative comparison. Meanwhile, the STING signaling activation efficiency of these SSAPIs was further evaluated in vivo using both B16F10 and CT26 tumor‐bearing murine mice models via intratumoral administration to eliminate additional impact factors from blood circulation and tumor accumulation.

FIGURE 1.

Selective STING‐activating polysaccharide immunomodulators (SSAPIs) for cancer therapy. (a) The schematic diagram and chemical structures of the polysaccharide immunomodulators, DAMX, DESX, DEEX, and DSSX, with selective STING activation capability via defined linkers (L1 to L4) with definite release kinetics. (b) Selective STING‐mediated immune activation of macrophage, dendritic cell, and tumor cell via the SSAPIs with varied activation capacities. The immune activation capacities were determined from the in vitro cell experimental results. (c) The SSAPIs activate the STING signaling pathway with defined drug release behaviors. Illustration of broad STING activation capacity across macrophage, dendritic cell, and tumor cell to reverse TIME to potentiate cancer immunotherapy.

2. Results

2.1. Design, Synthesis, and Characterization of SSAPIs

To design a suitable platform for selective STING activation in immune cell and tumor cell, several requirements need to be fulfilled. Considering the massive cell number of tumor cell, it is necessary to target immune cell to improve the drug delivery efficacy for broad immune activation. Meanwhile, nanoparticle self‐assembly driven by hydrophobic/hydrophilic or electrostatic interactions interferes with the drug release kinetics of the STING agonist, which should be precluded in the immunomodulator design. Finally, programmed drug release can be achieved via defined linkers that have definite cleavage mechanism in the reported literatures, which will reach solid conclusion for the evaluation of STING activation efficiency.

Based on the above principles, we chose dextran (Dex) as a nanocarrier for the delivery of the model synthetic STING agonist (DMXAA), and further selected ester, amide, and disulfide bonds as the covalent linkers, which had defined release kinetics and were widely employed for the development of macromolecular prodrugs [34, 35]. Dex is a water‐soluble polysaccharide with neutral charge, which can target antigen‐presenting cells (APCs) via mannose and scavenger receptors, including macrophage and dendritic cell [32, 36, 37]. Thus, the direct conjugation between the carboxyl group of DMXAA and the hydroxyl group of Dex provided the ester bond SSAPI, termed as DESX (Figure 2), which could be rapidly cleaved by various esterase and protons. To prepare the amide linker conjugate, Dex was modified with primary amines via the copper‐free thiol‐ene click reaction [37], and subsequently reacted with DMXAA to obtain the amide linker SSAPI (DAMX). Then, DMXAA was modified with 1,6‐hexanediol or bis(2‐hydroxyethyl) disulfide (Scheme S1) before conjugating to amino dextran via the carbamate linkage, forming another two SSAPIs, termed as DEEX and DSSX. These SSAPIs were designed to show accelerated drug release from immune cell to tumor cell to precisely control the STING activation efficiency in different cell populations. Detailed synthetic procedures could be found in the Experimental section in the Supporting Information.

FIGURE 2.

The synthetic routes for SSAPIs with defined linkers. (a) The synthetic route for DAMX with an amide linker via amino dextran prepared by the thiol‐ene click reaction. (b) The synthetic route for DESX with an ester linker under the catalyst of CDI for direct conjugation. (c) The synthetic route for DEEX with a diester linker under the catalyst of p‐nitrophenyl chloroformate. (d) The synthetic route for DSSX with a disulfide linker under the catalyst of p‐nitrophenyl chloroformate. DMXAA was selected as the model STING agonist and dextran was utilized as the polysaccharide carrier.

The chemical structures of SSAPIs and the small molecular prodrugs were analyzed via diverse techniques, such as ¹H and ¹³C nuclear magnetic resonance (NMR) technique, high‐resolution mass spectrometry (HRMS), and Fourier transform infrared spectroscopy (FTIR). Both the 1,6‐hexanediol and bis(2‐hydroxyethyl) disulfide were connected to the carboxyl group of DMXAA to obtain the small molecular prodrugs, DMX‐Hex‐OH and DMX‐SS‐OH, as confirmed by ¹H and ¹³C NMR and HRMS (Scheme S1, Figures S1–S4). The chemical structures of SSAPIs were further characterized by NMR and FTIR. Commercial Dex was modified with primary amines using the thiol‐ene click reaction, which was confirmed via the ¹H NMR technique (Figure S5). For the polysaccharide immunomodulators, the typical NMR signals of DMXAA could be detected at around 7.31–8.13 ppm, revealing the successful conjugation of DMXAA to the polysaccharide (Figure 3a). Meanwhile, the typical signals of DMXAA at around 1410 cm⁻¹ in the FTIR spectra further proved the successful preparation of the four SSAPIs (Figure 3b–e).

FIGURE 3.

Characterization of the SSAPIs. (a) The ¹H NMR spectra of DAMX, DESX, DEEX, and DSSX in DMSO‐d₆ . The FTIR spectra of DAMX (b), DESX (c), DEEX (d), and DSSX (e) in comparison with the carrier Dex. Dynamic light scattering (f) and Zeta potentials (g) of the SSAPIs in PBS with concentrations at around 0.2–0.3 mg mL⁻¹. The drug release behaviors of DAMX (b), DESX (c), DEEX (d), and DSSX (e) in PBS within 72 h, n = 3. A GSH PBS solution (5 mM) was employed to reveal the redox‐responsive drug release kinetics of DSSX.

These SSAPIs were water‐soluble, and could be used directly by dissolving in water solutions or buffers [32, 38]. The size and Zeta potential were measured by dynamic light scattering (DLS) in PBS. The hydrodiameters of DAMX, DESX, DEEX, and DSSX were close at round 15.3 to 17.3 nm because of the same drug nanocarrier as single‐molecule macromolecular prodrugs (Figure 3f). The SSAPIs were polysaccharide‐drug conjugates with good water solubility to eliminate the process of macromolecular self‐assembly, thus their morphologies could not be detected via transmission electron microscope (TEM) [10, 38]. The Zeta potentials of DAMX, DESX, DEEX, and DSSX were similar and close to around −6 mV, and DSSX showed a lower Zeta potential, which might be ascribed to the effect of the disulfide bond, but the difference was quite small (Figure 3g). The drug loadings of the SSAPIs were measured via a UV–vis standard curve (Figure S6), and provided in details along with the diameters in Table S1. Finally, the drug release kinetics were also measured in PBS (Figure 3h–k). DAMX with stable amide linker displayed a slow drug‐release rate, and the drug release speed increased heavily for DESX. Meanwhile, the drug release rate decreased for DEEX as compared with DESX, which might be ascribed to the hydrophobicity of the hexyl linker. DSSX exhibited a slow drug release kinetics in PBS, however, the drug release speed increased greatly with the catalysis of glutathione (GSH), which could trigger drug release by breaking the disulfide bond. Meanwhile, the drug release behaviors of the SSAPIs were further evaluated in pH 5.5 buffer to mimic the tumor acidic microenvironment, illustrating an accelerated release rate relative to PBS (Figure S7). Thus, by tuning the covalent linker, the release kinetics of DMXAA could be well controlled to achieve preferential STING activation in immune cell and tumor cell.

2.2. Selective APC Activation In Vitro

The activation of STING signaling pathway can stimulate macrophage M1 polarization and dendritic cell maturation to reverse TIME [24, 25]. The cell toxicity of the polysaccharide immunomodulators along with DMXAA were first measured, which displayed almost no cytotoxicity against macrophage cells even at high drug concentrations (Figures S8, S9). Thus, the potency of these polysaccharide immunomodulators for macrophage reprogramming was further investigated in vitro. The concentration of nitric oxide (NO) in the cell culture media was measured after coculturing for 24 h via a commercial kit as the overexpressed inducible nitric oxide synthase (iNOS) of M1 macrophage could greatly increase the cellular level of NO [39]. Among these four SSAPIs, only DESX significantly increased the concentration of NO in the cell culture medium, and DSSX displayed a relatively higher concentration as compared with DMXAA (Figure 4a). DAMX had the lowest NO concentration since the amide linker was quite stable in macrophage cells. Subsequently, the typical cell surface marker, CD86, was detected via flow cytometry under the same incubation condition. The CD86 expression level for the DESX group was the highest among all treated groups after 24 h incubation (Figure 4b), and the DSSX treatment group showed increased CD86 expression as compared with DAMX and DEEX. Also, the CD86 expression level of DAMX was the lowest among these SSAPIs because of the slow drug release behavior. The mRNA levels of inflammatory cytokines were further evaluated via the reverse transcription‐polymerase chain reaction (RT‐PCR) technique. The sequences for the primers of the targeted genes were given and listed in Table S2. The mRNA levels of iNOS, IL‐1β, and TNF‐α were the highest for DESX, and DSSX displayed increased expression among the other two SSAPIs (Figure 4d–f). Meanwhile, the protein expression level of the proinflammatory cytokine IL‐6 was further measured via the enzyme‐linked immunosorbent assay (ELISA), which illustrated the optimized macrophage M1 polarization for DESX among these treatment groups (Figure S10). Collectively, the above results indicated that the SSAPI DESX with an ester linker benefited for proinflammatory macrophage polarization in vitro.

FIGURE 4.

Selective APC activation via the SSAPIs. (a) The concentrations of NO in macrophage cell culture medium for different treatment groups of SSAPIs. Flow cytometry analysis (b) and the quantification (c) of the percentage of CD86⁺ macrophage cells for different treatment groups. The mRNA levels of iNOS (d), IL‐1β (e) and TNF‐α (f) of macrophage cells for different treatment groups. Flow cytometry analysis (g) and the quantifications (h) of the percentage of CD80⁺CD86⁺ BMDCs for different treatment groups. The mRNA levels of CXCL9 (i), IFN‐β (j), and TNF‐α (k) of BMDCs treated with different SSAPIs. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The activation of the STING signaling pathway can also promote dendritic cell maturation to present tumor‐specific antigens to T cells to initiate immune response against cancer cells [8, 14]. Thus, the effect of these SSAPIs for dendritic cell maturation was also investigated using bone marrow‐derived dendritic cells (BMDCs). Bone marrow cells were harvested from C57BL/6 mice and induced into BMDCs before incubation with different drug formulations for further analysis. As shown in Figure 4g–h, all polysaccharide immunomodulators increased the maturation (CD80⁺CD86⁺) of BMDCs after coculturing for 24 h. However, DAMX and DEEX showed a weak increasement of CD80⁺CD86⁺ positive BMDCs of 26.2% and 23.7% respectively, while DSSX further enhanced the level of CD80⁺CD86⁺ BMDCs to around 32.9%. Importantly, DESX exhibited the highest level of CD80⁺CD86⁺ positive BMDCs (45.3%), which was the highest among all treatment groups. Meanwhile, the typical inflammatory cytokines and chemokines of BMDCs were analyzed by the RT‐PCR technique since the activation of STING signaling pathway would enhance their expressions in a STING‐dependent manner [15, 16]. The mRNA expression levels of the typical downstream effectors, such as CXCL9, CXCL10, and IFN‐β, were the highest for BMDCs treated with the DESX SSAPI (Figure 4i–k). DSSX displayed moderated enhancement of CXCL9, CXCL10, and IFN‐β expressions in comparison with the other treatment groups. Lastly, the proinflammatory cytokine, TNF‐α, was investigated and exhibited similar trend as these genes (Figure 4l). Taken together, these results demonstrated the preferential STING activation of macrophages and dendritic cells via these SSAPIs.

2.3. Selective STING Activation

The activation of the STING pathway induces the down‐stream activation of TBK1 and IRF3, leading to immune cell reprograming and TIME reversal [12, 14]. To explore the difference of STING activation efficiency of these SSAPIs, Western blot analysis was utilized to measure the protein expressions of STING/p‐STING, TBK1/p‐TBK1, and IRF3/p‐IRF3 in macrophage and B16F10 tumor cell. As shown in Figure 5a, DESX induced better efficiency for the activation of STING and p‐STING, and DSSX displayed a relatively moderate efficiency for STING activation in macrophages, which was clearly observed in the quantifications of p‐STING/STING (Figure S11), and was in accordance with the results for macrophage M1 polarization efficacy in vitro (Figure 4). As a result, the down‐stream activation of TBK1 and IRF3 for DESX was more significant than the other three immunomodulators, further demonstrating the improved macrophage M1 reprogramming efficiency for DESX. These results revealed that SSAPI with an ester linker activated the STING pathway in macrophage more potently and effectively.

FIGURE 5.

Selective STING activation in macrophage and B16F10 tumor cell. (a) The expression of p‐STING, STING and downstream p‐TBK1 and p‐IRF3 of macrophage cells incubated with diverse SSAPIs. The mRNA levels of IFN‐β (b), IFN‐γ (c), and CXCL10 (d) of macrophage cells for different treatment groups. n = 3. (e) The expression of p‐STING, STING and downstream p‐TBK1 and p‐IRF3 of B16F10 tumor cells incubated with diverse drug formulations. The mRNA levels of IFN‐β (f), IFN‐γ (g), and iNOS (h) of B16F10 tumor cells for different treatment groups. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The activation of the STING signaling pathway further leads to the expression of the downstream inflammatory cytokines and chemokines to reverse TIME and recruit immune cells [15, 18]. Interferon (IFN) as one of the most important downstream cytokines for STING activation can regulate both innate and adaptive immunity for antitumor therapy against a variety of cancers. Thus, the mRNA expression levels of IFN‐β and IFN‐γ of macrophage cells treated with different SSAPIs were measured via the RT‐PCR technique. Similarly, DESX exhibited the highest expression levels of IFN‐β and IFN‐γ among these treatment groups, and DSSX displayed moderate improved expression of IFN‐β as compared with other groups (Figure 5b,c). Meanwhile, the typical chemokine, CXCL10, was also investigated via the RT‐PCR and ELISA techniques. DESX induced the most potent mRNA expression of CXCL10 among these treatment groups as observed previously (Figure 5d). Meanwhile, the protein expression level of CXCL10 of DESX was the highest among these SSAPIs (Figure S12). These results further demonstrated the optimal STING signaling activation of macrophage cells via the DESX SSAPI.

The activation of the STING pathway in tumor cell can reverse TIME to improve cancer therapy [30]. So, the activation of STING/p‐STING, TBK1/p‐TBK1, and IRF3/p‐IRF3 in B16F10 tumor cell was illustrated by Western blot. As shown in Figure 5e, DSSX exhibited the most potent STING activation efficiency as compared to DAMX, DESX, and DEEX, which significantly induced STING over activation. The comparison could be clearly observed in the quantifications of the expression levels of p‐STING/STING (Figure S13), demonstrating that DSSX with a disulfide linker could achieve the most potent activation of the STING signaling pathway in B16F10 tumor cell [25]. Thus, the downstream genes of IFN‐β and IFN‐γ along with the inflammatory cytokine, iNOS, were further investigated using the PCR technique, which illustrated the superior efficiency of DSSX among these SSAPIs for B16F10 tumor cell (Figure 5f–h). However, DESX only induced moderate enhancement of these three genes. The enhanced STING activation of DSSX in B16F10 tumor cells was ascribed to the much higher levels of redox species (e.g. GSH) in tumor cells as reported in other literatures [40]. To further prove this, a cell‐permeable and irreversible inhibitor of γ‐glutamylcysteine synthetase, l‐buthionine‐(S,R)‐sulfoximine (BSO), was employed to reduce the cellular GSH level in B16F10 tumor cells. This treatment significantly decreased the activation efficacy of DSSX on B16F10 cells, as evidenced by the down‐regulated mRNA expression levels of IFN‐β, IFN‐γ, and IL‐6 (Figure S14). Clearly, the STING activation efficiency in macrophage and B16F10 tumor cell was selectively altered using these linker‐defined SSAPIs.

2.4. Antitumor Therapy Via The SSAPIs

The activation of the STING signaling pathway will stimulate the downstream IRF3 and NF‐κB activation to produce type 1 interferons (IFN‐I) and proinflammatory cytokines/chemokines to enhance cancer immunotherapy efficiency [12]. Since the STING activation of DEEX was not effective in both immune cell and B16F10 tumor cell, we decided to eliminate DEEX for in vivo antitumor therapy. An important factor affecting the selective STING activation for different cell populations is the administration route, where intravenous and intraperitoneal administration bring additional considerations about the circulation, accumulation, and stability of the SSAPIs before entering solid tumors [41]. To investigated the impact of selective STING activation on antitumor efficacy, these SSAPIs were intratumorally administrated into B16F10 tumors to avoid the differences in blood circulation stability, tumor penetration, and accumulation. When the tumors grew to around 80–100 mm³, the SSAPIs were injected intratumorally with a dose of 2.0 mg kg⁻¹ DMXAA every other day (Figure 6a), and the tumor volume and body weight were measured accordingly. At the end of therapy, mice treated with different formulations were sacrificed, and major organs along with tumor and blood were harvested for further analysis.

FIGURE 6.

SSAPIs enhanced the antitumor therapeutic efficacy against B16F10 melanoma via selective immune activation. (a) The treatment schedule for C57BL/6 mice bearing B16F10 tumors via intratumoral administration of different formulations with a dose of 2.0 mg kg⁻¹ DMXAA every other day. The individual tumor growth curves of mice bearing B16F10 tumors treated with (b) PBS, (c) DMXAA, (d) DAMX, (e) DESX, or (f) DSSX. The average tumor growth curves (g) of mice treated with different drug formulations. The tumor weights (h) and tumor inhibition rates (i) of mice at the end of therapy. (j) The H&E, TUNEL, and CD8⁺ T cell immunofluorescence staining images of tumor tissue sections at the end of therapy. Scale bar: 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

From the individual tumor growth curves, DAMX with a stable amide linker exhibited similar tumor growth inhibition ability as compared with DMXAA, demonstrating that amide linker could be cleaved in tumor cells to release DMXAA (Figure 6b–f). Meanwhile, DESX with an ester linker improved the efficacy for tumor growth inhibition as compared with DAMX, which was ascribed to the relatively efficient drug release via the ester linker. Importantly, DSSX exhibited the strongest tumor growth inhibition with six out of eight complete tumor remission mice at the end of treatment, indicating the robust STING activation capability for both APCs and tumor cell. The average tumor growth curves, tumor weights, and tumor inhibition rates of different treatment groups further demonstrated the antitumor efficacy for the SSAPIs, especially for DSSX with broad STING activation capability across immune cell and tumor cell (Figure 6g–i). Moreover, from the H&E and TUNEL sections of dissected tumors (Figure 6j), DSSX induced large area of tumor cell apoptosis or death to inhibit tumor growth, and DESX exhibited moderate effect on tumor cell death, which was more effective as compared with DAMX. The activation of tumor STING would reverse tumor microenvironment to increase CD8⁺ T cell infiltration, which was further illustrated via immunofluorescence staining [22]. Apparently, DSSX generated large amount of CD8⁺ T cells at tumor regions, and DESX displayed much less amount of CD8⁺ T cells in tumor tissues. These results indicated that DSSX exhibited superior tumor growth inhibition by efficiently activating the STING signaling pathway across APC and tumor cell.

2.5. Antitumor Immune Responses

The activation of the STING signaling pathway can reverse TIME to promote dendritic cell maturation and T cell infiltration [16, 19]. To reveal the impact on major immune cells, SSAPIs were injected intratumorally for a single injection with a dose of 3.0 mg kg⁻¹ DMXAA. After 72 h, mice were sacrificed, and tumors and lymph nodes were harvested for immune cell analysis via multicolor flow cytometry.

As one of the most important antigen‐presenting cells, dendritic cell dominates the antigen presentation process to stimulate T cells for antitumor immunotherapy [27]. After a single treatment, DAMX could also increase the percentage of matured dendritic cells to around 24% as compared with the PBS control group (14%) (Figure 7a,b). DESX with the ester linker further improved the percentage of matured dendritic cells to around 29%, which displayed enhanced dendritic cell activation efficiency as compared with DMXAA and DAMX. Importantly, DSSX significantly enhanced the efficacy for dendritic cell maturation (38%) after a single injection, which was the highest among all treatment groups. Antigen presentation by dendritic cell will activate cytotoxic T cell and promote T cell tumor infiltration to recognize and kill cancer cells [16, 22]. The T cell population in tumors was further measured by multicolor flow cytometry. DAMX improved the CD8⁺ T cell population as compared with the PBS group, but the enhancement was less than free drug DMXAA (Figure 7c,d). However, both DESX and DSSX further increased the population of CD8⁺ T cell in tumor tissues, and DSSX exhibited the highest level of CD8⁺ T cell (∼60%). Meanwhile, the population of CD4⁺ T cell for all the treatment groups decreased to less than 35%, and DSSX further reduced the percentage of CD4⁺ T cell to around 19%. Thus, the ratio of CD8⁺ to CD4⁺ T cell also enhanced for these drug formulations as compared with the PBS group. DESX and DSSX exhibited increased ratio of CD8⁺ and CD4⁺ T cell (Figure 7f), especially for DSSX with the highest ratio of 3.1. Meanwhile, the expression levels of typical cytokine and chemokine, C‐X‐C motif chemokine ligand 10 (CXCL10) and interferon‐beta (IFN‐β), in tumor tissues were revealed via the PCR technique, which could drive immune cell tumor infiltration and induce tumor suppression [18]. As expected, DSSX significantly increased the mRNA levels of CXCL10 and IFN‐β in tumor tissues as in comparison with the other groups (Figure 7g,h), while DESX achieved superior mRNA levels of CXCL10 and IFN‐β as compared with DMXAA and DAMX. Finally, the protein levels of typical STING‐dependent inflammatory cytokines, TNF‐α and interferon‐gamma (IFN‐γ), of dissected tumor tissues were also detected via ELISA. Similarly, DSSX induced the highest levels of IFN‐γ and TNF‐α expressions in the dissected tumor tissues, and DESX displayed moderate increment of IFN‐γ and TNF‐α as compared with DMXAA and DAMX (Figure 7i,j). Collectively, these results revealed that DSSX with efficient STING activation across APC and tumor cell generated superior immune responses for tumor immunotherapy.

FIGURE 7.

SSAPIs selectively enhanced antitumor immune responses. Multicolor flow cytometric analysis (a) and quantifications (b) of the population of mature dendritic cells (CD11c⁺CD80⁺CD86⁺) in tumor draining lymph nodes. Multicolor flow cytometry analysis (c) and quantifications (d, e) of CD8⁺ T (CD45⁺CD3⁺CD8⁺) and CD4⁺ T (CD45⁺CD3⁺CD4⁺) cells in dissected tumor tissues. (f) The ratio of CD8⁺ and CD4⁺ T cells from flow cytometry analysis n = 4. The mRNA levels of IFN‐β (g) and CXCL10 (h) in tumor tissues for different treatment groups. Cytokine expression levels of IFN‐γ (i) and TNF‐α (j) in tumor tissues were measured via Elisa kit after different treatments n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The activation of the STING signaling pathway across broad cell populations exhibited improved antitumor therapeutic efficacy and immune responses, which were illustrated both in vitro and in vivo. These SSAPIs were prodrugs that displayed no pharmaceutical activities for STING activation, which required the covalent bond breakage and drug‐release processes [10, 16]. With broad STING‐mediated immune activation, the covalent bonds were cleaved in both immune cell and tumor cell, leading to increased amount of DMXAA in these cells. Thus, we set out to measure the total amount of DMXAA in tumors and also in major organs to validate this hypothesis. When mice B16F10 tumors were close to 250 mm³, DMXAA and the other three SSAPIs were injected intratumorally with a dose of 10 mg kg⁻¹, and mice were sacrificed at 6 and 12 h postinjection. The amount of DMXAA in tumors and major organs were analyzed via the HPLC technique (Figure S15). As shown in Figure S16, DESX showed much higher concentration of DMXAA as compared with DMXAA and DAMX groups, while DSSX further enhanced the DMXAA concentration in tumors at 6 h. The trend was familiar at 12 h as compared with 6 h, but the concentrations of DMXAA for all groups decreased, and the amounts of DMXAA in other major organs were quite low. These results could provide some evidence for the observed antitumor efficacy of these SSAPIs by activating immune cell and tumor cell.

To further elucidate the mechanism underlying preferential STING activation across different cell populations, we examined the cellular uptake of Cy5.5‐labeled SSAPIs using flow cytometry. Cell numbers and the concentration of Cy5.5 were kept constant in all uptake experiments. After 2 h of coincubation, the flow fluorescence intensities revealed that, for each cell type tested (Dendritic cells, M2 macrophages, and B16F10 tumor cells), there was no significant difference in the uptake of DAMX, DESX, and DSSX (Figure S17). In contrast, for each SSAPI, the fluorescence intensities of dendritic cells and M2 macrophages were significantly higher than that of B16F10 cells. In parallel, to evaluate the intracellular drug release profiles, the levels of DMXAA in B16F10 tumor cells following incubation with the various SSAPIs were quantified by HPLC at 2 and 4 h. For DAMX, the intracellular DMXAA amount was comparable to that of free DMXAA at 2 h, but was significantly elevated at 4 h (Figure S18). DESX yielded higher intracellular DMXAA levels than DAMX at both time points, and DSSX further increased the DMXAA accumulation relative to DESX. Given that the cellular uptake efficiencies of DAMX, DESX, and DSSX were similar in B16F10 cells (Figure S17), the pronounced differences in intracellular DMXAA concentrations suggest that drug release is predominantly governed by the linker‐controlled liberation process.

To investigate the generality and long‐term impact, a CT26 tumor‐bearing mice model was employed to evaluate the antitumor efficacy, survival, and immunological memory effect (Figure 8). Mice were treated with the same drug formulations, doses, administration route, and frequency as that of B16F10 melanoma. From the individual tumor growth curves, DMXAA attenuated tumor growth as compared with the PBS group (Figure 8b,c). DAMX further improved CT26 tumor growth suppression (Figure 8d), but without considerable difference as compared with DMXAA, ascribing to insufficient drug release process. DESX exhibited improved antitumor efficacy with two out of eight complete tumor remission, and DSSX significantly boosted antitumor efficacy with six out of eight complete tumor remission (Figure 8e,f). The inhibition impact of different treatment groups could be clearly observed from the average tumor growth curves (Figure 8g). The survival rates of mice treated with different formulations were monitored throughout the process, and DSSX remarkably prolonged the survival of mice with 100% survival rate over 60 days (Figure 8h). To illustrate impact of immunological memory, six tumor‐free mice from DSSX group were re‐challenged alongside age‐matched naïve mice. The re‐challenged tumor growth of DSSX treated tumor‐free mice was dramatically inhibited as compared with the naïve mice (Figure 8i–k). Meanwhile, the six re‐challenged mice remained survival and tumor‐free state for over 1 month (Figure 8l), demonstrating strong systemic immunological memory effect.

FIGURE 8.

SSAPIs exhibited enhanced tumor growth inhibition, prolonged survival, and durable immunological memory against re‐challenge. (a) Schematic illustration of the experimental timeline for survival monitoring and re‐challenge. The individual tumor growth curves of mice bearing CT26 tumors treated with (b) PBS, (c) DMXAA, (d) DAMX, (e) DESX, and (f) DSSX with equivalent dose of 2.0 mg kg⁻¹ DMXAA. (g) The average tumor growth curves of different treatment groups. (h) The percent survival of CT26 tumor‐bearing mice of different treatment groups (n = 8). The individual tumor growth curves in naïve (i) and DSSX (j) groups (n = 6; the day of tumor re‐inoculation was treated as day 0). The average tumor growth curves (k) and survival curves (l) in naïve and DSSX groups postrechallenge (n = 6). Survival analysis in h and l was conducted via a log‐rank test with the corresponding P values.

2.6. In Vivo Biosafety

To evaluate the biosafety of these SSAPIs in vivo, the body weights of mice treated with different drug formulations were monitored during the treatment period. At the end of therapy, mice blood was collected for biochemical analysis of AST, ALT, BUN, and CREA to investigate the effect on liver and kidney functions. Lastly, mice major organs, such as liver, heart, spleen, kidney, and lung, were harvested for histological analysis.

During the treatment period, there was no obvious difference of mice body weights for these treatment groups (Figure 9a). However, at the end of treatment, the mice body weight of the DSSX group did not increase as other groups, which was ascribed to the complete remission of six out of eight tumors after treatment. Meanwhile, the major organs of the treatment groups exhibited no damaged lesions or morphological abnormality from their H&E staining images (Figure 9b), demonstrating no apparent cytotoxicity for these treatment groups. Considering the importance of liver as the major organ for drug metabolism, the polarization of liver macrophage was investigated via the PCR technique. DMXAA induced increased mRNA level of iNOS in liver as compared with the SSAPIs, demonstrating the potential side effect of liver macrophage activation (Figure 9c), which was significantly reduced via the SSAPIs [38]. Finally, blood analysis with ALT, AST, BUN, and CREA revealed no statistical difference between these treatment groups or in comparison with the control group (Figure 9d–g). Some mice displayed evaluated levels for ALT, AST, or CREA, especially for the DMXAA group, but they were still within the normal range, which could be attributed to the heterogeneity of mice individuals. Overall, histological and blood analysis illustrated the good biosafety of these SSAPIs in vivo, which could further decrease the potential side effect of activating liver macrophage.

FIGURE 9.

In vivo biosafety evaluation. (a) Body weights of B16F10 tumor‐bearing mice treated with diverse drug formulations. n = 8. (b) The H&E staining of mice major organs (heart, liver, spleen, lung, and kidney) of different treatment groups at the end of therapy. (c) The mRNA level of iNOS of mice livers for different treatment groups. n = 3. (d–g) Blood serum levels of ALT (d), AST (e), BUN (f), and CREA (g) of mice for different drug formulations. n = 5. Scale bar: 100 µm. *p < 0.05, **p < 0.01.

3. Discussion

The STING signaling pathway plays a crucial role in regulating the innate immune responses, which has become a promising therapeutic target for cancer therapy [8, 42]. Activation of STING located in the (ER) triggers a cascade immune reaction of downstream pathways of the kinase TANK‐binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), which further induces the secretion of the transcription of type I interferons (IFNs), inflammatory cytokines, and chemokines [12, 43]. Natural and synthetic STING agonists have been discovered and utilized for preclinical or clinical trials, however, there is still no clinically approved STING agonists yet [44]. The toxicity of STING agonist is the major limitation, leading to severe side effects such as fever, chills, and cytokine syndromes. STING agonists commonly shows low stability against a variety of enzymes, low bioavailability, and tumor accumulation, which significantly reduce the immune activation efficiency in vivo. Finally, the tumor heterogeneity and immune activation variability compromise the efficacy for cancer therapy [45]. It is of great demand to develop innovative strategies to enhance the STING activation efficacy and reduce the toxicity.

The engineering of functional DDSs for STING agonists represents a powerful methodology to solve the existing challenges. DDSs delivering STING agonists typically exhibit improved tumor accumulation and penetration owing to their passive or active targeting to solid tumors, such as polymeric nanoparticles [16], supramolecular cyclic dinucleotide nanoparticles [18], PEG‐lipid nanodiscs [19], pH‐responsive micelles [13], spatiotemporally‐tailored STING nano‐immunomodulators [46], mesoporous polydopamine‐based nanoparticles [47], hybrid codelivery systems [48], and so on. By coating with diverse cell membranes, the hybrid DDSs realize elongated blood circulation, tumor targeting or penetration, including tumor cell membrane [49], dendritic cell‐derived vesicle [50], T cell membrane [51], red blood cell membrane [52], virus‐mimicking nanovaccine [53], and so on. The toxicity of the above systems has been partially resolved as compared with free STING agonists because of the targeting and controlled release functions of these DDSs. One concern is the increased accumulation in major organs, like liver and spleen, which may bring certain risk of immune activation in these organs. The major concern is the potential cell or immunogenic toxicity of these DDSs, showing systemic cytotoxicity originated from the intrinsic systems or their metabolites or fragments [54]. An important aspect is to increase the STING activation efficacy in solid tumors to induce potent immune responses, which has been a hot research field recently. These innovative DDSs illustrate the importance of targeting a variety of immune cell, such as tumor‐associated macrophage [24, 25], and dendritic cell [26, 39]. In fact, it has been demonstrated that the activation of various immune cell via the STING signaling generates strong immune responses to reverse TIME [14, 24]. Meanwhile, the activation of tumor cell STING signaling prompts the downstream pathway to produce IFNs and proinflammatory cytokines, which creates a kind of ‘hot tumor’ environment for improved cancer therapy [28, 30]. There exists a debate whether to activate the STING signaling in immune cell, such as dendritic cell and macrophage, or in tumor cell in order to accomplish efficient immunotherapy. In fact, it is a great challenge to quantify the contribution of the activation of the STING signaling of different cell populations to the efficacy of antitumor therapy.

In this work, we developed a platform to quantify the STING activation capability across diverse cell populations in solid tumor via a type of selective STING‐activating polysaccharide immunomodulators (SSAPIs). Dextran was utilized as the nanocarrier of STING agonist DMXAA since it can target immune cell, such as macrophage and dendritic cell, to balance the difference of cell number between immune cell and tumor cell in solid tumor [36]. Four typical covalent linkers with defined drug‐release mechanisms were designed to tune the STING activation capability of immune cell and tumor cell, including the amide linker, the ester linker and the disulfide linker. Cell experiments illustrated that the amide SSAPI (DAMX) had low STING activation efficiency for both immune cell and tumor cell. Although, the ester SSAPI (DESX) exhibited efficient STING activation in macrophage and dendritic cells but moderate efficacy in tumor cell. Meanwhile, the disulfide SSAPI (DSSX) displayed potent STING activation in tumor cell and improved efficacy in APC cells. As a result, DSSX with strong and broad STING activation capability significantly inhibited tumor growth with six out of eight complete tumor remission in B16F10 and CT26 tumor bearing mice models by reprogramming tumor TIME. The precise cell‐type‐specific release mechanism remains incompletely resolved, potentially due to differential levels of cellular redox species and enzymes. Elucidating these contributing factors represents a valuable direction for future research.

Compared with previously reported DDSs for STING agonist delivery, the SSAPIs exhibit several key features: (i) Selective STING activation across different cell populations by incorporating with well‐characterized linkers, (ii) elimination complications associated with nanoparticle assembly and disassembly via polysaccharide‐drug conjugates, (iii) the polysaccharide itself as both the cell‐targeting ligand and drug nanocarrier. Meanwhile, these SSAPIs do present challenges, such as the long‐term stability concerns, a relatively short blood circulation time, and the potential drug resistance as a monotherapy. This work did not develop novel multifunctional linkers to control the activation of STING signaling in a specific type of cell population in heterogenous tumor since only defined linkers with well‐known release mechanisms could bring to solid conclusions. This work emphasizes the importance of activating the STING signaling pathway across broad cell populations to accomplish superior immune responses for cancer therapy.

4. Conclusion

In summary, we have developed a type of selective STING‐activating polysaccharide immunomodulators (SSAPIs) to quantitatively compare the STING activation efficiency across tumor cell, macrophage, and dendritic cell. These SSAPIs were macromolecular single‐molecular prodrugs with similar physicochemical properties, while exhibiting distinct drug release kinetics by tuning the defined covalent linkers. The STING activation efficiency was investigated in macrophage, dendritic cell, and tumor cell in vitro, demonstrating preferential activation of macrophage and dendritic cell via DESX and tumor cell via DSSX. In both B16F10 and CT26 tumor‐bearing mice models, DSSX displayed much superior tumor growth inhibition efficacy with six out of eight complete tumor remissions as compared with the other SSAPIs, ascribing to the broad STING activation across immune cell and tumor cell. Lastly, these SSAPIs decreased the activation of liver macrophage as compared with the free STING agonist. Thus, this work highlights the conception of activation of the STING signaling pathway across multiple cell populations for cancer therapy.

5. Methods

5.1. CD86 and NO Measurements

M2 macrophages were seeded into a 24‐well plate with a density of 1 × 10⁵ cells/well, and allowed to adhere overnight. The cells were treated with free DMXAA or the SSAPIs at 35 µM for 24 h. After incubation, cells were harvested, and stained with a phycoerythrin (PE)‐conjugated anti‐mouse CD86 antibody (BD Biosciences, 553692) for 30 min at 4°C. After washing with PBS supplemented with 0.1% BSA, cells were centrifuged at 300 g for 5 min (4°C) and re‐suspended in PBS for quantitative flow cytometric analysis. Supernatants were collected and centrifuged for 5 min at 10 000 g (4 °C) to remove any cell debris. Nitric oxide level was determined by Griess reaction (S0021s, Beyotime) according to manufacturer's protocol.

5.2. RT‐PCR Analysis

M2 macrophages were seeded in a 12‐well plate with a density of 8 × 10⁵ cells/well. B16F10 cells were seeded in a 12‐well plate with a density of 2 × 10⁶ cells/well. These cells were treated with different drug formulations for 24 h, and total RNAs were isolated by RNA Extraction Kit (Vazyme, RC102) followed by DNase treatment to eliminate genomic DNA contamination. Then, RNA (1 µg) was reverse transcribed into cDNA with a cDNA Synthesis Kit (BeyoRTTM, D7178L) in a 20 µL reaction volume, quantified via the SYBR method. Finally, the machine‐measured data were analyzed for changes in mRNA levels by the 2^−∆∆Ct method [38]. For the study of redox‐responsive drug release, B16F10 cells were pretreated with l‐buthionine‐(S,R)‐sulfoximine (BSO) at a final concentration of 100 µM for 24 h to deplete intracellular GSH, and the mRNA levels of IFN‐β, IFN‐γ, and IL‐6 were determined using the same method.

5.3. Western Blot Analysis

The expressions of STING, p‐STING, p‐TBK1, and p‐IRF3 of M2 macrophages and B16F10 melanoma cells were assessed by Western blot. Cells were treated with 35 µM DMXAA or DMXAA‐equivalent SSAPIs for 24 h. After washing, cells were lysed and total proteins were quantified. Equal amounts of denatured protein (100°C, 5 min) were separated by 10% SDS‐PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA/TBST for 1 h, then incubated with primary antibodies (1:1000) at 4°C overnight. The HRP‐conjugated secondary antibodies (1:1000, goat antirabbit/mouse IgG) were applied for 2 h. Protein bands were detected using ECL reagents and imaged via a ChemiDoc MP System (Bio‐Rad).

5.4. Analysis of BMDCs Via Flow Cytometry

Primary BMDCs isolated from C57BL/6 male mice (8‐week‐old) underwent 7‐day induction in RPMI‐1640 complete medium containing 20% FBS, 1% penicillin‐streptomycin, and cytokines (20 ng mL⁻¹ GM‐CSF + 5 ng mL⁻¹ IL‐4, PeproTech). Then, cells were treated with different drug formulations for 24 h. After washing, the single cell suspensions were stained with CD11c (Biolegend, 117309), CD80 (BD, 561954), and CD86 (BD, 563692) antibodies for flow cytometric analysis.

5.5. Ethical Statement

All animal experiments were conducted strictly in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. The animal study was approved by the Institutional Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (Ethics Number: PZSHUTCM2307160003, PZSHUTCM2406020001).

5.6. Animal Models and Treatments

Five‐week‐old male C57BL/6 and BALB/c mice were purchased from Shanghai SLAC Laboratory Animal Company and kept in a SPF animal facility in the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. Mice were subcutaneously injected with 5 × 10⁵ B16F10 cells in 100 µL RPMI‐1640 basic medium to establish the tumor model. Tumor volume was measured by a Vernier caliper and calculated as (Leng×Width²)/2. When mice volume was around 80–100 mm³, mice were randomly separated into five groups: PBS, DMXAA, DAMX, DESX, and DSSX. Mice were intratumorally injected with different formulations at a dose of 2.0 mg kg⁻¹ DMXAA every other day for four times. Tumor volume and body weight were recorded every other day. After therapy, mice were sacrificed, and major organs, tumors, lymph nodes and serum were collected for further analysis. Biochemical analysis was employed for mice serum to evaluate liver and kidney functions. H&E and TUNEL tests were performed for mice tumors or major organs for histopathological assay.

To establish the CT26 tumor model, 1 × 10⁶ CT26 cells in 100 µL RPMI‑1640 basic medium was subcutaneously injected into the right flanks of BALB/c mice. For therapeutic studies, mice were randomly divided into five groups: 1) PBS, 2) DMXAA, 3) DAMX, 4) DESX, 5) DSSX, and intratumorally injected with equivalent doses of DMXAA at 2.0 mg kg⁻¹. Treatments were repeated every other day for a total of four times. The tumor volume and mice weight were monitored throughout the experiment using the same method for B16F10 tumor therapy. Tumor volumes were recorded until tumors exceeded 1500 mm³, and Kaplan–Meier survival curves were plotted with the log‐rank test for significance investigation. Mice surviving DSSX treatment were rechallenged with 1.5 × 10⁶ CT26 cells, and age‐matched naïve mice were employed as controls. Mice volume were measured until humane endpoints.

5.7. Analysis of Immune Cells Via Flow Cytometry

Tumor tissues were cut into pastes and lysed for about 2 h at 37 °C on a shaker. The cellular fluids were passed through a 70 µm filter and centrifuged for 3 min at 1000 rpm. The cells were washed with PBS before staining with CD45 (BD, 557659), CD3 (BD, 553061), CD4 (BioLegend, 100512), and CD8 (BioLegend, 100734) at 4°C for 30 min. Finally, the samples were measured via multicolor flow cytometric analysis. Tumor draining lymph nodes were removed and grounded to obtain cell suspensions. The fluids were filtered via 70 µm cell strainers to prepare single cell suspensions and further stained with CD11c (Biolegend, 117309), CD80 (BD, 561954), and CD86 (BD, 563692) antibodies for multicolor flow cytometric analysis. The flow cytometry gating strategies for DCs and T cells were provided in Figures S19 and S20.

5.8. Biodistribution Analysis

When tumor volume reached approximately 250 mm³, different drug formulations were administered intratumorally at a dose of 10 mg kg⁻¹. At 6 and 12 h postinjection, mice were sacrificed and major organs, including tumor, heart, liver, spleen, lung, and kidney, were promptly harvested. Different tissue samples with known weights were homogenized, centrifuged at 12 000 rpm for 30 min (4°C). The supernatants were dried using a centrifugal vacuum concentrator, redissolved in 100 µL of a methanol/water mixture (8:1, v/v, containing 1% DMSO), and centrifuged at 12 000 rpm for 30 min. The final supernatants were collected and subjected to HPLC analysis. The concentration (µg/mg) was calculated using the following formula:

Tissue concentration (µg/mg) = [Measured concentration (ng/mL) × Total homogenate volume (mL)] / Tissue weight (mg)

5.9. Safety Assessment

Since the start of treatment, mice body weights were measured every other day. At the end of therapy, serum was collected to measure the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), and creatinine (CREA) by biochemical analysis. Dissected organs were fixed via paraformaldehyde (4%), put in paraffin, and cut into 5 µm sections for H&E staining. Liver tissues were utilized for RT‐PCR analysis of M1 macrophage marker, iNOS.

5.10. Statistical Analysis

Cell experimental results were illustrated as mean ± SD (n = 3 unless specified). All animal statistical data were presented as mean ± SD unless otherwise stated, and analyzed by two‐tailed Student's t test via Microsoft Excel or Origin 2020. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) were defined as statistically significant.

Author Contributions

Q.Z., J.M., and D.X. performed the experiments, analyzed the data and wrote the manuscript. M.Z., S.C., J.W., W.C., and J.G. helped with the animal experiments. M.Z. also helped with cell experiments. S.M., G.G., and X.Z. provided the conceptual advice and supervised the study. L.X. and H.D. conceived and designed the experiments, analyzed the data, and revised the manuscript. H.D. also supervised the study.

Supporting Information

The Supporting Information file contains: Schemes S1, Figures S1–S20, Tables S1,S2, and supporting methods describing the synthesis of the polysaccharide nano‐immunomodulators, drug release, and additional cell experimental methods.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: adma74178‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.