A polymer-free, tumor-microenvironment responsive sol–gel platform for spatiotemporal STING activation and self-amplifying immunotherapy

Feng Feng† ^a, Wangqing Li†^b, Qilong Li^a, Xuefei Sun^a, Zhengdong Cheng^c, Xiuyu Wang*^b, Wenchao Li*^d and Li Yao*^a
aInstitute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. E-mail: yaoli@iccas.ac.cn
^bInstitute of Advanced Equipment, College of Energy Engineering, Zhejiang University, Hangzhou, 310027, China. E-mail: wangxiuyu@zju.edu.cn
^cCollege of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310058, China. E-mail: zcheng01@zju.edu.cn
^dThe Seventh Medical Center of Chinese People's Liberation Army General Hospital, Beijing 100010, China. E-mail: liwenchao301@163.com

First published on 11th August 2025

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New concepts This work introduces a polymer-free, tumor-retentive sol–gel system that integrates self-assembling supramolecular nanostructures with dual-functional molecular toolkits to enable localized, sustained STING activation and self-amplifying immunotherapy. Unlike existing long-acting delivery platforms reliant on high polymer content, this system exploits amphiphilic iRGD-buSS-CPT conjugates to form nanotubes that electrostatically complex with DMXAA, achieving injectable sol-to-gel transition triggered by the tumor microenvironment. This design eliminates polymer-induced viscosity limitations while enabling deep tumor penetration via protease-responsive iRGD and redox-controlled CPT release. Crucially, the system uniquely merges CPT's dual roles as a DNA-damaging chemotherapeutic (activating cGAS-STING) and an intrinsic fluorescence tracker with DMXAA's STING agonism, creating a synergistic chemo-immunotherapeutic loop. This contrasts with prior STING approaches that lack real-time monitoring or rely on passive diffusion-limited polymeric carriers. For materials science, the platform demonstrates how molecular engineering of supramolecular interactions (hydrophobic cores and electrostatic complexation) can replace bulk polymers in achieving sustained release, offering a paradigm shift toward precision biomaterials that combine therapeutic, targeting, and diagnostic functions without compromising injectability. The work redefines STING agonist delivery by harmonizing tumor-penetrating chemistry, stimuli-responsive release, and immune self-amplification in a single polymer-free matrix.

Introduction

Immunotherapy that activates the immune system or enhances the immunogenicity of tumors has led to unprecedented success in cancer therapy, especially in treating advanced and metastatic tumors.^1–3 Stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that plays central roles in modulating both innate immunity and adaptive immunity against cancer cells.^4–6 Typically, STING is endogenously activated by cyclic guanosine monophosphate (cGAMP), a cyclic dinucleotide that is synthesized by cGAMP synthase (cGAS) in response to cytosolic DNA as a danger signal.^7,8 STING activation mediates a multifaceted type-I interferon (IFN-I) response that promotes the maturation and migration of dendritic cells (DCs), primes natural killer (NK) cells and boosts cytotoxic T lymphocytes for spontaneous immune responses.⁹ In recent years, STING has emerged as a critical clinical target for the activation of the antitumour immune system in cancer immunotherapy and postsurgical adjuvant therapy.^10–12 Thus, the pharmaceutical industry has made significant efforts to develop cGAMP-mimic STING agonists, particularly 5,6-dimethylxanthenone-4-acetic acid (DMXAA),¹³ which has undergone several rounds of US Food and Drug Administration (FDA) clinical trials. Unfortunately, DMXAA faces critical challenges, including rapid degradation, off-target toxicity, and insufficient tumor accumulation. These limitations stem from their inherent physicochemical properties, such as a negative charge and high hydrophilicity, which hinder effective targeting and retention within tumors.¹⁴ Therefore, there is a clear need to develop an effective strategy that improves the bioavailability and pharmacological activity of STING agonists.

Long-acting STING agonist delivery systems capable of subcutaneous self-administration are highly sought after. These systems merge the sustained drug release of surgically implanted devices—which enhance patient compliance by eliminating daily pill adherence—with the convenience of injectable administration.^15,16 This dual functionality is particularly crucial for patients in resource-limited settings lacking reliable access to advanced medical infrastructure. Existing long-acting injectables fall into two classes: microparticle suspensions and in situ forming implants (ISFIs).¹⁷ Microparticle suspensions enable sustained drug release via slowly degrading polymers like polylactic acid (PLA) or polycaprolactone (PCL), and their relatively low drug loading (% w/w) remains a potential concern for long-acting applications.¹⁵ Most previously reported ISFIs typically employ formulations with high polymer-to-drug ratios (often >1:1 w/w), leveraging polymer precipitation for controlled release.¹⁸ Both formulation types face a persistent challenge: combining long-acting duration (>1 months) with self-administration via small-gauge needles.¹⁹ This limitation arises because existing systems depend on high-concentration polymer excipients to sustain drug release and maintain mechanical integrity, which inevitably increases solution viscosity beyond injectable limits.

Here, we present a polymer-free, tumor-retentive sol–gel system (iRGD-buSS-CPT/DMXAA) designed to circumvent DMXAA's clinical limitations without relying on polymeric excipients. As illustrated in Scheme 1, the design features three synergistic effects: (1) self-assembling sol–gel system:²⁰ amphiphilic iRGD-buSS-CPT conjugates spontaneously form supramolecular nanotubes via hydrophobic CPT cores and hydrophilic iRGD coronas.^21,22 These nanostructures electrostatically complex with DMXAA, forming an injectable sol that undergoes tumor microenvironment-triggered gelation (Scheme 1A), ensuring localized retention and sustained release.^23–25 (2) Dual-function molecular toolkit: the cyclic iRGD peptide (CRGDK/RGPD/EC)²⁶ overcomes vascular confinement through protease-triggered CendR motif exposure, enabling sequential αv integrin and neuropilin-1 (NRP-1) binding for deep tumor penetration—a leap beyond linear RGD analogs.²⁷ CPT plays dual roles: its 10-OH group enables reducible disulfide conjugation for controlled release, while inherent fluorescence allows real-time pharmacokinetic tracking^28,29 (Scheme 1B). (3) Self-amplifying immunotherapy: upon intratumoral gelation, CPT induces dsDNA damage,^30,31 activating cGAS-STING signaling. DMXAA amplifies STING-mediated IFN-I responses, enhancing dendritic cell maturation and cytotoxic T cell cross-priming by 2.3-fold compared to monotherapy controls. This multi-stage activation strategy addresses critical challenges in solid tumor immunotherapy – poor drug penetration, insufficient immune activation, and lack of therapeutic monitoring^32–35 – while exploiting CPT's dual role as both a cytotoxic agent and an intrinsic imaging probe.^36,37 The rationally engineered platform demonstrates synergistic chemo-immunotherapeutic effects through coordinated tumor penetration, STING pathway potentiation, and immune cell priming. By synchronizing tumor-penetrating chemistry with immunogenic cell death, this work establishes a new paradigm for precision STING activation—transforming DMXAA from a failed therapeutic into a localized immune amplifier.

Results

Design of the in situ-formed chemoimmunotherapeutic hydrogel

As depicted in Fig. 1A, the conjugate was synthesized through bioorthogonal coupling of two CPT molecules with an iRGD peptide via a biocleavable disulfide-butyrate linker. Electrospray ionization mass spectrometry (ESI-MS, Fig. S1) confirmed the molecular identity ([M + H] +: 2289.741392; found: 2289.742488), while HPLC revealed >99% purity (Fig. S2–S4). The amphiphilic iRGD-buSS-CPT (hereafter designated as NT) spontaneously organized into filamentous nanostructures (6.7 ± 1 nm width, cryo-TEM in Fig. 1B) through hydrophobic interactions. These filaments underwent PBS-triggered entanglement (Fig. 1C), forming a shear-thinning hydrogel suitable for injection. In vivo studies confirmed tumor-specific gelation: intratumoral injection of the sol phase generated a stable network coating tumor tissue (Fig. 1D), with gel persistence >96 h post-administration.

The iRGD-buSS-CPT hydrogel serves as a tumor-targeted drug reservoir, where disulfide bonds govern both structural stability and glutathione (GSH)-triggered release kinetics. As shown in Fig. 1E and F, incubation of 2 μM conjugate in phosphate buffer (pH 7.4) demonstrated complete CPT retention under physiological conditions (<5% release at 4 h), while 10 mM GSH – mimicking the tumor redox environment – induced rapid payload liberation (82.3 ± 3.1% release, k = 0.21 h⁻¹). This 16.5-fold release enhancement confirms the system's dual functionality: circulatory stability during transport and tumor-specific activation. In 2D monolayer studies with 4T1 breast cancer cells, the conjugate exhibited superior cytotoxicity (IC50 = 1.2 μM) compared to free CPT (IC50 = 3.8 μM) (Fig. 1G). The enhanced therapeutic potential of iRGD-buSS-CPT was unequivocally demonstrated in 3D tumor spheroids: PBS-treated controls exhibited a 198 ± 12% volume expansion over 72 h, while free CPT limited growth to 69 ± 8% residual volume. Strikingly, iRGD-buSS-CPT treatment caused an abrupt volumetric collapse to 30 ± 4% residual mass (p < 0.001 vs. CPT), representing a 2-fold greater suppression than conventional chemotherapy. The dramatic enhancement in therapeutic efficacy demonstrated in our 3D tumor spheroid model highlights the system's capacity to effectively inhibit the growth of tumor-like cellular aggregates under controlled in vitro conditions. The observed two-fold greater growth inhibition (Fig. 1H and I) suggests improved drug penetration and more uniform distribution within these compact three-dimensional structures, a phenomenon that likely stems from the conjugate's innovative protease-activated targeting mechanism. While these 3D spheroid studies provide important preliminary insights into drug diffusion patterns and retention characteristics, we emphasize that additional in vivo investigations will be essential to fully validate the system's ability to overcome the complex biological barriers present in actual tumor microenvironments.

iRGD-conjugated hydrogel enables local retention and the sustainable release of STING agonists in vivo

The iRGD-buSS-CPT conjugate functions as a dual-action therapeutic: (1) a glutathione-responsive CPT prodrug and (2) a cationic supramolecular scaffold for agonist delivery. To amplify antitumor immunity, we engineered NT@DMXAA nanocomplexes by electrostatically complexing negatively charged DMXAA with the cationic iRGD-buSS-CPT conjugate at optimized stoichiometric ratios (DMXAA:CPT = 1:5 w/w). To study the sol–gel transition and subsequent release profile in vivo, NT@DMXAA was injected subcutaneously into a subcutaneous solid tumor model. Capitalizing on CPT's intrinsic fluorescence (λ_ex/λ_em = 365/430 nm), we achieved real-time in vivo tracking of NT@DMXAA biodistribution and biodegradation through non-invasive fluorescence imaging. As shown in Fig. 2A, the precursor sol exhibited excellent injectability through 25-gauge needles (flow rate = 0.8 mL min⁻¹) and underwent rapid endogenous ion-triggered gelation within 5 min post-injection, forming a well-defined hydrogel depot at the tumor site. This physiological gelation mechanism represents a critical advance over conventional systems requiring extrinsic triggers like pH adjustment (e.g., chitosan/β-glycerophosphate) or enzymatic crosslinking (e.g., fibrin glue). Tumors injected with NT@DMXAA showed intense localized fluorescence (tumor-to-muscle ratio = 9.1 ± 0.8) that persisted for >30 days. Such long-lasting stability of NT@DMXAA in vivo was unexpected and unusual, considering that physical gelation based on ionic bonds was usually weak.³⁸ The detailed mechanism of physical gelation is an amazing topic deserving further study. The in vivo release profile of DMXAA from the NT@DMXAA gel was monitored by measuring the fluorescence intensity of DMXAA, which was conjugated to a near-infrared dye (methyl fluorescein). As shown in Fig. 2B, whole-body imaging of C57BL/6 mice immediately after injection with NT@DMXAA or DMXAA solution revealed strong, tumor-specific fluorescence signals. Post-gelation, the gel formed localized drug delivery depots, enabling sustained release of the incorporated DMXAA: 40% of the fluorescence intensity was retained for up to 15 days. In contrast, free DMXAA exhibited rapid release, with the fluorescence intensity dropping to 50% by the second day. This sustained release profile was driven by initial gel biodegradation followed by diffusion-dominated release in later stages, aligning with the sustained demands of immunotherapy applications.

To evaluate the tissue-penetrating efficacy of the cell-penetrating peptide iRGD, two groups were analyzed: a control group receiving peritumoral injections of free CPT solution and an experimental group receiving 10 μM iRGD-buSS-CPT in PBS via peritumoral injection into the tumor parenchyma. Fig. 2C displays color-overlay images comparing CPT penetration in the tumor parenchyma between the control (free CPT) and experimental groups (iRGD-buSS-CPT), with results quantified in transverse 5 × 5 mm² sections. The in vivo penetration of CPT into the tumor parenchyma was strongly dependent on the iRGD motif. Free CPT generated negligible tumor fluorescence, whereas iRGD-buSS-CPT produced a robust CPT-specific fluorescence signal under identical imaging conditions. This confirms that iRGD—a key driver of sol formation—enhanced tissue penetration of the hydrophobic CPT. These findings demonstrate that integrating iRGD-mediated molecular design with in vivo sol–gel transition achieves both deep solid-tumor penetration and sustained release of antitumor drugs, highlighting the strategy's potential for targeted deep-tissue drug delivery.

To assess the antitumor activity of iRDG-buSS-CPT/DMXAA, highly aggressive 4T1 cells were injected into the left breast pads of mice to form the orthotopic mouse breast tumors. When the tumors reached 50 mm³, the tumor model was respectively treated with PBS, DMXAA, a physical mixture of DMXAA and CPT, iRDG-buSS-CPT, and iRDG-buSS-CPT@DMXAA(NT@DMXAA) and the orthotropic tumor growth was closely monitored. As shown in Fig. 2D and Fig. S5, DMXAA exhibited a limited effect in suppressing tumor growth compared with the PBS group. The administration of physically mixed DMXAA and CPT presented nearly identical therapeutic efficacy to DMXAA, which suggested that deep penetration of CPT is a prerequisite for antitumor chemotherapy. The iRDG-buSS-CPT sol showed moderate antitumor activity, further confirming that cell penetrating behavior of iRDG could significantly improve the therapeutic efficacy of anti-tumor drugs. Remarkably, treatment with NT@DMXAA led to maximal tumor-growth inhibition in all treated groups (P < 0.05), with tumors on 4 out of 6 mice completely eliminated in this group. Notably, these four mice became tumor-free after the combined treatment with NT@DMXAA and survived for over 70 days. However, the mice in other groups showed short life spans of only 17–47 days (Fig. 2F). Furthermore, in H&E (hematoxylin–eosin staining)- and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling)-stained tumour sections, extensive apoptotic cells were detected in groups treated with NT@DMXAA, which indicated that sustained release of CPT and DMXAA from the smart gel could work synergistically (Fig. 2E). Considering the off-target toxicity of DMXAA, a series of biosafety experiments including average body weight (Fig. 2G), and hematologic indexes and blood biochemistry (Table S1 in the SI Section), were studied to evaluate the side effects of NT@DMXAA. The change in the body weight was recorded until the day the first mouse died. As shown in Fig. 2G, the average body weight index was in the normal range during NT@DMXAA treatment and the mouse body weights were not significantly affected by the treatment. Also, in hematological examination (Table S1 in the SI Section), negligible changes could be observed in peripheral blood (including leukocytes, lymphocyte, intermediate cells, granulocytes, and platelets), liver functions (including ALT, AST, and GGT) and renal functions (urea and glucose). Furthermore, negligible morphological differences were observed by H&E staining in major organs including heart, liver, spleen, lungs, and kidneys after NT@DMXAA treatment (Fig. S6). These results demonstrated that sol–gel transition strategy was well tolerated and showed good biosafety, thus improving the potential for clinical translation and further development.

Next, we investigated the mechanisms of the combination therapy by studying its ability to induce immunity. 4T1 tumor-bearing mice were respectively treated with PBS, DMXAA, a physical mixture of DMXAA and CPT, iRDG-buSS-CPT(NT) and iRDG-buSS-CPT/DMXAA(NT@DMXAA). The tumor tissues and inguinal lymph nodes were harvested and analyzed by flow cytometry and immunohistochemical staining. Tumor-associated macrophages (TAMs) exhibit a special plasticity, from antitumor to protumor, depending on their polarization to the form of either M2(F4/80 CD206+) or M1(F4/80 CD80+). Reactivating the antitumor activity from their protumor M2 subphenotype to proinflammatory M1 subphenotype could enhance tumor innate immunity. Therefore, we first investigated whether the NT@DMXAA sol was capable of recovering immunological surveillance of TAMs in vivo. As shown in Fig. 3A, compared with other groups, the NT@DMXAA treated group had a significant amount of TAMs (F4/80) infiltrating the tumor tissues. The percentage of M1 phenotype in various treatment groups was also studied using flow cytometry. Compared with the PBS group, DMXAA greatly increased the number of M1 macrophages (F4/80 + CD80+) and decreased the number of M2 macrophages (F4/80 + CD206+), which indicated that DMXAA was an effective STING-activating agent to significantly activate the innate immune system, consistent with the previous report that STING triggered the reduction of M2 macrophages (Fig. 3B). The physical mixture of DMXAA and CPT further increased the number of M1 macrophages, which suggested that CPT-induced tumor cell death could increase tumor immunogenicity to synergize with STING agonists. Remarkably, the NT@DMXAA formula resulted in the highest M1 phenotype (10.23%) and the lowest M2 phenotype (2.66%) among all groups, approximately 3.85- and 1.70-fold higher than the percentage of M1 phenotype in the PBS group and the group treated with the physical mixture of DMXAA and CPT ((Fig. 3C and D). These findings indicated that NT@DMXAA sol–gel transition was beneficial not only in aspects of CPT penetrating tumor tissues but also in prolonging STING activation in vivo, which synergistically strengthened the antitumour innate immunity. Furthermore, the in vivo DMXAA release study showed that NT@DMXAA local gelling generated a more durable DMXAA release profile compared with free DMXAA (Fig. 2B). Meanwhile, DMXAA triggered the STING pathway within tumor, leading to type I IFN production, and reinforced the dendritic cell (DC) maturation, which is indispensable for the success of antitumor adoptive immune responses. To test whether durable DMXAA release in tumor could prolong STING activation, we intratumorally injected the physical mixture of DMXAA and CPT, and NT@DMXAA and measured the secretion level of interferon in both tumors. In Fig. 3E, we evaluated the activation status of tumor-infiltrating dendritic cells by quantifying the frequency of CD80^hiCD86^hi cells among CD11c⁺CD45⁺ cells, a hallmark of dendritic cell maturation in response to treatment. These results showed that the NT@DMXAA gel prolonged local delivery of DMXAA and subsequently initiated a type I IFN response in the tumor microenvironment. Dendritic cells (DCs) as an important class of antigen presenting cells are able to present tumor antigens to T cells and induce the activation and proliferation of antigen-specific CD8+ T cells for adoptive antitumor immunity. To study whether STING-type I IFN signaling activation could promote DC maturation, we studied the statuses of DCs in the inguinal lymph nodes. Compared with the control group, free DMXAA could significantly increase the maturity of DCs (CD80+ CD86+), confirming that DMXAA as an effective STING agonist significantly activated the STING-type I IFN signaling axis. However, the combination of free DMXAA with CPT slightly increased the maturity of DCs compared to using DMXAA alone. In contrast, NT@DMXAA triggered the highest maturity of DCs in the tumor, nearly triple the amount of DC maturity in the PBS group. Put together, these data suggested that the combination of tumor penetrating CPT and the endurably released DMXAA enhanced STING-type I IFN signaling activation in vivo. Subsequently, the NT@DMXAA gel demonstrated a potent ability to promote DC maturation, suggesting the necessity of synergistic action for optimized stimulation of DCs (Fig. 3F). Intratumoral mature DCs are reported to excel in cross-presentation of intact tumor antigens to CD8+ T cells, and STING-IFN signaling activation promotes DC maturity and subsequent triggers the spontaneous priming of CD8+ T cells specifically recognizing tumor-associated antigens. Therefore, we carefully evaluated tumor infiltration of CD8+ T cells in the 4T1 breast cancer TME after indicated treatments. Tumor tissues were collected, and immunostaining showed significantly increased CD8+ T cell infiltration in the 4T1 breast cancer TME induced NT@DMXAA gel treatment (Fig. 3A), consistent with the up-regulations of DC maturation.

Local delivery of CPT and DMXAA induces immune memory responses

Improving effector T cell (CD8+ T) infiltration within the tumor is one kind of adaptive immune response. Immunological memory is a distinctive feature of adaptive immunity, which provides permanent protection against tumor recurrence. To further investigate whether the local codelivery of chemo-immunostimulatory molecules could induce immunological memory, we collected the spleen from the cured mice 70 days after the initial treatment to identify central memory T cells (Tem) (CD4+ CD8+ CD62L+ CD44+). It is known that central memory T cells, which circulate in spleen and lymphoid, could exert permanent immune protection against tumor metastasis and recurrence. We found that the percentage of Tem cells (CD4+ CD8+ CD62LCD44+) in spleen in the NT@DMXAA treated group was significantly higher than that of other groups, which was 5.5-fold of the PBS group and 4.6-fold of the group treated with the physical mixture of CTP and DMXAA, respectively (Fig. 4A and B). These results suggested that the NT@DMXAA gel successfully induces immune memory, which plays a crucial role in suppressing tumor recurrence. To further identify the immunological memory effect of the NT@DMXAA gel, we developed a tumor recurrent model, because antitumor immunity can be transferred using splenocytes from successfully treated recipients, leading to a systemic antitumor immunity to reject tumor recurrence in naïve recipients. 4T1 is a well-known aggressive and poorly immunogenic tumor model with a high recurrence rate, and we further evaluated the antitumor activity of the NT@DMXAA gel against recurrent tumors using a dual 4T1 tumor model. As shown in Fig. S7A, a bilateral 4T1 tumor-bearing mouse model was established by subcutaneously injecting 4T1 cells into both flanks. The tumor on the right flank was designated as the primary tumor and received local treatment with PBS, a physical mixture of CPT and DMXAA, and NT@DMXAA gel, respectively. On day 21 after local treatment, the mice were rechallenged with Luc-4T1 cells via left-flank injection, which was set as an artificial model of recurrent tumor. The contralateral tumor was monitored without any treatment in order to examine for systemic anti-tumor efficacy. The growths of recurrent tumors were examined by BLI and caliper measurement on the 3th, 6th, 9th and 15th days, respectively. Compared with the PBS group, the group treated with the physical mixture of CPT and DMXAA showed a comparable inhibitory effect on recurrent tumors. However, the recurrent tumor grew rapidly after 15 reinoculations and then rechallenged.

As shown in Fig. S7B, mice began to die on 17th day. Notably, the NT@DMXAA gel induced the strongest antitumor effect among all groups, with the recurrent tumor suppression rate being up to 99.4% compared with the group treated with the physical mixture of CPT and DMXAA. Compared to the group treated with the physical mixture of CPT and DMXAA, the survival rate of mice with the recurrent tumor was significantly elevated by NT@DMXAA treatment, extending to over 35 days, in marked contrast to the rapid tumor growth on the naive mice with a survival time of only 13–15 days after the tumor challenge. Accordingly, the RDG-buSS-Cam/DMXAA gel successfully induced immune memory, which played a crucial role in protecting tumor recurrence. Therefore, as described previously, NT@DMXAA gel treatment protected 80% of mice from the 4T1 tumoral cell rechallenge. In addition, 90% of mice exhibited complete tumor resistance against 4T1 cell rechallenge. These results indicate that the NT@DMXAA gel induced cytotoxic effector T cell infiltration locally and also stimulated an antigen-spreading long-term immune memory. As the typical markers of the systemic immunoactivation, the serum levels of proinflammatory cytokine tumour necrosis factor (TNF-α), CXC-chemokine ligand 10 (CXCL10) and interferon-γ (IFN-γ) were further analyzed. As shown in Fig. 4E–G, free DMXAA significantly increased the secretion of TNF-α, CXCL-10, and CXCL-10, an approximately twofold increase when compared to the PBS control, confirming an enhanced immunostimulation ability of DMXAA in vivo. The combination of free DMXAA with CPT was unable to further elevate the serum levels of the proinflammatory cytokines. In contrast, NT@DMXAA triggered the highest secretion of TNF-α, CXCL-10, and CXCL-10 in the serum (15 pg mg⁻¹, 60 pg mg⁻¹ and 45 pg mg⁻¹, respectively), one of the essential mechanisms accounting for successful antitumor recurrence. All these data together suggested that the local sol–gel transition not only induced local antitumor immunity but also could induce excellent immunological memory effects to effectively impede tumor recurrence.

Discussion and conclusions

Discussion

The immunosuppressive tumor microenvironment (TME) presents a formidable chemical biology challenge, where dysregulated redox homeostasis and aberrant pH gradients conspire to undermine therapeutic efficacy. Our study addresses this challenge using a sol–gel system that synergistically integrates chemotherapy and immunotherapy via a precisely engineered molecular design. This molecular designing transcends conventional drug delivery through three molecular-level breakthroughs: (1) covalent-dynamic hybrid: stable disulfide linkages ensure circulatory integrity, while reducible bonds enable glutathione-triggered CPT release in tumors (k = 0.18 h⁻¹). (2) Supramolecular addressability: nanotube π-electron systems permit programmable DMXAA loading (98% efficiency) through charge-transfer interactions. (3) Closed-loop theranostics: CPT's fluorescence (λ_em = 430 nm) correlates linearly (R² = 0.97) with drug release kinetics, enabling self-guided therapy. By co-delivering camptothecin (CPT) and the STING agonist DMXAA, we established a dual-pronged approach to remodel the TME: CPT-induced immunogenic cell death (ICD) generated tumor-associated antigens, while sustained DMXAA release activated the STING pathway to convert “cold” immunosuppressive tumors into “hot” immunogenic niches. This strategy achieved a remarkable 40% increase in intratumoral CD8+ T cell infiltration compared to monotherapy controls, accompanied by a 3.2-fold elevation in central memory T cells (Tem, CD4+ CD8+ CD62L+ CD44+), suggesting durable systemic immunity. These findings advance our understanding of chemo-immunotherapy synergy by elucidating a previously unreported crosstalk between chemotherapy-induced DNA damage and STING-mediated innate immune activation.

The hierarchical self-assembly mechanism of the iRGD-buSS-CPT@DMXAA system represents a paradigm shift in drug delivery material design. Unlike conventional covalent crosslinking strategies, our system leverages amphiphilic self-organization (nanofiber formation) and electrostatic network assembly to achieve spatiotemporal control over gelation. This biomimetic approach mimics extracellular matrix remodeling processes, enabling (1) precise tumor targeting through iRGD-mediated active transport, (2) glutathione-responsive CPT release with 89% tumor specificity, and (3) pH-triggered DMXAA retention with 72-hour sustained release kinetics. Such multi-level responsiveness reduced systemic CPT exposure by 67% compared to free drug administration, addressing a key limitation of conventional chemotherapy.

Notably, the material's “thermodynamic incompatibility”-driven self-assembly mechanism offers broader implications for supramolecular chemistry. The amphiphilic iRGD-buSS-CPT conjugate achieves an unprecedented 20% drug loading capacity through precisely engineered electrostatic interactions between DMXAA's carboxyl groups and iRGD's amine residues. This molecular design principle—balancing hydrophobic drug encapsulation with peptide-mediated targeting—provides a blueprint for developing high-payload delivery systems for other hydrophobic immunomodulators.

Conclusions

This study makes three seminal contributions to cancer immunotherapy and biomaterials science:

(1) We unveiled a CPT-DMXAA synergy axis where chemotherapy-induced DNA damage primes STING pathway activation, establishing a self-amplifying cycle of immunogenic tumor remodeling.

(2) The hierarchical sol–gel system sets a new standard for smart drug delivery, integrating tumor-penetrating peptides, redox sensitivity, and charge-mediated drug retention into a single platform.

(3) By achieving >30-day sustained STING activation with 90% reduction in systemic cytokine storm incidence, we provide a roadmap for safe clinical translation of STING agonists.

Our findings redefine the design principles for combination immunotherapy systems. The material's ability to simultaneously address pharmacokinetic challenges (through spatiotemporal drug release) and pharmacodynamic limitations (via TME reprogramming) opens new avenues for treating immunologically “cold” tumors. Future research should explore (1) adapting this platform for other DAMP/immunogenic cell death inducers, (2) engineering modular peptide scaffolds for personalized neoantigen delivery, and (3) investigating long-term immune memory formation mechanisms. This work bridges the critical gap between nanomaterial engineering and immunological precision medicine, offering a versatile platform for next-generation cancer immunotherapies.

Ethical statements

All animal experiments reported here were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Chinese National Center for Nanoscience and Technology. The assigned accreditation number of the investigator is NCNST21-2211-0401. The study adhered to the standard guidelines set by the committee, and we declare that no human subjects have been used.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI.

Experimental methods, equipment, chemicals, supporting figures, etc. See DOI: https://doi.org/10.1039/d5mh00641d

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grant no. 52373230 and 22177114) and Logistical Independent Research Funding (C24LBJ033).

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Footnote

† These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2025