Remodeling Tumor Microenvironment Using Prodrug nMOFs for Synergistic Cancer Therapy

Remodeling Tumor Microenvironment Using Prodrug nMOFs for Synergistic Cancer Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Remodeling Tumor Microenvironment Using Prodrug nMOFs for Synergistic Cancer Therapy Junliang Dong, Jindong Ding, Shifan Luo, Ruoshui Li, Yi Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5402726/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 14 You are reading this latest preprint version Abstract Metal-organic frameworks (MOFs) hold tremendous potential in cancer therapy due to their remarkable structural and functional adaptability, enabling them to serve as nanocarriers for biopharmaceuticals and nanoreactors for organizing cascade bioreactions. Nevertheless, MOFs are predominantly utilized as biologically inactive carriers in most cases. Developing nanoscale prodrug MOFs suitable for biomedical applications remains a huge challenge. In this study, we have designed a novel prodrug nano-MOFs (DCCMH) using metformin (Met) and α-cyano-4-hydroxycinnamic acid (CHCA) as ligands for coordination self-assembly with Cu 2+ , followed by loading of DOX and surface modification with HA. Upon internalization by cancer cells, DCCMH releases Cu 2+ , CHCA, Met, and DOX in response to high levels of GSH-H 2 O 2 within the tumor microenvironment (TME); Cu 2+ depletes GSH and generates Cu + that subsequently catalyzes H 2 O 2 to hydroxyl radical through a Fenton reaction; CHCA induces a further decrease in intracellular pH and promotes Fenton reactions by inhibiting lactate efflux; Met up-regulates tyrosine kinase activity and enhances the chemotherapy of DOX. With the abilities to synergistically combine chemo/chemodynamic therapy and remodel the TME, the DCCMH nMOFs inhibit murine hepatoma effectively. This study presents a feasible strategy for fabricating prodrug nano-MOFs which are capable of remodeling TME to improve efficacy through synergistic cancer therapy. Prodrug metal-organic frameworks GSH-responsive degradation redox homeostasis TME-remodeling synergistic chemo/chemodynamic therapy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The tumor microenvironment (TME) plays a pivotal role in fostering the proliferation and dissemination of malignant cells [ 1 , 2 ]. It is characterized by elevated levels of hydrogen peroxide (H 2 O 2 ), overexpressed glutathione (GSH), moderate acidity, hypoxia, and vigorous metabolism [ 3 , 4 ]. To effectively eradicate tumors, it is imperative to remodel the TME in conjunction with tumor treatment strategies [ 5 – 7 ]. Numerous methodologies have been documented for restructuring the TME, encompassing the enhancement of hypoxia conditions [ 8 ], regulation of the tumor extracellular matrix [ 9 ], utilization of tumor-associated fibroblast-targeting nanotherapy [ 10 ], and reconfiguration of the tumor vascular system [ 11 ]. However, solely remodeling the TME is not sufficient to eradicate cells, the incorporation of other therapeutic agents is also needed [ 12 – 18 ]. The field of nanotechnology has witnessed noteworthy advancements, particularly in the realm of nanomedicines with multimodal synergistic therapy [ 19 – 24 ]. A particularly auspicious approach in this domain is the utilization of nanoscale metal-organic frameworks (nMOFs), which are hybrid materials renowned for their stable structures, controllable components, and efficient encapsulation of small molecule drugs [ 25 – 27 ]. These distinctive properties have propelled nMOFs into the limelight of nanomedicine, garnering considerable attention and acclaim [ 28 – 30 ]. nMOFs, known for their self-assembled structures composed of metal ions and organic ligands [ 31 ], have exhibited immense potential in synergistically inhibiting tumor growth by combining the anticancer properties of metal ions with the delivery of chemotherapy (CT) and immunotherapy drugs [ 32 – 34 ]. However, the current nMOFs suffer from limited bioavailability, primarily attributed to the use of inactive and inflexible precursor ligands. To tackle this, researchers have recently proposed the incorporation of biologically active drug molecules as precursor ligands, giving rise to prodrug nMOFs [ 35 ]. These prodrug nMOFs selectively release their encapsulated drugs under specific stimuli, effectively preventing premature drug leakage and enhancing the overall anticancer efficacy of the components [ 36 , 37 ]. Despite the promising nature of this approach, the applications of prodrug nMOFs in antitumor combination therapy remain under-reported. To enhance the efficacy of prodrug nMOFs, we advocate for their construction in conjunction with the remodeling of the tumor microenvironment (TME). By specifically targeting and modifying the TME, we can create a more conducive environment for the release and action of the drugs encapsulated within the prodrug nMOFs. This innovative approach holds the potential to augment the therapeutic effects of nMOFs and elevate their overall antitumor activity. Herein, we successfully synthesized GSH-responsive prodrug nMOFs through the coordination of CuCl 2 ·2H 2 O with α-cyano-4-hydroxycinnamic acid (CHCA) and metformin hydrochloride (Met) under solvothermal conditions. The chemotherapeutic agent, doxorubicin (DOX), was encapsulated into the nanoporous structure, and the hyaluronic acid (HA) was surface-functionalized onto the nMOFs to improve biocompatibility and tumor-specific targeting. As depicted in Scheme 1 : (1) The as-prepared HA-coated and Dox-loaded α-cyano-4-hydroxycinnamic acid/Cu 2+ /metformin coordination nanoplatforms were termed DCCMH nMOFs, which were internalized by cancer cells via the specific interaction between HA and receptors on the cell membrane. (2) Subsequently, the nMOFs underwent gradual degradation within cancer cells by high concentrations of GSH, leading to the liberation of DOX, CHCA, Met, and Cu 2+ . (3) The GSH-mediated reduction of Cu 2+ efficiently generated Cu + ions, which in turn activated a Fenton-like reaction. This catalytic process facilitated the conversion of intracellular H 2 O 2 into highly toxic hydroxyl radicals (·OH). (4) The monocarboxylic acid transporter (MCT) inhibitor, CHCA, effectively impeded the efflux of lactic acid, thereby inducing an elevation in intracellular acidity. Consequently, this enhanced the efficiency of the Fenton-like reaction, intensifying oxidative damage to tumor cells. (5) Met triggered the up-regulation of AMP-activated protein kinase (AMPK), causing disruption to normal metabolic pathways and augmenting the chemotherapeutic sensitivity of tumor cells to DOX. The synergistic effect resulting from the depletion of GSH, regulation of acidity/metabolism, and the reinforcement of chemo/chemodynamic therapy (CT/CDT) induced substantial mitochondrial damage and disturbed the redox homeostasis of cancer cells, ultimately exerting robust inhibitory effects on tumors. Results and discussions Characterizations of DCCMH CCM was synthesized using CuCl 2 ·2H 2 O, α-cyano-4-hydroxycinnamic acid (CHCA), and metformin (Met) via a solvothermal method. Subsequently, DOX was loaded into CCM to obtain DCCM. Finally, hyaluronic acid (HA) was modified onto the surface of DCCM through electrostatic interactions, forming DCCMH. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed that CCM and DCCMH nanoparticles (NPs) possessed a distinctive porous snowflake-like morphology, measuring approximately 225 ± 10 nm in diameter, with a spatial lattice dimension of 0.5 nm for CCM (Figs. 1 a and 1 b). The similar morphology of DCCM and CCMH (HA was modified onto the surface of CCM) was observed ( Figure S1 ). Elemental analysis through energy-dispersive X-ray spectroscopy (EDS) and element mapping confirmed the presence of C, N, O, and Cu elements within the CCM NPs (Fig. 1 c). Dynamic light scattering (DLS) analysis demonstrated the hydrodynamic diameters of 190.6 nm, 223.2 nm, and 248.4 nm, with polydispersity indices (PDI) of 0.085, 0.123, and 0.118 for CCM, DCCM, and DCCMH NPs, respectively (Fig. 1 d). Notably, the sizes of the modified nanoparticles exhibited a gradual increase, accompanied by a corresponding decrease in zeta potential (Fig. 1 e), due to the loading of DOX and surface modification of HA. The Fourier-transform infrared (FTIR) spectra revealed the coordination of copper ions with Met and CHCA in CCM, as evidenced by the blue-shifted characteristic peaks. Specifically, the peaks at 1300 cm − 1 (-C-N), 1582 cm − 1 (-COO), 1644 cm − 1 (C = NH), 2208 cm − 1 (-C ≡ N), 3300 cm − 1 (Ph-OH), and 3450 cm − 1 (-NH 3 ) were indicative of these coordination interactions (Fig. 1 f). The X-ray diffraction (XRD) pattern unequivocally demonstrated the crystalline nature of CCM, exhibiting a spatial configuration similar to CuO and CuN crystals and a characteristic XRD peak at a 2θ angle of 8° (Figs. 1 g and S2), which combined with the high-resolution transmission (HR-TEM) images demonstrated the MOF structure. Furthermore, we determined the specific surface area of CCM and DCCM using the Brunauer-Emmett-Teller (BET) method ( Figure S3 ). CCM showed a surface area of 46.28 m 2 g − 1 with a pore volume of 1.5 cm 3 g − 1 , which decreased to 26.39 m 2 g − 1 with a pore volume of 0.052 cm 3 g − 1 after loading with DOX (DCCM), further suggesting the characteristically porous MOF structure of the as-prepared CCM. Owing to the porous crystalline bulk structure, the calculated loading and encapsulation efficiencies of DOX in CCM nMOFs were determined to be 8.12% and 48.63%, respectively. The FTIR spectra displayed in Figure S4 confirmed the successful HA-functionalization and DOX-loading. The characteristic UV–Vis absorption band of DOX was observed in DCCMH (Fig. 1 h). Moreover, to elucidate the element valence states, as shown in Fig. 1 i, the X-ray photoelectron spectroscopy (XPS) analysis implied the characteristic signals of Cu, C, O, and N within the DCCMH. Notably, by examining the high-resolution spectra of Cu2p orbitals, distinct characteristic peaks were observed at 932.5 eV and 952.5 eV, corresponding to the electron transitions of the 2p3/2 and 2p1/2 orbitals of Cu + ; the satellite peaks located at 961.1 eV and 941.1 eV indicated the presence of Cu 2+ . GSH depletion, ·OH generation, and cytotoxicity assessments The preservation of an optimal redox equilibrium is paramount for the survival of cells, and any disruption to this delicate balance can effectively impede the growth and proliferation of cancer cells [ 4 ]. Consequently, we assessed the capability of DCCMH to deplete GSH and generate ·OH. Figure 2 a demonstrates a gradual decline in GSH content with increasing concentrations of DCCMH. This reaction can primarily be ascribed to the reduction of Cu 2+ within DCCMH to Cu + by GSH ( Figures S5a and S5b ). As GSH is consumed, DCCMH NPs undergo complete degradation within 6 h when the GSH concentration is 10 mM and the DCCMH concentration is 80 µg mL − 1 ( Figure S5c ). Subsequently, as nMOFs degrade, the encapsulated drugs are rapidly released, resulting in the release of up to 78% of DOX (Fig. 2 b). This underscores the superior GSH-dissipative and responsive drug-release behavior of DCCMH NPs, as well as their capacity to disrupt the redox equilibrium of the TME by depleting GSH. Furthermore, we analyzed the release of CHCA and Met by observing their characteristic UV-Vis absorption spectra. To prevent any interference from the absorption spectrum of DOX on the determination of CHCA and Met absorption spectra, we opted to observe the changes in the absorption spectra of CCM without the presence of DOX before and after the addition of GSH. Subsequently, it was observed that the distinct absorption peaks of CHCA and Met were present in the absorption spectra after the degradation of CCM NPs in the GSH solution, indicating the successful release of CHCA and Met from the degraded CCM ( Figure S6 ). This observation further verifies that the structure and effectiveness of Met and CHCA remained intact during the high-temperature synthesis of CCM. Then, the ·OH produced by a Fenton-like reaction of H 2 O 2 with Cu + was measured using methylene blue (MB) as a probe [ 38 ]. The MB degradation was investigated in the MB, MB + H 2 O 2 , MB + H 2 O 2 + DCCMH, and MB + H 2 O 2 + DCCMH + GSH groups, respectively, as shown in Fig. 2 c. The more significant decrease of the MB absorbance in the MB + H 2 O 2 + DCCMH + GSH group than in other groups indicates the remarkable chemdynamic effect of DCCMH under the condition when both GSH and H 2 O 2 are present. To effectively eliminate cancer cells, it is crucial to maintain continuous production of ·OH, as they possess a short half-life and easily lose activity [ 39 ]. Consequently, we investigated the ·OH production over time. As demonstrated in Fig. 2 d, in the presence of H 2 O 2 (10 mM) and GSH (10 mM), the absorbance of MB gradually decreased, signifying that ·OH was continuously and gradually generated in this system, ensuring its sustained cytotoxicity. Electron spin resonance (ESR) spectroscopy further validated the efficient production of ·OH, using DMPO as a trapping agent (Fig. 2 e). Upon the addition of DCCMH and GSH, the appearance and gradual enhancement of ·OH (1:2:2:1) signals were observed. These results emphasize the catalytic potential of DCCMH as an antitumor nanomedicine for remodeling the TME by disrupting redox homeostasis and continuously generating cytotoxic ·OH within the simulated TME. Subsequently, the MTT assay was conducted to appraise the cytotoxicity of DCCMH towards cancer cells. As delineated in Figs. 2 f and 2 g, diverging from DOX, DCCMH exhibited negligible toxicity towards the human normal hepatic (HL7702) cells, whilst manifesting considerable cytotoxicity towards human hepatocellular carcinoma (HepG2) cells (Figs. 2 h and 2 i). DOX indiscriminately attacks various cells, whereas the specificity of DCCMH towards HepG2 cells, which overexpress the CD44 receptors on the surface, facilitates its specific uptake in HepG2 cells. Consequently, within the acidic environment of cancer cells and in the presence of high concentrations of GSH-H 2 O 2 , DCCMH rapidly degrades and releases synergistic anticancer drugs. The viability of HepG2 cells gradually declined with escalating drug concentration and incubation duration, wherein a mere 14.5% of HepG2 cells persevered following 24 h of incubation with 80 µg mL − 1 of DCCMH (containing 6.4 µg mL − 1 of DOX). Furthermore, the IC50 of DCCMH on HepG2 cells stood at 11.9 µg mL − 1 , underscoring its exceedingly potent capacity to annihilate hepatocellular carcinoma cells. Furthermore, DCCMH evinced commendable lethality against three additional cancer cell strains, namely Hela cells, U87MG cells, and 4T1 cells ( Figure S7 ). In vitro therapeutic efficacy Next, we thoroughly elucidated the mechanism of action of DCCMH on HepG2 cells. Initially, we quantified the cellular internalization of DCCNH using advanced techniques such as flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) analysis. Remarkably, the results depicted in Figs. 3 a and S8 demonstrated the profound translocation of DOX from DCCMH into the nucleus. Furthermore, the fluorescence signals emitted by DOX exhibited a steady augmentation within the HepG2 nucleus over time, indicative of a time-dependent elevation in the uptake of DCCMH NPs by HepG2 cells. Notably, the uptake of DCCMH reached its zenith after 6 h, astonishingly presenting a 9-fold higher fluorescence signal in comparison to the control group ( Figure S9 ). This enhanced cellular uptake of DCCMH NPs by HepG2 cells can predominantly be attributed to passive uptake based on the enhanced permeability and retention effect of nanomedicines as well as highly selective affinity-based active targeted uptake between HA in DCCMH and CD44 receptors abundantly expressed on the surface of HepG2 cells, which was proved by the CD44 blocking assay using HA ( Figure S10 ) [ 40 ]. Given the considerable degradation of DCCMH induced by GSH in solution, we proceeded to assess the depletion of intracellular GSH by DCCMH. As depicted in Fig. 3 b, the levels of intracellular GSH decreased by 45% in the treatment groups of CCMH and DCCMH in comparison to the PBS group. Following the degradation of CCMH and DCCMH, their constituents CHCA, Met, and Cu + were gradually liberated into the cancer cells. CHCA is specifically employed to inhibit the efflux of intracellular lactate [ 16 , 17 ]. Hence, we scrutinized the intra/extracellular levels of lactate and the intracellular changes in acidity. As illustrated in Fig. 3 c, the groups treated with CCMH and DCCMH exhibited significant alterations in the intra/extracellular content of lactic acid compared to the PBS and DOX groups. Notably, the DCCMH group manifested a remarkable twofold increase in intracellular lactic acid content, while simultaneously experiencing a substantial reduction of approximately 80% in extracellular lactic acid. The accumulation of intracellular lactic acid consequently led to a decline in intracellular pH, which was measured using the pH-sensitive fluorescent probe BCECF that exhibits green fluorescence positively correlated with pH level. As demonstrated in Fig. 3 d, the CCMH and DCCMH groups displayed low fluorescence intensities of BCECF compared to the PBS and DOX groups. The Fenton reaction is more likely to occur under acidic conditions, therefore, the elevation of acidity in cancer cells is beneficial for the intracellular Fenton reaction to occur [ 20 ]. The underlying principle of CDT revolves around the Fenton-like reaction for the intracellular generation of substantial quantities of toxic ·OH, which effectively induces oxidative damage to the mitochondria, ultimately leading to the eradication of cancer cells [ 40 ]. In light of this, we assessed the intracellular ·OH levels and mitochondrial membrane potential changes. As illustrated in Figs. 3 e and S11, the CCMH and DCCMH groups exhibited heightened DCFH fluorescence signals and generated significantly more ·OH compared to the PBS and DOX groups. Quantitative analysis of the average fluorescence intensity of DCFH revealed 73.68% in the DCCMH group, which was approximately 15-fold higher than that of the PBS group (Fig. 3 f). By the efficient reduction-degradation process in cancer cells and the subsequent increase in acidity, the Cu + ions released by DCCMH reacted efficiently with H 2 O 2 , continuously releasing reactive oxygen species (ROS) and facilitating a highly effective CDT effect. The changes in mitochondrial membrane potential, assessed using the membrane potential detection kit (JC-1), confirmed the damage inflicted upon cancer cells by CDT action (Figs. 3 g and S12). Analysis of the fluorescence ratio of JC-1 dimers to JC-1 monomers (R/G) demonstrated a gradual decrease in the R/G ratio across the different incubation groups, as depicted in Fig. 3 h. This decline in the R/G ratio signifies a reduction in mitochondrial membrane potential and an enhancement of oxidative stress in cancer cells. Furthermore, it has been discovered that metformin can modulate the activity of tyrosine kinase in cancer cells, particularly in digestive carcinoma, thereby promoting cancer therapy [ 12 – 14 ]. As shown in Fig. 3 i, compared with the PBS and DOX groups, both CCMH and DCCMH significantly up-regulated AMPK expression in HepG2 cells, thereby exerting a profound influence on cell metabolism and synergistically enhancing chemotherapy. Subsequently, cell viability and apoptosis were evaluated using live/dead staining analysis and apoptosis assay. HepG2 cells were subjected to fluorescence imaging after staining with Calcein-AM (indicating live cells, depicted as green fluorescence) and propidium iodide (PI) (indicating dead cells, depicted as red fluorescence). Notably, HepG2 cells treated with DCCMH exhibited a significantly higher proportion of deceased cells in comparison to the control groups (Figs. 3 j and S13). Additionally, DCCMH treatment led to a remarkable increase in the apoptosis rate of HepG2 cells (67.1%), as quantitatively assessed via flow cytometry (Fig. 3 k). This increase was predominantly observed in the initial stage of apoptosis, as indicated by the presence of cells in the Q3 region (Fig. 3 l). Concurrently, the apoptosis cycle assay revealed that DCCMH could impede the progression of cancer cells in the G2/M phase ( Figure S14 ). The amalgamation of TME-remodeling, Cu + -mediated CDT, and DOX-mediated chemotherapy synergistically contributed to the potentiation of apoptotic signaling, resulting in a heightened incidence of programmed cancer cell demise. In vivo therapeutic efficacy To evaluate the synergistic therapeutic efficacy in vivo , we first assess the biosafety of DCCMH. The hemolysis test ( Figure S15 ) revealed that DCCMH exhibited negligible hemolytic activity on red blood cells across various concentrations (5–160 µg mL − 1 ). Remarkably, even at a high concentration of 160 µg mL − 1 , the corresponding hemolysis rate was merely approximately 3%, underscoring the excellent biosafety profile of DCCMH. Subsequently, blood samples of mice with different treatments were collected for biochemical analysis and hematological evaluations. Notably, there were no significant differences between the experimental groups and the control group in terms of liver function, renal function, or hematological parameters ( Figures S16 and S17 ). The biodistribution and clearance dynamics of DCCMH NPs within H22 tumor-bearing mice were meticulously examined through the strategic integration of Cy5.5 onto the DCCMH nanoplatform. Notably, the fluorescence signals emitted by the DCCMH-Cy5.5 exhibited a gradual and substantial accumulation within the tumor milieu over an 8 h period, ultimately culminating in an extensive and enduring retention of up to 28 h ( Figures S18a and S18b ). Ex vivo fluorescence imaging of major organs further substantiated this observation, as evidenced by the conspicuously robust fluorescence signals discernible within the tumor regions post-intravenous administration of DCCMH-Cy5.5 after 28 h ( Figure S18c ). These results demonstrated that DCCMH could accumulate effectively in tumor regions for a long time, which was conducive to its long-term therapy for cancers. The in vivo therapeutic efficacy of the combination treatment was then assessed through the intravenous administration of DCCMH using H22 tumor-bearing mice. The H22 cell line is a widely utilized murine hepatocellular carcinoma cell line that overexpresses the CD44 receptor. It is suitable for establishing a fully immunocompetent hepatocellular carcinoma xenograft model [ 41 – 43 ]. Once the tumor size reached approximately 100 mm 3 , the H22 tumor-bearing mice were randomly divided into four groups (n = 5/group): (I) PBS (blank control), (II) DOX, (III) CCMH, (IV) DCCMH. The drugs were administered intravenously every 4 days (2 mg kg − 1 , 100 µL), and the mice were monitored for 18 consecutive days, with photographs taken, weights recorded, and tumor volumes measured every 3 days (Figs. 4 a and S19-22). Monotherapy exhibited limited efficacy due to drug resistance and the rapid stress response of cancer [ 44 ]. Remarkably, the DCCMH treatment effectively suppressed tumor growth, exhibiting an inhibition rate of 74.24%, surpassing other treatment modalities with statistically significant differences (Figs. 4 b-e, S23, and S24). The alterations in body weight exhibited no noteworthy aberrations across all experimental cohorts (Fig. 4 f), signifying the absence of evident systemic toxicity. To more intensively assess the synergistic anticancer efficiency and mechanisms of DCCMH, the combination index (CI, CI 1 indicates antagonism) and the activity of AMPK enzymes in isolated H22 tumor tissue were analyzed [ 45 ]. The results showed that the CI of DCCMH was 0.79, which strongly proved that DCCMH achieved good synergism of CDT and CT. As shown in Fig. 4 g, compared with the PBS and DOX groups, both CCMH and DCCMH significantly up-regulated AMPK enzyme activity in H22 tumors, further promoting chemotherapeutic efficacy. Additionally, histopathological analyses of extracted tumor tissues were performed using hematoxylin & eosin (H&E), terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), and Ki67 staining to assess morphology, apoptosis, and proliferation of tumor cells (Fig. 4 h). The H&E-stained tumor sections exhibited a significant decrease in cell density in the DCCMH treatment group. The intense green fluorescence observed in TUNEL staining and the weak red fluorescence observed in Ki67 staining suggested that DCCMH-mediated synergistic therapy induced significant apoptosis and inhibition of tumor cell proliferation. The main organs including the heart, liver, spleen, lungs, and kidneys were subjected to histological examination through H&E staining after all interventions, and the results indicated no significant organ toxicity within all experimental groups ( Figure S25 ). Conclusion In summary, we designed and prepared a novel nano-MOF using prodrugs endowed with the remarkable ability to modulate the tumor microenvironment. The as-obtained nano-prodrug MOFs, denoted as DCCMH, exhibit a gradual degradation upon exposure to high concentrations of GSH, leading to the release of Cu 2+/+ , CHCA, Met, and DOX upon internalization by cancer cells. Subsequently, a cascade of anti-tumor actions is triggered: (1) Cu 2+ proficiently depletes intracellular GSH and catalyzes a Fenton-like reaction, generating a continuous supply of highly toxic ·OH species to inflict oxidative damage upon cancer cells; (2) CHCA effectively hampers the exocytosis of intracellular lactic acid, thereby inducing an elevation in intracellular acidity, which in turn promotes the Fenton-like reaction; (3) Met activates the intracellular AMPK signaling pathway, which reduces the tolerance of tumor cells to DOX. Through comprehensive in vitro and in vivo investigations, we have demonstrated that DCCMH exhibits a homogeneous morphology, high stability, and excellent biosafety profile. Moreover, these properties contribute to the overall efficacy of synergistic tumor microenvironment remodeling and augmented CT/CDT, resulting in a substantial inhibition of tumor growth. This groundbreaking study unveils a novel engineering approach for the development of prodrug-loaded nMOFs, paving the way for highly efficient combination therapies targeting hepatocellular carcinoma tumors. Experimental section Materials and chemicals All reagents used in this study were of analytical grade and were obtained from reputable suppliers. α-cyano-4-hydroxycinnamic acid (CHCA), CuCl 2 ·2H 2 O, glutathione (GSH), hydrogen peroxide (H 2 O 2 , 30%), and methylene blue (MB) were bought from Aladdin-Reagent Co. Ltd. (China). Metformin (Met) and hyaluronic acid (HA, Mw < 10 kDa) were obtained from Coolaber Science & Technology Co. (Beijing, P. R. China). Doxorubicin (DOX) was bought from HEOWNS-Reagent Co. Ltd. (Tianjin, P. R. China). Dimethyl sulfoxide (DMSO) was bought from Chengdu Chron Chemical Co. Ltd. Hoechst 33258, reduced GSH assay kit, Annexin V-FITC/PI kit, mitochondrial membrane potential kit (JC-10 Assay), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), and fetal bovine serum (FBS) were purchased from Solarbio Science & Technology Co., Ltd. The Lactic acid content kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, P. R. China). Preparation of CCM NPs CuCl 2 ·2H 2 O (0.1 mM), CHCA (0.02 mM), and Met (0.08 mM) were mixed and dissolved in 4 mL solution ( V H2O : V ethanol = 1:1) with 30 µL of triethylamine, followed by ultrasonication to fully dissolve and transferred to a 15 mL Teflon-lined autoclave and heated at 100 ℃ for 3 h. After the reaction, the samples (CCM NPs) were washed with ultrapure water and methanol to remove impurities. Preparation of DCCM and DCCMH NPs DOX (1 mL, 10 mg mL − 1 ) was added drop by drop to aqueous CCM (5 mL, 2 mg mL − 1 ) under ultrasound. The mixture was then stirred for 24 h and collected by centrifugation at 12000 rpm for 15 min. The resulting precipitate, referred to as DCCM, was washed three times with water. For the preparation of DCCMH NPs, HA (1 mL, 10 mg mL − 1 ) was added drop by drop to aqueous DCCM (5 mL, 2 mg mL − 1 ) under ultrasound, the mixture was stirred for 12 h and the precipitate was collected by centrifugation at 12000 rpm for 15 min. The resulting precipitate, referred to as DCCMH, was washed three times with water. DOX loading and release from DCCMH NPs To determine the drug loading efficiency (DLE%) of DCCMH, the centrifugal supernatants obtained during the loading of DOX were collected. The concentration of DOX in the supernatants was measured using a standard curve of DOX determined by UV–Vis spectroscopy. The DLE% and encapsulation efficiency (EE%) of DCCMH were calculated using the following equations (1) and (2). Drug release was assessed under both the physiological and the tumor microenvironments. Specifically, 2 mg of DCCMH NPs were suspended in 4 mL of different phosphate-buffered solutions (pH5.0 with 0 mM GSH, and pH5.0 with 10 mM GSH) and stirred at 37 ℃. At each time interval, 1.5 mL of release medium was extracted to determine the percentage of DOX released via ultraviolet-visible spectrophotometry analysis. The sample was then returned to its original release system for further evaluation. GSH depletion and ·OH generation The reduction in GSH levels caused by varying concentrations of DCCMH nanoparticles was assessed using the reduced GSH assay kit. Briefly, the 0.5 mL of different concentrations of DCCMH solution were mixed with 0.5 mL of GSH (20 mM) solution, and the mixture was incubated at 37 ℃ for 6 h. Subsequently, the solutions were centrifuged at 12000 rpm for 10 min, and the absorbance of the supernatant was measured at 412 nm using UV–Vis spectroscopy. The production of ·OH was measured using methylene blue (MB) as a probe. A solution of MB (10 µg mL − 1 ) was added to PBS, H 2 O 2 , H 2 O 2 + DCCMH, and H 2 O 2 + DCCMH + GSH solutions (H 2 O 2 : 10 mM, DCCMH: 100 µg mL − 1 , GSH: 10 mM) and stirred for various durations. The bleaching of MB was monitored by measuring its absorbance at 665 nm using UV–Vis spectroscopy. Additionally, electron spin resonance (ESR) spectroscopy was employed to verify the generation of ·OH. A mixture of 10 µL of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 1 mL of the DCCMH suspension (H 2 O 2 : 10 mM, DCCMH: 100 µg mL − 1 ) was measured using an EMX plus spectrometer (Bruker, Germany). Cell culture and cellular uptake HL7702 cells, HepG2 cells, Hela cells, U87MG cells, and 4T1 cells were purchased from KeyGEN BioTECH Co. (Nanjing, China). Cells were cultured in high glucose medium DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ under 5% CO 2 . For cellular uptake tests, the HepG2 cells were seeded into 6-well plates or CLSM culture dishes and incubated with DCCMH (50 µg mL − 1 ) for different durations (1, 3, 6, and 9 h). Subsequently, the cells were harvested and resuspended in PBS. Flow cytometry was employed to measure the fluorescence intensity of the cells. Additionally, the cells were washed twice with PBS and stained with Hoechst 33258 for 10 min. CLSM was used to capture images of the stained cells (Hoechst 33258: λ ex = 405 nm, λ em = 460 nm; DOX: λ ex = 488 nm, λ em = 590 nm). Cytotoxicity assay Cells were seeded into 96-well plates at a density of 7.0 × 10 3 cells per well and incubated with various drugs (DOX, CCMH, or DCCMH) for 24 or 48 h. After that, 20 µL of thiazolyl blue tetrazolium bromide (MTT) solution (5 mg mL − 1 ) was added to each well, and cells were further incubated for 4 h. Next, the resulting formazan crystals were dissolved using 100 µL of dimethyl sulfoxide, and the absorbance of each well was measured at 490 nm using a microplate reader (FLUOstar Omega). Intracellular GSH depletion and ROS generation HepG2 cells were seeded into T25 culture flasks and allowed to grow for 24 h, then the cells were treated with PBS, DOX (4 µg mL − 1 ), CCMH (46 µg mL − 1 ), and DCCMH (50 µg mL − 1 ), respectively, for 12 h. After removing the culture medium, the cells were collected and subjected to three cycles of freezing and thawing using liquid nitrogen and 37 ℃ water, respectively. The resulting cell lysates were then centrifuged, and the absorbance at 412 nm was measured using a microplate reader (FLUOstar Omega). HepG2 cells were seeded into 6-well plates at a density of 2.0 × 10 5 cells per well for 24 h. To measure the level of ROS in the cells, similarly, the cells were cultured with different drugs for 12 h, and after removing the culture medium, 1 mL of DCFH-DA (10 µM) solution was added to each well and cultured for another 30 min. Then, the cells were washed three times with PBS to remove excess DCFH-DA and fluorescence images were captured using CLSM. In vitro lactic acid and pH measurements HepG2 cells were seeded into 6-well plates at a density of 2.0 × 10 5 cells per well and incubated with various drugs (PBS, DOX: 4.0 µg mL − 1 , CCMH: 46 µg mL − 1 , DCCMH: 50 µg mL − 1 ) for 12 h, respectively. Then, both the cells and the culture medium were collected for the measurement of lactic acid levels using lactic acid detection kits, following the manufacturer's instructions. HepG2 cells were cultured in CLSM culture dishes overnight, then the cells were treated with different drugs including PBS, DOX (4.0 µg mL − 1 ), CCMH (46 µg mL − 1 ), and DCCMH (50 µg mL − 1 ) for 12 h. Subsequently, the cells were washed with PBS and incubated with the pH fluorescent probe (BCECF-AM, 5 µM in PBS) for 30 min, and observed using a CLSM. The culture media with a pH of 7.4 was used as standard samples for comparison. Mitochondrial membrane potential measurement The HepG2 cells were seeded into CLSM culture dishes and cultured overnight. After treatment with PBS, DOX (4.0 µg mL − 1 ), CCMH (46 µg mL − 1 ), and DCCMH (50 µg mL − 1 ) for 12 h, respectively, the cells were incubated with JC-1 dye for 30 min to measure the MMP through CLSM analysis. Live/dead cell staining and apoptosis assay HepG2 cells were seeded into CLSM culture dishes with a density of 1.0 × 10 5 cells per dish for 24 h, and then treated with PBS, DOX (4.0 µg mL − 1 ), CCMH (46 µg mL − 1 ), and DCCMH (50 µg mL − 1 ) for 12 h, respectively. Then, the cells were stained with Calcein-AM (2 µM) and PI (4 µM) for 30 min to measure the cell viabilities through CLSM analysis. HepG2 cells were cultured with different drugs (PBS, DOX: 4.0 µg mL − 1 , CCMH: 46 µg mL − 1 , DCCMH: 50 µg mL − 1 ) for 24 h. Afterward, all of the treated cells were collected, washed, and stained with FITC/PI for 20 min. Then, the intracellular fluorescence signals of FITC/PI were measured by flow cytometry, to determine the apoptosis and necrosis rate of HepG2 cells. Hemolysis assay 1 mL of red blood cell suspension (0.2%, v/v) was mixed with 1 mL of different concentrations of DCCMH and incubated at 37 ℃ for 4 h. The cells incubated in double distilled water and PBS were used as positive and negative controls, respectively. After centrifugation at 3000 rpm for 6 min, the optical density (OD) at 540 nm of each solution was measured using the microplate reader. The hemolysis rate was calculated according to the following formula (3). In vivo antitumor evaluation Female Balb/c mice were subcutaneously inoculated with 0.1 mL of H22 cell suspension (1.0 × 10 6 cells) into their right leg to establish the tumor model. Subsequently, once the tumor volume reached 50–70 mm 3 , the mice were randomly divided into four groups with five mice in each group: Control (PBS), DOX, CCMH, and DCCMH. The respective formulations were intravenously injected every 4 days for a total of 18 days, with a dose of 1.6 mg kg − 1 of DOX, 18.4 mg kg − 1 of CCMH, and 20 mg kg − 1 of DCCMH. The tumor size and body weight of the mice were measured once every three days. After treatment for 18 days, tumors in all treatment groups were harvested for H&E staining, TUNEL staining, and Ki67 staining. All animal experiments were carried out under the protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University. Long-term in vivo biosafety evaluation After the in vivo tumor treatment with different formulations including PBS, DOX (1.6 mg kg − 1 ), CCMH (18.4 mg kg − 1 ), and DCCMH (20 mg kg − 1 ), the major organs (heart, liver, spleen, lungs, and kidneys) of mice were harvested and conducted H&E staining for the histological analysis. The blood samples were collected from the mouse orbital venous plexus for blood biochemistry and blood routine examinations. Statistical analysis The one-way analysis of variance (ANOVA) statistical method was performed to evaluate the experimental data. A p-value of 0.05 was selected as the significance level and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively. Declarations Supporting Information Supporting Information is available from the Online Library or the author. Acknowledgements This work was supported by the National Natural Science Foundation of China (22171230, 82001956), the National University of Singapore (NUHSRO/2020/133/Startup/08, NUHSRO/2023/008/NUSMed/TCE/LOA, NUHSRO/2021/034/TRP/09/Nanomedicine), National Medical Research Council (MOH-001388-00, MOH-001041, CG21APR1005), Singapore Ministry of Education (MOE-000387-00), National Research Foundation (NRF-000352-00), the Project of Science and Technology of Social Development in Shaanxi Province (2023YBSF-151, 2021SF-120), and 2023 Hangzhou West Lake Pearl Project Leading Innovation Youth Team Project (TD2023017). The authors thank the Teaching and Research Core Facility at the College of Life Science, the Life Science Research Core Services, and the Northwest A&F University for helping with characterizations including SEM, TEM, and CLSM, etc . Author contributions Junliang Dong formulated the conceptual framework and established the experimental procedures, conducted in vitro and in vivo experiments, analyzed data, drafted the manuscript, compiled all figures, and revised the manuscript based on the feedback from Wenjing Sun, Xiaoyuan Chen, and Zhichao Pei. Jindong Ding assisted in writing and revising the manuscript. Shifan Luo, Ruoshui Li, Yi Wang, and Bing Xiao participated in conducting the research. Yuxin Pei, Wenjing Sun, Xiaoyuan Chen, and Zhichao Pei collaboratively reviewed and edited the manuscript, and provided financial support. All authors collectively analyzed the findings, revised the manuscript, and unanimously endorsed the final version. Data availability No datasets were generated or analyzed during the current study. The animal experimental was carried out following the guidelines of the Institutional Animal Care and Use Committee of Zhejiang University. 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Supportinginformation.doc Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 09 Dec, 2024 Reviews received at journal 07 Dec, 2024 Reviews received at journal 05 Dec, 2024 Reviews received at journal 04 Dec, 2024 Reviews received at journal 26 Nov, 2024 Reviewers agreed at journal 23 Nov, 2024 Reviewers agreed at journal 22 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers invited by journal 20 Nov, 2024 Editor assigned by journal 08 Nov, 2024 Submission checks completed at journal 08 Nov, 2024 First submitted to journal 06 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5402726","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381708531,"identity":"14d7d8c9-e540-4827-9800-7d8f0116c54a","order_by":0,"name":"Junliang Dong","email":"","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Junliang","middleName":"","lastName":"Dong","suffix":""},{"id":381708532,"identity":"6f8c4a76-ba17-4ec2-bf35-6d164ee97062","order_by":1,"name":"Jindong Ding","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jindong","middleName":"","lastName":"Ding","suffix":""},{"id":381708533,"identity":"dfe88740-7f65-4559-9f48-9f7d261c4256","order_by":2,"name":"Shifan Luo","email":"","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Shifan","middleName":"","lastName":"Luo","suffix":""},{"id":381708534,"identity":"0619411c-6124-402f-a091-a6c7d609ba46","order_by":3,"name":"Ruoshui Li","email":"","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Ruoshui","middleName":"","lastName":"Li","suffix":""},{"id":381708535,"identity":"7748a6d3-0be0-4e21-b1c2-2f1be64ce9c7","order_by":4,"name":"Yi Wang","email":"","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wang","suffix":""},{"id":381708537,"identity":"35c3237f-fc5d-4a01-9d6e-978006e3c0fd","order_by":5,"name":"Bing Xiao","email":"","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Xiao","suffix":""},{"id":381708538,"identity":"5b905a27-a621-4842-9ceb-bb59d8d47794","order_by":6,"name":"Yuxin Pei","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Pei","suffix":""},{"id":381708542,"identity":"89611ab0-fe73-41c8-9b32-d64696be9119","order_by":7,"name":"Xiaoyuan Chen","email":"","orcid":"","institution":"Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Chen","suffix":""},{"id":381708544,"identity":"fbd25dbf-a00c-466a-8605-3511f0b41c01","order_by":8,"name":"Wenjing Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RMQrCQBBA0ZFAbBZsJ4h6hZEFoyB6lYiQSm1sUklEiI1YK3qIHGFFME0OELtUsbGwtNNVUm+0E9xfTTEPBgZAp/vVkJBVyj6QnEv+Z8Rr162V+IZA7HFKnPdYTBqb8Qk7AQ788yWdMujWQmFkqYpQMnHRkmS+cxzOwOWhMG1SEhy1khdZVB0hyXEQCmai+rCcBNbBl+RRTCCRBGPkDA2QRBQTiq/2HT2sI3Ohuach3x7Nlvqw5YhzpBnrR1FGV69XW0eLTH2YzMg3THo/0yjal5VuOU0/WNbpdLo/7Al62UR1peCvUQAAAABJRU5ErkJggg==","orcid":"","institution":"ZJU-Hangzhou Global Scientific and Technological Innovation Center","correspondingAuthor":true,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Sun","suffix":""},{"id":381708546,"identity":"1ae0e041-eadb-4de8-92de-8a93dc6ae4f3","order_by":9,"name":"Zhichao Pei","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Pei","suffix":""}],"badges":[],"createdAt":"2024-11-06 12:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5402726/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5402726/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03202-7","type":"published","date":"2025-02-19T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71101604,"identity":"9d9ab474-d128-4735-b3a5-8c4b8be1582f","added_by":"auto","created_at":"2024-12-11 06:54:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":390575,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of nMOFs. (a) SEM images and (b) TEM images of CCM and DCCMH NPs (insert: the HR-TEM image of CCM). (c) Elemental mapping of DCCMH NPs. (d) The distribution of hydrodynamic diameters and (e) zeta potential of CCM, DCCM, CCHM, and DCCMH. (f) FTIR spectra of Met, CHCA, and CCM. (g) The XRD pattern of CCM NPs. (h) The UV–Vis absorption spectra of DOX, CCMH, and DCCMH. (i) The XPS spectrum of CCM NPs (insert: the high-resolution spectra of the Cu2p orbitals).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/f266e5cb4304fbbdaeeee848.png"},{"id":71101603,"identity":"7ac126c2-53d5-4706-8fb5-978e200b2702","added_by":"auto","created_at":"2024-12-11 06:54:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294856,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The relative GSH content treated with different concentrations of DCCMH for 6 h. (b) The DOX release profile of DCCMH under different conditions. (c) The UV–Vis absorption spectra of MB, MB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+DCCMH, and MB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+DCCMH+GSH solutions. (d) The degradation of MB at different time intervals mediated by DCCMH. (e) The ESR spectra of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+DCCMH, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+DCCMH+GSH using DMPO as the trapping agent. (f-i) Cell viability assay of HL7702 cells and HepG2 cells incubated with different samples at varying concentrations for (f) 24 h of HL7702 cells, (g) 48 h of HL7702 cells, (h)\u003cstrong\u003e \u003c/strong\u003e24 h of HepG2 cells, (i) 48 h of HepG2 cells (n = 6). (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/2fcdb8c8fdf657e2e9d2a3a2.png"},{"id":71101595,"identity":"e3f1f1de-34a4-4208-ad53-8dc6f8e3eca5","added_by":"auto","created_at":"2024-12-11 06:54:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":984055,"visible":true,"origin":"","legend":"\u003cp\u003e(a) CLSM images depicting the internalization of DCCMH by HepG2 cells at varying time intervals. (b) The intracellular total GSH levels under different groups of incubation (n = 6). (c) The intra/extracellular lactic acid content under different groups of incubation (n = 6). (d) CLSM images of HepG2 cells treated with different samples and stained with pH fluorescent probe (BCECF), Scale bar: 100 μm. (e) The intracellular ROS detection through DCFH-DA staining, Scale bar: 50 μm. (f) The mean fluorescence intensity values of intracellular DCFH (n = 3). (g) CLSM images of HepG2 cells treated with different samples and stained with JC-1, Scale bar: 100 μm. (h) Changes in the mitochondrial membrane potential of HepG2 cells (n = 6). (i) Analysis of AMPK enzyme activity in different treatment groups (n = 6). (j) Live/dead staining images of HepG2 cells after different treatments, Scale bar: 100 μm. (k) Flow cytometry analysis of HepG2 cell apoptosis. (l) Analysis of apoptosis time in different groups (Concentrations of drugs selected for cellular experiments: DOX: 4.8 μg mL\u003csup\u003e-1\u003c/sup\u003e, CCMH: 55.2 μg mL\u003csup\u003e-1\u003c/sup\u003e, and DCCMH: 60 μg mL\u003csup\u003e-1\u003c/sup\u003e). (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/3af4552ea3f0e9a7a79903f9.png"},{"id":71101594,"identity":"2ec7ff25-b010-429f-84ba-e59866786e33","added_by":"auto","created_at":"2024-12-11 06:54:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1870976,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the therapeutic intervention in the H22 subcutaneous tumor-bearing mouse model. (b) Respective tumor growth curves for each tumor-bearing mouse in different groups. (c) The average tumor growth patterns exhibited by H22 tumor-bearing mice following different 18-day treatments. (d) The weight of extracted H22 tumors after 18-day treatments. (e) The photographs of extracted H22 tumors. (f) The changes in body weight of H22-tumor-bearing mice in different treatment groups. (g) Analysis of AMPK enzyme activity in H22 tumor tissue from different treatment groups (n = 5). (h) H\u0026amp;E staining, TUNEL labeling, and Ki-67 staining of tumors in mice subjected to various treatments for 18 days. All Error bars represent mean ± S.D. n = 5 biologically independent samples. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/09310a8bb5ffe07352552b4c.png"},{"id":77054323,"identity":"11abd7dd-3dbe-4214-9547-9467bdddd596","added_by":"auto","created_at":"2025-02-24 16:31:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4487098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/3d0495df-84e7-46d6-8fed-b3072a4411ed.pdf"},{"id":71101596,"identity":"6e1a1011-9dcc-4d51-9ff6-95db3a18bdf0","added_by":"auto","created_at":"2024-12-11 06:54:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2151154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Schematic illustration of (a) the fabrication procedure of the HA-coated and DOX-loaded α-cyano-4-hydroxycinnamic acid/Cu\u003csup\u003e2+\u003c/sup\u003e/metformin coordination nanoMOFs (DCCMH nMOFs), and (b) the underlying mechanism of DCCMH for synergistic cancer therapy.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/f0f9ea50aa8bc77837985c18.png"},{"id":71101591,"identity":"4d6e1a05-d9cd-4f27-8f03-c58f3168920c","added_by":"auto","created_at":"2024-12-11 06:54:36","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14003200,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-5402726/v1/2b0e4a20c83c6d5c61fc64d1.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Remodeling Tumor Microenvironment Using Prodrug nMOFs for Synergistic Cancer Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tumor microenvironment (TME) plays a pivotal role in fostering the proliferation and dissemination of malignant cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is characterized by elevated levels of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), overexpressed glutathione (GSH), moderate acidity, hypoxia, and vigorous metabolism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To effectively eradicate tumors, it is imperative to remodel the TME in conjunction with tumor treatment strategies [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Numerous methodologies have been documented for restructuring the TME, encompassing the enhancement of hypoxia conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], regulation of the tumor extracellular matrix [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], utilization of tumor-associated fibroblast-targeting nanotherapy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and reconfiguration of the tumor vascular system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, solely remodeling the TME is not sufficient to eradicate cells, the incorporation of other therapeutic agents is also needed [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The field of nanotechnology has witnessed noteworthy advancements, particularly in the realm of nanomedicines with multimodal synergistic therapy [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A particularly auspicious approach in this domain is the utilization of nanoscale metal-organic frameworks (nMOFs), which are hybrid materials renowned for their stable structures, controllable components, and efficient encapsulation of small molecule drugs [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These distinctive properties have propelled nMOFs into the limelight of nanomedicine, garnering considerable attention and acclaim [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003enMOFs, known for their self-assembled structures composed of metal ions and organic ligands [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], have exhibited immense potential in synergistically inhibiting tumor growth by combining the anticancer properties of metal ions with the delivery of chemotherapy (CT) and immunotherapy drugs [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the current nMOFs suffer from limited bioavailability, primarily attributed to the use of inactive and inflexible precursor ligands. To tackle this, researchers have recently proposed the incorporation of biologically active drug molecules as precursor ligands, giving rise to prodrug nMOFs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These prodrug nMOFs selectively release their encapsulated drugs under specific stimuli, effectively preventing premature drug leakage and enhancing the overall anticancer efficacy of the components [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Despite the promising nature of this approach, the applications of prodrug nMOFs in antitumor combination therapy remain under-reported. To enhance the efficacy of prodrug nMOFs, we advocate for their construction in conjunction with the remodeling of the tumor microenvironment (TME). By specifically targeting and modifying the TME, we can create a more conducive environment for the release and action of the drugs encapsulated within the prodrug nMOFs. This innovative approach holds the potential to augment the therapeutic effects of nMOFs and elevate their overall antitumor activity.\u003c/p\u003e \u003cp\u003eHerein, we successfully synthesized GSH-responsive prodrug nMOFs through the coordination of CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO with α-cyano-4-hydroxycinnamic acid (CHCA) and metformin hydrochloride (Met) under solvothermal conditions. The chemotherapeutic agent, doxorubicin (DOX), was encapsulated into the nanoporous structure, and the hyaluronic acid (HA) was surface-functionalized onto the nMOFs to improve biocompatibility and tumor-specific targeting. As depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: (1) The as-prepared HA-coated and Dox-loaded α-cyano-4-hydroxycinnamic acid/Cu\u003csup\u003e2+\u003c/sup\u003e/metformin coordination nanoplatforms were termed DCCMH nMOFs, which were internalized by cancer cells \u003cem\u003evia\u003c/em\u003e the specific interaction between HA and receptors on the cell membrane. (2) Subsequently, the nMOFs underwent gradual degradation within cancer cells by high concentrations of GSH, leading to the liberation of DOX, CHCA, Met, and Cu\u003csup\u003e2+\u003c/sup\u003e. (3) The GSH-mediated reduction of Cu\u003csup\u003e2+\u003c/sup\u003e efficiently generated Cu\u003csup\u003e+\u003c/sup\u003e ions, which in turn activated a Fenton-like reaction. This catalytic process facilitated the conversion of intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into highly toxic hydroxyl radicals (\u0026middot;OH). (4) The monocarboxylic acid transporter (MCT) inhibitor, CHCA, effectively impeded the efflux of lactic acid, thereby inducing an elevation in intracellular acidity. Consequently, this enhanced the efficiency of the Fenton-like reaction, intensifying oxidative damage to tumor cells. (5) Met triggered the up-regulation of AMP-activated protein kinase (AMPK), causing disruption to normal metabolic pathways and augmenting the chemotherapeutic sensitivity of tumor cells to DOX. The synergistic effect resulting from the depletion of GSH, regulation of acidity/metabolism, and the reinforcement of chemo/chemodynamic therapy (CT/CDT) induced substantial mitochondrial damage and disturbed the redox homeostasis of cancer cells, ultimately exerting robust inhibitory effects on tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussions","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizations of DCCMH\u003c/h2\u003e \u003cp\u003eCCM was synthesized using CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, α-cyano-4-hydroxycinnamic acid (CHCA), and metformin (Met) via a solvothermal method. Subsequently, DOX was loaded into CCM to obtain DCCM. Finally, hyaluronic acid (HA) was modified onto the surface of DCCM through electrostatic interactions, forming DCCMH. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed that CCM and DCCMH nanoparticles (NPs) possessed a distinctive porous snowflake-like morphology, measuring approximately 225\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm in diameter, with a spatial lattice dimension of 0.5 nm for CCM (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The similar morphology of DCCM and CCMH (HA was modified onto the surface of CCM) was observed (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Elemental analysis through energy-dispersive X-ray spectroscopy (EDS) and element mapping confirmed the presence of C, N, O, and Cu elements within the CCM NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Dynamic light scattering (DLS) analysis demonstrated the hydrodynamic diameters of 190.6 nm, 223.2 nm, and 248.4 nm, with polydispersity indices (PDI) of 0.085, 0.123, and 0.118 for CCM, DCCM, and DCCMH NPs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Notably, the sizes of the modified nanoparticles exhibited a gradual increase, accompanied by a corresponding decrease in zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), due to the loading of DOX and surface modification of HA. The Fourier-transform infrared (FTIR) spectra revealed the coordination of copper ions with Met and CHCA in CCM, as evidenced by the blue-shifted characteristic peaks. Specifically, the peaks at 1300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-C-N), 1582 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-COO), 1644 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;NH), 2208 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-C\u0026thinsp;\u0026equiv;\u0026thinsp;N), 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ph-OH), and 3450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-NH\u003csub\u003e3\u003c/sub\u003e) were indicative of these coordination interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The X-ray diffraction (XRD) pattern unequivocally demonstrated the crystalline nature of CCM, exhibiting a spatial configuration similar to CuO and CuN crystals and a characteristic XRD peak at a 2θ angle of 8\u0026deg; (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and S2), which combined with the high-resolution transmission (HR-TEM) images demonstrated the MOF structure. Furthermore, we determined the specific surface area of CCM and DCCM using the Brunauer-Emmett-Teller (BET) method (\u003cb\u003eFigure S3\u003c/b\u003e). CCM showed a surface area of 46.28 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a pore volume of 1.5 cm\u003csup\u003e3\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which decreased to 26.39 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a pore volume of 0.052 cm\u003csup\u003e3\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after loading with DOX (DCCM), further suggesting the characteristically porous MOF structure of the as-prepared CCM. Owing to the porous crystalline bulk structure, the calculated loading and encapsulation efficiencies of DOX in CCM nMOFs were determined to be 8.12% and 48.63%, respectively. The FTIR spectra displayed in \u003cb\u003eFigure S4\u003c/b\u003e confirmed the successful HA-functionalization and DOX-loading. The characteristic UV\u0026ndash;Vis absorption band of DOX was observed in DCCMH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Moreover, to elucidate the element valence states, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, the X-ray photoelectron spectroscopy (XPS) analysis implied the characteristic signals of Cu, C, O, and N within the DCCMH. Notably, by examining the high-resolution spectra of Cu2p orbitals, distinct characteristic peaks were observed at 932.5 eV and 952.5 eV, corresponding to the electron transitions of the 2p3/2 and 2p1/2 orbitals of Cu\u003csup\u003e+\u003c/sup\u003e; the satellite peaks located at 961.1 eV and 941.1 eV indicated the presence of Cu\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGSH depletion, ·OH generation, and cytotoxicity assessments\u003c/h3\u003e\n\u003cp\u003eThe preservation of an optimal redox equilibrium is paramount for the survival of cells, and any disruption to this delicate balance can effectively impede the growth and proliferation of cancer cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, we assessed the capability of DCCMH to deplete GSH and generate \u0026middot;OH. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea demonstrates a gradual decline in GSH content with increasing concentrations of DCCMH. This reaction can primarily be ascribed to the reduction of Cu\u003csup\u003e2+\u003c/sup\u003e within DCCMH to Cu\u003csup\u003e+\u003c/sup\u003e by GSH (\u003cb\u003eFigures S5a and S5b\u003c/b\u003e). As GSH is consumed, DCCMH NPs undergo complete degradation within 6 h when the GSH concentration is 10 mM and the DCCMH concentration is 80 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cb\u003eFigure S5c\u003c/b\u003e). Subsequently, as nMOFs degrade, the encapsulated drugs are rapidly released, resulting in the release of up to 78% of DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This underscores the superior GSH-dissipative and responsive drug-release behavior of DCCMH NPs, as well as their capacity to disrupt the redox equilibrium of the TME by depleting GSH. Furthermore, we analyzed the release of CHCA and Met by observing their characteristic UV-Vis absorption spectra. To prevent any interference from the absorption spectrum of DOX on the determination of CHCA and Met absorption spectra, we opted to observe the changes in the absorption spectra of CCM without the presence of DOX before and after the addition of GSH. Subsequently, it was observed that the distinct absorption peaks of CHCA and Met were present in the absorption spectra after the degradation of CCM NPs in the GSH solution, indicating the successful release of CHCA and Met from the degraded CCM (\u003cb\u003eFigure S6\u003c/b\u003e). This observation further verifies that the structure and effectiveness of Met and CHCA remained intact during the high-temperature synthesis of CCM. Then, the \u0026middot;OH produced by a Fenton-like reaction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e with Cu\u003csup\u003e+\u003c/sup\u003e was measured using methylene blue (MB) as a probe [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The MB degradation was investigated in the MB, MB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DCCMH, and MB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DCCMH\u0026thinsp;+\u0026thinsp;GSH groups, respectively, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. The more significant decrease of the MB absorbance in the MB\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DCCMH\u0026thinsp;+\u0026thinsp;GSH group than in other groups indicates the remarkable chemdynamic effect of DCCMH under the condition when both GSH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are present. To effectively eliminate cancer cells, it is crucial to maintain continuous production of \u0026middot;OH, as they possess a short half-life and easily lose activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consequently, we investigated the \u0026middot;OH production over time. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) and GSH (10 mM), the absorbance of MB gradually decreased, signifying that \u0026middot;OH was continuously and gradually generated in this system, ensuring its sustained cytotoxicity. Electron spin resonance (ESR) spectroscopy further validated the efficient production of \u0026middot;OH, using DMPO as a trapping agent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Upon the addition of DCCMH and GSH, the appearance and gradual enhancement of \u0026middot;OH (1:2:2:1) signals were observed. These results emphasize the catalytic potential of DCCMH as an antitumor nanomedicine for remodeling the TME by disrupting redox homeostasis and continuously generating cytotoxic \u0026middot;OH within the simulated TME.\u003c/p\u003e \u003cp\u003eSubsequently, the MTT assay was conducted to appraise the cytotoxicity of DCCMH towards cancer cells. As delineated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, diverging from DOX, DCCMH exhibited negligible toxicity towards the human normal hepatic (HL7702) cells, whilst manifesting considerable cytotoxicity towards human hepatocellular carcinoma (HepG2) cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). DOX indiscriminately attacks various cells, whereas the specificity of DCCMH towards HepG2 cells, which overexpress the CD44 receptors on the surface, facilitates its specific uptake in HepG2 cells. Consequently, within the acidic environment of cancer cells and in the presence of high concentrations of GSH-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, DCCMH rapidly degrades and releases synergistic anticancer drugs. The viability of HepG2 cells gradually declined with escalating drug concentration and incubation duration, wherein a mere 14.5% of HepG2 cells persevered following 24 h of incubation with 80 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of DCCMH (containing 6.4 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of DOX). Furthermore, the IC50 of DCCMH on HepG2 cells stood at 11.9 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, underscoring its exceedingly potent capacity to annihilate hepatocellular carcinoma cells. Furthermore, DCCMH evinced commendable lethality against three additional cancer cell strains, namely Hela cells, U87MG cells, and 4T1 cells (\u003cb\u003eFigure S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIn vitro therapeutic efficacy\u003c/h3\u003e\n\u003cp\u003eNext, we thoroughly elucidated the mechanism of action of DCCMH on HepG2 cells. Initially, we quantified the cellular internalization of DCCNH using advanced techniques such as flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) analysis. Remarkably, the results depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and S8 demonstrated the profound translocation of DOX from DCCMH into the nucleus. Furthermore, the fluorescence signals emitted by DOX exhibited a steady augmentation within the HepG2 nucleus over time, indicative of a time-dependent elevation in the uptake of DCCMH NPs by HepG2 cells. Notably, the uptake of DCCMH reached its zenith after 6 h, astonishingly presenting a 9-fold higher fluorescence signal in comparison to the control group (\u003cb\u003eFigure S9\u003c/b\u003e). This enhanced cellular uptake of DCCMH NPs by HepG2 cells can predominantly be attributed to passive uptake based on the enhanced permeability and retention effect of nanomedicines as well as highly selective affinity-based active targeted uptake between HA in DCCMH and CD44 receptors abundantly expressed on the surface of HepG2 cells, which was proved by the CD44 blocking assay using HA (\u003cb\u003eFigure S10\u003c/b\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the considerable degradation of DCCMH induced by GSH in solution, we proceeded to assess the depletion of intracellular GSH by DCCMH. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the levels of intracellular GSH decreased by 45% in the treatment groups of CCMH and DCCMH in comparison to the PBS group. Following the degradation of CCMH and DCCMH, their constituents CHCA, Met, and Cu\u003csup\u003e+\u003c/sup\u003e were gradually liberated into the cancer cells. CHCA is specifically employed to inhibit the efflux of intracellular lactate [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Hence, we scrutinized the intra/extracellular levels of lactate and the intracellular changes in acidity. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the groups treated with CCMH and DCCMH exhibited significant alterations in the intra/extracellular content of lactic acid compared to the PBS and DOX groups. Notably, the DCCMH group manifested a remarkable twofold increase in intracellular lactic acid content, while simultaneously experiencing a substantial reduction of approximately 80% in extracellular lactic acid. The accumulation of intracellular lactic acid consequently led to a decline in intracellular pH, which was measured using the pH-sensitive fluorescent probe BCECF that exhibits green fluorescence positively correlated with pH level. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the CCMH and DCCMH groups displayed low fluorescence intensities of BCECF compared to the PBS and DOX groups. The Fenton reaction is more likely to occur under acidic conditions, therefore, the elevation of acidity in cancer cells is beneficial for the intracellular Fenton reaction to occur [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe underlying principle of CDT revolves around the Fenton-like reaction for the intracellular generation of substantial quantities of toxic \u0026middot;OH, which effectively induces oxidative damage to the mitochondria, ultimately leading to the eradication of cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In light of this, we assessed the intracellular \u0026middot;OH levels and mitochondrial membrane potential changes. As illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and S11, the CCMH and DCCMH groups exhibited heightened DCFH fluorescence signals and generated significantly more \u0026middot;OH compared to the PBS and DOX groups. Quantitative analysis of the average fluorescence intensity of DCFH revealed 73.68% in the DCCMH group, which was approximately 15-fold higher than that of the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). By the efficient reduction-degradation process in cancer cells and the subsequent increase in acidity, the Cu\u003csup\u003e+\u003c/sup\u003e ions released by DCCMH reacted efficiently with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, continuously releasing reactive oxygen species (ROS) and facilitating a highly effective CDT effect. The changes in mitochondrial membrane potential, assessed using the membrane potential detection kit (JC-1), confirmed the damage inflicted upon cancer cells by CDT action (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and S12). Analysis of the fluorescence ratio of JC-1 dimers to JC-1 monomers (R/G) demonstrated a gradual decrease in the R/G ratio across the different incubation groups, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh. This decline in the R/G ratio signifies a reduction in mitochondrial membrane potential and an enhancement of oxidative stress in cancer cells. Furthermore, it has been discovered that metformin can modulate the activity of tyrosine kinase in cancer cells, particularly in digestive carcinoma, thereby promoting cancer therapy [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, compared with the PBS and DOX groups, both CCMH and DCCMH significantly up-regulated AMPK expression in HepG2 cells, thereby exerting a profound influence on cell metabolism and synergistically enhancing chemotherapy.\u003c/p\u003e \u003cp\u003eSubsequently, cell viability and apoptosis were evaluated using live/dead staining analysis and apoptosis assay. HepG2 cells were subjected to fluorescence imaging after staining with Calcein-AM (indicating live cells, depicted as green fluorescence) and propidium iodide (PI) (indicating dead cells, depicted as red fluorescence). Notably, HepG2 cells treated with DCCMH exhibited a significantly higher proportion of deceased cells in comparison to the control groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej and S13). Additionally, DCCMH treatment led to a remarkable increase in the apoptosis rate of HepG2 cells (67.1%), as quantitatively assessed via flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). This increase was predominantly observed in the initial stage of apoptosis, as indicated by the presence of cells in the Q3 region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el). Concurrently, the apoptosis cycle assay revealed that DCCMH could impede the progression of cancer cells in the G2/M phase (\u003cb\u003eFigure S14\u003c/b\u003e). The amalgamation of TME-remodeling, Cu\u003csup\u003e+\u003c/sup\u003e-mediated CDT, and DOX-mediated chemotherapy synergistically contributed to the potentiation of apoptotic signaling, resulting in a heightened incidence of programmed cancer cell demise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIn vivo therapeutic efficacy\u003c/h3\u003e\n\u003cp\u003eTo evaluate the synergistic therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e, we first assess the biosafety of DCCMH. The hemolysis test (\u003cb\u003eFigure S15\u003c/b\u003e) revealed that DCCMH exhibited negligible hemolytic activity on red blood cells across various concentrations (5\u0026ndash;160 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Remarkably, even at a high concentration of 160 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the corresponding hemolysis rate was merely approximately 3%, underscoring the excellent biosafety profile of DCCMH. Subsequently, blood samples of mice with different treatments were collected for biochemical analysis and hematological evaluations. Notably, there were no significant differences between the experimental groups and the control group in terms of liver function, renal function, or hematological parameters (\u003cb\u003eFigures S16 and S17\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e The biodistribution and clearance dynamics of DCCMH NPs within H22 tumor-bearing mice were meticulously examined through the strategic integration of Cy5.5 onto the DCCMH nanoplatform. Notably, the fluorescence signals emitted by the DCCMH-Cy5.5 exhibited a gradual and substantial accumulation within the tumor milieu over an 8 h period, ultimately culminating in an extensive and enduring retention of up to 28 h (\u003cb\u003eFigures S18a and S18b\u003c/b\u003e). \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging of major organs further substantiated this observation, as evidenced by the conspicuously robust fluorescence signals discernible within the tumor regions post-intravenous administration of DCCMH-Cy5.5 after 28 h (\u003cb\u003eFigure S18c\u003c/b\u003e). These results demonstrated that DCCMH could accumulate effectively in tumor regions for a long time, which was conducive to its long-term therapy for cancers.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e therapeutic efficacy of the combination treatment was then assessed through the intravenous administration of DCCMH using H22 tumor-bearing mice. The H22 cell line is a widely utilized murine hepatocellular carcinoma cell line that overexpresses the CD44 receptor. It is suitable for establishing a fully immunocompetent hepatocellular carcinoma xenograft model [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Once the tumor size reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, the H22 tumor-bearing mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;5/group): (I) PBS (blank control), (II) DOX, (III) CCMH, (IV) DCCMH. The drugs were administered intravenously every 4 days (2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 100 \u0026micro;L), and the mice were monitored for 18 consecutive days, with photographs taken, weights recorded, and tumor volumes measured every 3 days (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and S19-22). Monotherapy exhibited limited efficacy due to drug resistance and the rapid stress response of cancer [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Remarkably, the DCCMH treatment effectively suppressed tumor growth, exhibiting an inhibition rate of 74.24%, surpassing other treatment modalities with statistically significant differences (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-e, S23, and S24). The alterations in body weight exhibited no noteworthy aberrations across all experimental cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), signifying the absence of evident systemic toxicity. To more intensively assess the synergistic anticancer efficiency and mechanisms of DCCMH, the combination index (CI, CI\u0026thinsp;\u0026lt;\u0026thinsp;1 indicates synergism, CI\u0026thinsp;=\u0026thinsp;1 indicates additive effect, and CI\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates antagonism) and the activity of AMPK enzymes in isolated H22 tumor tissue were analyzed [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The results showed that the CI of DCCMH was 0.79, which strongly proved that DCCMH achieved good synergism of CDT and CT. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, compared with the PBS and DOX groups, both CCMH and DCCMH significantly up-regulated AMPK enzyme activity in H22 tumors, further promoting chemotherapeutic efficacy.\u003c/p\u003e \u003cp\u003eAdditionally, histopathological analyses of extracted tumor tissues were performed using hematoxylin \u0026amp; eosin (H\u0026amp;E), terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), and Ki67 staining to assess morphology, apoptosis, and proliferation of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). The H\u0026amp;E-stained tumor sections exhibited a significant decrease in cell density in the DCCMH treatment group. The intense green fluorescence observed in TUNEL staining and the weak red fluorescence observed in Ki67 staining suggested that DCCMH-mediated synergistic therapy induced significant apoptosis and inhibition of tumor cell proliferation. The main organs including the heart, liver, spleen, lungs, and kidneys were subjected to histological examination through H\u0026amp;E staining after all interventions, and the results indicated no significant organ toxicity within all experimental groups (\u003cb\u003eFigure S25\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we designed and prepared a novel nano-MOF using prodrugs endowed with the remarkable ability to modulate the tumor microenvironment. The as-obtained nano-prodrug MOFs, denoted as DCCMH, exhibit a gradual degradation upon exposure to high concentrations of GSH, leading to the release of Cu\u003csup\u003e2+/+\u003c/sup\u003e, CHCA, Met, and DOX upon internalization by cancer cells. Subsequently, a cascade of anti-tumor actions is triggered: (1) Cu\u003csup\u003e2+\u003c/sup\u003e proficiently depletes intracellular GSH and catalyzes a Fenton-like reaction, generating a continuous supply of highly toxic ·OH species to inflict oxidative damage upon cancer cells; (2) CHCA effectively hampers the exocytosis of intracellular lactic acid, thereby inducing an elevation in intracellular acidity, which in turn promotes the Fenton-like reaction; (3) Met activates the intracellular AMPK signaling pathway, which reduces the tolerance of tumor cells to DOX. Through comprehensive \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e investigations, we have demonstrated that DCCMH exhibits a homogeneous morphology, high stability, and excellent biosafety profile. Moreover, these properties contribute to the overall efficacy of synergistic tumor microenvironment remodeling and augmented CT/CDT, resulting in a substantial inhibition of tumor growth. This groundbreaking study unveils a novel engineering approach for the development of prodrug-loaded nMOFs, paving the way for highly efficient combination therapies targeting hepatocellular carcinoma tumors.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e\n\n "},{"header":"Experimental section","content":"\u003ch2\u003eMaterials and chemicals\u003c/h2\u003e\n\u003cp\u003eAll reagents used in this study were of analytical grade and were obtained from reputable suppliers. \u0026alpha;-cyano-4-hydroxycinnamic acid (CHCA), CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, glutathione (GSH), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 30%), and methylene blue (MB) were bought from Aladdin-Reagent Co. Ltd. (China). Metformin (Met) and hyaluronic acid (HA, Mw\u0026thinsp;\u0026lt;\u0026thinsp;10 kDa) were obtained from Coolaber Science \u0026amp; Technology Co. (Beijing, P. R. China). Doxorubicin (DOX) was bought from HEOWNS-Reagent Co. Ltd. (Tianjin, P. R. China). Dimethyl sulfoxide (DMSO) was bought from Chengdu Chron Chemical Co. Ltd. Hoechst 33258, reduced GSH assay kit, Annexin V-FITC/PI kit, mitochondrial membrane potential kit (JC-10 Assay), 2\u0026prime;,7\u0026prime;-Dichlorofluorescin diacetate (DCFH-DA), and fetal bovine serum (FBS) were purchased from Solarbio Science \u0026amp; Technology Co., Ltd. The Lactic acid content kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, P. R. China).\u003c/p\u003e\n\u003ch3\u003ePreparation of CCM NPs\u003c/h3\u003e\n\u003cp\u003eCuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO (0.1 mM), CHCA (0.02 mM), and Met (0.08 mM) were mixed and dissolved in 4 mL solution (\u003cem\u003eV\u003c/em\u003e\u003csub\u003eH2O\u003c/sub\u003e: \u003cem\u003eV\u003c/em\u003e\u003csub\u003eethanol\u003c/sub\u003e = 1:1) with 30 \u0026micro;L of triethylamine, followed by ultrasonication to fully dissolve and transferred to a 15 mL Teflon-lined autoclave and heated at 100 ℃ for 3 h. After the reaction, the samples (CCM NPs) were washed with ultrapure water and methanol to remove impurities.\u003c/p\u003e\n\u003ch2\u003ePreparation of DCCM and DCCMH NPs\u003c/h2\u003e\n\u003cp\u003eDOX (1 mL, 10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added drop by drop to aqueous CCM (5 mL, 2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) under ultrasound. The mixture was then stirred for 24 h and collected by centrifugation at 12000 rpm for 15 min. The resulting precipitate, referred to as DCCM, was washed three times with water. For the preparation of DCCMH NPs, HA (1 mL, 10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added drop by drop to aqueous DCCM (5 mL, 2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) under ultrasound, the mixture was stirred for 12 h and the precipitate was collected by centrifugation at 12000 rpm for 15 min. The resulting precipitate, referred to as DCCMH, was washed three times with water.\u003c/p\u003e\n\u003ch2\u003eDOX loading and release from DCCMH NPs\u003c/h2\u003e\n\u003cp\u003eTo determine the drug loading efficiency (DLE%) of DCCMH, the centrifugal supernatants obtained during the loading of DOX were collected. The concentration of DOX in the supernatants was measured using a standard curve of DOX determined by UV\u0026ndash;Vis spectroscopy. The DLE% and encapsulation efficiency (EE%) of DCCMH were calculated using the following equations (1) and (2). Drug release was assessed under both the physiological and the tumor microenvironments. Specifically, 2 mg of DCCMH NPs were suspended in 4 mL of different phosphate-buffered solutions (pH5.0 with 0 mM GSH, and pH5.0 with 10 mM GSH) and stirred at 37 ℃. At each time interval, 1.5 mL of release medium was extracted to determine the percentage of DOX released \u003cem\u003evia\u003c/em\u003e ultraviolet-visible spectrophotometry analysis. The sample was then returned to its original release system for further evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003ch2\u003eGSH depletion and \u0026middot;OH generation\u003c/h2\u003e\n\u003cp\u003eThe reduction in GSH levels caused by varying concentrations of DCCMH nanoparticles was assessed using the reduced GSH assay kit. Briefly, the 0.5 mL of different concentrations of DCCMH solution were mixed with 0.5 mL of GSH (20 mM) solution, and the mixture was incubated at 37 ℃ for 6 h. Subsequently, the solutions were centrifuged at 12000 rpm for 10 min, and the absorbance of the supernatant was measured at 412 nm using UV\u0026ndash;Vis spectroscopy.\u003c/p\u003e\n\u003cp\u003eThe production of \u0026middot;OH was measured using methylene blue (MB) as a probe. A solution of MB (10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to PBS, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DCCMH, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DCCMH\u0026thinsp;+\u0026thinsp;GSH solutions (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: 10 mM, DCCMH: 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, GSH: 10 mM) and stirred for various durations. The bleaching of MB was monitored by measuring its absorbance at 665 nm using UV\u0026ndash;Vis spectroscopy. Additionally, electron spin resonance (ESR) spectroscopy was employed to verify the generation of \u0026middot;OH. A mixture of 10 \u0026micro;L of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 1 mL of the DCCMH suspension (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: 10 mM, DCCMH: 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was measured using an EMX plus spectrometer (Bruker, Germany).\u003c/p\u003e\n\u003ch2\u003eCell culture and cellular uptake\u003c/h2\u003e\n\u003cp\u003eHL7702 cells, HepG2 cells, Hela cells, U87MG cells, and 4T1 cells were purchased from KeyGEN BioTECH Co. (Nanjing, China). Cells were cultured in high glucose medium DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e. For cellular uptake tests, the HepG2 cells were seeded into 6-well plates or CLSM culture dishes and incubated with DCCMH (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for different durations (1, 3, 6, and 9 h). Subsequently, the cells were harvested and resuspended in PBS. Flow cytometry was employed to measure the fluorescence intensity of the cells. Additionally, the cells were washed twice with PBS and stained with Hoechst 33258 for 10 min. CLSM was used to capture images of the stained cells (Hoechst 33258: \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;405 nm, \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;460 nm; DOX: \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;488 nm, \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;590 nm).\u003c/p\u003e\n\u003ch2\u003eCytotoxicity assay\u003c/h2\u003e\n\u003cp\u003eCells were seeded into 96-well plates at a density of 7.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and incubated with various drugs (DOX, CCMH, or DCCMH) for 24 or 48 h. After that, 20 \u0026micro;L of thiazolyl blue tetrazolium bromide (MTT) solution (5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to each well, and cells were further incubated for 4 h. Next, the resulting formazan crystals were dissolved using 100 \u0026micro;L of dimethyl sulfoxide, and the absorbance of each well was measured at 490 nm using a microplate reader (FLUOstar Omega).\u003c/p\u003e\n\u003ch2\u003eIntracellular GSH depletion and ROS generation\u003c/h2\u003e\n\u003cp\u003eHepG2 cells were seeded into T25 culture flasks and allowed to grow for 24 h, then the cells were treated with PBS, DOX (4 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CCMH (46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and DCCMH (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively, for 12 h. After removing the culture medium, the cells were collected and subjected to three cycles of freezing and thawing using liquid nitrogen and 37 ℃ water, respectively. The resulting cell lysates were then centrifuged, and the absorbance at 412 nm was measured using a microplate reader (FLUOstar Omega). HepG2 cells were seeded into 6-well plates at a density of 2.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well for 24 h.\u003c/p\u003e\n\u003cp\u003eTo measure the level of ROS in the cells, similarly, the cells were cultured with different drugs for 12 h, and after removing the culture medium, 1 mL of DCFH-DA (10 \u0026micro;M) solution was added to each well and cultured for another 30 min. Then, the cells were washed three times with PBS to remove excess DCFH-DA and fluorescence images were captured using CLSM.\u003c/p\u003e\n\u003ch2\u003eIn vitro lactic acid and pH measurements\u003c/h2\u003e\n\u003cp\u003eHepG2 cells were seeded into 6-well plates at a density of 2.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated with various drugs (PBS, DOX: 4.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CCMH: 46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, DCCMH: 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 12 h, respectively. Then, both the cells and the culture medium were collected for the measurement of lactic acid levels using lactic acid detection kits, following the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003eHepG2 cells were cultured in CLSM culture dishes overnight, then the cells were treated with different drugs including PBS, DOX (4.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CCMH (46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and DCCMH (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 12 h. Subsequently, the cells were washed with PBS and incubated with the pH fluorescent probe (BCECF-AM, 5 \u0026micro;M in PBS) for 30 min, and observed using a CLSM. The culture media with a pH of 7.4 was used as standard samples for comparison.\u003c/p\u003e\n\u003ch2\u003eMitochondrial membrane potential measurement\u003c/h2\u003e\n\u003cp\u003eThe HepG2 cells were seeded into CLSM culture dishes and cultured overnight. After treatment with PBS, DOX (4.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CCMH (46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and DCCMH (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 12 h, respectively, the cells were incubated with JC-1 dye for 30 min to measure the MMP through CLSM analysis.\u003c/p\u003e\n\u003ch2\u003eLive/dead cell staining and apoptosis assay\u003c/h2\u003e\n\u003cp\u003eHepG2 cells were seeded into CLSM culture dishes with a density of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per dish for 24 h, and then treated with PBS, DOX (4.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CCMH (46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and DCCMH (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 12 h, respectively. Then, the cells were stained with Calcein-AM (2 \u0026micro;M) and PI (4 \u0026micro;M) for 30 min to measure the cell viabilities through CLSM analysis.\u003c/p\u003e\n\u003cp\u003eHepG2 cells were cultured with different drugs (PBS, DOX: 4.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CCMH: 46 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, DCCMH: 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 24 h. Afterward, all of the treated cells were collected, washed, and stained with FITC/PI for 20 min. Then, the intracellular fluorescence signals of FITC/PI were measured by flow cytometry, to determine the apoptosis and necrosis rate of HepG2 cells.\u003c/p\u003e\n\u003ch2\u003eHemolysis assay\u003c/h2\u003e\n\u003cp\u003e1 mL of red blood cell suspension (0.2%, v/v) was mixed with 1 mL of different concentrations of DCCMH and incubated at 37 ℃ for 4 h. The cells incubated in double distilled water and PBS were used as positive and negative controls, respectively. After centrifugation at 3000 rpm for 6 min, the optical density (OD) at 540 nm of each solution was measured using the microplate reader. The hemolysis rate was calculated according to the following formula (3).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" alt=\"\"\u003e\u003c/p\u003e\n\u003ch2\u003eIn vivo antitumor evaluation\u003c/h2\u003e\n\u003cp\u003eFemale Balb/c mice were subcutaneously inoculated with 0.1 mL of H22 cell suspension (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) into their right leg to establish the tumor model. Subsequently, once the tumor volume reached 50\u0026ndash;70 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly divided into four groups with five mice in each group: Control (PBS), DOX, CCMH, and DCCMH. The respective formulations were intravenously injected every 4 days for a total of 18 days, with a dose of 1.6 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of DOX, 18.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of CCMH, and 20 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of DCCMH. The tumor size and body weight of the mice were measured once every three days. After treatment for 18 days, tumors in all treatment groups were harvested for H\u0026amp;E staining, TUNEL staining, and Ki67 staining. All animal experiments were carried out under the protocols approved by the Institutional Animal Care and Use Committee of Zhejiang University.\u003c/p\u003e\n\u003ch2\u003eLong-term in vivo biosafety evaluation\u003c/h2\u003e\n\u003cp\u003eAfter the \u003cem\u003ein vivo\u003c/em\u003e tumor treatment with different formulations including PBS, DOX (1.6 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CCMH (18.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and DCCMH (20 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the major organs (heart, liver, spleen, lungs, and kidneys) of mice were harvested and conducted H\u0026amp;E staining for the histological analysis. The blood samples were collected from the mouse orbital venous plexus for blood biochemistry and blood routine examinations.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe one-way analysis of variance (ANOVA) statistical method was performed to evaluate the experimental data. A \u003cem\u003ep-value\u003c/em\u003e of 0.05 was selected as the significance level and the data were indicated with (*) for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, (**) for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and (***) for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Online Library or the author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (22171230, 82001956), the National University of Singapore (NUHSRO/2020/133/Startup/08, NUHSRO/2023/008/NUSMed/TCE/LOA, NUHSRO/2021/034/TRP/09/Nanomedicine), National Medical Research Council (MOH-001388-00, MOH-001041, CG21APR1005), Singapore Ministry of Education (MOE-000387-00), National Research Foundation (NRF-000352-00), the Project of Science and Technology of Social Development in Shaanxi Province (2023YBSF-151, 2021SF-120), and 2023 Hangzhou West Lake Pearl Project Leading Innovation Youth Team Project (TD2023017). The authors thank the Teaching and Research Core Facility at the College of Life Science, the Life Science Research Core Services, and the Northwest A\u0026amp;F University for helping with characterizations including SEM, TEM, and CLSM, \u003cem\u003eetc\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJunliang Dong\u003c/strong\u003e formulated the conceptual framework and established the experimental procedures, conducted \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, analyzed data, drafted the manuscript, compiled all figures, and revised the manuscript based on the feedback from Wenjing Sun, Xiaoyuan Chen, and Zhichao Pei. \u003cstrong\u003eJindong Ding\u003c/strong\u003e assisted in writing and revising the manuscript. \u003cstrong\u003eShifan Luo, Ruoshui Li, Yi Wang, \u003c/strong\u003eand\u003cstrong\u003e Bing Xiao\u003c/strong\u003e participated in conducting the research. \u003cstrong\u003eYuxin Pei, Wenjing Sun, Xiaoyuan Chen, \u003c/strong\u003eand\u003cstrong\u003e Zhichao Pei \u003c/strong\u003ecollaboratively reviewed and edited the manuscript, and provided financial support. All authors collectively analyzed the findings, revised the manuscript, and unanimously endorsed the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003eThe animal experimental was carried out following the guidelines of the Institutional Animal Care and Use Committee of Zhejiang University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to publish this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnderson NM, Simon MC. The tumor microenvironment. 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ACS Appl Mater Interfaces. 2020;12:12591\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prodrug metal-organic frameworks, GSH-responsive degradation, redox homeostasis, TME-remodeling, synergistic chemo/chemodynamic therapy.","lastPublishedDoi":"10.21203/rs.3.rs-5402726/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5402726/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetal-organic frameworks (MOFs) hold tremendous potential in cancer therapy due to their remarkable structural and functional adaptability, enabling them to serve as nanocarriers for biopharmaceuticals and nanoreactors for organizing cascade bioreactions. Nevertheless, MOFs are predominantly utilized as biologically inactive carriers in most cases. Developing nanoscale prodrug MOFs suitable for biomedical applications remains a huge challenge. In this study, we have designed a novel prodrug nano-MOFs (DCCMH) using metformin (Met) and α-cyano-4-hydroxycinnamic acid (CHCA) as ligands for coordination self-assembly with Cu\u003csup\u003e2+\u003c/sup\u003e, followed by loading of DOX and surface modification with HA. Upon internalization by cancer cells, DCCMH releases Cu\u003csup\u003e2+\u003c/sup\u003e, CHCA, Met, and DOX in response to high levels of GSH-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e within the tumor microenvironment (TME); Cu\u003csup\u003e2+\u003c/sup\u003e depletes GSH and generates Cu\u003csup\u003e+\u003c/sup\u003e that subsequently catalyzes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to hydroxyl radical through a Fenton reaction; CHCA induces a further decrease in intracellular pH and promotes Fenton reactions by inhibiting lactate efflux; Met up-regulates tyrosine kinase activity and enhances the chemotherapy of DOX. With the abilities to synergistically combine chemo/chemodynamic therapy and remodel the TME, the DCCMH nMOFs inhibit murine hepatoma effectively. This study presents a feasible strategy for fabricating prodrug nano-MOFs which are capable of remodeling TME to improve efficacy through synergistic cancer therapy.\u003c/p\u003e","manuscriptTitle":"Remodeling Tumor Microenvironment Using Prodrug nMOFs for Synergistic Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-11 06:53:46","doi":"10.21203/rs.3.rs-5402726/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-09T18:25:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-07T13:19:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-06T01:32:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-04T14:28:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T08:06:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24079524474842011588676783511628883031","date":"2024-11-23T11:59:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46681300805099891988676175030054281456","date":"2024-11-23T03:03:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157186184895741706441344926518347606408","date":"2024-11-21T01:37:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2371671926573915472289044971065023786","date":"2024-11-21T00:34:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116090416475151535418440346486473507436","date":"2024-11-20T23:13:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-20T21:23:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T09:01:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-08T09:00:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-11-06T12:13:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b50fd01-0bf4-44e6-a69d-a39faf0d8a2a","owner":[],"postedDate":"December 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:25:05+00:00","versionOfRecord":{"articleIdentity":"rs-5402726","link":"https://doi.org/10.1186/s12951-025-03202-7","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-02-19 15:57:38","publishedOnDateReadable":"February 19th, 2025"},"versionCreatedAt":"2024-12-11 06:53:46","video":"","vorDoi":"10.1186/s12951-025-03202-7","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03202-7","workflowStages":[]},"version":"v1","identity":"rs-5402726","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5402726","identity":"rs-5402726","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}