Cascade amplification of tumor chemodynamic therapy and starvation with re- educated TAMs via Fe-MOF based functional nanosystem

doi:10.21203/rs.3.rs-2494616/v1

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Cascade amplification of tumor chemodynamic therapy and starvation with re- educated TAMs via Fe-MOF based functional nanosystem

https://doi.org/10.21203/rs.3.rs-2494616/v1

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published 11 Apr, 2023

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Tumor microenvironment is characterized by the high concentration of reactive oxygen species (ROS), which is an effective key used to open the pandora's box against cancer. Herein, a tumor-targeted nanosystem HFNP@GOX@PFC composed of ROS-cleaved Fe-based metal-organic framework, hyaluronic acid (HA), glucose oxidase (GOX) and perfluorohexane (PFC) has been developed for tumor cascade amplified starvation and chemodynamic therapy (CDT). In response to the high concentration of hydrogen peroxide (H₂O₂) intratumorally, HFNP@GOX@PFC endocytosed by tumor cells can specially be disassembled and release GOX, PFC and Fe²⁺, which can collectively starve tumor and self-produce additional H₂O₂ via competitively glucose catalyzing, supply oxygen to continuous support GOX-mediated starvation therapy, initiate CDT and cascade amplify oxidative stress via Fe²⁺-mediated Fenton reaction, leading to the serious tumor damage with activated p53 signal pathway. Moreover, HFNP@GOX@PFC also significantly initiates antitumor immune response via re-educating tumor-associated macrophages (TAMs) by activating NF-κB and MAPK signal pathway. In vitro and in vivo results collectively demonstrate that nanosystem not only continuously initiates starvation therapy, but also pronouncedly cascade-amplify CDT and polarize TAMs, consequently efficiently inhibiting tumor growth with good biosafety. The functional nanosystem combined the cascade amplification of starvation and CDT provides a new nanoplatform for tumor therapy.

Cascade amplification

tumor starvation

chemodynamic therapy

re-education macrophages

Fe-MOF nanosystem

Chemodynamic therapy (CDT) driven by Fenton/Fenton-like reaction is known as a potential tumor therapeutic modality with minimal side effects [1, 2]. Generally, nanocatalysts and H₂O₂ are the two key initiators and limitation factors of CDT [3, 4]. Benefiting from the high catalytic activity, iron (Fe)-based nanoparticles have been frequently used to construct CDT platform against tumors [5]. However, the inadequate and limited content of endogenous H₂O₂ in tumors seriously restricts the efficiency of CDT with the unsatisfactory therapy effect, owing to the tumor heterogeneity. Besides, the overexpressed reducing substances in tumor microenvironment, such as glutathione, can counteract the production of hydroxyl radical (·OH) and extra reduce the CDT therapeutic efficacy [6]. Therefore, it is necessary to fabricate a functional Fe-based nanosystem integrating with H₂O₂ self-supply capacity for improved tumor CDT efficiency. For one thing, the excess H₂O₂ generated by nanosystem converts the reductive property of glutathione and cascade amplifies CDT efficiency [7]. For another thing, the excess ROS also possesses the initiation antitumor immune response potential via re-educating tumor-associated macrophages (TAMs) [8], devoting to effective tumor killing.

Typically, glucose oxidase (GOX) seems to be a good choice to solve the shortage of endogenous H₂O₂. GOX is an endogenous oxidoreductase, which can effectively catalyze glucose to gluconic acid and generate abundant H₂O₂ [9]. Notably, glucose, the substrate of GOX and nutritional supplier, plays a vital role in tumor growth. The proliferation of tumor cells mainly depends on aerobic glycolysis, which makes tumor cells more sensitive to the change in glucose concentration [10]. It is reported that cancer cells need to burn almost 20-fold glucose to get the same amount of energy compared with normal cells [11]. Therefore, the GOX combined with the CDT strategy can not only provide enough endogenous H₂O₂ for cascade amplifying CDT efficiency, but also significantly consume glucose for tumor starvation therapy, displaying the potential antitumor effect [10, 12, 13]. However, the catalysis activity mediated by GOX is severely limited by the relatively low delivery efficiency and hypoxia in vivo, the significant characteristics of tumor microenvironment. Thus, efforts to improve the delivery efficiency of GOX and self-supply exogenous O₂ simultaneously are critical for the combined tumor CDT and starvation therapy triggered by GOX.

Metal-organic frameworks (MOFs) as applicable nanocarriers have been frequently reported for drug delivery with superior delivery efficiency, resulting from the natural advantages of adjustable pores, high surface area, diverse structure and easy functionalization [14, 15], which could protect the biologically active drugs (e.g. enzyme, GOX, etc) from inactivation caused by the external environment, so as to safely deliver drugs to tumors with high efficiency. Inspired by the high Fenton reaction activity of Fe-based nanoparticles and excellent drug delivery capacity, Fe-based MOF is thus recommended as a class of natural CDT initiators and GOX carriers. Considering the perfluorohexane (PFC) has been extensively evaluated as a substitute oxygen carrier, it could effectively self-supply exogenous O₂ to overcome above mentioned hypoxia and GOX-mediated starvation therapy limitation obstacles. Hence, Fe-based MOF co-loaded with GOX and PFC may serve as a promising platform able to self-supply exogenous O₂ and H₂O₂, which could effectively cascade amplify tumor CDT and starvation therapy efficiency with potential TAMs regulation, realizing the desired cancer damage. However, poor-degradability, off-targeting capacity and associated potential biosafety have always restricted the application of the Fe-based MOF system. Therefore, a new solution to improve the biocompatible and active targeting of Fe-MOFs for tumor CDT and starvation therapy is urgently needed.

Based on the above considerations, we herein developed a tumor-targeted functional nanosystem based on ROS-responsive degradable Fe-MOF co-loaded GOX & PFC for the cascade amplified CDT and starvation therapy with TAMs polarization. The ROS-responsive degradable Fe-MOF was first synthesized through hydrothermal reaction composed of ROS-sensitive agent thioketal (TK) and Fe²⁺ ion. Subsequently, GOX as exogenous H₂O₂ supplier and starvation inducer and PFC as O₂ provider are co-loaded into Fe-MOF. Finally, the tumor targeting molecular hyaluronic acid (HA) is introduced into the surface of drug loaded Fe-MOF, harvesting the resultant functional nanosystem (HFNP@GOX@PFC). After it is injected into the venous system, HA-functionalized Fe-MOF nanosystem could prolong circulation time and accumulate in the tumor site through passive targeting of the enhanced permeability and retention effect (EPR) and active targeting of HA receptor-ligand mediated endocytosis. HFNP@GOX@PFC uptake by tumor cells are then disassembled in response to the relatively high concentrations of ROS in the cytoplasm, leading to the release of O₂ supplier PFC, H₂O₂ supplier and starvation inducer GOX, and CDT trigger agent Fe²⁺. Subsequently, plenty of O₂ self-generated by PFC reduces hypoxia. Moreover, GOX competitively reacts with intracellular glucose and sufficient O₂ generated by PFC to starve tumor and produce abundant H₂O₂, which not only accelerate the disassembly of HFNP@GOX@PFC and drug release, but also promote the re-education of TAMs and provide the fuel for CDT. The released Fe²⁺ reacts with fuel of H₂O₂ self-supplied by GOX to cascade amplify CDT and induce effective tumor damage. The above continuous “Self-Amplified” process can proceed continuously in the tumor microenvironment to boost tumor killing efficacy with activated p53 signal pathway through combining starvation and CDT. More importantly, ROS generated by above cycle could additionally boost tumor immune response via re-educating TAMs by activating NF-κB and mitogen-activated protein kinase (MAPK) signal pathways. Eventually, the multiple therapies induce the superior tumor therapy effect (Fig. 1). Hence, we hypothesized that HFNP@GOX@PFC functional nanosystem could achieve desired antitumor therapy by combining starvation and CDT with TAMs re-education.

Preparation and characterization of HFNP@GOX@PFC nanosystem

The degradable Fe-MOF nanoparticles (FNP) were synthesized by conjugation of Fe²⁺ with ROS-cleaved linker TK using hydrothermal reaction. In order to improve biocompatibility and tumor targeting capacity, HA coat-layer was introduced into the surface of FNP nanoparticles, which was used to prolong the circulation in blood vessels and endowed HFNP nanosystem with the tumor targeting features. Transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurement, X-ray diffraction (XRD), Fourier Transform Infrared Spectrometer (FTIR), ultraviolet and visible spectrophotometry (UV-vis) were employed to monitor the functionalization of HFNP nanosystem. Firstly, both FNP and HFNP exhibited the distinctively spherical morphology with good monodispersity detected by TEM (Fig. 2a, b), and the uniform sizes were respectively distributed at 130 ± 9.2 and 158 ± 6.5 nm, which was consistent with the corresponding DLS results (Fig. 2c). As companying with the introduction of HA, the size of HFNP displayed an obvious increase, indicating the successful modification of HA. Simultaneously, the zeta potential of HFNP was also significantly reduced from − 22 ± 1 to -25 ± 0.4 mV after the conjugation of HA (p < 0.01, Additional file 1: Fig. S1), suggesting the successful construction of HFNP nanosystem again. Secondly, XRD results showed that the obvious peaks of 5.58°, 8.62° and 10.1° were observed in FNP sample (Additional file 1: Fig. S2), attributed to the typical structure of Fe-MOF nanoparticles [16]. After conjugation with HA molecule, similar peaks of 5.62°, 8.64° and 10.08° were also displayed in HFNP sample, indicating again the synthesis of FNP. More importantly, the new peak of 25.18° was shown in the XRD spectrum of HFNP, it was ascribed to the characteristic peak of HA, which was consistent with previous research [17]. The result further confirmed the successful modification of HA and fabrication of HFNP. Thirdly, both the FTIR spectrum of FNP and HFNP showed that the typical characteristic peak of carboxyl at 1637 cm^− 1 and 1425 cm^− 1 [18], owing to the introduction of the abundant TK ligand with carboxyl in the synthesis of FNP and HFNP. Furthermore, the distinctively characteristic peaks of HA (1051 cm^− 1, 1085 cm^− 1 and 1150 cm^− 1) were displayed in the FTIR spectrum of HFNP (Additional file 1: Fig. S3), attributing to the C-O-C stretching peaks of glycosyl agent in the HA molecule [17, 19]. It thus implied that the HA layer was actually conjugated on the surface of FNP. The results collectively suggested that the ROS-responsive degradable Fe-MOF nanosystem HFNP with HA modification was successful construction.

Subsequently, the drug loading content and drug loading efficiency of GOX and PFC in HFNP@GOX@PFC nanosystem were detected via UV-vis spectrophotometry and gas chromatography (GC). As shown in Fig. 2d, both HFNP@GOX and HFNP@GOX@PFC groups without H₂O₂ treatment had no obvious GOX release, proved by the negligible absorption intensity of GOX at 276 nm, indicating the successfully GOX loading in the nanosystem. Furthermore, the loading content and encapsulation efficiency of GOX in HFNP were calculated as 2.42% and 82.5% based on the associating standard curve (Additional file 1: Fig. S4). Moreover, after treatment with H₂O₂, both HFNP@GOX and HFNP@GOX@PFC groups showed the typical GOX absorption peak at 276 nm compared with GOX group, implying the effective drug release. Furthermore, the loading content and efficiency of PFC in HFNP@GOX@PFC nanosystem were measured with GC. HFNP@GOX@PFC exhibited the obvious elution peak of PFC at 1.152 min the same as PFC (Additional file 1: Fig. S5), indicating the successful encapsulation of PFC. The loading content and encapsulation efficiency of PFC in HFNP@GOX@PFC were determined as 32.37% and 78.07%, according to the corresponding standard curve (Additional file 1: Fig. S6). These results indicated that the nanosystem had been fabricated successfully. Additionally, the stability of HFNP@GOX@PFC nanosystem was evaluated. As shown in Fig. 2e, f, the size & zeta potential of HFNP@GOX@PFC had no significant change, despite the short-term (0–24 h) in 10% fetal bovine serum of PBS or long-term (1–14 day) in PBS incubation, proving that HFNP@GOX@PFC had great biostability & storage stability, and it was conducive to the drug delivery as nanocarriers for tumor therapy in vivo and in vitro.

The vital features of HFNP@GOX@PFC nanosystem are ROS-responsive Fe-MOF disassembly, drug release, self-produced H₂O₂ and cascade amplification of CDT. Firstly, TEM, DLS and UV-vis spectrophotometry were used to monitor the ROS-responsive Fe-MOF disassembly & drug release trait. As shown in Fig. 2g, h, both the morphology of FNP and HFNP were obviously changed from the initial sphere to collapse structure when exposed to H₂O₂ (Fig. 2a, B vs. Figure 2g, h), attributing to the ROS-responsive disassembly. It is consistent with the related DLS results (Fig. 2i). The reason could be explained that the key group of the linker agent TK is S-C-S, which could actually cleave in response to H₂O₂ stimuli. Thus, FNP and HFNP composed of Fe²⁺ ions and TK linker could be rapidly disintegrated triggered by H₂O₂, which is beneficial for the following drug release. Furthermore, both the ROS-responsive Fe²⁺ and GOX release behaviours of HFNP@GOX@PFC nanosystem were quantitatively measured using o-phenanthroline method and UV-vis spectrophotometry. The intracellular ROS concentration of tumor cells was simulated through H₂O₂ to analyse the drug release of nanosystem. As shown in Fig. 3a, there was barely any Fe²⁺ release in the control group (PBS) upon 48 h, indicating the good encapsulation, stability and potential biocompatibility of HFNP@GOX@PFC nanosystem (owing to no leakage during the blood circulation). In contrast, the release amount of Fe²⁺ was dramatically increased in the presence of H₂O₂, showing the H₂O₂ concentration-dependence drug release pattern (Fig. 3a, 0.1 M H₂O₂ vs. 1 M H₂O₂), suggesting that HFNP@GOX@PFC nanosystem actually possesses the ROS-responsive drug release capability. At the same time, GOX also showed H₂O₂ concentration-dependent release behaviour, and the release amount reached 69% and 91% (almost complete drug release) in 0.1 mM H₂O₂ and 1 mM H₂O₂ treatment groups, while the release amount of GOX was negligible in control group as well (Fig. 3b). The result further confirmed again the effective ROS-responsive drug release features of HFNP@GOX@PFC nanosystem with good stability. Notably, H₂O₂ led to the disintegration of HFNP@GOX@PFC nanosystem and the release of Fe²⁺ with H₂O₂-dependent pattern, which benefited for the subsequent CDT treatment.

Secondly, UV-vis spectrophotometry was used to characterize the self-produced H₂O₂ capability of HFNP@GOX@PFC nanosystem, since H₂O₂ possesses the natural characteristic peak at 240 nm. As shown in Fig. 3c, GOX loaded nanoparticles (HFNP@GOX and HFNP@GOX@PFC) co-incubated with glucose exhibited the obviously characteristic peak of H₂O₂ compared to control (PBS) and negative HFNP groups, confirming the self-generation of H₂O₂. It also indicated that the catalytic activity of GOX was not affected by the encapsulation of the HFNP@GOX@PFC nanosystem. Furthermore, the self-supply H₂O₂ capability of the HFNP@GOX@PFC nanosystem displayed a time-dependent pattern (Fig. 3d), and the yield of H₂O₂ at 6 h incubation was about 160 times that of 1 h, proving again self-produced H₂O₂ feature of HFNP@GOX@PFC nanosystem. It could be explained that the GOX loaded HFNP@GOX@PFC nanocarrier could continuously generate H₂O₂ under glucose via GOX-mediated catalytic reaction, and the self-supply dose of H₂O₂ was increased with time. More importantly, the self-generation of H₂O₂ would not only accelerate nanocarriers disassembly and drug release in turn, but also provide the fuel “H₂O₂ and Fe²⁺” for Fenton reaction, leading to the cascade amplification of CDT and desired antitumor effect.

Thirdly, the generation level of ·OH as the direct indicator labelled CDT was investigated via Hydroxyl Free Radical assay Kit for evaluating the cascade amplification of CDT. As shown in Fig. 3e, compared with control (PBS), HFNP, HFNP@GOX and HFNP@GOX@PFC groups co-incubated with H₂O₂ all showed the typical ·OH peaks without glucose input, which further proved the successful construction of Fe²⁺-based MOF with good Fenton reaction ability. More importantly, the production of ·OH increased sharply after adding glucose, because the catalytic activity of GOX was amplified under glucose and produced much more H₂O₂, so as to cascade amplify the Fenton reaction of the nanosystem. Considering the good stability, ROS-responsive Fe-MOF disassembly, drug release, self-produced H₂O₂ and cascade amplification of CDT performance of the nanosystem, it inspired us to further explore the antitumor effect in vitro and in vivo.

Cytotoxicity of HFNP@GOX@PFC nanosystem

The cytotoxicity of HFNP@GOX@PFC nanosystem against the HC11 normal breast cells and 4T1 breast cancer cells were both detected by CCK-8 assay. After treatment with a series of concentration HFNP@GOX@PFC (from 7.5 to 120 µM of Fe²⁺) for 24 h, HC11 cells showed good cell viability similar to that of the control (Fig. 3f), indicating the good biocompatibility. Only the moderate cytotoxicity exhibited in the largest drug dosage treatment (240 µM and 480 µM). On the contrary, 4T1 tumor cells showed obvious Fe²⁺ dose-dependent cytotoxicity when the concentration ranges from 30 µM to 480 µM. More importantly, the cell viability dropped sharply upon the Fe²⁺ dose exceed to 60 µM (p < 0.01), and the IC50 was 59.18 × 10^− 6 M. It was suggested that the HFNP@GOX@PFC nanosystem possesses the superior cytotoxicity against tumor cells, and the friendly cell viability for normal cells. The reasons may be attributed to the HA-endowed actively targeting endocytosis in the tumor cells and the cascade activation of the CDT combined starvation therapy in vitro, leading to the effective tumor killing.

Furthermore, the tumor killing effect of HFNP@GOX@PFC was investigated using CCK-8 assay as well. Typically, 4T1 cells were co-cultured with different treatment groups for 24 and 48 h, respectively. Compared with control (PBS), free PFC treatment displayed no significant cytotoxicity (Fig. 3g), indicating the biosafety of PFC. Both GOX and HFNP caused the moderate cytotoxicity after treatment for 24 h, and the tendency became more obvious as the incubation time extended to 48 h, owing to the inherent starvation therapy mediated by GOX and CDT cytotoxicity executed by Fe²⁺-mediated Fenton reaction [10, 20]. Moreover, GOX loaded groups (HFNP@GOX and HFNP@GOX@PFC) showed higher cytotoxicity than free GOX and blank HFNP upon 24 h or 48 h, benefiting from the superior drug delivery efficiency and improved biocompatibility. More importantly, HFNP@GOX@PFC induced the most serious cytotoxicity among all the treatment groups despite of incubation time, indicating the excellence tumor killing effect. The reason could be explained that HFNP@GOX@PFC nanosystem with high delivery efficiency could effectively accumulated into tumor cells, and the release of O₂ supplier PFC, H₂O₂ supplier plus starvation inducer GOX and CDT trigger agent Fe²⁺ could collectively kill tumor cells via initiation the cascade amplification tumor starvation and CDT therapy, leading to the highest tumor damage.

Tumor-targeted cell uptake of HFNP@GOX@PFC nanosystem

In order to reveal the active targeting endocytosis of HFNP@GOX@PFC nanosystem with high efficiency above mentioned, GOX conjugated with FITC were first loaded into the nanosystem. Subsequently, 4T1 cells were treated with FNP@GOX@PFC and HFNP@GOX@PFC for 12 h, and the cellular uptake degree of nanosystem were distinctly observed detected by CLSM. As shown in Fig. 3h, both the FNP@GOX@PFC and HFNP@GOX@PFC nanosystem labelled with green fluorescence were uptake by tumor cells, shown by the obvious green fluorescence distributed in the cytoplasm. Furthermore, the amount of HFNP@GOX@PFC endocytosed by tumor cells was significantly higher than that of FNP@GOX@PFC without HA conjugation, which was confirmed by the correspondingly quantitative analysis (about 2.3-fold, p < 0.01, Fig. 3i). The result suggested that HA-modified nanosystem could effectively uptake by 4T1 cells overexpressed HA receptors via the active targeting endocytosis pathway based on the HA receptor-ligand interaction effect, which was consistent with a previous study [16].

The ROS self-generation and hypoxia suppression of HFNP@GOX@PFC nanosystem

As the realization of the desired antitumor effect is closely with the activity of GOX and PFC cargoes, which was used to start the designed starvation and Fenton-based CDT therapy in HFNP@GOX@PFC nanosystem. The intratumoral activity of GOX and PFC were subsequently investigated. Since the GOX-mediated tumor starvation and PFC-mediated O₂ supply and CDT amplification were accompanied by ROS generation and hypoxia suppression, which were respectively investigated in the following study. For one thing, 4T1 cells were treated with administrations and the ROS generation level were monitored by FCM using the typically ROS probe of DCFH-DA. As shown in Fig. 4a, compared with control (PBS), HFNP treatment induced the obvious fluorescence signal of DCF, indicating the generation of ROS, which was owed to the production of ·OH via Fenton reaction mediated by Fe²⁺ [20]. Furthermore, the amount of ROS generation generated by HFNP@GOX was significantly higher than HFNP group, benefitting from the introduction of GOX. Moreover, HFNP@GOX@PFC induced the highest ROS level in tumor cells among all treatments (Additional file 1: Fig. S7), implying the effective ROS self-generation capacity. The reason could be explained that the introduction of O₂ supplier PFC in HFNP@GOX@PFC nanosystem could provide the endogenous O_2, which was the key fuel of GOX-mediated starvation, leading to the dramatically H₂O₂ self-generation and ·OH accumulation via the cascade amplification of Fe²⁺-based Fenton reaction. These results directly illustrated that HFNP@GOX@PFC nanosystem is capable of the ROS self-generation features, which was helpful for the improved tumor killing.

For another thing, the hypoxia suppression induced by HFNP@GOX@PFC nanosystem were simultaneously investigated. Since the hypoxia level of tumor cells is positively associated with the expression of the typically hypoxia protein CD47 [8], its expression was thus monitored for evaluation the hypoxia suppression in tumor cells using Western blot and Image J software. After treatment for above groups for 24 h, there was no obvious difference in the CD47 expression of 4T1 cells between control (PBS) and HFNP group (Fig. 4b), suggesting that the introduction of HFNP without O₂ supplier PFC and O₂ consumer GOX loading has no effect on the hypoxia suppression, resulting from the inactivation of O₂ generation. On the contrary, the expression of CD47 was significantly up-regulated in H₂O₂ supplier of GOX loaded HFNP@GOX group owing to the additional O₂ exhaustion, which was the indispensable fuel of the catalytic reaction mediated by GOX. Furthermore, HFNP@GOX@PFC group induced the sharply downregulation of the CD47 expression (p < 0.01, Fig. 4c), compared with other groups. Accordingly, the FCM result confirmed the above phenomenon, with a significant reduction in CD47 expression on the HFNP@GOX@PFC group (Fig. 4d and Additional file 1: Fig. S8). Benefiting from the relatively higher loading content of PFC than GOX (32.37% vs. 2.42%, Additional file 1: Fig. S6 and Fig. S4), the self-supply amount of the endogenous O₂ generated by PFC in HFNP@GOX@PFC nanosystem is correspondingly higher than that of O₂ exhaustion mediated by GOX, leading to the effective hypoxia suppression, as shown by the lowest CD47 expression in 4T1 cells. The result further confirmed that the HFNP@GOX@PFC nanosystem not only self-supply O₂ in tumor cells and relieve its hypoxic state, but also self-generate ROS via initiating the starvation mediated by GOX, and cascade amplify CDT effect mediated by Fe²⁺-based Fenton reaction, leading to the desired tumor killing.

The in vitro tumor apoptosis of HFNP@GOX@PFC nanosystem

In order to further explore the antitumor effect in vitro, the apoptosis ratio of 4T1 cells treated with above experiment group was subsequently detected using Annexin V-FITC/PI kit (NeoBioscience, China) and analyzed by FCM. As shown in Fig. 4e, blank HFNP group caused a moderate apoptosis ratio compared to control (PBS), attributed to the limited CDT efficiency in presence of the insufficient H₂O₂. Furthermore, HFNP@GOX treatment group caused more severe apoptosis compared to HFNP group (p < 0.01, Fig. 4f), owing to the introduction of starvation therapy mediated by GOX. Moreover, the HFNP@GOX@PFC nanosystem induced the highest apoptosis rate compared with other groups, as confirmed by the quantitative analysis (Fig. 4f). The reasons could be explained that the introduction of O₂ supplier-PFC could effective self-supply the endogenic O₂ and relieve intracellular hypoxia, which were conducive to promote the GOX-based starvation reaction and generate the abundant H₂O₂, followed by accelerating MOF disassembly, drug release and cascade boosting CDT, consequently leading to the effective tumor apoptosis. Subsequently, the expression levels of the typical proteins closely associated with apoptosis pathway were measured with western blotting and qPCR assays for revealing the apoptosis mechanism induced by HFNP@GOX@PFC nanosystem [21, 22]. Compared to control, HFNP, HFNP@GOX and HFNP@GOX@PFC groups certainly downregulated the expression of anti-apoptotic BCL-2 & CytC genes and upregulated the expression of pro-apoptotic BAX, Casp3 genes & p53 protein (Fig. 4g, h and Additional file 1: Fig. S9), in an order of HFNP < HFNP@GOX < HFNP@GOX@PFC, confirming again the superior tumor killing efficiency of HFNP@GOX@PFC nanosystem. It was explained that taking the combined advantages of the self-supply O₂ via PFC, the dramatically H₂O₂ self-generation via GOX-based catalytic reaction, the ·OH accumulation via the cascade amplification of Fenton reaction, and the improved delivery efficiency via HA-targeted strategy, HFNP@GOX@PFC nanosystem induced the most serious cell apoptosis in vitro.

Re-education of macrophage and anti-metastasis in vitro

As the level of H₂O₂ is closely associated with the polarization of tumor-associated macrophages (TAMs) [8], and HFNP@GOX@PFC nanosystem actually possess the ROS self-generation features, as revealed by above result (Fig. 3c-e and Fig. 4a). Thus, the HFNP@GOX@PFC nanosystem may play a key role in the re-education of TAMs. Based on above consideration, 4T1 tumor cells and IL4-pretreated RAW264.7 macrophages were firstly cocultured in the Transwell, and 4T1 cells were then treated with various administrations, FCM was finally used to monitor the re-education degree of macrophages. The coculturation platform was illustrated in Fig. 5a, and the gate strategy of FCM was shown in Additional file 1: Fig. S10. After above administrations, the percentage of M1 macrophages were all significantly increased compared with control (PBS), in an order of HFNP < HFNP@GOX < HFNP@GOX@PFC (p < 0.01, Fig. 5b, c), indicating the effective TAMs polarization. It was attributed to the difference ROS generation capacity (Fig. 3c-e and Fig. 4a). Notably, the introduction of O₂ supplier PFC and H₂O₂ supplier GOX in HFNP@GOX@PFC nanosystem could provide the sufficient fuel (O₂ and H₂O₂) for GOX-mediated catalytic reaction and Fe²⁺-based Fenton reaction, leading to the abundant production of ROS (including H₂O₂ and ·OH) and the highest re-education number of TAMs.

As the polarization of TAMs is positively associated with the special cytokine secretion [23]. In general, TNF-α and IL-6 are considered to be the special markers of M1 macrophages, and IL-10 is considered to be the special marker of M2 macrophages [24]. In order to further clarify the re-education degree of TAMs, Elisa assay were employed to investigate expression change of above cytokine mentioned. As shown in Fig. 5d, the expression of TNF-α and IL-6 in HFNP and HFNP@GOX groups were obviously higher than control (PBS), and HFNP@GOX@PFC group induced the highest expression among all the treatments. On the contrary, the expression of IL-10 in treatment groups displayed the decrease tendency, in an order of HFNP@GOX@PFC < HFNP@GOX < HFNP, and HFNP@GOX@PFC group caused the lowest expression than other treatment groups, confirming again the great TAMs polarization capability. It was consistent with previous FCM analysis on the re-education of TAMs (Fig. 5b). These results directly confirmed that HFNP@GOX@PFC nanosystem is capable of the re-education of TAMs features, which was helpful for the immune activation and tumor killing.

As mentioned above, we have confirmed that HFNP@GOX@PFC nanosystem can induce the re-education of TAMs, but the molecular mechanism needs further explored in the following study. It is reported that ROS may serve as secondary messenger to promote the regulation of downstream pathways, such as NF-κB and MAPK, which are closely associated with macrophage polarization [25]. Therefore, 4T1 and RAW264.7 cells were co-cultured in the Transwell chamber and treated with HFNP, HFNP@GOX and HFNP@GOX@PFC, respectively. The key proteins of SYK, PLCγ2, ASK1, pp38 and p38 associated with NF-κB and MAPK pathways were measured with Western blot. As shown in Fig. 5e, the expression of SYK, PLCγ2, ASK1 and pp38 positive regulatory proteins associated with TAMs polarization in HFNP group were obviously higher than control (PBS), and HFNP@GOX group displayed higher expression than HFNP group. Importantly, HFNP@GOX@PFC group induced the highest expression among all the treatments (p < 0.01, Fig. 5f). It was attributed to the highest ROS generation of HFNP@GOX@PFC nanosystem. These results proved that the molecular mechanism of TAMs re-education of HFNP@GOX@PFC nanosystem was indeed achieved by ROS-activated NF-κB and MAPK signalling pathways.

Since TAMs could involve directly or indirectly in malignant tumor behaviours of metastasis [26], we additionally investigated the wound healing, migration and invasion behaviour of 4T1 cells in Transwell co-culturation system containing RAW 264.7 cells after above administrations (Fig. 5g), detected by the optical microscope. As for the wound healing assay, compared with the control group (PBS), the wound closure rate of HFNP, HFNP@GOX and HFNP@GOX@PFC groups obviously decreased to 85.9%, 68.9% and 42.6% (Fig. 5h and Additional file 1: Fig. S11), respectively. It was due to that the addition of PFC accelerated the generation of ROS and improved the re-education of macrophages, because the pro-inflammatory M1 macrophages can suppress to tumor cells and anti-inflammatory M2 macrophages can be conducive to tumor metastasis and progress [27]. As for migration and invasion assay, compared with control, HFNP, HFNP@GOX and HFNP@GOX@PFC groups could dramatically down-regulate migration ratio to 73.3%, 63.4% and 55.2% (Fig. 5h and Additional file 1: Fig. S12), and invasion rate to 94.1%, 79.9% and 73.9% (Fig. 5h and Additional file 1: Fig. S13) as well, suggesting again the effective anti-tumor invasion and migration capability of HFNP@GOX@PFC nanosystem. Above results collectively suggested that the HFNP@GOX@PFC uptake by tumor cells with high efficiency can significantly self-supply H₂O₂ and O₂ via delivering GOX and PFC, which could not only accelerate the disintegration of the HFNP@GOX@PFC nanosystem and drug release, but also cascade amplify the GOX-mediated tumor starvation and CDT therapy via Fe²⁺-based Fenton reaction with re-educated TAMs, consequently devoting to the effective antitumor effect in vitro.

In vivo antitumor effect of HFNP@GOX@PFC nanosystem

Inspired by the excellent antitumor effect of the HFNP@GOX@PFC nanosystem in vitro, we further studied its antitumor effect in vivo in 4T1 tumor-bearing mouse models. After various administrations, the tumor volume, body weight and survival rate of the tumor-bearing mouse were significantly changed. In detail, HFNP group induced moderate tumor growth inhibition (Fig. 6a) compared with control (saline), attributing to the insufficient CDT. Furthermore, the inhibition effect of tumor growth in HFNP@GOX group was higher than of HFNP, owing to the additional starvation therapy mediated by GOX. Moreover, the HFNP@GOX@PFC group generated the most serious tumor growth inhibition, resulting from the combined effect between GOX-provided starvation therapy and cascade amplification CDT mediated by Fe²⁺-based Fenton reaction. Additionally, the re-education of TAMs mediated by HFNP@GOX@PFC nanosystem may also play an essential role in the overall antitumor effect, which was investigated in the following section. The tumor volume analysis (Fig. 6b) further confirmed the similar inhibition tendency of the tumor growth. More importantly, compared with other groups, HFNP@GOX@PFC group significantly prolonged the survival time of tumor-bearing mice (Fig. 6c) without weight loss (Additional file 1: Fig. S14), displaying the highest survival rate as 83.3% upon 36 days, suggesting the excellent anti-tumor efficacy and good biosafety in vivo.

Subsequently, H&E, TUNEL and immunofluorescence staining (IFC) assays were performed to further reveal the tumor killing in vivo. Compared with saline, HFNP, HFNP@GOX and HFNP@GOX@PFC groups all led to different degree of tumor tissues damage, in an order of HFNP < HFNP@GOX < HFNP@GOX@PFC, demonstrated by the chromatin condensation and distinctly tissue structure damaged in H&E images, and abundant magenta dots labelled the damaged DNA in TUNEL images (Fig. 6d). Besides, the IFC analysis of Ki67 in the tumor tissues also verified that HFNP@GOX@PFC group could effectively reduce the expression of Ki67 (Fig. 6d), confirming again the excellent antitumor effect in vivo of HFNP@GOX@PFC nanosystem. It may be attributed to the combined therapy advantages between the GOX-mediated starvation therapy and cascade amplification CDT provided by Fe²⁺-based Fenton reaction with potential TAMs re-education in HFNP@GOX@PFC nanosystem. The results collectively suggested again that HFNP@GOX@PFC nanosystem certainly was capable of the excellent antitumor efficiency in vivo.

In order to clarify the antitumor mechanism of HFNP@GOX@PFC nanosystem, we firstly investigated hypoxia suppression in vivo, as the level of endogenous O₂ was closely with GOX-mediated starvation effect, and the H₂O₂ produced by GOX also affected the subsequent CDT and the overall antitumor effect. The IFC analysis of hypoxia marker CD47 in tumor sections after various administrations was investigated for characterizing the hypoxia suppression in vivo. As shown in Fig. 6d, the expression of CD47 in HFNP group exhibited the relatively high dose, as shown by the bright green fluorescence, and it was no different from that of control (saline) group, indicating the inherent tumor hypoxia and the little hypoxia suppression without additional O₂ supply or exhaustion, Furthermore, O₂ consumer and H₂O₂ supplier of GOX loaded HFNP@GOX group displayed the much higher expression of CD47 than HFNP group, and the reason could be attributed the fact that the GOX indeed induces the H₂O₂ generation with O₂ deletion via the catalytic reaction, leading to the increased hypoxia in the tumor, as shown by the higher expression of CD47. Importantly, HFNP@GOX@PFC group with high PFC loading content (32.37%) displayed the lowest expression of CD47 among all groups (p < 0.01, Additional file 1: Fig. S15), implying the effective hypoxia suppression. It was explained that the abundant introduction of O₂ supplier-PFC could significantly relieve the hypoxia of tumor cells. These results were consistent with the western blotting and FCM analysis of CD47 expression in vitro (Fig. 4b-d and Additional file 1: Fig. S8). Therefore, the results collectively suggested that the HFNP@GOX@PFC nanosystem could self-supply O₂ and H₂O₂ with high efficiency, which could not only relieve tumor hypoxia, but also contribute to the realization of the cascade amplification the CDT and GOX-mediated tumor starvation, leading to the most effective antitumor effect in vivo.

We next studied the polarization degree of TAMs in vivo for further revealing the antitumor mechanism of HFNP@GOX@PFC nanosystem, as the self-generation of ROS could actually influence the re-education of TAMs and devote to the overall antitumor performances in vivo. Accordingly, the IFC analysis of the typical marker CD86 labelled M1 macrophages in tumor sections after various administrations was detected by CLSM for evaluating the polarization degree of TAMs in vivo. As shown in Fig. 6d, compared with control (saline), HFNP induced the moderate TAMs re-education, as shown by the suitable up-regulation of the CD86 expression, owing to the limited CDT and insufficient ROS generation. Moreover, the number of M1-macrophages in HFNP@GOX group was obviously higher than HFNP group, attributing to the additional H₂O₂ generation mediated by GOX and its promotion effect on TAMs polarization. More importantly, HFNP@GOX@PFC group induced the highest TAMs polarization, shown by the brightest fluorescence of CD86 among all treatment groups (Additional file 1: Fig. S16). It was explained that the delivery of PFC could significantly promote the GOX-mediated tumor starvation with the abundant H₂O₂ generation via supplying the plenty of O₂, which could not only accelerate MOF disassembly, drug release, and cascade amplify CDT via provided the sufficient fuel of Fe²⁺ and H₂O₂, but also enhanced the polarization of TAMs, consequently contributing to the desired antitumor effect in vivo.

Finally, the blood biosafety of the tumor-bearing mouse was investigated for evaluating the overall biosafety of the HFNP@GOX@PFC nanosystem in vivo by hematology analysis. As shown in Additional file 1: Fig. S17, compared with control (saline), most of the measured parameters for HFNP@GOX@PFC treated displayed no discernible changes. Excitingly, the major tissues (heart, liver, spleen, kidney and lung) also had no obvious damage in HFNP@GOX@PFC group (Additional file 1: Fig. S18), and the body weight steadily increased with the feeding time after HFNP@GOX@PFC treatments as well (Additional file 1: Fig. S14), confirming the good biocompatibility of the HFNP@GOX@PFC nanosystem. Based on the above results, the excellent antitumor effect of the HFNP@GOX@PFC nanosystem with good biosafety could be explained as follows (Fig. 6e): (1) the HA-modified functional nanosystem HFNP@GOX@PFC based on ROS- responsive degradable Fe-MOF could effectively uptake by 4T1 cells via the active targeting endocytosis pathway; (2) the endocytosed nanosystem could rapidly degradable and release cargoes of the O₂ supplier PFC, H₂O₂ supplier plus starvation inducer GOX and CDT trigger agent Fe²⁺ in response to the relatively high concentrations of ROS in the cytoplasm; (3) GOX from HFNP@GOX@PFC nanosystem could effectively consume intracellular glucose and O₂ to starve tumor cells and generate plenty of H₂O₂, and the PFC from nanosystem could self-supply abundant O₂ for further promoting GOX-mediated tumor starvation and H₂O₂ generation. More importantly, the self-generation of H₂O₂ could not only accelerate HFNP@GOX@PFC disassembly and cargoes release, but also cascade amplify the CDT via Fe²⁺-based Fenton reaction; (4) ROS generated by above cycle reactions could excitingly re-educate TAMs, which could further improve tumor killing. The above works could collectively devote to the superior tumor therapy effect. The in vitro and in vivo data suggested that HFNP@GOX@PFC nanosystem could achieve effective antitumor therapy with good biocompatibility by the cascade amplification tumor starvation mediated by GOX and CDT with TAMs re-education.

In summary, a tumor-targeted HFNP@GOX@PFC nanosystem composed of ROS-cleaved Fe-based metal-organic framework, HA, GOX and PFC have been developed for tumor cascade amplified starvation and CDT with TAMs re-education. The nanosystem possesses several advantages: (1) HFNP@GOX@PFC nanosystem could specially accumulate in the tumor site via the HA receptor-ligand mediated targeting endocytosis pathway; (2) HFNP@GOX@PFC nanosystem uptake by tumor cells are then disassembled in response to ROS in the cytoplasm of tumor cells, leading to the release of O₂-supplier PFC, H₂O₂ supplier and starvation inducer GOX, and CDT trigger agent Fe²⁺; (3) PFC can reduce the hypoxia and promote the initiation of catalytic reaction mediated by GOX. GOX can competitively react with intracellular glucose and sufficient O₂ to starve tumor and produce abundant H₂O₂, which could further facilitate nanosystem disassembly, drug release and oxidative tumor killing. Fe²⁺ reacts with H₂O₂ to cascade amplify CDT and induce effective tumor damage; (4) ROS generated by the above cycle could additionally boost tumor immune response via re-educating TAMs by activating NF-κB and MAPK signal pathway, leading to the superior tumor therapy effect. The in vitro and in vivo studies had shown that HFNP@GOX@PFC nanosystem had excellent stability, biocompatibility and antitumor effect by combining starvation and CDT with TAMs re-education. Given the above advantages, this work exhibits that HFNP@GOX@PFC nanosystem is a potent self-motivation vehicle combining the cascade amplification of starvation and CDT with TAMs re-education against malignant tumors.

Materials

Ferrous acetate (FeAc₂·4H₂O) and thioketal (TK) were respectively purchased from Sigma-Aldrich (Beijing, China) and Ruixi Bio-Technology Co., Ltd (Xi’an, China). Hyaluronic acid (HA, 36 kDa) was offered by Shandong Freda Biochem Co., Ltd (Shandong, China). Perfluorohexane (PFC) and Glucose oxidase (GOX) were purchased from Macklin Biochem-Technology (Xi’an, China) and Yuanye Bio-Technology Co., Ltd (Shanghai, China). Interleukin-6 (IL-6), Interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China). Anti-CD11b, anti-F4/80 and anti-CD86 were purchased from BioLegend Co., Ltd (California, America). All the reagents were analytical grade.

Preparation of HFNP@GOX@PFC nanosystem

Firstly, FeAc₂·4H₂O (1 mmol) and TK (1 mmol) were dissolved in 15 mL N, N-Dimethylformamide (DMF) in presence of acetic acid (1 mL), the mixture solution was then placed into a Teflon-liner steel autoclave at 65 ℃ for 2 h. Next, the obtained sediment was collected by centrifugation at 5000 rpm min^− 1 for 10 mins. The sediment was added to 100 mL ultrapure water overnight, and the blank Fe-MOF (named as FNP) was harvested via centrifugation.

Subsequently, FNP (20 mg), GOX (1.5 mg) and PFC (500 µL) were dissolved in PBS (10 mL) and stirred overnight at room temperature. The drug loaded FNP (donated FNP@GOX@PFC) was obtained by centrifugation and lyophilization. Lastly, FNP@GOX@PFC (30 mg) was resuspended in the ultrapure water (4 mL), HA (60 mg) was then added into the above mixture with stirring at 40 ℃ for 4 h. The mixture was centrifuged and freeze-dried to obtain the resultant HFNP@GOX@PFC. Notably, the blank nanosystem without drug loading was donated as HFNP.

Meanwhile, the drug loading content (DLC) and efficiency (DLE) of GOX in HFNP@GOX@PFC nanosystem were monitored by the ultraviolet and visible spectrophotometry (UV-vis) at 276 nm and the corresponding standard curve, according to previous literature [8]. Besides, the DLC and DLE of PFC in HFNP@GOX@PFC were determined by gas chromatography (GC). Briefly, 50 µL of HFNP@GOX@PFC was dissolved in 50 µL of acetonitrile and immersed in ultrasonic for 10 s. Then, 1,1,1,3,3-pentafluorobutane (200 µL) was added to the solution under ultrasonic for 10 s. Afterward, the mixture was centrifuged at 1500 g for 2 mins at 4 ℃ and stored at -20 ℃ overnight. 0.5 µL of sample solution was then detected by the GC and calculated the concentration of PFC based on its standard curve. Notably, pure PFC was treated in the same way to obtain the standard curve. DLC and DLE were calculated as follows:

DLC = (WT – WF) / WNP × 100%

DLE = (WT − WF) / WT × 100%

where WT is the total weight of GOX or PFC fed, WF is the weight of nonencapsulated free GOX or PFC, and WNP is the weight of drug loaded nanosystem.

Characterization of materials

The size and zeta potential of nanoparticles are measured by dynamic light scattering (DLS). The morphology and structure of FNP and HFNP nanosystem were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD), respectively. The conjugation and functionalization processes of HFNP nanosystem were analyzed by Fourier Transform Infrared Spectrometer (FTIR). The drug release behavior of HFNP@GOX@PFC nanosystem was characterized by UV-vis spectrophotometry.

Stability of HFNP@GOX@PFC

Typically, HFNP@GOX@PFC nanosystem (1 mg mL^− 1) was firstly co-cultured with 10% FBS in PBS solution at 37 ℃ for various time intervals (0 h, 2 h, 4 h, 8 h, 10 h, 12 h and 24 h), DLS was used to detect its size and potential change for evaluation the stability of HFNP@GOX@PFC in the short term. As for long-term stability, HFNP@GOX@PFC nanosystem (1 mg mL^− 1) were cultured in PBS solution at 4 ℃ for various time intervals (1 d 3 d, 5 d, 7 d, 9 d, 11 d and 14 d), DLS was used to monitor the stability of nanosystem using above same method.

Drug release behaviour

HFNP@GOX@PFC nanoparticles (1 mg) were dispersed into 5 mL PBS solution containing different concentrations of H₂O₂ (0 mM, 0.1 mM and 1 mM) and transferred to dialysis bag (MWCO = 14 kDa). Then, the dialysis bag was immersed in 29 mL PBS solution under gently stirring, 0.8 mL of PBS was taken out at the certain time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h) and supplemented with the same amount of fresh PBS containing different doses of H₂O₂. The release content of GOX and Fe²⁺ were respectively monitored by the analyzed UV-vis at 276 nm and 506 nm using o-phenanthroline method [16].

Self-supplying H₂O₂ detection and self-amplified generation of ·OH

In order to detect the self-generation of H₂O₂ from HFNP@GOX@PFC nanosystem, PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC were respectively added into 8 mL PBS containing glucose (1 mg mL^− 1). The mixture was then incubated at room temperature under gently stirring, 0.8 mL of PBS was taken out at 2 h and detected by UV-vis spectrophotometry for characterizing the generation of H₂O₂ at 240 nm. Besides, the samples harvested from HFNP@GOX@PFC treatment at the certain time points (0 h, 1 h, 2 h, 4 h and 6 h) were also monitored by the UV-vis spectrophotometry for investigating the time-dependent H₂O₂ generation.

For self-amplification generation of ·OH detection, PBS (1 mL), HFNP (1 mg), HFNP@GOX (1 mg), HFNP@GOX@PFC (1 mg) and HFNP@GOX@PFC (1 mg) plus glucose (1 mg mL^− 1) were respectively mixed with 0.1 mM H₂O₂ solution. The above mixture was then stirred under room temperature for 2 h. Next, the samples were detected by UV-vis spectrophotometry at 550 nm using Hydroxyl Free Radical assay kit (Beorebo), according to the manufacturer's protocols.

Cell culture

4T1 breast cancer cells, HC11 mammary gland cells and RAW264.7 macrophages are supplied by Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (w/v) penicillin (100 U mL^− 1)/streptomycin (100 µg mL^− 1) with 5% CO₂ at 37 ℃.

In vitro cytocompatibility and cytotoxicity

As for the dose-dependent cytotoxicity, 4T1 cells and normal HC11 cells seeded on the 24-well plates (1×10⁴ cells well^− 1) were respectively treated with HFNP@GOX@PFC at different doses (equivalent of 0 µM, 7.5 µM, 15 µM, 30 µM, 60 µM, 120 µM, 240 µM, 480 µM Fe²⁺) for 24 h. The medium was replaced by the mixture of fresh medium (200 µL) and CCK-8 (20 µL) and incubated for another 2 h. Finally, the supernatant was translated to 96-well plates and the absorbance was recorded at 450 nm by spectrophotometric microplate reader (Bio-Rad 680, USA). IC50 was calculated according to the cytotoxicity result.

As for the time-dependent cytotoxicity, 4T1 cells were treated with PBS, GOX (1.5 µM), PFC (4.85 µM), HFNP (3.3 mg mL^− 1), HFNP@GOX (3.4 mg mL^− 1, equivalent of 59.18 µM Fe²⁺) and HFNP@GOX@PFC (5.06 mg mL^− 1, equivalent of 59.18 µM Fe²⁺) for 24 h and 48 h, respectively. CCK-8 kit was used to determine the corresponding cell viability, according to the above method.

Tumor targeting cellular uptake in vitro

In order to visualize the tumor targeting endocytosis of 4T1 cells against nanosystem, 4T1 cells (1 × 10⁵ cells well^− 1) inoculated in 6-well plates were co-cultured with FNP@GOX@PFC and HFNP@GOX@PFC for 12 h, respectively. Notably, GOX loaded into the above nanosystem was pre-labeled with FITC before drug loading, thus exhibiting green fluorescence upon laser irradiation. Then, the cell was washed, fixed and permeabilized, the nuclei were stained with DAPI and the cytoskeletons were stained with Tetramethyl Rhodamine Isothiocyanate (TRITC). Finally, these cells were imaged by confocal laser scanning microscopy (CLSM) to observe the tumor targeting endocytosis behaviour and the cellular distribution of the nanosystem.

Cellular ROS detection

Briefly, 4T1 cells (1 × 10⁵ cells well^− 1) were inoculated in 6-well plates and treated with PBS, HFNP (3.3 mg mL^− 1), HFNP@GOX (3.4 mg mL^− 1) and HFNP@GOX@PFC (5.06 mg mL^− 1) for 24 h. DCFH-DA probes were used to mark the generation of intracellular ROS, which was imaged by CLSM and quantitively analyzed by Image J software. The detailed procedures were referred to a previous literature [28].

The Flow cytometry (FCM) analysis of the hypoxia, TAMs polarization and apoptosis

For the hypoxia suppression study, 4T1 cells (1 × 10⁵ cells well^− 1) inoculated in 6-well plates was treated with PBS, HFNP (3.3 mg mL^− 1), HFNP@GOX (3.4 mg mL^− 1) and HFNP@GOX@PFC (5.06 mg mL^− 1) for 24 h. The cells were then washed with PBS, collected by centrifugation, followed by staining with CD47 (hypoxia mark) antibody labelled with PE. Finally, the cells were monitored by FCM.

For TAMs polarization study, RAW264.7 was pre-treated with IL-4 for 24 h, and then co-cultured with 4T1 cells in the Transwell chamber. Meanwhile, the 4T1 cells seeded in the lower chamber were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. Subsequently, RAW264.7 cells seeded in the upper chamber were collected, co-stained with PerCP-Cy5.5-CD11b, AF488-F4/80 and PE-CD86 antibodies, followed by FCM analysis.

As for the apoptosis assay, 4T1 cells (1 × 10⁵ cells well^− 1) inoculated in 6-well plates were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. The cells were then washed with PBS twice, collected by centrifugation, followed by staining with Annexin V-FITC/PI kit, the detailed procedures could refer to our previous literature [29].

Enzyme linked immunosorbent assay (ELISA)

Briefly, the RAW264.7 cells were seeded in the upper chamber of the Transwell, and the 4T1 cells seeded in the lower chamber were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. The supernatant was collected and measured the level of TNF-α, IL-6 and IL-10 to evaluate the polarization degree of TAMs using ELISA Kit (Elabscience), according to the manufacturer's protocols.

Western blot analysis

4T1 cells (1×10⁵ cells well^− 1) inoculated in 6-well plates were incubated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. Subsequently, cells were lysed, collected and determined the total protein concentration using BCA kit. The expression of p53 protein was measured using Western blot and Image J software. Besides, 4T1 cells treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h were also collected and analyzed the expression of CD47 protein using above method.

For another thing, RAW264.7 and 4T1 cells were co-cultured in the Transwell chamber, and the 4T1 cells seeded in the lower chamber were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. Then RAW264.7 cells were collected to analyze the expression levels of SYK, PLCγ2, ASK1, pp38 and p38 proteins. The detailed experiment procedures were according to previous literature [30].

Quantitative real-time polymerase chain reaction (qPCR) analysis

4T1 cells (1×10⁵ cells well^− 1) inoculated in 6-well plates were incubated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC, respectively. After 24 h of incubation, the total RNA was extracted with Trizol reagent, and then reverse transcription using the reverse transcription Kit (TransGen Biotech). The qPCR was carried out for analysis of the mRNA level of BAX, BCL-2, Caspase-3 (Casp3) and Cytochrome C (CytC) according to previous literature [31]. The sequences of primers were listed in Additional file 1: Table S1.

Wound healing, migration and invasion assays

RAW264.7 and 4T1 cells were first co-cultured in the Transwell chamber, and the 4T1 cells seeded in the lower chamber were treated with PBS, HFNP, HFNP@GOX and HFNP@GOX@PFC for 24 h, respectively. The supernatant of the co-culture system containing various cytokines secreted by TAMs was collected and donated as Solution 1 for further use.

As for the wound healing assay, 4T1 cells (1×10⁵ cells well^− 1) were inoculated in 6-well plates. After the cell density reached 70–80%, a p200 pipette tip was used to produce a scratching wound. Next, the cell medium was removed and added above collected Solution 1 for 24 h co-incubation. The wound healing levels were finally imaged by a microscope (Olympus, Japan).

As for migration and invasion assays, 4T1 cells (1×10⁵ cells well^− 1) inoculated in 6-well plates were firstly treated with Solution 1 for 24 h. On the one hand, 4T1 cells (5×10⁴ cells) harvested from above system were then seeded in the upper chamber of the Transwell and cultured with serum-free medium, 500 µL of the complete medium was added into the lower chambers. On the other hand, 4T1 cells harvested from above system were seeded in the upper chamber of Transwell pre-coated with Matrigel (BD Biosciences), the medium was chosen as the serum-free medium, and 500 µL of the complete medium was added into the lower chambers. After treatment for 48 h, the cells on the upper station of the chamber were wiped, while cells on the lower surface came from above two systems were stained with crystal violet and observed by microscope to evaluate the migration and invasion level of tumor cells. The detailed experiment procedures were referred to a previous study [32].

Construction of the 4T1-bearing mice and in vivo antitumor efficacy

All animal experiments were strictly conducted in accordance with the Institutional guidelines for animal experimentation and were approved by the Animal Ethics Committee of the Northwestern Polytechnical University. BALB/c mice (5–6 weeks old) were bought from Beijing Institution for Drug Control, China. The model of tumor-bearing mice was constructed by subcutaneous injection of 4T1 cells (1×10⁶) into the right flank groin of the Balb/c mice. After the tumor size reached about 50 mm³, tumor-bearing mice were randomly divided into 4 groups (n = 6) and intravenously injected with Saline, HFNP, HFNP@GOX and HFNP@GOX@PFC with an equal amount of Fe²⁺ (60 µmol kg^− 1), respectively. The above treatments were repeated 3 times per week. The body weight and tumor volume were recorded every 2 days. The calculation formula of tumor volume was as follows: tumor volume = ab²/2 (a: the maximum dimension of the tumor, b: the minimum dimension of the tumor). The mice were euthanized after 18 days, blood was collected for analysing the biochemical blood index. The survival rate of mice was monitored for another 18 days after the last administration.

In vivo histological examination and immunofluorescence assay

After 18-days treatment, the tumor and major tissue including heart, liver, spleen, kidney and lung were collected and sliced for H&E examinations. The morphological changes of these organs after administration were observed by microscope. Besides, the tumor sections were also stained with TUNEL apoptosis detection kit (Beyotime) and imaged by CLSM. As for the immunofluorescence study, tumor sections were blocked with 5% bovine serum albumin, incubated with CD47, CD86 or Ki67 antibodies, and then stained with Alexa Fluor 488-labeled IgG according to the manufacturer's instructions (Immunol Fluorescence Staining Kit, Beyotime, China). Sections were then stained with DAPI and imaged by CLSM.

Statistics analysis

We performed the statistical analysis with the software of Origin (version 9.0) through Student’s t-test and one-way analysis of variance (ANOVA). All data were expressed as means ± standard deviation (SD). P values of * P < 0.05, ** P < 0.01 and *** P < 0.001, respectively. The confidence levels of 95% and 99% were regarded as a significant difference.

Author Contributions

Xinmin Zheng: Investigation, Data curation, Writing-original draft. Xiang Li: Investigation, writing-original draft. Siyu Meng: investigation, writing-original draft. Guolin Shi: Investigation. Hui Li: Investigation. Huiping Du: Investigation. Liangliang Dai: Conceptualization, Supervision, Writing-original draft. Hui Yang: Validation, Supervision.

Funding

This work was financially supported by the National Natural Science Foundation of China (52003223), the Key R & D Program of Shaanxi Province (2022SF-012), the Fundamental Research Funds for the Central Universities (D5000220120) and the Young Talent fund of University Association for Science and Technology in Shaanxi (20200302), China.

Availability of data and materials

All data generated and analyzed during this research are included in this published article and additional file.

Ethics approval and consent to participate

Consent for publication

All authors agreed to publish this manuscript.

Conflicts of interest

There are no conflicts to declare.

Author details

¹ School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China. ² Institute of Medical Research, Northwestern Polytechnical University, Xi’an 710072, China.

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Cascade amplification of tumor chemodynamic therapy and starvation with re- educated TAMs via Fe-MOF based functional nanosystem

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results And Discussion

Preparation and characterization of HFNP@GOX@PFC nanosystem

Cytotoxicity of HFNP@GOX@PFC nanosystem

Tumor-targeted cell uptake of HFNP@GOX@PFC nanosystem

The ROS self-generation and hypoxia suppression of HFNP@GOX@PFC nanosystem

Conclusions

Materials And Methods

Materials

Preparation of HFNP@GOX@PFC nanosystem

Characterization of materials

Stability of HFNP@GOX@PFC

Drug release behaviour

Self-supplying H₂O₂ detection and self-amplified generation of ·OH

Cell culture

Cellular ROS detection

The Flow cytometry (FCM) analysis of the hypoxia, TAMs polarization and apoptosis

Enzyme linked immunosorbent assay (ELISA)

Western blot analysis

Quantitative real-time polymerase chain reaction (qPCR) analysis

Wound healing, migration and invasion assays

Statistics analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Cascade amplification of tumor chemodynamic therapy and starvation with re- educated TAMs via Fe-MOF based functional nanosystem

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results And Discussion

Preparation and characterization of HFNP@GOX@PFC nanosystem

Cytotoxicity of HFNP@GOX@PFC nanosystem

Tumor-targeted cell uptake of HFNP@GOX@PFC nanosystem

The ROS self-generation and hypoxia suppression of HFNP@GOX@PFC nanosystem

Conclusions

Materials And Methods

Materials

Preparation of HFNP@GOX@PFC nanosystem

Characterization of materials

Stability of HFNP@GOX@PFC

Drug release behaviour

Self-supplying H2O2 detection and self-amplified generation of ·OH

Cell culture

Cellular ROS detection

The Flow cytometry (FCM) analysis of the hypoxia, TAMs polarization and apoptosis

Enzyme linked immunosorbent assay (ELISA)

Western blot analysis

Quantitative real-time polymerase chain reaction (qPCR) analysis

Wound healing, migration and invasion assays

Statistics analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Self-supplying H₂O₂ detection and self-amplified generation of ·OH