3.1 Preparation and characterization of M-FNM.
Figure 1A illustrated the schematic representation of the creation of the biocompatible and targeted iron delivery system, M-FNM. To begin, solvothermal reduction was utilized to create the Fe3O4 cores, followed by repeated coating procedures to produce FNM nanoparticles[16]. Mannose was then modified and applied to the surface of FNM to improve tumor targeting and transmembrane administration effectiveness. To clarify their morphology, TEM results showed the structure of Fe3O4, FNM, and M-FNM were spheric structure (Fig. 1B, Figure S1), with average diameters of approximately 85, 95, and 110 nm, respectively. The thickness of the NH2-MIL-100 layer was roughly 10 nm (Fig. 1C, Figure S2). Additionally, mannose modification resulted in a slightly larger size of M-FNM. High-resolution TEM (HRTEM) imaging confirmed the crystalline structure of M-FNM, displaying a lattice fringe of 0.18 nm (Fig. 1B). The BET surface area measurement indicated that M-FNM had a porosity of 174.6 m2/g, favorable for the exchange and diffusion of ROS (Fig. 1D). Further investigation revealed that the hole size was approximately 9.3 nm, demonstrating potential for facilitating the transport of anticancer medications (Figure S3). In aqueous solution, M-FNM's zeta potential was found to be -23.3 mV (Figure S4). Moreover, XRD analysis confirmed the crystal structure of M-FNM, with all diffraction peaks of M-FNM nanoparticles corresponding to the standard data of mannose, Fe3O4 (JCPDS file 19–0629, magnetite), and NH2-MIL100 (Fig. 1E).
To gain further insight into the structural information on M-FNM, Fourier transform infrared (FT-IR) analysis was conducted, which revealed that a distinct peak at 1000 cm− 1, corresponding to mannose, in comparison to the spectrum of FNM. This indicated the the mannose modification on FNM was successful (Fig. 1F). The successful decoration of mannose in the M-FNM backbone was further validated by the clear shift observed in the X-ray photoelectron spectroscopy (XPS) spectrum, which showed the presence of N, Fe, O, and C (Fig. 1G). The N 1s spectrum exhibited three peaks, with the dominant peak being the C-N bond at 399.87 eV, and the other two peaks representing the C = N bond at 401.4 eV and the NH- bond at 402.1 eV (Fig. 1H). The high-resolution spectra of Fe 2p displayed two sub-peaks at 724.48 and 710.4 eV, which were assigned to Fe (III) 2p1/2 and Fe (II) 2p3/2, respectively (Fig. 1I). The asymmetric O 1s spectrum displayed three peaks at 532.4, 531.4, and 530.6 eV, indicating the presence of -NO, O = C-C, and -OH, respectively (Fig. 1J). Furthermore, the C 1s spectrum was deconvoluted into three peaks at 284.8 eV, 286.5 eV, and 288.8 eV, corresponding to C-C, C = C, and O-C = O, respectively (Fig. 1K). The X-ray energy spectrum mapping profile also revealed a homogeneous distribution of N, Fe, O, and C, which further supported the synthesis of M-FNM (Fig. 1M).
3.2 Cellular uptake and cytotoxicity effects of M-FNM
The cellular uptake of M-FNM is the foremost process in its anti-cancer effect. Therefore, in this study, we investigated the uptake behavior and pathway of CAL27 cells by monitoring the fluorescence intensity of FITC labeled on nanoparticles (Fig. 2A). After incubating CAL27 cells with FITC-labeled M-FNM for 1 h, a we observed bright green fluorescence in the cytoplasm, indicating that CAL27 cells internalize M-FNM easily (Fig. 2B). We compared the cellular uptake efficiency of FNM and M-FNM and found that M-FNM exhibited a stronger fluorescence intensity, indicating that modification of mannose could increase the transmembrane efficienc (Fig. 2B, C). To clarify the role of MR in increasing transmembrane efficiency, we used mannan, an inhibitor of MR, to pretreat cells and inhibit binding of mannose to MR[17, 18]. Our results showed that pretreatment of mannan significantly decreased the cellular uptake efficiency of M-FNM (Fig. 2B, C).
The inhibitory effect on cell proliferation activity is a key basis for evaluating anti-tumor effects in vitro. Following treatment with 300 µg/mL Mannose, Fe3O4, FNM, and M-FNM, the viability of CAL27 cells was reduced to 67.89%, 59.34%, 57.08%, and 32.84%, respectively (Fig. 2D). M-FNM demonstrated an extraordinary killing power (IC50 ≈ 80 µg/mL), and the killing effect was concentration-dependent, according to the results of the CCK-8 assays (Figure S5). Given the positive anti-proliferative impact, Annexin V/PI was initially used to verify the antineoplastic effect. Compared to other groups, the number of fluorescent cells staining by Annexin V/PI in M-FNM group increased more significantly (Fig. 2E). Conditional responsive degradation of MOF is also a vital feature in achieving targeted ions delivery. MIL-100 is broken down by the reductive action of lysosomes' abundant cysteine[19] Therefore, we experimentally added cysteine to clarify the impact of MIL-100 degradation on the inhibitory effect of M-FNM. In further Annexin V/PI staining, M-FNM caused cells to exhibit a vacuolar death pattern, which is not a characteristic of apoptotic cells. Subsequently, with the treatment of cysteine, there was a clear promotion of the formation of more bubble-like cells in M-FNM + Cysieine group (Figure F). Moreover, CCK-8 results showed that the addition of cysteine further inhibited cell proliferation, a similar trend was observed in Annexin V/PI staining (Fig. 2G). These results indicated that cysteine involved in the degradation of M-FNM and contributed to its ability to inhibit cell proliferation. Together, M-FNM relied on MR-mediated endocytosis to enter cells, further was degraded and mediated cell death under the action of cysteine in lysosomes.
3.3 M-FNM induced GSDMD-mediated pyroptosis in CAL27 cells
Upon investigating the mode of CAL27 cell death mediated by M-FNM, pyroptosis was identified as the key characteristic through the formation of transmembrane pores, leading to cellular swelling and large bubbles as evident (Fig. 3A). Hence, cell morphology observation was initially introduced as the most intuitive method. The cells treated by M-FNM showed swelling characteristics and large bubbles, in sharp contrast to the control group (Fig. 3B). CDT can convert endogenous H2O2 into OH, which is the most harmful ROS due to the triggering of pyroptosis[20]. As a result, we investigated and discovered that M-FNM considerably raised the level of intracellular ROS (Fig. 3C, D) Lactate dehydrogenase (LDH) an IL-1β inflammatory cytokines are released from the cells when pyroptosis occurs[21–23]. Similarly, under the treatment of M-FNM, the concentration of LDH and IL-1β in the supernatant of CAL27 cells dramatic increased, respectively (Fig. 3F, S6). Furthermore, apoptosis-associated speck-like protein (ASC) forms macromolecular dimers in the cytoplasm during inflammasome activation when Caspase-1 separating from inflammasomes[24]. Immunofluorescence research revealed that M-FNM also promoted the formation of ASC specks (Fig. 3G). To investigated Caspase-1 has been activated and subsequently cleave GSDMD into N-GSDMD, western blot analysis as authoritative method was initiated to assess protein expression. As western blot findings showed, elevated expression of cleaved Caspase-1 and N-GSDMD proteins was observed in the M-FNM group, underpinning the permeabilization of the plasma membrane and the formation of membrane pores. The high expression of ASC and cleaved Caspase-1 in the M-FNM group is basis for Caspase-1 activation. IL-1β is one of the substrates of Caspase-1, and the increased expression of mature form of IL-1β (p17) also proved Caspase-1 activation (Fig. 3E). Meanwhile, for the phenotypic analysis of the antitumor activity of M-FNM, a multicellular spheroids (MSCs) tumor model was established using CAL27 cells. MSCs have more pronounced solid tumor associations[25]. When compared to the control group, CLSM images showed that M-FNM increased the expression of N-GSDMD and cleaved Caspase-1 in MSCs (Fig. 3I). However, the CCK-8 result demonstrated that the cell viability was preserved when Z-YVAD-FMK (a caspases inhibitor) was used, further demonstrating that M-FNM induced pyroptosis (Fig. S7). Due to the admission of iron ions into cells by M-FNM, the cell viability was not restored under the influence of liproxstatin-1 (a ferroptosis inhibitor), demonstrating that ferroptosis is not the form of cell death induced by M-FNM (Figure S8). Moreover, following M-FNM treatment, the expression level of N-GSDMD in CLSM images of tumor tissue was highly elevated (Fig. 3H). These findings also demonstrated the role of M-FNM in mediating pyroptosis and the Caspases family's predominance in the pyroptosis process. Overall, we concluded that M-FNM kills tumor cells by triggering pyroptosis.
3.4 M-FNM activated the PERK pathway to induce pyroptosis
Considering the results of M-FNM triggering pyroptosis, the upstream regulatory process mediating pyroptosis was still unknown. Therefore, the mechanism by which M-FNM causes pyroptosis was investigated (Fig. 4A). ROS is one of the stimulators of ER stress, a cellular defence mechanism for maintaining intracellular homeostasis[26]. In details, PERK is a key sensor of ER stress, which can sense intracellular ROS levels and mediate ER stress[27]. Among them, the leakage of calcium ions into the cytoplasm is a sign of ER stress occurrence[28, 29]. As expected, calcium ions in the cytoplasm increased in response to M-FNM dramatically (Fig. 4B, C). These results suggest the cascade reaction of Caspase-1 and the incidence of pyroptosis are mediated by ER stress[29–31].
Evaluating the expression levels of p-eIF2α and other pathway proteins is the gold standard for determining whether the PERK signaling pathway is activated[32]. Elevated expression of p-eIF2α, PERK, ATF4, and CHOP was observed in M-FNM group, but total-eIF2α did not alter (Fig. 4F). Thus, western blot results showed that PERK signaling pathway was activated by M-FNM. Meanwhile, increasing the expression of CHOP will help to activate Caspase-1[33]. According to CLSM images, the green fluorescence signal in the M-FNM group confirmed the upregulation of cleaved Caspase-1 expression. (Fig. 4D). To investigated the role of the PERK pathway in Caspase-1 activation, 4-PBA, a small molecule inhibitor for the PERK pathway was chosen[34, 35]. The expression of N-GSDMD, cleaved Caspase-1, and L-1β was verified through western blot and L-1β ELISA assay, respectively. The 4-PBA-related group showed inhibited the expression of above proteins (Fig. 4H), and there was also a substantial decrease in IL-1β secretion in the cell supernatant (Fig. 4E). Additionally, it was discovered that the 4-PBA interfered with the pyroptosis caused by M-FNM from the results of cell morphology and Annexin V/PI staining (Fig. 4G). To summarize, M-FNM induced the activation of the PERK-eIF2α-ATF4-CHOP pathway in CAL27 cells. Cleaved Caspase-1 cleaved the full length GSDMD into N-GSDMD with membrane breaking activity and immature IL-1β into mature IL-1β, Therefore, ER stress and PERK-eIF2α-ATF4-CHOP pathway played a key role in the the M-FNM-induced pyroptosis.
3.5 Tumor targeting and inhibitory effect of M-FNM
The effective accumulation of drugs in the local tumor is the basic for anti-tumor effect, and it also avoids damage to normal tissue[36, 37]. To verify the targeting effect in vivo, FNM and M-FNM nanoparticles were labeled with Cy5.5 and injected into CAL27 tumor-bearing mice (BALB/c-nu) through tail vein, respectively. At 6 h after injection, ISVS analysis revealed that the M-FNM group was more enriched in the tumor than FNM group. At 8 h after injection, the M-FNM group could still detect a significant fluorescence intensity in tumor (Figure S9A). The above results indicated that M-FNM possessed excellent targeting effect on CAL27 tumors. Additionally, the tumor and vital organs of the mice after 8 h were collected, and the fluorescence results showed that consistent with the in vivo imaging results, the M-FNM group had more aggregation in the tumor. In contrast to other organs, the accumulation of M-FNM in the kidney suggesting that the metabolism of M-FNM may be completed in kidney (Figure S9B).
To evaluate the anti-tumor effect of M-FNM in vivo, we created a 5-week-old male BALB/c-nu mouse model of the CAL27 subcutaneous tumor. In Figure S10, the treatment strategy is displayed. Subsequently, after nanoparticles administration, tumor volume was monitored and validated, which is an important indicator for evaluating treatment ability. After 12 days of treatment, the average tumor volume of the mice was 355 mm3, which is much smaller than other groups (Figure S11). Digital photos of free tumors for each growth data were presented in Figure S12. Due to the lack of involvement of a sound immune system in nude mice, the above experiments were unable to fully evaluate the anti-tumor effect of M-FNM. Therefore, to investigated the effectiveness of immunotherapy, a 5-week-old male C57BL/6J mouse model of SCC-7 subcutaneous tumor was developed (Fig. 5A). Following M-FNM therapy, SCC-7 cells also undergone pyroptosis (Figure S13). Tumor growth was significant inhibition in the M-FNM therapy group. After 12 days of treatment, the mice's typical tumor volume was 242 mm3. Compared with the Fe3O4 group with mild tumor inhibition, the average volume of mice treated with M-FNM decreased by 441 mm3 (Fig. 5B, Figure S14), suggesting the M-FNM group produced a notable inhibitory impact on SCC-7 tumors. For histopathological evaluation, Hematoxylin and Eosin (H&E) staining examination was performed to determine the degree of cell damage and necrosis. Only the SCC-7 cells in the control group and the Mannose group retained their normal morphology, whereas the M-FNM group showed the most histological damage (Fig. 5D). Immunofluorescence analysis was also used to detect the expression of pyroptosis related proteins to gain a deeper understanding of the underlying mechanism of cell death. After M-FNM treatment, the expression of cleaved Caspase-1 was detected to increase, indicating pyroptotic cell death (Fig. 5E). However, neither of the two in vivo models that M-FNM treated resulted in a substantial change in mouse body weight (Fig. 5C, Figure S15).
3.6 M-FNM boosted anti-tumor immunity in vivo
According to previous studies, pyroptosis is advantageous for triggering an anticancer response by recruiting and activating immune cells[38] (Fig. 6A). DAMPs are released by tumor cells during pyroptosis, which will cause a rise in the infiltration of mature dendritic cells (DC) and further T cell activation[38]. To examined the potential role of M-FNM-induced pyroptosis in activation of anti-tumor immunity, DC infiltration was investigated by immunofluorescence staining. The findings showed that M-FNM could efficiently promote the infiltration of CD11c+ DC (Fig. 6D, E). Mice treated with FNM displayed less effective tumor inhibition and pyroptosis induction, which resulted in less effective CD8+/CD4+ T cells infiltration. Much greater percentages of CD8+/CD4+ T cells were dramatically increased in the tumor after M-FNM therapy (Fig. 6D, F, G). The proportion of Treg cells in the M-FNM group was significantly lower than that of the other groups (Fig. 6D, H).
Furthermore, the ELISA method was utilized to quantitatively assess the concentration of cytokines in mouse serum. The serum of mice treated with FNM and M-FNM groups displayed significantly higher levels of IL-1β and TNF-α than those of the PBS, Mannose, and Fe3O4 groups. Notably, M-FNM proved to be more effective in eliciting an anti-tumor immune response in mice compared to other therapies, as evidenced by the higher levels of cytokine production observed (Fig. 6B, C). Given that the biological toxicity of nanomaterials is crucial in translating from experimental settings to practical applications, we conducted biocompatibility analysis in vivo using H&E staining. The main organs (including the heart, liver, spleen, lung, and kidney) were stained with H&E, and no substantial inflammatory lesions or pathological alterations were observed in any group, which indicates the good biocompatibility of M-FNM (Figure S16).
Transition metals, such as Fe, are the most common constituent elements of nanozymes, and are reliable candidates for CDT of malignant tumors.[39]. During this process, endogenous H2O2 is transformed into hydroxyl radicals (• OH), and there is a change in the valence state from ferrous to trivalent iron[40]. Despite iron being a key candidate for CDT of tumors, the targeting effect on occult malignant tumors, such as Oral Squamous Cell Carcinoma, is still insufficient and the impact is limited. Additionally, delivery efficiency is a scientific issue that requires special attention[41]. In this study, we successfully prepared a Fe3O4-containing MOF that is surface-modified with mannose. The mannose modification has significantly improved targeting and relied on MR-mediated endocytosis to improve transmembrane transport efficiency and enhance therapeutic efficacy. This modification is based on the high local expression of MR in malignancies,[13] which was highly expressed in the CAL27/SCC-7 cell models used in this study. More abundant iron ions guarantee sustained and efficient catalysis of H2O2, thereby maintaining the continuous production of hydroxyl radicals and mediating tumor regression.
Lysosomes are an inevitable process after internalization of nanomaterials by cells, and the unique lysosomal environment has both positive and negative effects on the functional performance of nanomaterials[42, 43]. Therefore, the M-FNM prepared in this study can cleverly respond to cysteine in lysosomes to achieve its own anti-tumor effect. Subsequently, a pharmacological mechanism of M-FNM’s anticancer is pyroptosis. Pyroptosis is distinguished by the activation of Caspase-1 and the increase of N-GSDMD expression[44]. However, the development of anticancer medications that target pyroptosis is severely constrained due to the incomplete elucidation of the upstream mechanism of pyroptosis. Our findings showed ER stress and the PERK pathway are vital for M-FNM-mediated pyroptosis. Furthermore, other anticancer nanomaterials may be able to regulate pyroptosis by targeting the PERK pathway.
According to in vivo studies, the anti-tumor effect of M-FNM in the BALB/c tumor bearing mouse model has not been fully demonstrated, due to the lack of immune cells. M-FNM converts "cold" tumors into "hot" tumors by recruiting immune cells, prohibiting the growth of tumors in C57BL/6J tumor bearing mice. Therefore, in the context of future therapeutic transformation therapies, M-FNM-induced pyroptosis may make tumors more susceptible to immune checkpoint inhibitors. However, in the following studies, we will further consider forming new combinations with immune checkpoint inhibitors and investigate the synergistic effect of magnetism and mannose targeting to acquire more accurate tumor targeting effects.