Persistent glutathione-depleting MFO@MIL nanoreactors enhance the antitumor efficiency of a skin scaffold

Abstract

The efficacy of reactive oxygen species (ROS)-related skin tumor therapies is significantly restricted by intracellular overexpressed glutathione (GSH) which is a free radical scavenger. Herein, a GSH-depleting and high ROS production nanoreactor (MFO@MIL) is constructed by in situ loading manganese ferrite (MnFe₂O₄) onto an iron-based metal organic framework (MIL-101). The MFO@MIL is then incorporated into polycaprolactone (PCL) to prepare a porous skin scaffold, aiming to continuously release MFO@MIL and simultaneously regulate intracellular reducibility and ROS yield to enhance anti-tumor efficacy. Particularly, MnFe₂O₄ with GSH peroxidase-like activity can persistently deplete GSH to reduce its consumption of hydroxyl radicals (˙OH), which are produced by the Fenton reaction between MIL-101 and hydrogen peroxide (H₂O₂). Meanwhile, the depletion process of MnFe₂O₄ to GSH will produce Mn²⁺, which collaborates with MIL-101 to catalyze H₂O₂ to produce ˙OH, remarkably increasing ˙OH yield and enhancing anti-tumor efficacy. The results showed that the depletion rate of GSH using the scaffold reached 84.4% within 24 hours. The ˙OH yield of the scaffold was significantly higher than that of the scaffold loaded with MIL-101 alone. Systematic cell experiments demonstrated the powerful anti-tumor efficacy of the scaffold. This study proposes a feasible strategy to enhance ROS-based anti-tumor efficacy.

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1. Introduction

Skin tumors are some of the most common human malignant tumors, and the number of patients has increased rapidly in recent years.^1–3 Surgical excision is the main treatment modality for skin tumors, but the circulating tumor cells continue to induce tumor recurrence and increase the risk of remote metastasis after surgery.^4,5 Fortunately, there is a high level of hydrogen peroxide (H₂O₂) in the acidic tumor microenvironment, which can be catalyzed by peroxidase-like (POD-like) nanozymes to produce toxic hydroxyl radicals (˙OH) to induce apoptosis of tumor cells.^6–8 However, nearly all POD-like nanozymes present merely single catalytic activities. Under the complex and dynamic tumor microenvironment characterized by hypoxia and overexpressed glutathione (GSH), this leads to a relatively inadequate anti-tumor capacity.

Iron-based metal–organic framework MIL-101 (MIL) possesses peroxidase enzyme activity, degradability, high specific surface area and adjustable pore size, so it has been frequently utilized as a nanozyme and drug carrier in various anti-tumor therapies in recent years.^9–11 Yang et al. demonstrated that MIL as a nanozyme catalyzed endogenous substances to generate ˙OH and induced tumor cell apoptosis.¹² Li et al. synthesized MIL as a carrier to deliver Juglone, and the results showed that MIL improved anti-tumor performance through pH-responsive degradation, selective release of Juglone and catalytic ˙OH generation.¹³ Notably, overexpressed GSH in the tumor microenvironment is a free radical scavenger that can directly neutralize the as-produced reactive oxygen species (ROS, such as ˙OH, singlet oxygen and superoxide radicals), significantly reducing the anti-tumor effects.^14,15 Therefore, it is urgently necessary to develop a nanoreactor capable of consuming GSH to avoid its scavenging of ROS.

Recently, MnFe₂O₄ nanoparticles with GSH peroxidase-like activities have been used to consume GSH in the tumor microenvironment.^16–18 However, these attempts were subject to several limitations, including the consumption of MnFe₂O₄, instantaneous GSH depletion, or necessary external near-infrared light activation. Since MnFe₂O₄ will gradually consume itself while depleting GSH, and moreover, the regeneration mechanism within tumor cells will replenish the lost GSH to maintain the redox balance, it is impossible to obtain a long-term GSH-depleting capacity. Therefore, it is crucial for anti-tumor therapy to explore practical strategies that can continuously deplete GSH and disrupt the intracellular redox balance.

In this study, an MFO@MIL nanoreactor was synthesized by loading MnFe₂O₄ onto the MIL, and then compounded with PCL to prepare a porous skin scaffold using selective laser sintering (SLS) technology. Especially, the gradual degradation of the PCL/MFO@MIL scaffold will continuously supply the MFO@MIL nanoreactor, which is capable of consuming GSH via MnFe₂O₄ to reduce its consumption of pre-generated ˙OH. Meanwhile, the released Mn²⁺ ions will collaborate with MIL to catalyze H₂O₂ to produce more ˙OH, disrupting the intracellular redox balance and potentially realizing long-term anti-tumor treatment (Scheme 1). The morphology, GSH consumption capacity, ˙OH generation ability and anti-tumor efficiency of the scaffolds have been thoroughly studied.

Scheme 1 MFO@MIL synthesis process and scaffold anti-tumor mechanism.

2. Materials and methods

2.1. Materials

Ferric chloride hexahydrate (FeCl₃·6H₂O, 98%), manganese chloride tetrahydrate (MnCl₂·4H₂O, 99%), terephthalic acid (H₂BDC, 99%), N,N-dimethylformamide (DMF, AR, 99.5%), hydrogen peroxide aqueous solution (H₂O₂, 30%), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and 3,3′,5,5′-tetramethylbenzidine (TMB) were bought from Aladdin Chemistry Co. Ltd. PCL powder was obtained from Shenzhen Polymtek Biomaterial Co., Ltd. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS, pH = 7.4 and pH = 5) were purchased from Gibco (Invitrogen, USA). The human osteosarcoma cell line MG-63 was provided by the Testing Center of Center South University. The above chemicals were applied as received without further purification.

2.2. Synthesis of the MIL and MFO@MIL

MIL was synthesized based on previous studies with some modifications.¹⁹ The method was as follows: 1.76 g FeCl₃·6H₂O and 0.54 g terephthalic acid (H₂BDC) were added to 80 mL DMF and sonicated until a homogeneous suspension was formed, and then the suspension was transferred to an autoclave to heat for 15 h at 120 °C. After natural cooling and centrifugation, the product was transferred to methanol solution and stirred for three days, and the methanol was replaced daily to remove unformed material. Thereafter, the MIL was obtained after centrifugation and drying.

The synthesis procedure of MFO@MIL was as follows: 0.7 g MIL was added to 45 mL deionized water, then added 0.273 g MnCl₂·4H₂O and 1.082 g FeCl₃·6H₂O and stirred for 1 h. Subsequently, the prepared NaOH solution (6 M) was dropped into the suspension to enable pH = 11 and continuously stirred for 1 h. Then placed in an autoclave and heated to 200 °C for 12 h. After centrifugation, cleaning and drying, MFO@MIL composite powder was obtained.

2.3. Characterization

The morphologies of the MIL and MFO@MIL powders were characterized via scanning electron microscopy (SEM, EVO 18, Zeiss, Germany). The internal conditions and elemental distribution of MFO@MIL were characterized via transmission electron microscopy (TEM) and selected area electron diffractometer (SAED). The crystal structures of MIL and MFO@MIL nanoparticles were analyzed via X-ray diffraction (XRD, D/MAX-RA, Japan). The elemental composition of MFO@MIL was determined by X-ray photoelectron spectroscopy (XPS, Escalab 250Xi, Thermo Fisher Scientific, USA).

2.4. Fabrication of skin scaffolds

Briefly, 0.25 g of MFO@MIL powders and 4.75 g of PCL powders were dispersed in 30 mL of ethanol, followed by stirring and sonication for 1 h. Then, the PCL/MFO@MIL powders were obtained after centrifugation, drying, and grinding. Finally, the PCL/MFO@MIL porous scaffold was prepared by using the SLS technique. The processing parameters were maintained as follows: laser power of 6 W, scanning speed of 400 mm s⁻¹, monolayer thickness of 0.15 mm, and line spacing of 0.24 mm. In addition, PCL and PCL/MIL scaffolds as control groups were also prepared using the same process.

2.5. Degradation and ion release of scaffolds

The biodegradation properties of the scaffolds were investigated by immersion experiments in PBS (pH = 7.4). Before immersion, the scaffolds were weighed and recorded as W_y. Then, the scaffolds were immersed in PBS solution at 37 °C and incubated for 7, 14, 21, and 28 days in a shaker. Thereafter, the scaffolds were washed, dried, weighed and recorded as W_t. The mass loss of the scaffolds was calculated according to the following equation:

The released concentrations of Mn²⁺ and Fe³⁺ from the PCL/MFO@MIL scaffold were quantitatively detected by inductively coupled plasma emission spectrometry (ICP-OES, Spectro Blue Sop, Germany). Briefly, 300 mg of the scaffolds were immersed in centrifuge tubes containing 8 mL PBS buffer, and the solutions were replaced with fresh PBS solution at each specified time (1, 3, 5, 7, 14, 21, and 28 days). Then, the concentrations of Mn²⁺ and Fe³⁺ in the solution at each time point were determined by ICP-OES. The released amounts in each time interval were summed to calculate the cumulative concentrations of Mn²⁺ and Fe³⁺ released over 4 weeks.

2.6. Catalytic activity of scaffolds

The catalytic activity of PCL/MFO@MIL scaffolds was investigated using TMB as a substrate. The scaffolds were first soaked in PBS buffer for 3 days, followed by the addition of prepared TMB (6.4 mM) and H₂O₂ (200 μM) solutions. After a certain reaction time, the absorption spectra in the range of 350–750 nm were recorded using a microplate reader. The ˙OH generation induced by PCL/MFO@MIL scaffold was carried out in PBS solution in the presence of H₂O₂ and using OPD as the substrate. After different reaction times, the absorbance of the color reactions was detected using a microplate reader.

The generation of ˙OH was evaluated by electron paramagnetic resonance (EPR) using 5,5-dimethyl-1-oxypyrroline (DMPO) as a trapping agent using an EPR spectrometer (EMXplus-6/1, Bruker, Germany). The PCL/MIL and PCL/MFO@MIL scaffolds were added into aqueous solution (pH = 6.5) containing 5 mL of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO, 0.02 mM) and H₂O₂ (200 μM), respectively. The solution was then analyzed to obtain ESR spectra.

In general, benzoic acid is a very weak fluorescent substance, which can react with ˙OH to produce hydroxybenzoic acid with strong fluorescence.^20,21 The fluorescence intensity of hydroxybenzoic acid in different scaffold groups was measured using ultraviolet-visible spectroscopy at 410 nm. The difference ΔF between fluorescence intensity F₀ and blank reference solution (acidic solution of benzoic acid) was calculated to indirectly obtain the apparent production of ˙OH oxidized in the Fenton reaction, that is, the content of benzoic acid oxidized by ˙OH is directly proportional to the production of ˙OH in the Fenton reaction system.

The GSH-depleting property of the PCL/MFO@MIL scaffold was evaluated using DTNB chromogen, which could react with GSH to produce a yellow product.^22,23 Briefly, PCL/MFO@MIL scaffolds were respectively introduced into five tubes containing 1 mL of GSH solution (10 mM). At different time points (0, 4, 8, 12, 24 h), 100 μL of solution was extracted from each tube and inoculated with 50 μL of DTNB (1 mM) for 5 minutes. Subsequently, the absorbance profiles of the resulting solutions at 250–550 nm were determined to assess the residual GSH concentration. As a control, the color change of the solution without scaffold was studied by the same process.

2.8. Anti-tumor efficiency of scaffolds

Human osteosarcoma MG-63 cells were selected to study the antitumor efficiency of the scaffolds. The cells were cultured in high-Dulbecco's modified Eagle's medium (H-DMEM) containing 10% FBS in a cell incubator containing 5% CO₂ at 37 °C. The PCL, PCL/MIL, and PCL/MFO@MIL scaffolds (∅8 × 2 mm³) were sterilized under ultraviolet radiation for 3 h.

The intracellular ROS levels were assessed using DCFH-DA as the fluorescent probe. MG-63 cells were seeded in 48-well plates at a density of 5 × 10³ cells per well. After 1 day of culture, the cells were incubated with the scaffold for another day at 37 °C. Then, 200 μL of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) was added to each well and incubated for 30 min. Cells were washed with serum-free DMEM to fully remove DCFH-DA which did not enter the cells. Finally, the intracellular ˙OH level was assessed via fluorescence microscopy (BX53F2, OLYMPUS, Japan). The fluorescence quantitative data were analyzed using ImageJ software.

Cell viability of MG-63 cells in different scaffold groups was assessed using live/dead staining kits (Beyotime, China). Specifically, MG-63 cells were seeded in 48-well plates at a density of 5 × 10³ cells per well. After culturing for 1 day, the cells were cocultured with the scaffold, and DMEM was refreshed every day. At each set time point, cytocalciferin acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) were used to stain live and dead cells to visualize cell behavior. Then, the fluorescence images were obtained using a fluorescence microscope.

A cell counting kit-8 (CCK-8) assay was performed to quantitatively analyze cell viability in each scaffold group. MG-63 cells were cultured in 96-well plates with different scaffold groups. After incubating for 1, 3 and 5 days, the cells were stained by a CCK-8 reagent, and the optical density (OD) value was detected by using a microplate reader at 450 nm.

MG-63 cell apoptosis treated by scaffolds was assessed using the Annexin V-PE apoptosis detection kit (Beyotime, China), with subsequent analysis using immediate flow cytometry. Specially, MG-63 cells were seeded in 6-well plates and incubated with different scaffolds for 5 days. The cells were trypsinized, rinsed, and resuspended, followed by culturing with Annexin V-PE. Thereafter, the cells were washed and analyzed using a flow cytometer (EasyCell 204A1, Wellgrow, China).

2.9. Statistical analysis

All the quantitative data were presented as mean values ± standard deviation (SD) for n ≥ 3 independent experiments. The statistical difference between groups was analyzed using the Student's t-test, in which *p < 0.05, **p < 0.01 and ***p < 0.001 were recognized as statistical differences.

3. Results and discussion

3.1. Microstructural characterization studies of MFO@MIL

The microstructures of the MIL and MFO@MIL nanoparticles were analyzed using SEM and TEM (Fig. 1). It could be clearly seen that the MIL showed a typical octahedral structure (Fig. 1a), which had a smooth surface with an average edge length in the μm-scale. The morphological structures of MFO@MIL are exhibited in Fig. 1b, with many nanoparticles adhering to the surface compared to MIL. The typical lattice spacing of these nanoparticles were 0.167, 0.150 and 0.257 nm, which were consistent with the (333), (440) and (311) crystal planes of MnFe₂O₄ (PDF#38-0430), respectively (Fig. 1c). Moreover, the diffraction rings of MFO@MIL were also consistent with the typical crystal planes of MnFe₂O₄ (Fig. 1d). Besides, the EDS-elemental mapping images of the MFO@MIL confirmed the uniform distribution of Mn and Fe elements.

Fig. 1 The microstructures of the (a) MIL and (b) MFO@MIL nanoparticles. (c) High-resolution image and lattice spacing of MFO@MIL nanoparticles. (d) Diffraction rings of MFO@MIL. (e) and (f) Mn and Fe elemental distribution of MFO@MIL.

To further confirm the crystal structures of MFO@MIL, XRD tests of MIL and MFO@MIL were performed (Fig. S1, ESI†). Compared with MIL, there were some new characteristic peaks at 29.59°, 34.81°, 42.40°, 56.01°, and 61.38° presented in the MFO@MIL pattern, which corresponded to the (202), (311), (400), (333) and (440) crystal planes of MnFe₂O₄ (PDF#38-0430), respectively. Combined with TEM results, the MnFe₂O₄ was successfully loaded onto MIL.

The chemical compositions and electronic states of the MFO@MIL were characterized via XPS tests. As shown in Fig. 2a, the Mn, Fe, and O elements were clearly visible. Among them, the high-resolution spectra of Mn 2p showed two typical peaks centered at 641.28 eV and 652.98 eV (Fig. 2b), which could be attributed to Mn 2p_3/2 and Mn 2p_1/2.²⁴ The Mn 2p_3/2 peak can be delineated into two peaks: Mn²⁺ (641.28 eV) and Mn³⁺ (644.68 eV).²⁵ In the meantime, the satellite peak at 647.68 eV also indicated the presence of Mn²⁺. So as to analyze the bonding of O elements, peak fitting was performed on the XPS spectrum of O 1s (Fig. 2c). The main peaks appearing at 529.98 eV and 531.98 eV corresponded to the lattice O of metal oxides (Fe–O and Mn–O, denoted as O_Latt) and the adsorbed O (denoted as O_Ads).²⁶ With regard to the XPS spectrum of Fe 2p (Fig. 2d), two main binding energy peaks at 710.98 eV and 724.38 eV could be attributed to Fe 2p_3/2 and Fe 2p_1/2,²⁷ respectively. Two pairs of binding energy peaks located at 710.98 eV and 724.38 eV, and 713.98 eV and 727.48 eV were ascribed to Fe²⁺ and Fe³⁺.^24,28 Moreover, the satellite peak at 718.78 eV indicated the exclusive presence of the Fe³⁺ in the MnFe₂O₄.²⁴ The above results further confirmed the successful synthesis of MFO@MIL nanoparticles.

Fig. 2 (a) Survey XPS spectra of MFO@MIL nanoparticles; (b)–(d) deconvolution of XPS spectra of Mn 2p, O 1s, and Fe 2p, respectively.

3.2. Structure and degradation behavior of skin scaffolds

The synthesized MFO@MIL nanoparticles were compounded with PCL powders and fabricated into porous skin scaffolds using SLS technology (Fig. 3a–c). It could be seen that the PCL/MFO@MIL scaffold exhibited superior flexibility, and the pore size ranged from 320 to 800 μm, which was suitable for cell adhesion and growth. For comparison, PCL and PCL/MIL scaffolds were also prepared using the same process. Their degradation capacities and ion release behaviors were then evaluated by weight loss and ion detection (Fig. 3d–f). Clearly, all scaffolds degraded gradually with time. The degradation rate of PCL scaffolds was the lowest, with a mass loss of only 3.27 ± 0.27% after 4 weeks. Notably, the incorporation of MIL and MFO@MIL nanoparticles significantly accelerated the degradation of scaffolds; especially, the mass loss of PCL/MFO@MIL scaffolds reached 17.07 ± 0.29% after 4 weeks. This might be because the nanoparticles occupied numerous spaces in the scaffolds, and their release increased the area where water molecules attack PCL molecular chains, speeding up the hydrolysis of PCL.

Fig. 3 (a)–(c) The porous PCL/MFO@MIL scaffold prepared by SLS technology. (d) Weight loss of scaffolds with time. (e) and (f) The non-cumulative and cumulative release amount of Fe³⁺ and Mn²⁺ ions from PCL/MFO@MIL scaffold with degradation.

The degradation of PCL/MFO@MIL scaffold was accompanied by the release of Fe³⁺ and Mn²⁺ ions; hence, the ion release amounts were recorded (Fig. 3e and f). The non-cumulative release and cumulation release curve showed sustained release of Fe³⁺ and Mn²⁺ ions with the scaffold degradation, which was conducive to the long-term antitumor activity.

3.3. Catalytic activity of scaffolds

It is well known that high levels of H₂O₂ and GSH exist in the tumor microenvironment.^29,30 Especially, the H₂O₂ can convert Fe³⁺ and Mn²⁺ ions released from the scaffold into Fe²⁺ and Mn⁴⁺ ions, respectively, in which the Fe²⁺ ions will further undergo the Fenton reaction with H₂O₂ to produce highly toxic ˙OH to damage tumor cells.³¹ Meanwhile, Mn⁴⁺ ions can deplete GSH, greatly reducing its antioxidant efficacy.³² Moreover, this depletion process will convert Mn⁴⁺ into Mn²⁺, which may cooperate with Fe²⁺ to continuously catalyze H₂O₂ to generate ˙OH, significantly improving the antitumor efficiency of the scaffold. Hence, the POD-like catalytic activity and GSH depletion capacity of the scaffold was measured systematically.

To verify the POD-like catalytic activity of PCL/MFO@MIL scaffold, 3,3′,5,5′-tetramethylbenzidine (TMB) was used as a substrate by using a typical colorimetric method. In the presence of H₂O₂, the released Fe³⁺ and Mn²⁺ ions from scaffold could oxidize TMB to generate blue oxTMB with characteristic absorbances of 370 and 652 nm.^33,34 As shown in Fig. 4a, the absorbance was almost negligible in the absence of scaffold, indicating that no oxidation reaction occurred between TMB and H₂O₂. Apparently, the two absorbance peaks at 370 and 652 nm were observed after adding PCL/MFO@MIL scaffold into TMB-H₂O₂ mixture solution, which confirms its POD-like catalytic activity.

Fig. 4 In vitro characterization of the catalytic activity of PCL/MFO@MIL scaffolds against H₂O₂. (a) The UV-vis absorption spectra of the oxidation of TMB (oxTMB) by different reaction systems. (b) UV-vis absorption spectra of oxOPD catalyzed using the PCL/MFO@MIL scaffold at varied times. (c) Schematic diagram of the PCL/MFO@MIL scaffold simulating POD catalytic process.

The catalytic activity of the PCL/MFO@MIL scaffold was further evaluated by detecting the steady-state catalytic kinetics in a reaction system containing the PCL/MFO@MIL scaffold, o-phenylenediamine (OPD) and H₂O₂ at various times (0, 4, 8, 16, and 24 h) at room temperature (Fig. 4b). Obviously, the absorbance variation of the reaction solution was time-dependent, and the characteristic peak of oxOPD intensity at 417 nm became significantly stronger with time. This confirmed the scaffold could consistently and steadily produce ˙OH. The possible reaction mechanism was similar to the oxidation of TMB, in which the O–O bond in the H₂O₂ molecule was broken to ˙OH, following by oxidation of TMB and OPD to oxTMB and oxOPD (Fig. 4c).

The ˙OH generation capacities of PCL/MIL and PCL/MFO@MIL scaffold were evaluated by EPR. As depicted in Fig. 5a, the characteristic 1 : 2 : 2 : 1 absorbance peak of DMPO–˙OH in the PCL/MFO@MIL group was significantly higher than that in PCL/MIL group, indicating that the introduction of MnFe₂O₄ effectively promoted the production of ˙OH.

Fig. 5 (a) EPR spectra recorded for ˙OH of PCL/MIL and PCL/MFO@MIL groups. Instantaneous concentrations of ˙OH in (b) PCL/MIL and (c) PCL/MFO@MIL scaffold groups at different H₂O₂ contents. (d) Absorbance of the PCL/MFO@MIL scaffold after co-culturing with GSH solution with time. (e) Photograph of GSH solution soaked with PCL/MFO@MIL. (f) The GSH depletion principle diagram.

The fluorescence intensity of hydroxybenzoic acid formed after the interaction of different benzoic acid concentrations with PCL/MIL scaffold (Fig. 5b) or PCL/MFO@MIL scaffold (Fig. 5c) was measured at 410 nm. In general, the fluorescence intensity is proportional to the content of ˙OH. It could be clearly seen that the absorbance of solution increased with the increase of H₂O₂ concentration and the fluorescence intensity of PCL/MFO@MIL scaffold group was higher than that of the PCL/MIL scaffold group.

As a rich endogenous antioxidant, GSH in the tumor microenvironment can effectively clear ˙OH and inhibit cell damage induced by ˙OH.³⁵ Hence, the GSH consumption capacity of the PCL/MFO@MIL scaffold was studied. The scaffold was co-cultured with GSH solution, and the absorbance of the solution was measured at different reaction times. Clearly, the characteristic peak intensity of GSH decreased with time, and the absorbance was only about 0.5 after 24 h (Fig. 5d), which was about 87.5% lower than the initial value. Meanwhile, it could also be intuitively seen that the color of the reaction solution changed from yellow to transparent over time (Fig. 5e). The results confirmed the superior GSH consumption capacity of the scaffold. Mechanically, Mn²⁺ ions released from the scaffold reacted with H₂O₂ to transform into Mn⁴⁺ (Fig. 5f), which could oxidize GSH to GSSG due to the excellent oxidation properties, thus achieving the consumption of GSH.

3.4. Anti-tumor capacity of the scaffold

High ˙OH levels and GSH consumption will induce apoptosis of tumor cells, thus the anti-tumor capacity of the scaffold was further studied. The intracellular ROS levels were assessed using DCFH-DA as the fluorescent probe, which could be oxidized by ROS and emits green fluorescence.^36,37 As illustrated in Fig. 6a, an inconspicuous green fluorescence was observed in the PCL group, indicating that almost no ˙OH was produced in the MG-63 cells. For the PCL/MIL group, weak green fluorescence was detected. Significantly, the PCL/MFO@MIL group presented the strongest green fluorescence at any time point, proving that plenty ROS was generated inside the cells. Furthermore, ImageJ software was used to quantitatively analyze the intracellular ROS levels by calculating the mean fluorescence intensity (Fig. 6b). According to the results, the fluorescence intensity in the PCL/MFO@MIL group was higher than that in other groups. This was mainly due to the consumption of Mn⁴⁺ ions on endogenous GSH, which reduced the antioxidant capacity of cells and enabled the continuous generation of ˙OH via Fenton reaction.

Fig. 6 (a) Detection of intracellular ROS levels stained with DCFH-DA after co-incubation with PCL, PCL@MIL, and PCL/MFO@MIL scaffolds. (b) The mean fluorescence intensity of intracellular ROS.

To determine the anti-tumor properties of the scaffolds, the scaffold and MG-63 cells co-culture experiments were performed and subsequently stained with Calcein-AM and PI. In general, Calcein-AM can enter living cells to hydrolyze, generate calcein and emit green fluorescence, while PI can only enter the nucleus of dead cells to emit red fluorescence.^38,39 Therefore, the green and red fluorescence intensity can qualitatively analyze the cell activity. As shown in Fig. 7a, the cell viability in the PCL group did not decrease significantly after 5 days, while the PCL/MIL group led to partial cell death. Notably, the number of live cells in the PCL/MFO@MIL group decreased significantly, accompanied by a remarkable increase in the number of dead cells.

Fig. 7 Anti-tumor properties of different scaffolds. (a) Live–dead fluorescent staining of MG-63 cells. (b) The results of CCK-8 assay after co-culturing scaffolds and MG-63 cells for 1, 3 and 5 days, respectively. (c)–(e) MG-63 cells apoptosis after treatment with PCL, PCL/MIL and PCL/MFO@MIL groups.

The CCK-8 assay was used to quantitatively analyze the killing effect of the scaffolds on MG63 cells.^40,41 It could be obviously seen that the optical density (OD) values in the PCL group tended to increase with time, while the OD value in the PCL/MIL group decreased at day 5. Notably, the OD value in PCL/MFO@MIL group was lowest at any time, confirming the superior killing effect of the scaffold on MG-63 cells (Fig. 6b). This could be attributed to the consumption of endogenous antioxidant GSH by the MnFe₂O₄, which maximized the antitumor effect of ˙OH.

Flow cytometry assay was performed to quantitatively evaluate the ability of different scaffolds to induce cell apoptosis. As observed in Fig. 7c–e, the proportion of cell apoptosis reached 82.30% in PCL/MFO@MIL groups, which was distinctly higher than that in PCL and PCL/MIL groups. Combined with the previously results, it indicated that the PCL/MFO@MIL scaffold induced tumor cell death through apoptosis.

4. Conclusions

In summary, MFO@MIL nanoparticles with GSH-depleting and high ˙OH production capacities were successfully synthesized using in situ loading technology, followed by fabricating PCL/MFO@MIL scaffolds using SLS. The scaffolds presented powerful POD-like activities and GSH-depleting capabilities due to the continuous release of MFO@MIL nanoparticles, and Fe³⁺ and Mn²⁺ ions. In the tumor microenvironment, the scaffolds continuously catalyzed the generation of ˙OH from H₂O₂via the Fenton reaction. Meanwhile, the conversion from Mn⁴⁺ to Mn²⁺ led to GSH depletion, which significantly disrupted the redox balance and reduced the reducibility. Moreover, the depletion of GSH enabled the significant increase of intracellular ˙OH yield, ultimately triggering tumor cells apoptosis. Hence, the loading of MFO@MIL significantly enhanced POD-like activities and GSH-depleting capabilities of scaffold, enhancing its immense potential in antitumor therapy.

Data availability

All data are available from the corresponding authors upon reasonable request.

Conflicts of interest

The authors declare that they have no known competing financial interests that could appear to have influenced the work reported in this article.

Acknowledgements

This study was supported by the following funds: the Natural Science Foundation of China (52465041, 52105352, U24A20120, 52475362, and 52365046); JiangXi Provincial Natural Science Foundation of China (20224BAB214049 and 20224ACB204013); National Key Research and Development Program of China (Grant No. 2023YFB4605800); and Jiangxi Provincial Key Laboratory of Additive Manufacturing of Implantable Medical Device (2024SSY11161).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qm01014k

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