Proton Nanomodulators for Enhanced Mn 2+ -mediated Chemodynamic Therapy of Tumors via HCO 3 − Regulation

doi:10.21203/rs.3.rs-4555479/v1

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Research Article

Proton Nanomodulators for Enhanced Mn 2+ -mediated Chemodynamic Therapy of Tumors via HCO 3 − Regulation

https://doi.org/10.21203/rs.3.rs-4555479/v1

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Journal Publication

published 01 Nov, 2024

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Background

Mn²⁺-mediated chemodynamic therapy (CDT) has been emerged as a promising cancer therapeutic modality that relies heavily on HCO₃⁻ level in the system. Although the physiological buffers (H₂CO₃/HCO₃⁻) provide certain amounts of HCO₃⁻, the acidity of the tumor microenvironment (TME) would seriously affect the HCO₃⁻ ionic equilibrium (H₂CO₃ ⇌ H⁺ + HCO₃⁻). As a result, HCO₃⁻ level in the tumor region is actually insufficient to support effective Mn²⁺-mediated CDT.

Results

In this study, a robust nanomodulator MnFe₂O₄@ZIF-8 (PrSMZ) with the capability of in situ self-regulation HCO₃⁻ is presented to enhance therapeutic efficacy of Mn²⁺-mediated CDT. Under an acidic tumor microenvironment, PrSMZ could act as a proton sponge to shift the HCO₃⁻ ionic equilibrium to the positive direction, significantly boosting the generation of the HCO₃⁻. Most importantly, such HCO₃⁻ supply capacity of PrSMZ could be finely modulated by its ZIF-8 shell thickness, resulting in a 1000-fold increase in reactive oxygen species (ROS) generation. Enhanced ROS-dependent CDT efficacy is further amplified by a glutathione (GSH)-depletion ability and the photothermal effect inherited from the inner core MnFe₂O₄ of PrSMZ to exert the remarkable antitumor effect on mouse models.

Conclusions

This work addresses the challenge of insufficient HCO₃⁻ in the TME for Mn²⁺-mediated Fenton catalysts and could provide a promising strategy for designing high-performance Mn²⁺-mediated CDT agents to treat cancer effectively.

chemodynamic therapy

HCO3−

reactive oxygen species

multi-level enhanced

photothermal effect

Chemodynamic therapy (CDT) is a burgeoning form of cancer treatment through the Fenton reaction or Fenton-like reagents to generate site-directed toxic reactive oxygen species (ROS)^{[1, 2]}. Owing to the natural overabundance of H₂O₂ within tumor regions, the CDT strategy has many advantages, such as less reliance on external stimuli and low systemic toxicity compared to other cancer treatments^{[3, 4]}. Therefore, extensive efforts have been devoted to improve iron (Fe²⁺/Fe³⁺)-mediated CDT efficacy^[5]. Unfortunately, the acidic tumor microenvironment (TME) (pH ≈ 6.5) considerably deviates from the optimal catalytic window of Fe²⁺ (optimal pH range = 2.0–4.0)^[6], resulting in insufficient ROS production. With exceptional progress in Fenton catalysis, various transition metal-mediated CDT agents have been used to improve ROS generation efficacy ^[5]. Particularly, Mn²⁺ could generate ROS at neutral pH and mild temperatures owing to the octahedral Mn catalytic sites with high active site density^{[2, 7]}. These active sites are further stabilized through HCO₃⁻ ligands, and their catalytic effect is positively correlated with HCO₃⁻ concentration^[8]. Unfortunately, the acidic TME (pH ≈ 6.5) disrupts the HCO₃⁻ ionic equilibrium (H₂CO₃ ⇌ H⁺ + HCO₃⁻), reducing HCO₃⁻ supply to tumor regions and hindering Mn²⁺-mediated CDT efficacy. Therefore, developing a facile and robust strategy to efficiently elevate HCO₃⁻ levels in tumor regions and further enhances Mn²⁺-mediated CDT is desirable but remains underestimated.

The concept of a proton sponge with strong H⁺ capture capability has been widely applied in drug delivery and industrial catalysis^[9–11]. Some quaternary amine-containing compounds, such as amino acids, imidazole^{[12, 13]}, and polyetherimide^{[14, 15]}, can act as proton-trapping agents (PTAs) in acidic environments, thereby reducing H⁺ concentration. Among various PTAs, imidazole arouses our interest. Imidazole is able to form imidazolium salts via protonation^[16]. Moreover, imidazole as the ligand can coordinate with Zn²⁺ to create nanoscaled zeolitic imidazolate framework-8 (ZIF-8), which can grow on different substrates (metal or metal oxide nanoparticles (NPs) and form a shell with tunable thicknesses. These favorable features of imidazole provide the possibility for enhancing their tumor accumulation ability^{[17, 18]} and controlling proton levels^{[19, 20]}. Therefore, we hypothesize that introducing such PTAs (ZIF-8) into Mn²⁺-mediated CDT might be a potential strategy for improving HCO₃⁻ levels and CDT efficacy.

Herein, we developed a “proton sponge-like” MnFe₂O₄@ZIF-8 (PrSMZ) to restore tumor HCO₃⁻ microenvironment and improve CDT outcomes. As shown in Scheme 1A, PrSMZ comprises biocompatible MnFe₂O₄, which is capable of generating •OH and depleting glutathione (GSH)^[21], and pH-sensitive ZIF-8. Guided by an external magnetic field, PrSMZ effectively accumulates within the tumor region. Under acidic conditions, PrSMZ decomposes to release alkaline 2-methylimidazole (2-MIM) and Zn²⁺ (Scheme 1B). The protonation of alkaline 2-MIM further reduces H⁺ concentration and shifts the ionic equilibrium in the positive direction (ionization direction). Moreover, careful modulation of the shell thickness of ZIF-8 enhances HCO₃⁻ supply capacity, promoting ROS-based antitumor efficacy. Leveraging the near-infrared (NIR) photothermal effect of MnFe₂O₄ accelerates the chemical reaction rates (ionization equilibrium, •OH generation, and GSH depletion). Thus, this “proton sponge-like” nanomodulator presents ROS-driven GSH depletion for synergistic hyperthermia-accelerated CDT, exemplifying its potential as a multifunctional nanoplatform for efficient tumor inhibition. This work addresses the challenge of insufficient HCO₃⁻ in the TME for Mn²⁺-mediated Fenton catalysts and expands the application of ZIF-8 or pH-sensitive nanomaterials in biomedicine.

MnFe₂O₄ acts as Mn²⁺-mediated Fenton Reagent

Typically, citrate-stabilized MnFe₂O₄ was prepared through a hydrothermal method with slight modifications (Scheme 1A) ^[22]. Scanning electron microscopy (SEM) images shown in Fig. 1A and Figure S1 revealed a spherical morphology with uniform size (mean particle size = 150 ± 8.6 nm). Dynamic light scattering (DLS) (Figure S2) and zeta potential (Fig. 1B) data proved that MnFe₂O₄ were monodisperse and negatively charged (− 23.97 ± 0.97 mV)^[22]. Figure S3 demonstrates that a certain amount of Mn²⁺ was released under an acidic phosphate-buffered saline (PBS) solution (pH = 6.5), verifying the suitability of MnFe₂O₄ as Mn²⁺-mediated Fenton reagent. Negligible changes were observed in the characteristic absorption of MB^[23] (Figure S4) when MnFe₂O₄ was added into neutral (Fig. 1C) or even acidic PBS solution (Figure S5). These results align with previous reports indicating that pH changes alone are inadequate to induce Mn²⁺-mediated Fenton reactions^[8]. However, the introduction of HCO₃⁻ substantially decreased MB absorption as a function of HCO₃⁻ concentration (Figs. 1C-D and S6), thus indicating that HCO₃⁻ plays a pivotal role in enhancing MnFe₂O₄-mediated Fenton reactions. Moreover, polycrystalline-like Fe₃O₄ and ZnFe₂O₄ NPs with a similar size as MnFe₂O₄ (Figures S1 and S2) was developed as control samples ^[24]. Notably, neither low pH (Figures S5) nor HCO₃⁻ (Figs. 1C and S7) affected the Fenton activity of ZnFe₂O₄. In contrast, only very low pH (Figure S8) but not HCO₃⁻ (Figure S7) impacted the Fenton activity of Fe₃O₄. Collectively, these results demonstrate the indispensability and specificity of HCO₃⁻ in facilitating Mn²⁺-mediated Fenton activity.

Preparation and Characterization of PrSMZ

To achieve high HCO₃⁻ concentrations at the tumor site and further enhance Mn²⁺-mediated CDT efficacy, PrSMZ structures with different shell thicknesses were meticulously constructed. SEM and DLS data (Figs. 1A and S9) demonstrated the uniform morphology of PrSMZ, exhibiting an enlarged size of 230 nm. Transmission electron microscopy (TEM) images in Figs. 1A also showed a distinct core–shell structure. In addition, the shift in zeta potential of PrSMZ from − 24 to − 4 mV confirmed the successful coating of positively charged ZIF-8 (Fig. 1B)^[25]. Furthermore, the X-ray diffraction (XRD) pattern of PrSMZ presented characteristic peaks of MnFe₂O₄ and ZIF-8 (Fig. 1E), indicating that these coatings preserved their inherent crystal structure. Then, we finely tailored the shell thickness of PrSMZ to serve as a controlled proton nanomodulator. As shown in Fig. 1F, the ZIF-8 layer was adeptly grown into a shell structure on the surface of MnFe₂O₄, with the thickness adjustable from 30 to 120 nm as a function of the 2-MIM/Zn²⁺ ratio. Finally, thermogravimetric analysis (TGA) of PrSMZ revealed that the total amount of ZIF-8 coated on the surface of MnFe₂O₄ varied from 19–30.6% (Fig. 1G)^[26]. These results verify that the shell thickness of PrSMZ could be finely tailored, making them potential candidates for proton nanomodulation.

Regulation of HCO₃⁻ for enhancing Mn²⁺-mediated Fenton activity using PrSMZ

Abnormal cancer cell metabolism disrupts the H₂CO₃ ⇌ H⁺ + HCO₃⁻ ionization equilibrium, leading to low HCO₃⁻ levels within tumor regions^[27]. Therefore, promoting a positive shift in the HCO₃⁻ ionic equilibrium becomes pivotal for enhancing Mn²⁺-mediated Fenton activity (Fig. 2A). In general, ZIF-8 is a well-documented example of pH-sensitive nanomaterials, and its degradation could effectively deplete H⁺ concentration^[25]. As shown in Fig. 2B, PrSMZ exhibited no size alternation under pH 7.4, while it showed a dramatic decline in size within 12 h in the pH = 6.5 solution, demonstrating their effective pH-degradation ability. Then, the effects of PrSMZ with different ZIF-8 shell thicknesses on HCO₃⁻ regulation behavior were evaluated. Phenol red indicators were used to monitor pH-induced color changes, correlated with HCO₃⁻ concentration (Fig. 2A). The solution was yellow when naked MnFe₂O₄ was added (Fig. 2C) owing to the properties of the phenol red indicator (yellow in acids and red in alkali solutions). In addition, the pH of the above solution was determined using a pH meter measuring 6.62, which aligned with the phenol red indicator. In contrast, PrSMZ with different shell thicknesses presented distinct color changes. Figure 2C demonstrates that the color became red with increased ZIF-8 thickness (PrSMZ-1 to PrSMZ-8), demonstrating increased pH of solution because of proton depletion of PrSMZ. Correspondingly, the measured pH values changed from 6.94 to 7.48. These results collectively demonstrate that thicker ZIF-8 coatings rendered solutions more alkaline compared to naked MnFe₂O₄-treated. Moreover, according to the measured pH values, the calculated HCO₃⁻ from the HCO₃⁻ distribution fraction (Fig. 2A) improved from 62.6–92.8% after PrSMZ shell thickness regulation, verifying a substantial HCO₃⁻ regulation ability associated with different ZIF-8 thickness in PrSMZ.

We then explore the ROS generation of PrSMZ by MB oxidation experiments. Figure S10 revealed the MnFe₂O₄-treated group could only oxidize 15% MB within 1 h. In contrast, as ZIF-8 thickness on MnFe₂O₄ increased, additional MB molecules were oxidized. Notably, PrSMZ-8 (Fig. 2D) revealed a substantial reduction in MB content to 52%, suggesting a considerably high •OH generation owing to HCO₃⁻ regulation by a 120-nm-thick ZIF-8 shell. Electron spin resonance (ESR) results (Fig. 2E) verified the improved •OH generation ability after HCO₃⁻ regulation. Moreover, a three-dimensional correlation was established among shell thickness, HCO₃⁻ content, and ROS generation ability (Fig. 2F). In the X–Y plane (purple curve), a strong positive correlation between ZIF-8 thickness and HCO₃⁻ concentration was evident. Meanwhile, the Y–Z plane (blue curve) demonstrated a negative correlation between HCO₃⁻ concentration and residual MB, indicating that elevated HCO₃⁻ concentration resulted in enhanced •OH-mediated MB degradation. In the X–Z plane (green curve), altering ZIF-8 thickness from 30 to 120 nm led to varying residual MB levels from 84–64%, respectively, demonstrating the noticeable impact of ZIF-8 thickness adjustments on MB degradation. Above results verified that PrSMZ could act as proton nanomodulator to affect HCO₃⁻ content, meanwhile, these correlations are poised to enhance the Mn²⁺-mediated ROS generation performance in other strategies.

Then, the underlying mechanism of HCO₃⁻ regulation was further investigated. It is well-known that 2-MIM can remove a proton and form an electron-rich structure that binds with electron-deficient Zn²⁺ in ZIF-8. Therefore, the disassembly of ZIF-8 restores its precursors (Zn²⁺ and 2-MIM), which initially consumes a certain amount of H⁺. Moreover, the released 2-MIM functions as a base, perpetuating H⁺ depletion via an acid–base interaction (Fig. 2G). Altogether, the proton nanomodulator ability of PrSMZ arises from continuous H⁺ depletion through two steps, thereby promoting the ionic equilibrium to produce additional HCO₃⁻ and improve Mn²⁺-mediated •OH generation. Considering its proton modulating capacity and the suitable size for in vivo application, PrSMZ-2 was finally selected for the following studies.

GSH-depletion and Photothermal Properties of PrSMZ

GSH holds the intracellular redox homeostasis and protect cancer cells from ROS-related cell damage^[28]. Therefore, depleting GSH is an effective method to indirectly enhance the antitumor effects. After incubation of PrSMZ-2 with GSH, the fluorescence of the DTNB solution sharply decreased in a PrSMZ concentration-dependent manner, demonstrating the GSH-depletion property of PrSMZ (Figure S11-12). X-ray photoelectron spectroscopy analysis of Mn 2p of MnFe₂O₄ shown in Figure S13 revealed that the peaks located at 641.1 and 642.7 eV were assigned to Mn²⁺ and Mn⁴⁺, respectively, confirming the existence of multivalent Mn. The coexistence of multivalent Mn elements provides the potential for GSH depletion through redox reactions facilitated by PrSMZ^[7]. Furthermore, PTT-induced hyperthermia displays a great role in catalysis reaction^[2]. We then demonstrated that ZIF-8 coating would not damage the photothermal effect of PrSMZ (Figure S14). Meanwhile, the photothermal performance of MnFe₂O₄ (Figure S15) and PrSMZ-2 (Figure S16) were also investigated. The temperature rapidly increased by 30°C at a PrSMZ-2 concentration of 200 µg mL^− 1, whereas the water temperature only increased by 3.2°C. Eventually, the photothermal effects and absorption spectra remained stable after varied post-irradiation durations (Figures S17–S18). Lastly, we investigated the effect of hyperthermia on •OH generation. Residual MB upon PrSMZ treatment at 45°C was lower compared to that at 37°C (Figure S19), indicating enhanced MB oxidation ability at high treatment temperatures. Concurrently, PrSMZ-induced hyperthermia also contributed to GSH depletion (Figure S19). Overall, our PrSMZ achieves HCO₃⁻ regulation and results in a GSH-depletion effect, synergistically enhancing hyperthermia-accelerated •OH generation.

In Vitro Cellular Cytotoxicity

Encouraged by the admirable •OH generation performance, in vitro cytotoxicity of PrSMZ NPs was evaluated. No detectable cytotoxicity was observed in cancerous 4T1 cell lines (Fig. 3A) even after incubation with 200 µg mL^− 1 MnFe₂O₄ for 24 h, whereas PrSMZ showed a dose-dependent cell-killing behavior under the same conditions (Fig. 3A). Moreover, the PrSMZ + NIR group showed pronouncedly reduced cell viability on 4T1 cell lines, attributed to synergistic hyperthermia-accelerated CDT, exemplifying the cell-killing effects. (Figs. 2E and 3A). However, with the addition of Vitamin C, a ROS scavenger, cell viability recovered to a high level, ≈ 60%, suggesting the hyperthermia only play a limited role for cell-killing. Altogether, these results demonstrate ROS as the root cause of cellular damage, with ROS elimination causing reverse cell-killing behavior.

Then, the effect of HCO₃⁻ regulation on Mn²⁺-mediated •OH generation in vitro was investigated. Confocal microscopy results shown in Fig. 3C demonstrated that the intracellular fluorescence intensity of DCF by different treatments, indicative of efficient ROS generation^[29]. Notably, the PrSMZ-treated group exhibited a markedly stronger green signal compared to naked MnFe₂O₄, indicating that the thick ZIF-8 shell benefited ROS production. In addition, flow cytometry quantified ROS generation, revealing a staggering 1000-fold

enhancement signals compared to the control group (Fig. 3D). These data suggest that the ZIF-8 shell is important in enhancing Mn²⁺-mediated •OH generation. To observe the living and dead cell distribution, 4T1 cells were treated with different PrSMZ formulations for 12 h and co-stained with dyes. Results in Fig. 3E demonstrated massive cell death in the PrSMZ-8 group, attributed to amplified intracellular ROS levels. However, cell viability was notably higher in other groups. Collectively, HCO₃⁻ regulation substantially amplified Mn²⁺-mediated ROS generation. A correlation among ZIF-8 shell thickness (X axis), relative ROS generation (Y axis), and cell viability (Z axis) was established to prove the HCO₃⁻ regulation effect of PrSMZ in vitro. With increased ZIF-8 shell thickness, additional ROS generation was observed (X–Y plane, purple curve), correlating with reduced cell viability (Y–Z plane, blue curve), as shown in Fig. 3F. Therefore, a positive correlation was established between ZIF-8 shell thickness and their in vitro antitumor effect (X–Z plane, green curve). Furthermore, PrSMZ-induced mild hyperthermia effects were verified via ROS probe (Figure S20) and live/dead cell co-staining assays (Figure S21).

Finally, the influence of HCO₃⁻ regulation on cancer cells was further explored in detail. Generally, H⁺ depletion would shift H₂CO₃ toward HCO₃⁻ formation, consequently increasing HCO₃⁻ concentration (Figs. 2A and 3B) and would inhibit HCO₃⁻ efflux from cells through the carbonic anhydrase IX (CA IX) protein-mediated pathway^[30]. PrSMZ-treated cells exhibited diminished mRNA expression of CA IX (Fig. 3G), corroborating the hypothesis. Moreover, the high HCO₃⁻ concentration within the TME would accelerate HCO₃⁻ entry into cells. However, owing to the intracellular and extracellular proton exchange function, sodium bicarbonate cotransporter (NBC) protein mainly yielded to the activity of CA IX^[30]. Hence, PrSMZ groups displayed suppressed mRNA expression of NBC compared to the MnFe₂O₄ group (Fig. 3H). Therefore, PrSMZ not only regulates HCO₃⁻ levels within the TME but also effectively downregulates mRNA expressions of NBC and CA IX to regulate cellular HCO₃⁻ levels, which is beneficial for Mn²⁺-mediated CDT outcomes in vitro.

In vivo Biocompatibility and Biodistribution

Given the promising in vitro performance of PrSMZ, we intended to investigate it in vivo antitumor effect. Hemolysis analysis revealed that PrSMZ would not cause hemolysis even at 200 µg mL^− 1 (Fig. 4A). Furthermore, hematoxylin and eosin (H&E) staining of main organs demonstrated good biocompatibility of PrSMZ (Fig. 4B). Magnetization curves shown in Fig. 4C verified that PrSMZ was superparamagnetic, inheriting magnetic targeting and magnetic particle imaging (MPI) property from MnFe₂O₄^[22]. Figure 4D and Figure S22-23 depicts that the PrSMZ group revealed a remarkable Fe accumulation within the liver. In contrast, the PrSMZ + M group showed enhanced Fe content in tumor at 4 h postinjection (kept in a magnetic field for 4 hours) of PrSMZ. Guided by biodistribution results, in vivo mild photothermal experiments were performed on 4T1-tumor-bearing mice. Tumor temperature scarcely increased in the PBS group (Figs. 4E-F). However, the PrSMZ + M group displayed elevated brightness, and the temperature reached 48°C. These excellent NIR laser absorption ability of PrSMZ would induce mild hyperthermia effect within tumor region to improve the efficacy of ROS generation and GSH depletion for cancer therapy.

In vivo Tumor Elimination Assay

To evaluate the in vivo antitumor activity of PrSMZ-mediated CDT, 4T1-tumor-bearing mice were established and randomly divided into six groups for the treatments (Fig. 5A). Figures 5B-5C depict rapid tumor growth in the G1, G2, and G3 groups. And the G4 group showed uncontrollable tumor growth, while G5 exhibited considerable tumor growth suppression (Figs. 5B–5F). HCO₃⁻ regulation improved ROS catalysis, thus facilitating tumor growth suppression. Notably, the G6 group displayed the best tumor inhibition (Fig. 5D), eliminating three out of five tumors, indicating that NIR laser benefits CDT outcomes (Figs. 5B–5G). To further illuminate the therapeutic effect, immunohistochemical (IHC) analysis was performed, measuring CA IX protein expression. Unlike the G4 group, which negligibly affected CA IX protein levels, the G6 group demonstrated distinct brown staining, indicating the downregulation of CA IX proteins (Fig. 5H). In addition, qRT–PCR of tumors revealed downregulation trends for CA IX and NBC expression in G6 group-treated tumors (Figs. 5I–J). These results revealed that PrSMZ not only regulates HCO₃⁻ to improve ROS catalytic effect but also affects the mRNA expression of CA IX and NBC to amplify therapeutic

effect. Lastly, PrSMZ treatment showed negligible influence on healthy mice (Figs. 5K–N and S24). These data further support the capability of our “proton sponge-like” strategy for a GSH depletion–driven ROS synergistic hyperthermia-accelerated CDT, exemplifying an efficient tumor inhibition.

In summary, we developed a multifunctional proton nanomodulator, PrSMZ, consisting of the pH-sensitive ZIF-8 shell and the MnFe₂O₄ core for enhancing the therapeutic efficacy of Mn²⁺-mediated CDT. Under acidic conditions, PrSMZ exhibited excellent proton capture ability and promoted the HCO₃⁻ ionic equilibrium to the positive direction, improving HCO₃⁻ content from 62.6–92.8%. Benefiting from raised HCO₃⁻ level, Mn²⁺-mediated Fenton-like reaction was triggered effectively to produce a considerable amount of ROS. Notably, the proton modulating capability of PrSMZ is positively correlated with ZIF-8 shell thickness, resulting in a 1000-fold increase in ROS generation. Additionally, PrSMZ could not only regulate HCO₃⁻ levels within the TME but also effectively downregulate mRNA expressions of NBC and CA IX to regulate cellular HCO₃⁻. Upon reaching the tumor region with the assistance of an external magnetic field, PrSMZ-induced CDT acted synergistically with the GSH-depletion function and the photothermal effect elicited by PrSMZ to further amplify its therapeutic efficacy, substantially inhibiting tumor growth. This study provides a robust proton sponge-like strategy to address HCO₃⁻ deficiency within the TME, which would finally facilitate the development of novel Mn²⁺-mediated CDT agents and optimize CDT efficacy.

Materials. Mangnese chloride tetrahydrate (MnCl₂·4H₂O), Iron (III) chloride hexahydrate (FeCl₃·6H₂O) were purchased from Shanghai Aladdin Reagent Co., Ltd. Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O), Sodium acetate, Trisodium citrate dihydrate, sodium bicarbonate (NaHCO₃) were obtained from Shanghai Macklin Biochemical Co., Ltd. Ethylene glycol, 2-Methylimidazole, Phenol red, Ethanol, Methanol, Hydrogen peroxide (H₂O₂, 35%) were purchased from Beijing Yinuokai Technology Co., Ltd. All chemicals in this work were of analytical grade and used according to the received standards.

Characterization. The morphologies of MnFe₂O₄ and MnFe₂O₄@ZIF-8 were observed by SEM (SU8010, Japan). TEM images were recorded on a JEM 2100F (Japan Electronics Corporation). The XRD pattern was recorded on a D8 Advance diffractometer (Bruker, Germany). XPS analysis was carried out on a Thermo ESCALAB 250XI (Thermo Kalpha, USA). TG analysis was performed on a TG209F1 Thermogravimetric Analyzer (Netzsch, Germany). The hysteresis loop was detected on a PPMS DynaCool (Quantum Design, USA). The production of hydroxyl radicals was tested on an EMX Plus (Bruker, Germany).

The contents of Mn elements were measured using an inductively coupled plasma instrument (ICP-AES, JY 2000-2, Horiba, France). MPI performances of magnetic tracers were investigated using the magnetic particle imaging system (Magnetic Insight, Inc., USA). The frequency of MPI is 45 kHz; the magnetic gradient strength of MPI is 5.7 T m^− 1. The original data obtained from the MPI scanner were processed by Vivo Quant software.

Synthesis of MnFe₂O₄

Firstly, MnCl₂·4H₂O (0.396 g, 2 mmol) and FeCl₃·6H₂O (1.08 g, 4 mmol) were dispersed in ethylene glycol (20 mL) solution, and ultrasonically disperse uniformly. Subsequently, NaAc (1.20 g) and Na₃Cit·2H₂O (0.24 g) were added, and ultrasonically dispersed again to obtain a yellow-brown liquid. After the mixed solution was stirred vigorously for 30 min, it was sealed in a hydrothermal reactor and reacted in an oven at 200 ℃ for 12 h. After the reaction, it was cooled to room temperature, and the reaction product was separated with a permanent magnet, and washed with ethanol and deionized water several times.^[14]

Synthesis of PrSMZ Nanoparticles

Briefly, 5 mg MnFe₂O₄ were dispersed in 40 mL methanol solution containing 8.2 mg (0.1 mmol) 2-MIM, and ultrasonically disperse uniformly. Subsequently, 5 mL of methanol solution containing 29.7 mg (0.1 mmol) Zn (NO₃)₂·6H₂O was added to the above mixed solution by means of gradual dropwise addition method, and the reaction was maintained at room temperature for 2 h. After the reaction, the prepared nanoparticles were separated with permanent magnets and washed with methanol three times. The thickness of the ZIF-8 shell can be adjusted by adjusting the ratio of Zn²⁺ to 2-MIM as shown in Table 1.^[12b]

Table 1

The preparation recipes of the PrSMZ-n nanoparticle.
Sample	MnFe₂O₄(mg)	2-MIM(mg)	Zn(NO₃)₂·6H₂O (mg)	2-MIM/Zn²⁺
PrSMZ-1^a)	10	8.2	29.7	1:1
PrSMZ-2	10	16.4	29.7	2:1
PrSMZ-5	10	41	29.7	5:1
PrSMZ-8	10	65.6	29.7	8:1

a) Ratio of 2-MIN/Zn²⁺ employed in the assembly process.

Chemodynamic Activity of MnFe₂O₄

The MnFe₂O₄ (10 µg mL^− 1), H₂O₂ (10 mM), and MB (7 µg mL^− 1) were prepared in NaHCO₃ aqueous solution (5–50 mM); the total volume was 1 mL. Then the reaction system was shaken at 37°C in the dark. The solution was centrifuged at different time points of the reaction (0 h, 0.5 h, 1 h, 2 h, 3 h), and then the absorbance at 664 nm of the remaining MB in the supernatant was measured by an UV/vis spectrophotometer.^[13]

GSH Consumption

Firstly, 100 µL MnFe₂O₄ nanoparticles of different concentrations (0, 200, 500 and 1000 µg/mL) were incubated with 50 µL H₂O₂ (20 mM) and 500 µL GSH (2 mM) at 37 ℃ for 3 h. Then, the MnFe₂O₄ nanoparticles were magnetically separated, the supernatant was collected, and GSH was detected with GSH kit (Beyotime Biotechnology). Specifically, 10 µL of supernatant was added into 150 µL of GSH kit reaction mixture for 5 min and measured by UV–vis to determine GSH concentration.^[5]

Photothermal Effect and Thermal Stability of MnFe ₂ O ₄: Different concentrations (0-100 µg mL^− 1) of MnFe₂O₄ aqueous solutions were exposed to 808 nm laser (2 W cm^− 2) for 500 s. At the same time, an infrared thermal imaging camera is used to record the thermal imaging temperature results every 10 s. Then, the thermal stability of MnFe₂O₄ aqueous solution was detected. Typically, MnFe₂O₄ nanoparticle aqueous solution (100 µg mL^− 1) was irradiated with a 808 nm laser (2 W/cm²) for 400 s, and naturally cooled to room temperature. Then repeat the above heating and cooling cycle process three times. At the same time use the infrared thermal imaging camera to record the thermal imaging temperature results every 10 s.^[23]

Photothermal Effect of PrSMZ: Different concentrations (0-200 µg mL^− 1) of PrSMZ aqueous solutions were exposed to 808 nm laser (2 W cm^− 2) for 500 s. At the same time, an infrared thermal imaging camera is used to record the thermal imaging temperature results every 10 s.

Phenol Red Shows the Change of Solution pH

Different concentrations of 2-MIM, ZIF-8 and PrSMZ with different shell thickness were added to pH = 6.5 PBS solution. Subsequently, 20 µL of phenol red indicator with a mass fraction of 0.1% was added to each of the above systems. The pH meter is used to test the pH value of different system solutions. Finally, according to the distribution of carbonic acid at different pH, the percentage of HCO₃⁻ ion in the current solution is calculated.

Cytotoxicity of MnFe₂O₄ and PrSMZ

Cytotoxicity evaluation of MnFe₂O₄ and PrSMZ was performed on mouse fibroblast cells (3T3), and 4T1 mouse breast tumor cells, respectively. These cells were implanted in 96-well microtiter plates and allowed to grow overnight. Subsequently, the culture medium was substituted by fresh culture medium including MnFe₂O₄ or PrSMZ at diluted sample concentrations of 0, 5, 12.5, 25, 50, 100, and 200 µg mL^− 1, respectively. After the coincubation for 24 h, the culture medium was substituted by MTT (20 µg mL^− 1) culture solution and incubation for 4 h. Ultimately, dimethyl sulfoxide (150 µL) was added to each well. Cell viability was calculated by measuring the absorbance at λ = 490 nm to the control via a microplate reader.^[23]

In vitro Synergistic Therapeutic Efficacy

The 4T1 cancer cells were cultivated into 96-well plates. And then treated with PBS, laser, MnFe₂O₄, PrSMZ or PrSMZ plus laser irradiation (100 µg mL^− 1) at 37°C. Subsequently, the cells were irradiated with 808 nm laser (2 W cm^− 2) for 8 min. After that, the cells of each group were incubated with CA (4 µM) and PI (4 µM) staining reagents for 10 min and then observed using CLSM.

DCFH-DA Detection •OH: DCFH-DA ROS assay kit was utilized to monitor the intracellular ROS generation. 4T1 cells were implanted into 6-well plates and grew adherently to the wall. Fresh culture medium containing MnFe₂O₄ or PrSMZ (100 µg mL^− 1) was added, which was further cultured for 6 h. After washing three times with PBS, the cells were irradiated upon 808 nm laser (2 W cm^− 2) for 8 min. Then, 10 µM DCFH-DA was added to culture with 4T1 cells for another 30 min. The generation of free radicals was observed by CLSM.^[21]

Hemolysis Assay

Red blood cells (RBCs) were isolated from serum by centrifugation, washed, and diluted with PBS. Then, 0.2 mL of RBC suspension was mixed with 0.8 mL of PrSMZ (1 − 200 µg mL^− 1) in PBS.^[21]

0.8 mL of PBS solution (negative control group), or 0.8 mL of water (positive control group). After incubated at 37 ℃ for 3–4 h, these samples were centrifuged and the supernatants were collected to analyze by UV/vis absorbance at 541 nm.

Animal Experiment: Animal protocols related to this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (approval number: 20230458). All applicable institutional guidelines for the care and use of animals were followed. SPF female healthy BALB/c mice (4–5 weeks) were purchased from Beijing Huafukang Biotechnology Co. Ltd. (Beijing, China). The tumor model was established by the subcutaneous injection of 4T1 cells (5.0 × 10⁵ cells) into the right buttock of each female BALB/c mouse. A breast cancer can be successfully established in about a week. When the tumor volume reached 50–80 mm³, the 4T1-tumor-bearing mice were used for in vivo therapy experiments. The mice were randomly divided into 6 groups (n = 5/group), including (1) PBS, (2) NIR, (3) PrSMZ, (4) MnFe₂O₄ + M, (5) PrSMZ + M, and (6) PrSMZ + M + NIR, M means magnetic targeting. Tumor-bearing mice were injected with 100 µL MnFe₂O₄ or PrSMZ (10 mg/kg), and the mice were kept in a magnetic field for 4 hours. The mice were then irradiated with an 808 nm laser (2 W cm^− 2) for 8 min. During the treatment, the tumor volume was recorded every 2 days with a digital vernier caliper. The tumor volume is calculated by the following formula: V = length × width²/2. (Relative tumor volumes were calculated as V/V0, V0 was the tumor volume when the treatment was initiated). Besides, the body weights were also recorded during the treatment. All mice were sacrificed after 14 days of treatment; tumors and main organs were collected for H&E staining and analyzed with a microscope.

Statistical Analysis: The error of parallel test was calculated as mean ± SD. Statistical difference was calculated through two-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Supporting Information

The online version contains supplementary material available at (will be filled in by the editorial staff).

Ethics approval and consent to participate

The animal experimental was carried out following the guidelines of the Air Force Medical University Institutional Committee for the Care and Use of Laboratory Animals and were approved by the Committee on Ethical Use of Animals of Air Force edical University (approval number: 20230458).

Consent for publication

All authors agreed to publish this manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors have declared that no competing interest exists.

Funding and Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2022YFB3203800), National Natural Science Foundation of China (Nos. 82272159, 91959124 and 32371433), Innovation Capability Support Program of Shaanxi (No. 2022TD-52), the Guang Dong Basic and Applied Basic Research Foundation (No. 2023A1515030207), Dual-chain Integration Special Program of Qin chuang yuan Construction (No. 23LLRH0070), and the Open Project Program of the State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers (Xi jing Hospital of Digestive Diseases, Fourth Military Medical University) (No. CBSKL2022ZDKF14). Natural Science Basic Research Plan in Shaanxi Province of China (2024JC-YBMS-181), and the Fundamental Research Funds for the Central Universities (ZYTS24155) We acknowledge the Instrumental Analysis Center of Xidian University for providing test equipment.

Author contributions

Peng Yang, Xianghan Zhang, Ruili Zhang, Zhongliang Wang wrote the main manuscript text and prepared part of figures, Peng Yang, Shaojie Liu and Zhuang Chen prepared figures 1-2, Weijing Liu and Zuo Yang prepared scheme and figures 3, Haohao Yan, Zhiping Rao prepared figures 4-5, All authors reviewed and approved the manuscript.

Authors' information

¹Lab of Molecular Imaging and Translational Medicine (MITM), Engineering Research Center of Molecular & Neuroimaging, Ministry of Education, School of Life Science and Technology, Xidian University & International Joint Research Center for Advanced Medical Imaging and Intelligent Diagnosis and Treatment, Xi’an, Shaanxi, 710126 P. R. China

²Guangzhou Institute of Technology, Xidian University, Guangzhou, Guangdong, 510555 P. R. China

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Proton Nanomodulators for Enhanced Mn 2+ -mediated Chemodynamic Therapy of Tumors via HCO 3 − Regulation

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Results and Discussion

MnFe₂O₄ acts as Mn²⁺-mediated Fenton Reagent

Preparation and Characterization of PrSMZ

Regulation of HCO₃⁻ for enhancing Mn²⁺-mediated Fenton activity using PrSMZ

GSH-depletion and Photothermal Properties of PrSMZ

In Vitro Cellular Cytotoxicity

In vivo Biocompatibility and Biodistribution

In vivo Tumor Elimination Assay

Conclusion

Materials and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Proton Nanomodulators for Enhanced Mn 2+ -mediated Chemodynamic Therapy of Tumors via HCO 3 − Regulation

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Results and Discussion

MnFe2O4 acts as Mn2+-mediated Fenton Reagent

Preparation and Characterization of PrSMZ

Regulation of HCO3− for enhancing Mn2+-mediated Fenton activity using PrSMZ

GSH-depletion and Photothermal Properties of PrSMZ

In Vitro Cellular Cytotoxicity

In vivo Biocompatibility and Biodistribution

In vivo Tumor Elimination Assay

Conclusion

Materials and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

MnFe₂O₄ acts as Mn²⁺-mediated Fenton Reagent

Regulation of HCO₃⁻ for enhancing Mn²⁺-mediated Fenton activity using PrSMZ