Hybridized nanozymes for anti-osteosarcoma therapy via the Fenton reaction

Summary

Abnormal accumulation of hydrogen peroxide (H₂O₂) in the tumor microenvironment is associated with altered metabolism, abnormal proliferation of tumor cells, and changes in the tumor microenvironment. Based on this phenomenon, we have developed manganese-doped zeolitic imidazolate frameworks (Mn-ZIF) nanozymes, which exhibit superior peroxidase (POD)-like activity and enhanced cytotoxicity. Inside the tumor, the H₂O₂ is catalyzed by Mn-ZIF nanozymes through the Fenton reaction to generate more potent hydroxyl radicals (·OH), further increasing the local reactive oxygen species (ROS) levels in tumor cells and inducing tumor cell death. Meanwhile, the removal of H₂O₂ in the tumor microenvironment reduces tumor proliferation. We have confirmed the anti-tumor effect of these particles in an in situ osteosarcoma (OS) model, providing a direction for the future design of hybrid nanozyme drug delivery systems.

Graphical abstract

Highlights

• •The constructed Mn-ZIF nanozymes exhibited superior peroxidase (POD)-like activity
• •Mn-ZIF nanozymes achieves anti-tumor and bone protection through Fenton reaction
• •Mn-ZIF nanomases provide a good platform for future drug design for tumor therapy

Materials chemistry; Biomedical materials

Introduction

Osteosarcoma (OS) is the most common primary malignant solid tumor of the skeletal system. The incidence rate in the general population is 2–3 per million, with higher rates during adolescence, where males have a 1.4 times higher incidence rate than females.¹ OS primarily occurs in the long bones of the limbs, near the epiphyseal growth plate, with the most common sites being the femur, tibia, and humerus.² Therefore, the murine tibial in situ OS model is one of the most used animal models for studying OS. The main treatment for OS is surgical resection; however, local excision alone is insufficient^3,4 as 80–90% of seemingly localized cases will develop metastasis. Without combined chemotherapy, patients are likely to have a poor prognosis.² Although chemotherapy is widely accepted in OS treatment, the severe adverse reactions it causes, such as vomiting, fever, and liver and kidney toxicity, cannot be ignored.⁵ This prompts us to continue searching for safer and less toxic therapeutic approaches for OS.

In previous studies, researchers have discovered elevated levels of hydrogen peroxide (H₂O₂) within OS cells, which differ from normal cells.⁶ This is associated with the abnormal metabolic pathways in the tumor region. H₂O₂ can serve as an important substance within the tumor area, regulating various processes in OS, including proliferation, autophagy, apoptosis, and drug resistance, by influencing numerous classical pathways, thereby promoting the occurrence and development of OS.^7,8 Moreover, the high H₂O₂ state within tumor cells can diffuse into the tumor microenvironment, thereby affecting the survival state of OS. Thus, regulating the H₂O₂ levels within OS cells to induce tumor cell death and reverse the localized tumor microenvironment is a viable strategy. This can be achieved through the Fenton reaction, which catalyzes the high concentration of H₂O₂ in the tumor into more cell-damaging hydroxyl radicals (·OH).^{9,10,11,12,13} This increase in reactive oxygen species (ROS) contribute to tumor cell death and has a positive effect on reversing the accumulation of H₂O₂ in the tumor microenvironment.

Zeolitic imidazolate frameworks (ZIFs) belong to the subclass of metal-organic frameworks (MOFs) and are topological isomers synthesized from zinc ions and imidazole linkers.¹⁴ Relevant studies have shown that ZIFs can maintain structural stability in neutral pH environments but undergo dissolution in acidic environments.¹⁵ Additionally, ZIF-derived nanoparticles exhibit excellent cellular uptake properties.¹⁶ Therefore, developing ZIFs with peroxidase (POD)-like activity may enable long-term derived superoxide anion effects. Nanoparticles doped with manganese (Mn) have been shown to possess good POD-like activity.¹⁷ Thus, if Mn ions can be doped into ZIFs, it may produce MOFs with POD-like activity. Furthermore, the excellent carrier functionality of ZIFs may facilitate future loading of other antitumor drugs or nanoparticles. The great potential of ZIF-structured materials in anti-tumor immunotherapy has been demonstrated by Ding and Ge et al. The tumor-region responsiveness and good drug-carrying capacity of ZIF structures provide the basis for these effects.^15,18

This study aims to synthesize a novel MOF nanoparticle enzyme with POD-like activity by doping Mn metal elements. The results demonstrate that the synthesized Mn-doped ZIF (Mn-ZIF) exhibits high POD-like activity under H₂O₂ conditions and possesses advantages such as low dosage and excellent cellular uptake capability. In vivo experiments confirm that Mn-ZIF can generate ROS at the tumor site, leading to tumor cell death. This research provides a new strategy for using ZIFs in the therapy of tumors (Scheme 1).

Scheme 1.

Mn-ZIF nanozymes for anti-cancer therapy

Results and discussion

Synthesis and characterization of Mn-ZIF

In this study, we attempted to test whether the synthesis of ZIFs with enzyme-like activity was possible through the one-pot method by changing the metal ion composition.¹⁹ ZIF and Mn-ZIF were visible in transmission electron microscopy (TEM) images (Figures 1A and 1B) with clear morphology as previously described.²⁰ ROS at high levels can irreversibly inhibit tumor cells in the vicinity, and the high level of H₂O₂ in tumors provides the material conditions for the generation of ROS. Therefore, we detected the POD-like activity of ZIF and Mn-ZIF separately (Figure 1C), and the results confirmed that Mn-ZIF can undergo redox reaction with H₂O₂ to produce ·OH. The ·OH generation in tumors is also important for tumor therapy, so we determined the ·OH-generating capacity of the different formulations. H₂O₂/Mn²⁺ generates ·OH via the Fenton reaction. As shown in Figure 1D, the ·OH generation rate of the Mn-ZIF group (58.6 ± 3.3%) was significantly higher than that of the ZIF group (3.6 ± 0.9%). In addition, the ·OH was captured by 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO), and then the ability of the Mn-ZIF nanozymes to produce ·OH was detected by electron spin resonance (ESR). In the Mn-ZIF-treated samples, the signal intensity of ·OH was significantly higher than other groups, indicating its ability to produce more ·OH (Figure 1E). Next, Malvern particle size analysis showed that the average fluid dynamic diameter of ZIF and Mn-ZIF was 85.6 ± 6.32 nm and 90.67 ± 7.12 nm, respectively (Figure 1 F), which is beneficial for the accumulation of materials in the OS region through the enhanced permeability and retention effect (EPR) effect. In addition, zeta potential analysis showed that the average potential of ZIF and Mn-ZIF was −7.56 and −8.76 mV, respectively, which may be a result of good cell uptake characteristics (Figure 1G). These results indicate that the synthesized nanozyme has good POD-like activity, favorable particle size and zeta potential.

Figure 1.

Synthesis and characteristics of Mn-ZIF

(A and B) TEM images of (A) ZIF and (B) Mn-ZIF.

(C) POD-like activity of ZIF or Mn-ZIF using TMB as the chromogenic substrate.

(D and E) The ·OH generating capacity of ZIF and Mn-ZIF was determined by microplate reader and ESR.

(F) Particle size and PDI of synthesized nanoparticles.

(G) Zeta potential of ZIF and Mn-ZIF. Data are expressed as mean. All statistical data are presented as a mean ± standard deviation.

Next, to further verify their crystal morphology, we detected ZIF and Mn-ZIF through powder X-ray diffraction (XRD). The results showed that the data table of Mn-ZIF was similar to that of conventionally prepared ZIF8 (Figure 2A), and no impurity peak was observed, indicating good crystallinity of Mn-ZIF and almost no effect of Mn doping on the crystal integrity of ZIF. In addition, to verify the presence of Mn elements, energy-dispersive X-ray spectroscopy (EDS) and elemental mapping were used for analysis, which showed that Mn elements was present in the synthesized nanozymes (Figures 2B and 2C).

Figure 2.

Crystal structure and element distribution of Mn-ZIF

(A) XRD patterns of ZIF (green) and Mn-ZIF (blue).

(B and C) The elemental mapping and EDS confirm the existence of C, N, O, Zn, and Mn elements in Mn-ZIF.

Cytotoxicity, cell uptake and ROS clearance of Mn-ZIF

Tumor cell uptake of Mn-ZIF is a prerequisite for achieving nanozyme killing of tumors. After co-incubating FITC-labeled Mn-ZIF nanozymes (Mn-ZIF-FITC) with K7M2 cells, the process of cell uptake was observed. The results showed that compared with 0 h, the accumulation of Mn-ZIF-FITC inside tumor cells gradually increased when co-cultured with cells for 24 h (Figure 3A), indicating that more Mn-ZIF-FITC were taken up by tumor cells with time. After co-culturing for 72 h, the green fluorescence inside tumor cells further increased, which was attributed to the continuous uptake of small-sized nanozymes by tumor cells over a long incubation time (Figure 3A). Next, the cytotoxicity of Mn-ZIF nanozymes on OS cells was evaluated using CCK8 and live/dead staining methods. The results exhibited that when the concentration of Mn-ZIF reached 200 μg/mL after co-culturing for 72 h, significant inhibition of K7M2 cell proliferation was observed (Figure 3B). Live/dead staining also showed that at a concentration of 200 μg/mL Mn-ZIF, extensive cell death occurred in tumor cells after 72 h (Figure 3C). Effectively increasing ROS in tumors facilitates tumor suppression. So we next evaluated ROS by dichlorofluorescein diacetate (DCFH-DA). We co-cultured H₂O₂ (200 μM) with cells and nanozymes for 24 h. We found that the accumulation of ROS in the cells in the Mn-ZIF group was significantly increased compared to the other treatment groups (Figure 3D). Ex vivo biodistribution is crucial for assessing the anti-tumor effect and side effects of Mn-ZIF nanozymes. By injecting Mn-ZIF-FITC nanozymes intravenously and removing major organs and tumors after 0, 24, and 72 h, their distribution was observed. Strong fluorescent signals were observed in the liver, lungs, and kidneys after intravenous injection of Mn-ZIF-FITC nanozymes, suggesting that Mn-ZIF-FITC nanozymes are mainly dependent on the metabolism of these organs (Figure 3E). Notably, 24 h after intravenous injection, Mn-ZIF-FITC showed high fluorescence signals in tumors, which was attributed to enhanced permeability and retention effects (Figure 3E). The fluorescent signal in the tumor decreased with prolonged treatment time, which was attributed to the reaction of the nanozymes with H₂O₂ in the tumor as well as continued metabolism in other organs (Figure 3E).

Figure 3.

Toxicity and biodistribution of Mn-ZIF nanozyme in tumors

(A) Cell uptake of Mn-ZIF nanozyme.

(B) CCK8 assay to detect the toxicity of Mn-ZIF nanozyme to K7M2 cells at different time points.

(C) Live/dead staining to show the toxicity of Mn-ZIF nanozyme to tumor cells after 72 h.

(D) DCFH-DA images of osteosarcoma cells after different treatments.

(E) Fluorescent images of tissue distribution after intravenous injection of Mn-ZIF nanozyme. All statistical data are presented as a mean ± standard deviation (∗∗∗p < 0.001).

Distribution and metabolism of Mn-ZIF nanozyme in vivo

We analyzed the targeting effect of Mn-ZIF nanozyme on tumor regions in vivo using small animal live imaging (Figure 3D). As previously mentioned, due to the appropriate particle size of Mn-ZIF nanozyme, we observed good passive targeting effect on tumor regions. This is caused by the EPR effect resulting from the loose and deformed vessels in the tumor tissue. We can see that the nanoparticles distribute rapidly to various parts of the body shortly after injection, with the highest concentration in the kidneys, which may indicate that this material is metabolized through the kidneys. At 24 h after injection, the concentration of this nanomaterial in the tumor reached a peak. Until 72 h after injection, there was still accumulation in the tumor tissue, indicating the tumor retention ability of Mn-ZIF and providing guidance for further in vivo experimental design.

Therapeutic effect of Mn-ZIF nanozyme on mouse OS

We observed the anti-OS effect of Mn-ZIF nanozyme using a mouse K7M2 tibial OS model. We analyzed the growth curve of tumors in situ (Figure 4A). We observed that the Mn-ZIF group showed superior therapeutic effect, which was not observed in the ZIF group. This indicates that the Mn-induced Fenton reaction in the nanozyme plays a decisive role in inhibiting OS. Specifically, after two administrations (day 4), there were significant changes in tumor volume in different groups of mice. At day 6 after treatment, the difference in tumor volume between the groups increased further. This difference further expanded in subsequent experiments. Finally, the tumor volume in the control group and ZIF group was three times higher than that in the Mn-ZIF group.

Figure 4.

In vivo anti-tumor and bone protection effect of Mn-ZIF nanozyme

(A) Progression of tumor volume in each group of mice.

(B) Progression of tumor volume in each mouse of each group.

(C) In vivo photos of tumors in each group of mice.

(D) Weight of tumors in each group.

(E) Inhibition rate of tumors in each group.

(F) Condition of tibial plateau bone in tumor region of each group. All statistical data are presented as a mean ± standard deviation (∗∗∗p < 0.001).

In addition, we can see from the tumor inhibition curve that there is a general trend of comparison between groups with no significant outliers. This indicates that the therapeutic effect of this regimen is relatively stable (Figure 4B). There are individuals with continuous tumor volume decreases in the Mn-ZIF group, suggesting good prospects for this treatment regimen. After the end of the treatment period, we photographed the sacrificed mice (Figure 4C). We can see differences between groups consistent with the tumor inhibition curve. We further analyzed mouse OS ex vivo. The results exhibited that the average weight of tumors in the Mn-ZIF group was much lower than that in the other two groups (Figure 4D). The tumor inhibition rate could indicate the efficacy of different treatment groups on OS, so we further calculated the tumor inhibition rate. The results showed that there was no significant difference in tumor inhibition between the ZIF nanozyme group and the Control group, whereas the Mn-ZIF nanozyme group exhibited a tumor inhibition rate of 58 ± 7.5% compared with the other two groups, indicating that the Mn-ZIF nanozymes possessed a superior anti-OS effect (Figure 4E).

Effective inhibition of OS bone destruction by Mn-ZIF

We directly observed bone tissue changes in OS areas using Micro-CT scanning (Figure 4F). We can see that in the tibial plateau region, there is serious bone destruction in both the control group and ZIF group, which is very similar to pathological fractures in patients with OS. However, in specimens from the Mn-ZIF group, this damage is not obvious. This indicates that Mn-ZIF nanozyme has a protective effect on bone erosion by OS cells, which is very exciting. Subsequently, we conducted pathological observations on tumor specimens (Figure 5A). In both the control group and ZIF group, tumor cells are intact and growing actively, while after treatment with Mn-ZIF nanozyme, most cells in OS areas are dead, with a significantly reduced density of viable cells compared to the other two groups. A significant increase in ROS levels in Mn-ZIF treated tumors was also found by observing tumor sections (Figure 5A). This further explains how Mn-ZIF nanozyme achieves tumor treatment and bone protection.

Figure 5.

In vivo safety assessment

(A) H&E and DCFH-DA images of tumors.

(B) H&E images of organs after different treatments.

(C) Weight monitoring of mice after different treatments.

(D) Survival analysis of mice after different treatments. All statistical data are presented as a mean ± standard deviation (∗∗∗p < 0.001).

Survival rate and in vivo safety analysis of Mn-ZIF therapy

We used the same treatment protocol to treat another batch of mice to obtain survival data (Figure 5D). It can be observed that after Mn-ZIF nanozyme treatment, the survival time of OS mice was greatly prolonged. Although the gradual increase of OS led to all mice reaching the experimental endpoint within 7 weeks in the later stage, this result still prolonged the survival cycle of tumor-bearing mice by about 1.6 times. This indicates that Mn-ZIF nanozyme therapy can not only inhibit local tumors but also improve the survival status and quality of mice. At the same time, we analyzed the weight changes of mice in each group after administration (Figure 5C). We can see that there were no significant fluctuations or differences in weight among the three groups of mice. This indicates that all three treatment methods did not cause toxic effects on the mice, and this result is a significant advantage compared to previously used classic chemotherapy drugs. Organ damage is the most common side effect target organ for small molecule drugs.^21,22 We fixed, sliced, and stained the main organs of mice in each treatment group with H&E (Figure 5B). Under the microscope, no significant cell necrosis, fracture, or deformation was observed in the hearts, livers, spleens, lungs, or kidneys of mice in each treatment group. This result demonstrates the excellent safety and biocompatibility of Mn-ZIF nanozyme.

Limitations of the study

This study explored the anti-tumor efficacy of Mn-ZIF nanozymes using an in situ mouse osteosarcoma model. It would be advantageous for future work to establish human tumor models to further confirm their efficacy against osteosarcoma and provide better references for clinical treatment. Additionally, further investigations are needed to explore the inhibitory effects of Mn-ZIF nanozymes on other types of tumors.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Zn(NO3)2·6H2O	Aladdin Biochemical Reagents Inc(China)	CAS#10196-18-6
N, N-dimethylformamide	Aladdin Biochemical Reagents Inc(China)	CAS#68-12-2
2-methylimidazole	Aladdin Biochemical Reagents Inc(China)	CAS#693-98-1
Experimental models: Cell lines
K7M2 cells	Cell Bank of Chinese Academy of Science (Shanghai, China).	N/A
Experimental models: Organisms/strains
Female BALB/c mice	Charles River Laboratories (Beijing, China).	N/A
Software and algorithms
GraphPad Prism 9 software	GraphPad Prism Software Inc	https://www.graphpad.com/

Resource availability

Lead contact

The relevant experimental reagents, experimental methods, and related data of this study can be obtained by contacting Gaofeng Huang (huanggaofeng120@163.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

In situ OS model construction in mice

6-week-old female Balb/C mice were anesthetized by intraperitoneal injection of 4 mg/mL pentobarbital sodium (60 μL). The hair on the right hind limb was removed and the area was disinfected. The mouse was positioned in a supine position, and the right hind limb was bent to fully expose the patellar ligament. K7M2 cell suspension (30 μL containing 2 million cells) was injected through the patellar ligament into the tibia using a syringe. The needle was withdrawn, and pressure was applied to the injection site. The Medical Ethics Committee of Shanghai Sixth People's Hospital approved all the experimental procedures (KT2023439).

Method details

Synthesis and characterization of Mn-ZIFs

ZIF: Zn(NO₃)₂·6H₂O (0.002 mol, 0.59498 g) was fully dissolved in 10 mL N, N-dimethylformamide (DMF), and then added dropwise to a DMF solution (0.006 mol, 0.4926 g) containing dimethylimidazole (DMF). After stirring for 20 h, the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, washed thrice with alcohol, and vacuum dried.

Mn-ZIF: Mn(NO₃)₂·6H₂O (0.001 mol, 0.28704 g) and Zn(NO₃)₂·6H₂O (0.001 mol, 0.29749 g) were dissolved in 10 mL DMF, and the mixture was stirred for 30 minutes. Then, a DMF solution (0.006 mol, 0.4926 g) containing dimethylimidazole was added dropwise. The mixture was stirred for 20 h, and the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, washed thrice with alcohol, and vacuum dried.

POD-like activity of Mn-ZIFs

The pod-like activity of Mn-ZIFs was determined in the presence of H₂O₂ using TMB as substrate. Briefly, NaAc (pH = 4.55), TMB, Mn-ZIFs and H₂O₂ were mixed at room temperature. Absorption spectra of ZIF, H₂O₂, Mn-ZIF, and Mn-ZIF + H₂O₂ were determined by UV-Vis spectroscopy. The Mn²⁺/H₂O₂ system generated ·OH. The generating capacity of the Mn-ZIF for ·OH was investigated using ESR with DMPO as a probe.

Cell culture

The murine osteosarcoma K7M2 cell line was obtained from Cell Bank of Chinese Academy ofScience (Shanghai, China) . K7M2 cells were cultured DMEM supplemented with 10% (V/V) fetal bovine serum (FBS), penicillin (50 IU mL⁻¹), and streptomycin (50 IU mL⁻¹) at 37°C in 5% (V/V) carbon dioxide (CO₂).

Cell uptake

Mn-ZIF was mixed with FITC to obtain Mn-ZIF-FITC. K7M2 cells were co-cultured with Mn-ZIF-FITC for 0, 24, and 72 h. After washing the cells with PBS and fixing them with 4% paraformaldehyde, cell uptake was observed using an inverted fluorescence microscope.

In vitro cell toxicity assay

Cell toxicity of Mn-ZIF was evaluated using a CCK-8 assay (Beyotime, Beijing). After K7M2 cells adhered to the wall, they were incubated with DMEM containing different concentrations of Mn-ZIF for 1 and 3 days. Then, CCK-8 solution was added and incubated for 2 h. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader.

Live/dead staining assay

Live/dead staining assay was performed using a live/dead kit (Molecular Probes, CA) according to the manufacturer’s instructions. Briefly, K7M2s (1 ×104/mL) were cultured with different Mn-ZIF for 72 h. A total of 5 μL of calcein-AM (component A) and 20 μL of ethidium homodimer-1 (component B) were diluted into 10 mL of PBS to create the staining solution. The culture medium was removed before 200 μL of the staining solution was added directly onto the cells. After 30 min incubation at 25°C in the dark, the cells were imaged under a Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss Inc., Thornwood, NY).

Mouse grouping, treatment, and in situ tumor volume measurement

A total of 12 mice with a tumor volume of 400 mm³ in the in situ tumor area were randomly divided into Control, ZIF, and Mn-ZIF groups, with 4 mice in each group. Treatment was administered via tail vein injection every 4 days for a total of 4 times. The treatment doses were as follows: Control group - 200 μL saline; ZIF group - 200 mg/kg ZIF in 200 μL; Mn-ZIF group - 200 mg/kg Mn-ZIF in 200 μL. The experiment ended on day 14. Tumor volume was measured every other day during the treatment period using the formula: V = 4π/3ab², where a and b (mm) represent half of the length of the tumor along the tibia axis and the maximum length perpendicular to the tibia, respectively. After the experiment, the mice were euthanized by cervical dislocation. The heart, liver, spleen, lung, and kidney were collected from each group, fixed with 4% paraformaldehyde, and used for subsequent sectioning. The in situ tumor area of the right hind limb of the mice was photographed and weighed. The skeletal part was used for Micro-CT scanning.

Survival analysis

A total of 15 mice with a tumor volume of 400 mm³ in the in situ tumor area were randomly divided into Control, ZIF, and Mn-ZIF groups, with 5 mice in each group. The treatment regimen was the same as before. The survival status of the mice was observed for survival analysis.

Distribution and metabolism of materials in vivo

FITC-loaded Mn-ZIF was intravenously injected at a dose of 200 mg/kg in 200 μL. Mice were euthanized at 0, 24, and 72 h post-injection, and the heart, liver, spleen, lungs, and kidneys were collected for fluorescence imaging. The fluorescence images of each organ in each mouse were captured using a small animal live imaging system (INDEC BioSystems, USA).

Micro-CT

The excised right hind limb area of the mouse was fixed on the sample stage. Micro-CT imaging (Bruker, Skycan1172, Germany) was used to scan the OS area of the right tibia in the mouse, and three-dimensional reconstruction was performed using computer software (Bruker, Germany) to observe bone damage.

H&E staining and imaging

The in situ tumor tissue of the mice was fixed and decalcified with EDTA decalcification solution. The heart, liver, spleen, lungs, and kidneys collected from each group fixed with paraformaldehyde were embedded and sectioned. The tumor and organ sections were stained with H&E, and the tumor tissue as well as the heart, liver, spleen, lungs, and kidneys were observed under a Nikon Eclipse Ti microscope at 20x magnification for histological morphology.

Quantification and statistical analysis

All experiments were repeated at least three times. Data were expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism 9 software and statistical differences were analyzed by one-way ANOVA. p < 0.05 was considered as statistically significant. (∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001).

Published: March 11, 2024