Construction of genetically encoded biosensors for monitoring cytosolic and mitochondrial H₂O₂ in response to nanozymes in THP-1 cells

Abstract

Intracellular H₂O₂ levels are tightly regulated and can be modulated by various stimuli. A variety of nanozymes have been revealed with the ability to catalyze substrates of oxidoreductases, mostly including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), and some of them display multienzyme-like properties, which make them highly attractive for biomedical applications. However, the specific manifestations of nanozyemes within cells remain challenging to predict and detect. In this study, we developed a real-time, dynamic, and highly sensitive live-cell biosensor by expressing HyPer7 probe in the cytosol and mitochondria to monitor the cytosolic and mitochondrial H₂O₂ dynamics in a leukemia cell line THP-1. The successful expression of the probes in the cytosol and mitochondria was confirmed using confocal fluorescence microscopy. When the THP-1 cells were exposed to exogenous H₂O₂, the fluorescence intensity at 525 nm upon excitation with 405 nm lasers (referred to as F405) decreased, while that upon excitation with 488 nm lasers (referred to as F488) increased. Using this biosensor, we examined the dynamics of cytosolic and mitochondrial H₂O₂ in response to Daunorubicin, Fe₃O₄ nanozyme with Polyetherimide (PEI)- or Dextran (Dex)-modification, and Prussian blue nanozyme with different diameters. Results indicated that the particle size of PBNPs and surface modification of Fe₃O₄ play critical roles in their intracellular effects on the aspect of H₂O₂ modulation. The live-cell biosensors thus provide a powerful tool for detecting the variations of cytosolic and mitochondrial H₂O₂ in response to nanozymes, thereby facilitating a better understanding of the biological effects of nanozymes and their potential biomedical applications.

Graphical abstract

Keywords

H₂O₂ / Nanozymes / Oxidoreductase / Biosensors

1 INTRODUCTION

Reactive oxygen species (ROS), which originate from molecular oxygen and are produced through redox reactions or electronic excitation, have attracted widespread research attention due to their contentious effects (Sies et al. 2022). ROS are believed to play a role in oxygen toxicity owing to their heightened chemical reactivity. Moreover, they act as intracellular signaling molecules, taking part in various physiological and pathological processes (D'Autréaux and Toledano 2007; Sies et al. 2024). In recent years, it has become evident that using ROS as a blanket term is somewhat imprecise, given that each type of reactive oxygen species possesses distinct properties and functions (Murphy et al. 2022; Sies and Jones 2020).

Among the diverse ROS molecules, hydrogen peroxide (H₂O₂) stands out as the primary ROS involved in the redox regulation of biological activities. H₂O₂ serves as a versatile and pleiotropic physiological signaling agent, functioning as a second messenger in biological processes by reversibly oxidizing specific protein thiolates (Sies and Jones 2020). The intracellular concentration of H₂O₂ is maintained in the low nanomolar range (approximately 1–100 nmol/L) and is under tight control (Parvez et al. 2018). H₂O₂ is produced from various sources within cells, including specific enzymatic sources such as NADPH oxidases (NOXs) (Bedard and Krause 2007), as well as the mitochondrial electrons transport chains (Murphy 2009), and removed via several intrinsic anti-oxidant small molecules and enzymes, including the thioredoxin system and the glutathione system. Other oxidoreductase can also modulate the intracellular H₂O₂ concentration, including peroxidases (POD), superoxide dismutase (SOD), and catalase (CAT). These properties modulated ROS towards different directions, for example, POD catalyzes the oxidation of substrates in the presence of peroxides (mostly H₂O₂ with a few as organic hydroperoxides) (Jiang et al. 2019), therefore consuming hydrogen peroxide to generate other oxides, while SODs disproportionates superoxide radicals into oxygen and H₂O₂ (Jiang et al. 2019) to increase the concentration of hydrogen peroxide, and catalase accelerates the dismutation of H₂O₂ into water and oxygen (Jiang et al. 2019), therefore scavenging local H₂O₂.

It has been well documented that nanozymes hold the ability to catalyze specific biochemical reactions, showing effects similar to natural enzymes (Ren et al. 2022). The most commonly exhibited properties of nanozymes were oxidoreductase-like activity (Jiang et al. 2019), including peroxidases (POD), superoxide dismutase (SOD), and catalase (CAT). Many nanozymes, such as metal (Guan et al. 2024), metal oxides (Gao et al. 2007) and Prussian Blue nanoparticles (Zhang et al. 2016), even exhibit multienzyme-like properties in modulating ROS. However, the specific manifestations of nanozymes within cells remain difficult to predict and detect, because different physicochemical properties lead to differences in intracellular distributions and predominant activities. Therefore, it would be beneficial to monitor the dynamics of H₂O₂ of certain organelles for understanding the intracellular activity of nanozymes, especially those that exhibit oxidoreductase-like activities.

Monocytes as part of the innate immune system are one of the first immune cells that are at the sites of infections contributing to pathogen defense with phagocytosis, cytokine and reactive oxygen species (ROS) production. THP-1 monocytes, isolated from the peripheral blood of a boy with acute myeloid leukemia (Tsuchiya et al. 1980), are widely used as model systems for immunomodulation studies including drug and natural product testing (Schultze et al. 2017). At the same time, THP-1 is a cell line widely used in the investigations of acute myeloid leukemia (Lübbert et al. 1992).

Genetically encoded fluorescent protein sensors have provided major advances in cellular H₂O₂ detection (Bilan and Belousov 2018; Morgan et al. 2016). These probes contain a dithiol switch that changes the overall fluorescence of the probe depending on its oxidation status. High sensitivity and specificity for H₂O₂ have been achieved by coupling a redox-sensitive green fluorescent protein (GFP) mutant to a H₂O₂-sensitive thiol protein, such as oxyR (HyPer series) (Bilan and Belousov 2018), or to a peroxidase such as Orp1 or TSA2 (roGFP2-based probes) (Morgan et al. 2016). Among the several biosensors, HyPer7 is a pH-insensitive, genetically encoded H₂O₂ reporter, which consists of a cyclically permutated GFP with N- and C-terminal OxyR-RD domain derived from Neisseria meningitidis (Pak et al. 2020). Following oxidation by H₂O₂, HyPer7 forms an intramolecular disulfide bridge that alters the excitation spectra, and the maximum excitation of the HyPer7 chromophore shifts from 405 nm in the reduced state to 488 nm in the H₂O₂-oxidized state (Pak et al. 2020; Yang et al. 2023). Here, we constructed real-time, dynamic, and highly sensitive live-cell biosensors to monitor the cytosolic and mitochondrial H₂O₂ dynamics in a leukemia cell line THP-1, utilizing Hyper7 fused with subcellular localization guide peptides mitochondria localization sequence (MLS) and nuclear exclusion sequence (NES) to monitor cytosolic and mitochondrial H₂O₂ dynamics respectively in THP-1 cells, aiming to provide a powerful tool for detecting cytosolic and mitochondrial H₂O₂ in response to nanozymes.

2 MATERIALS AND METHODS

2.1 Cell culture

THP-1 cells were purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in modified RPMI medium (HyClone, Cytiva, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), and 100 μg/mL penicillin-streptomycin (HyClone) in a humidified atmosphere of 5% CO₂ at 37°C. HEK 293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS.

2.2 Construction of cell lines

HyPer7 was fused at the N–terminal of the protein with nuclear export sequence (NES, NSNELALKLAGLDINK) and mitochondrial localization sequence (MLS, MSVLTPLLLRGLTGSARRLPVPRAKIHSL) to express the sensor in the cytosol (CytoHyPer7), mitochondria (MitoHyPer7) of the cell, respectively. In brief, The THP-1 cells constructively expressed CytoHyPer7 and MitoHyPer7 were established by infecting with lentiviral carry the sequences. To obtain lentiviral, the sensor vectors (pLVX-NES-HyPer7, pLVX-MLS-HyPer7) together with three lentiviral packaging vectors (pLPI, pLPII, and pLPVSVG) were used to transfect HEK 293T cells at 50%–60% confluency by Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. The culture supernatant containing recombinant lentivirus was harvested after 72 h. Then THP-1 cells were seeded in six-well culture plates for lentiviral infection in the presence of 4 μg/mL of polybrene (Macgene, Beijing, China) followed by centrifugation at 1000g for 1 h at 37°C. Following the lentivirus infection, cells were cultured for 1 week in media containing 3 μg/mL puromycin. Afterward, the fluorescent cells were sorted by FACS Aria IIIu (BD Biosciences, Franklin Lakes, NJ, USA).

2.3 Nanozymes

Nanozymes used in this study included Prussian Blue Nanoparticles (PBNPs) and Fe₃O₄ nanoparticles, PtNPs, Au@Pt MnO₂, and MnBTC. PBNPs of 3.4 nm, referred to as ultrasmall Prussian Blue Nanoparticles (USPBNPs) were synthesized according to the procedure described previously (Qin et al. 2020). Briefly, to prepare PBNPs, 0.75 g of PVP and 0.0275 g of K₃[Fe(CN)₆] were dissolved in 10 mL of ethanol solution. After stirring at room temperature for half an hour, the mixture was heated at 80°C for 20 h. The blue product was collected by centrifugation and washed several times with double-distilled water (ddH₂O). PBNPs, PEI- and Dex-modified Fe₃O₄ nanoparticles were purchased from Nanjing NanoEast Biotech Co. LTD. Transmission electron microscopy (TEM) and Dynamic Light Scatter (DLS) were utilized to characterize the shape, size, and Zeta potential of the nanoparticles, TEM images of Fe₃O₄ were provided by the supplier. PtNPs and Au@Pt were synthesized by the methods described previously (Wen et al. 2020). MnO₂ and MnBTC were kindly gifted by Prof. Lianbing Zhang (Chen et al. 2024).

2.4 Fluorescence microscope

Fluorescence microscopes were utilized to observe the intracellular location of HyPer7. For THP-1-Mito-HyPer-7, the cells were stained with 200 nmol/L MitoTracker® Red CMXRos (#M7512, Invivogen) for 20 min according to the instructions before fixing. Both cells were then fixed with 1 mL of 100% pre-cooled methanol for 10 min and washed with PBS. The cells were then resuspended with 1 mL of PBS, and 200 μL of the cell suspension was subjected to cytospin. The cells were mounted with a mounting medium containing DAPI (#ZLI-9556, Zhongshan Golden Bridge) and covered with a coverslip for observation. The slides were observed and photographed using a confocal microscope (Leica TCS SP8 STED, Leica) under the conditions of Ex/Em = 350/450 nm (For DAPI), 488/525 nm (For HyPer7), and 577/602 nm (For MitoTracker® Red).

2.5 Flow cytometry

To detect the responsiveness of the biosensors to exogenous H₂O₂, THP-1-CytoHyPer7 cells and THP-1-MitoHyPer7 cells with a density of 4 × 10⁵ cell/mL were treated with H₂O₂ (10011218, Sinopharm Chemical Reagent Co., Ltd.) at concentrations ranging from 1 μmol/L to 400 μmol/L for 2 min. The fluorescence in the cells was detected by flow cytometry (CytoFLEX, Beckman Coulter) using 405 and 488 nm as excitation lights and collecting the emission light through 525/50 nm and 530/30 nm bandpass filters, respectively. Imaging flow cytometry (ImageStream^X MarkⅡ, Merk) was also used to photograph the emission fluorescence at 525 nm upon 488 nm excitation after being incubated with 100 μmol/L H₂O₂.

To monitor the dynamics of cytosolic and mitochondrial H₂O₂ induced by chemotherapeutics reagents and nanozymes, THP-1-CytoHyPer7 cells and THP-1-MitoHyPer7 cells with a density of 4 × 10⁵ cell/mL were incubated with different concentrations of chemotherapeutics reagents and nanozymes. After co-incubation for 6, 24, and 48 h, the cells were collected by centrifugation, washed once with PBS, and detected by flow cytometry. The experimental data were analyzed using FlowJo (V10).

2.6 Statistics

All data were expressed as the mean ± standard deviation (SD) for at least triplicate experiments. Statistical analysis was performed in Graphpad Prism 8.3.0. To compare the means of three or more groups defined by one factor, One-way ANOVA was employed and followed by Dunnett post-hoc test to compare the means of a prespecified pair of columns. P < 0.05 is considered statistically significant.

3 RESULTS AND DISCUSSION

3.1 Cytosolic and mitochondrial localization of Hyper7 probe in THP-1 cells

Hyper7 has been widely employed as H₂O₂ biosensors in model organisms including yeasts (de Cubas et al.2021; Kritsiligkou et al.2021, 2023), Arabidopsis thaliana (Dopp et al.2023), Mus musculus (Kano et al.2024; Li et al.2022), Zebrafish Danio rerio (Sergeeva et al.2025), as well as cell lines in culture including human umbilical vein endothelial cells (HUVEC) (Jacobs et al.2022; Waldeck-Weiermair et al.2022), mouse hepatocytes (AML12 cells) (Shashkovskaya et al.2023), hippocampal neurons (Kotova et al.2023), human iPCs derived spheroids (Usatova et al.2024). In this study, we applied Hyper7 to monitor the dynamics of cytosolic and mitochondrial H₂O₂ in THP-1 cells. The cells expressing Cyto-Hyper7 (upper column in Fig. 1) exclusively exhibited a uniform cytosolic distribution of fluorescent protein sensor signals (green in Fig. 1) compared to the DAPI-stained nucleus (blue in Fig. 1). Mito-Hyper7 (down column in Fig. 1) was intended to target the mitochondria and colocalize with Mito-Tracker Red (red in Fig. 1), and the PCC between the Mito-HyPer7 probe and MitoTracker signal was recorded as 0.90, indicating a collocation. These images clearly confirmed the correct distribution of Cyto-HyPer7 and Mito-HyPer7 in the cytosol and mitochondria, and the successful construction of the biosensors.

3.2 Responsiveness of the biosensors to exogenously added hydrogen peroxide

H₂O₂ can diffuse from extracellular space into cytosol and further into mitochondria (Pak et al.2020). To verify whether the biosensors can respond to the perturbation of H₂O₂, we added exogenously H₂O₂ to the culture medium, and results showed that externally added H₂O₂ caused a detectable oxidation of both probes. Upon oxidation, the excitation spectra of HyPer7 changed with a decrease at 405 nm and an increase of the 488 nm peak, while the emission spectra are similar in both states, peaking at 525 nm (Fig. 2A). Therefore, we detected fluorescence upon the excitation with lasers of 405 nm (referred to as F405) and 488nm (referred to as F488). It was shown that the fluorescence upon 405 nm excitation was reduced while that upon 488 nm was increased when the cells were incubated with exogenous H₂O₂ (Figs. 2B and 2C). Images acquired from the imaging flow cytometry (Figs. 2D and 2E) supported the results of flow cytometry, showing that the fluorescence of the cells exposed to 100 μmol/L H₂O₂ was brighter than that of the control cells.

Having confirmed that HyPer7 is expressed and responsive, we performed a titration experiment to determine the minimal amount of exogenous H₂O₂ that is required to elicit a detectable probe response and the maximal detectable H₂O₂ concentration. We used flow cytometry to evaluate the variation in fluorescence intensity after co-incubation with H₂O₂. For THP-1-CytoHyPer7, the minimal detectable amount of exogenous H₂O₂ was 10 μmol/L, and F488 increased along with the climb of the H₂O₂ concentration when the concentration of exogenous H₂O₂ ranged from 10 to 40 μmol/L, and when the exogenous H₂O₂ concentration exceeds 40 μmol/L, the fluorescence intensity reached a plateau without further enhancement (Fig. 3A). For THP-1-MitoHyPer7, the minimal detectable amount of exogenous H₂O₂ was 10 μmol/L, and the detectable range was 10 to 100 μmol/L (Fig. 3B). To make it simpler, we used the normalized fluorescence ratio of F488 to F405 (F488/F405) to characterize the relative H₂O₂ compared to the untreated cells (Figs. 3C and 3D), the maximal F488/F405 of THP-1-CytoHyPer7 and THP-1-MitoHyPer7 reached 6.83 and 9.49, respectively.

3.3 Monitor H₂O₂ level in response to chemotherapeutic agents

Most chemotherapeutic agents were reported to induce intracellular ROS accumulation through several mechanisms. Herein, we employed Daunorubicin (DNR) to monitor the dynamics of cytosolic and mitochondrial H₂O₂. It is well known that DNR plays its cytotoxicity by increasing intracellular ROS (Burt et al.2019), however, the subcellular compartment of ROS generation was unclear. Our results from the CCK8 assay indicated the IC₅₀ at 24 h was 161.4 nmol/L (Fig. 4A). By using the biosensors established in this study, we found out that in the dose range of 20 nmol/L to 200 nmol/L, the cytosolic and mitochondrial H₂O₂ remained unchanged after treated for 6 h and 24 h (Figs. 4B and 4C). Only a slight increase of F488/F405 was observed in the cytosolic H₂O₂ after treated for 48 h while a dramatic increase of 1.55 folds was observed in the mitochondrial H₂O₂ (Fig. 4D). It should be noted that in the 6-h experiment, dosages were increased up to 1.5 mmol/L. Results showed that the high doses of DNR increased cytosolic and mitochondrial H₂O₂ at the same time, and 1.5 mmol/L of DNR resulted in 1.33 folds and 1.21 folds of cytosolic and mitochondrial H₂O₂ compared with the untreated cells, respectively. These results indicated the long-term effects on H₂O₂ occurred primarily in mitochondria while the short-term and high-dose effects occurred in both mitochondria and cytosol. Moreover, the increase of cytosolic H₂O₂ induced by DNR could be partly reversed by N-acetylcysteine (NAC), indicating that the sensor cells were capable of sensing and detecting the attenuation of H₂O₂ induced by antioxidant (Fig. 4E).

Since ROS has been identified as one of the common mediators for chemo-resistance in leukemia (Trombetti et al. 2021), the constructed biosensors offer a powerful platform to monitor the dynamic of cytosolic and mitochondrial H₂O₂ in resistant or sensitive cells and to continuously monitor the adjustment of H₂O₂, which will be helpful to reveal the mechanism of chemotherapy resistance in leukemia.

3.4 Monitor H₂O₂ level in response to nanozymes

Next, we applied the biosensors to monitor H₂O₂ dynamics after co-incubation with several nanozymes that have multi-enzyme properties to affect intracellular H₂O₂ concentrations. Fe₃O₄ nanoparticle was the first nanozyme reported with POD-like activity (Gao et al.2007), and further investigations revealed its POD-like activity under the acidic environment (pH = 4.8) and CAT-like activity in neutral conditions (pH = 7.4) (Chen et al.2012). Herein, by using the biosensors, we detected the dynamics of cytosolic and mitochondrial H₂O₂ after incubation with Fe₃O₄ nanoparticles coated with PEI (referred to as PEI-Fe₃O₄) and Dextran (referred to as Dex-Fe₃O₄). The diameters of both Fe₃O₄ nanoparticles were less than 20 nm under TEM (Figs. 5A and 5B). Their hydrodynamic diameters were 46.57 ± 1.31 nm and 27.95 ± 1.29 nm determined by DLS. Dex-Fe₃O₄ was negatively charged with zeta potentials of −20.29 ± 5.00 mV, while PEI-Fe₃O₄ was positively charged with zeta potentials of 13.18 ± 3.39 mV. After co-incubation with Dex-Fe₃O₄ for 6 h, cytosolic and mitochondrial H₂O₂ were scavenged at the same time and decreased further after 24 h (Figs. 5C−5E). PEI-Fe₃O₄ acted differently from Dex-Fe₃O₄. The variation in mitochondrial H₂O₂ was less than 1% after incubation with PEI-Fe₃O₄ for 6 h, which can be considered unchanged (Fig. 5F). At the same time, the variation of cytosolic H₂O₂ was uncommon, that was, it decreased by 5% upon being treated with 10 mg/L PEI-Fe₃O₄, while as the concentration climbed to 20 mg/L and 40 mg/L, H₂O₂ gradually rose and became comparable to the control group in the 40 mg/L PEI-Fe₃O₄ group (Fig. 5F). After co-incubation for 24 h, the cytosolic H₂O₂ was diminished compared to the untreated cells (Fig. 5G). As the incubation time was extended to 48 h, the concentration of mitochondrial H₂O₂ decreased too (Fig. 5H). Therefore, it is plausible to consider that the overall H₂O₂ was attributed to both the nanozyme’s properties and the surface modification of the nanoparticles.

Surface modification-dependent effects (Fig. 5) highlight the need for quick screening for the intracellular effects of nanozymes, especially those that could modulate ROS. The biosensors provide a standardized system to guide surface engineering to minimize unintended ROS modulation.

PBNPs were reported for their ability of scavenging ROS both in tubes as well as in cells (Zhang et al.2016). In this study, we detect the intracellular effects of two PBNPs with different diameters by using the established biosensors. The Prussian Blue nanoparticles exhibit a sub-spherical shape under TEM with a diameter of about 60 nm, and the hydrodynamic diameter was determined to be 92.23 ± 4.33 nm (Fig. 6A). Ultrasmall Prussian Blue nanoparticles exhibit a cluster-like shape under TEM with a diameter of about 5 nm, and the hydrodynamic diameter was determined to be 34.73 ± 7.96 nm (Fig. 6B). PBNPs and USPBNPs were both negatively charged with zeta potentials of −12.08 ± 0.97 mV and −40.71 ± 1.43 mV, respectively. We found out that the PBNPs didn’t change cytosolic or mitochondrial H₂O₂ after 6 h incubation and eventually scavenged H₂O₂ by 13% after being treated for 48 h (Figs. 6C−6E). However, it is interesting to see that USPBNPs exhibited different overall effects on the intracellular H₂O₂. The cytosolic and mitochondrial H₂O₂ was elevated upon co-incubation with USPBNPs (Figs. 6F−6H), which suggested that the particle size of nanozyme also played a role in regulating intracellular H₂O₂, though the high POD-like and CAT-like activities were demonstrated in USPBNPs over PBNPs.

Mn-based and Pt-based nanozymes were also detected with the biosensors. After co-incubation for 24 h, the PtNPs caused an increase in both cytosolic and mitochondrial H₂O₂. MnO₂ and Au@Pt decreased cytosolic H₂O₂ without changing mitochondrial H₂O₂, while MnBTC increased mitochondrial H₂O₂ without changing cytosolic H₂O₂ (Fig. 7).

4 CONCLUSION

In summary, this work constructed the genetically encoded fluorescent sensors CytoHyper7 and MitoHyper7 to detect cytosolic and mitochondrial H₂O₂ levels. The performance of the sensors was characterized by fluorescent spectroscopy, and the responses to chemotherapeutics DNR and nanozymes of PBNPs with different diameters and iron oxide nanoparticles with different surface modifications were revealed. Results obtained by using the biosensors indicated that the particle size of PBNPs and surface modification of Fe₃O₄ play critical roles in their intracellular effects on the aspect of H₂O₂ modulation.

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