Synthesized manganese oxide nanorods: Fabrication, characterization, application in cardiomyocyte protection from oxidative stress during sepsis, and evaluation of biochemical aspects of hemoglobin interaction

2.1 Materials

Human hemoglobin (CAT Nr.: H7379), manganese (II) sulfate (MnSO₄, ≥99.99 % trace metals basis, CAT Nr: 229784), potassium permanganate (KMnO₄, ≥ACS reagent, ≥99.0 %, CAT Nr: 223468) were purchased from Sigma Aldrich (USA). All other chemicals used were of analytical grade.

2.2 Synthesis of Mn₃O₄ nanorods

Mn₃O₄ nanorods were prepared through the hydrothermal method as previously described with some modifications (El-Said et al., 2022). Briefly, MnSO₄ (1.69 g), KMnO₄ (3.1 g), and H₂O₂ (50 mL) were dissolved in deionized water (60 mL), followed by stirring for 60 min, transferring into the autoclave vessel (80 °C, 2 h). The samples were then washed with deionized water and ethanol and then dried in an oven at 80 °C (El-Said et al., 2022).

2.3 Characterization of synthesized Mn₃O₄ nanorods

The structural and colloidal features of synthesized Mn₃O₄ nanorods were determined through several analyses. Transmission electron microscopy (TEM) analysis was done at room temperature after drying a droplet of samples on the TEM grid. The image was then obtained using a JEOL 200CX at 80 kV. Dynamic light scattering (DLS) analysis was done at room temperature to determine the diameter and zeta potential value of synthesized Mn₃O₄ nanorods at aqueous using a Brookhaven Instrument. X-ray diffraction (XRD) analysis was done to determine the crystalline phase of fabricated Mn₃O₄ nanorods using a PANalytical X’Pert Pro with a Cu X-ray source (45 kV, 40 mA). UV–Vis spectrophotometry was used to determine the absorption characteristics of synthesized Mn₃O₄ NPs using a CARY 50–BIO UV–Vis spectrophotometer (Varian, Australia). FTIR analysis was also done on a Thermo scientific™ Nicolet iS™50 FTIR Spectrometer.

2.4 Preparation of solutions

Mn₃O₄ nanorods were first exposed to UV for 50 min and then suspended in cell culture medium or phosphate-buffered saline (PBS; 0.9 % NaCl in 10 mM sodium phosphate buffer, pH 7.4) followed by sonication (30 min at 50 W) for cell culture or protein binding assays, respectively. Hemoglobin solution was also prepared in PBS (0.9 % NaCl in 10 mM sodium phosphate buffer, pH 7.4).

2.5 Cell culture

AC16 human cardiomyocyte cells were used to explore the antioxidant activity of Mn₃O₄ nanorods. AC16 cells obtained from American Type Culture Collection (ATCC, Virginia 20110–2209, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and 1 % penicillin–streptomycin solution at 37 °C and 5 % CO₂.

2.6 Oxidative stress model stimulated by H₂O₂

After culturing the cells, sepsis-induced oxidative stress model was performed by incubating AC16 cells with a fixed concentration of H₂O₂ (0.5 mM) for 6 h. AC16 cells without any H₂O₂ addition were considered as a control group.

2.7 Cell viability assay

The MTT assay was performed to assess the influence of H₂O₂ and Mn₃O₄ nanorods on the viability of AC16 cells. In the nanorod-related analyses, cells were incubated with increasing concentrations of Mn₃O₄ nanorods (0.1–50.0 μg/mL) for 24 h. In protective assays, AC16 cells were pretreated with Mn₃O₄ nanorods in a fixed concentration of 5 μg/mL (the maximum concentration of Mn₃O₄ nanorods with no significant cytotoxicity) for 18 h, followed by addition with 0.5 mM H₂O₂ for additional 6 h. Cells without any treatment were used as a control group. The cells were then added with MTT (5.0 mg/mL) for 4 h followed by replacing the medium with DMSO (150 μL) for 10 min. Finally, the absorbance of samples was read at 570 nm on a microplate reader (Multiskan™ GO, Thermo Scientific, Waltham, MA, USA).

2.8 ROS assay

Intracellular ROS levels in AC16 cells (1 × 10⁴ cells/well, 96-well plate) were assessed using 2ʹ,7ʹ-dichlorofluorescein diacetate (DCF-DA) detection kit (Sigma-Aldrich, USA) based on the manufacturer’s protocols. Briefly, after pretreating the cells with Mn₃O₄ nanorods with a concentration of 5 μg/mL for 18 h and further incubation with 0.5 mM H₂O₂ for 6 h, the cells were added by 0.3 mL of DCFH-DA probe for 30 min. Afterward, the cells were exposed to washing at least two times with DMEM. The fluorescence intensity was then measured using a fluorometer (Invitrogen, Carlsbad, CA, USA) at 485Ex/535Em nm.

2.9 ELISA assay for measuring the content of SOD, CAT and GSH

AC16 cells were seeded (1 × 10⁶ cells/well in a 6-well plate) and treated. Then, the cells were lysed using RIPA lysis solution, and the levels of antioxidant enzymes [SOD (Catalog number: A001-1) and CAT (Catalog number: A007-2)], and non-enzymatic antioxidant [GSH (Catalog number: A006-1)] in AC16 cells were determined based on the protocols of assay kits (Nanjing Jiancheng Bioengineering Research Institute, Jiangsu, China).

2.10 Caspase-3 activity assay

The activity of caspase-3 was assessed using a Caspase-3 Activity Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, 40 μL of the cells (1 × 10⁴ cells/well in a 96-well plate) lysed using RIPA lysis solution lysates were added by 10 μL Ac-DEVD-PNA (2 mM) and incubated for 60 min. Finally, the activity of caspase-3 was determined at 570 nm on a microplate reader (Multiskan™ GO, Thermo Scientific, Waltham, MA, USA).

2.11 Fluorescence spectra

All fluorescence studies were performed on the F-7000 spectrofluorometer (Hitachi, Japan) employing a quartz cuvette of 1 cm path length. The excitation wavelength was fixed at 280 nm and the fluorescence spectra of hemoglobin (3 µM) either alone or with different concentrations of Mn₃O₄ nanorods (1–30  µM) was read in the wavelength range of 310–410 nm with a scan speed of 600 nm/min. The fluorescence experiments were done at 298 K, 305 K, and 310 K.

2.12 Circular dichroism (CD) studies

CD spectra of hemoglobin (3 µM) either alone or with a fixed concentration of Mn₃O₄ nanorods (30  µM) were read on JASCO J1500 CD spectrometer with a quartz cuvette of 1 mm path length. The wavelength range was set at 190–250 nm and the scan speed was fixed at 100 nm/min. All CD spectra were read at 298 K under continuous nitrogen flow.

2.13 Molecular docking study

A cylindrical (rod) Mn₃O₄ cluster with a 1 nm diameter and 4 nm length as well as a spherical Mn₃O₄ cluster with a dimension of 2 nm were constructed in Materials Studio software via repetition of the Mn₃O₄ oxide unit cell. All other Mn₃O₄ clusters were derived from cylindrical Mn₃O₄ clusters. The X-ray crystallographic 3D structure of human normal adult hemoglobin (PDB ID: 2H35) was downloaded from the online Protein Data Bank RCSB PDB (https://www.pdb.org). The docking simulations were performed on AutoDock Vina software. The docking box was centered on the protein structure and set to 80 x 70 x 80 Å with a spacing of 1 Å between grid points. The exhaustiveness parameter was set to 8 to ensure that the docking simulations were thorough. The binding energy and interaction forces were analyzed using Discovery Studio.

2.14 Statistical analysis

All assays in this study were done in triplicates and data were expressed as the mean ± standard deviation (SD) and the outcomes were analyzed by Student's t-test. The data were considered to be statistically significant at P≤0.05.

3.1 Characterization of Mn₃O₄ nanorods

As a result of the importance of metal oxide nanoparticles in the development of potential antioxidant platforms and evaluation of their pharmacokinetic properties, the current report attempted to explore the characterization and antioxidant/protein binding properties of Mn₃O₄ nanorods prepared by hydrothermal method.

The absorption spectrum of Mn₃O₄ nanorods was analyzed using a UV–Vis spectrometer, in order to reveal their optical characteristics. In Fig. 1A the UV–Vis spectrum of Mn₃O₄ nanorods showed an absorption peak in the range of 260 to 420 nm deriving from charge transferrin between O²⁻, Mn²⁺ and Mn³⁺ species (Vázquez-Olmos et al., 2005, Ghosh et al., 2017). Also, another absorption peak ranging from 420 nm to 700 nm can be associated with the d–d crystal field transitions observed on octahedral Mn³⁺ species (Vázquez-Olmos et al., 2005, Giri et al., 2014, Ghosh et al., 2017).

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Fig. 1

Characterization of Mn₃O₄ nanorods through the hydrothermal method. (A) UV–vis spectroscopy, (B) Fourier transform-infrared spectroscopy (FTIR), (C) X-ray diffraction (XRD), (D) TEM analysis, (E) Zeta-sizer analysis for hydrodynamic size determination, (F) Zeta-sizer analysis for zeta potential determination.

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The FTIR spectrum of Mn₃O₄ nanorods is shown in Fig. 1B. The successful synthesis of Mn₃O₄ nanorods was further supported by the bands presented in the range of 410 cm⁻¹ to 621 cm⁻¹. The Mn–O bond shows one stretching mode at around 621 cm⁻¹ in a tetrahedral environment and one distortion vibration at around 510 cm⁻¹ in an octahedral environment (Xing et al., 2011, Ghosh et al., 2017, Wang et al., 2021). Indeed, the peaks centered at around 510 cm⁻¹, 629 cm⁻¹ and 410 cm⁻¹ can be characteristics of the vibration of manganese species (Mn₃⁺) in an octahedral state (Ghosh et al., 2017, Wang et al., 2021). Moreover, the characteristic bands at around 3420 and 1619 cm⁻¹ were attributed to the hydroxyl groups (Shaik et al., 2021). These FTIR spectrum-based results are reliable with the data achieved from UV–vis characterization outcomes.

Furthermore, the crystallinity of Mn₃O₄ nanorods produced via hydrothermal method was assessed using XRD techniques as presented in Fig. 1C. XRD analysis indicated the presence of 13 distinct peaks attributed to 112, 200, 103, 211, 004, 220, 105, 312, 303, 321, 224, 400, and 411 crystalline phases (Fig. 1C), which is consistent with XRD values reported for Mn₃O₄ NPs (JCPDS No.24–0734) and previous studies (Shaik et al., 2021, Yewale et al., 2022).

Additionally, a TEM investigation was done to determine the shape and diameter of prepared Mn₃O₄ nanorods. The synthesized particles, as depicted in Fig. 1D, had a rod-like morphology and a diameter range of 10 to 75 nm. In general, it was realized that the size of synthesized nanorods was less than 80 nm. A variation in length was also found from 50 nm to greater amounts among the synthesized nanorods.

Furthermore, DLS analysis was used to determine the hydrodynamic radius of Mn₃O₄ nanorods, which revealed that the hydrodynamic size of prepared nanorods was 371.7 nm (Fig. 1E). According to the data, synthesized Mn₃O₄ nanorods may exhibit an agglomeration tendency in aqueous media. However, zeta potential analysis demonstrated that the charge distribution of fabricated Mn₃O₄ nanorods was about −43.3 mV (Fig. 1F), indicating the presence of a high surface charge to stabilize the nanoparticles against agglomeration (Yang et al., 2018). Therefore, it can be suggested that the prepared nanorods have good colloidal stability and dispersion properties.

In addition, Yang et al. reported the synthesis of Mn₃O₄ in the form of nanoparticles via laser ablation method and indicated the presence of large coercivity and exchange bias in the structure of synthesized NPs (Yang et al., 2019). Furthermore, El-Said et al. reported that the hydrothermal-based synthesis of Mn₃O₄ nanorods with an average diameter of 100 ± 30 nm, evidenced by SEM imaging (El-Said et al., 2022), which is almost comparable with our TEM data. However, the authors did not report any DLS data to enable us to compare the hydrodynamic radius of prepared nanorods.

3.2 Effects of Mn₃O₄ nanorods on the viability of AC16 cells

In order to choose a suitable and safe concentration of Mn₃O₄ nanorods for protective effects against oxidative damage in AC16 cells, the cell viability was assessed by a well-known MTT assay. As displayed in Fig. 2A, the cell viability significantly declined when treated with Mn₃O₄ nanorods at concentrations of 10 and 50 µg/mL in comparison with the control group. Whereas, no significant differences in AC16 cell viability were detected in the range of 0.1 to 5 µg/mL of Mn₃O₄ nanorod treatments. Therefore, 5 µg/mL of Mn₃O₄ nanorods was determined as the appropriate concentration for inducing protective effects against sepsis-induced oxidative damage model in vitro in this study.

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Fig. 2

AC16 cell viability in the presence of varying concentrations of Mn₃O₄ nanorods (0.1–50 µg/mL) after 24 h, evidenced by MTT assay (A). Protective effects of Mn₃O₄ nanorods (5 µg/mL) against H₂O₂ (0.5 mM)- induced cytotoxicity in AC16 cells, measured by MTT assay (B). Cells were pretreated with Mn₃O₄ nanorods (5 µg/mL) for 18 h and then added by H₂O₂ (0.5 mM) for additional 6 h. ROS assay for determination of oxidative stress induced by H₂O₂ (0.5 mM) and the protective effects of Mn₃O₄ nanorods pretreatment, assessed by DCF fluorescence intensity measurement (C). The contents of SOD (D), CAT (E), and GSH content (F) in AC16 cells treated with different samples, analyzed by ELISA assay. *p < 0.5, **p < 0.01, ***p < 0.001.

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3.3 Effects of Mn₃O₄ nanorods on H₂O₂-triggered AC16 cytotoxicity

H₂O₂ has been widely used to induce oxidative damage during sepsis in vitro (Ben Saad et al., 2019). Hence, in this study, H₂O₂ was used to trigger sepsis-induced oxidative stress model in AC16 cells, in vitro. Then, the protective effect of Mn₃O₄ nanorods on H₂O₂ (0.5 mM)- stimulated cytotoxicity and oxidative stress in AC16 cells were assessed by cell viability and DCF assays. As exhibited in Fig. 2B, in comparison with the control, the incubation of the cells with 0.5 mM H₂O₂ for 6 h significantly (***p < 0.001) mitigated the cell viability to 62.16 ± 6.47 %, while pretreatment with Mn₃O₄ nanorods for 24 h revealed a significant recovery (**p < 0.01) in the cell viability compared to the H₂O₂ group, indicating that Mn₃O₄ nanorods may potentially prevent cytotoxicity in AC16 cells. Indeed, in the H₂O₂/ Mn₃O₄ nanorods treated group, the cell viability was calculated to be 89.10 ± 4.29 %, which was about 1.43- folds higher than the H₂O₂ group (**p<0.01). This data might be attributed to the promising antioxidant activity of Mn₃O₄ nanorods during sepsis, which was well-supported by the outcome of Singh et al. (Singh et al., 2019) and Adhikari et al. (Adhikari et al., 2017).

3.4 Effects of Mn₃O₄ nanorods on the generation of intracellular ROS

The incubation of Ac16 cells with H₂O₂can result in stimulation of the generation of excessive ROS during sepsis. At normal conditions due to low activity of antioxidant enzymes in AC16 cells, the ROS generated inside the cells can attack the intracellular components and lead to oxidative stress/damage during sepsis. ROS, a crucial indicator of oxidative stress, is closely associated with heart disorders (Takano et al., 2003, Lin et al. 2013). Fig. 2C demonstrates the effects of Mn₃O₄ nanorods on ROS production of AC16 cells in response to H₂O₂-triggered oxidative stress. The level of ROS was significantly elevated in H₂O₂-treated cells (***p < 0.001), which were 5.38-fold higher compared to the control group. A probable suggestion for this phenomenon is that the addition of H₂O₂causes an oxygen reaction in the cells, leading to producing excessive amounts of ROS (Yoon et al., 2019). Whereas Mn₃O₄ nanorods pretreatment apparently (**p < 0.01) inhibited H₂O₂-triggered elevation of ROS level (Fig. 2C). These outcomes proposed that Mn₃O₄ nanorods played a protective effect in H₂O₂-induced AC16 cells by reducing the generation of ROS.

3.5 Effects of Mn₃O₄ nanorods on the levels of SOD, CAT and GSH

SOD, CAT, and GSH, crucial free radical scavengers, play a key role in inhibiting oxidative damage during sepsis (He, He et al. 2017, Kumar et al., 2018, Keshani et al., 2024). Importantly, SOD and CAT as antioxidant enzymes and GSH as a non-enzymatic antioxidant biomolecule can serve as free radical scavengers (Irato and Santovito 2021). Therefore, evaluating the levels of these biomolecules can be used as a potential indicator for determining the antioxidant activities of Mn₃O₄ nanorods. In this study, the influences of Mn₃O₄ nanorods on the levels of SOD, CAT and GSH in H₂O₂-triggered AC16 cells were assessed. As depicted in Fig. 2D, the SOD activity was significantly (*p < 0.05) declined in H₂O₂-triggered AC16 cells in comparison with the control cells, however promisingly increased in Mn₃O₄ nanorods-pretreated groups. The content of SOD was 83.52 ± 2.84 % in the Mn₃O₄ nanorods-pretreated groups, which was significantly higher than the H₂O₂ group (*p < 0.05), while lower than the control group (*p < 0.05).

CAT activity was also determined to be increased significantly in the Mn₃O₄ nanorods-pretreated group relative to the H₂O₂ group (**p < 0.01) (Fig. 2E), which well-agreed with the SOD results (Fig. 2D).

In Fig. 2F, the GSH level was detected to have a 2.18-fold reduction in the H₂O₂ group in comparison with the control (***p < 0.001). After pretreatment with Mn₃O₄ nanorods, the level of GSH was enhanced, with a GSH level of 74.21 ± 13.81 %, significantly higher than that of the H₂O₂ group (45.73 ± 7.61 %) (**p < 0.01), which was the same as the SOD data.

In general, synthesized Mn₃O₄ nanorods showed potential antioxidant activity. Indeed, the aforementioned outcomes manifested that incubation with H₂O₂ could remarkably decrease the levels of SOD, CAT and GSH, while pretreatment with Mn₃O₄ nanorods could apparently recover the levels of these biomolecules in H₂O₂-stimulated cells as a model of oxidative stress during sepsis, indicating that Mn₃O₄ nanorods pretreatment may protect the cells against oxidative damage during sepsis through intensifying cellular antioxidant activity.

3.6 Caspase-3 activity

One common feature of cardiomyocyte oxidative damage during sepsis is apoptosis (Zhang et al., 2024). In fact, a number of studies indicate that ratio of Bax/Bcl-2, overexpressed by an excessive amount of ROS, induces the initiation of apoptosis by elevation of caspase-3 activity (Li et al. 2021, Fan et al., 2023). It has been shown that some modified nanoparticles can mitigate oxidative stress and apoptosis in myocardial cells during sepsis (Xiao and Chen 2020, Wang et al., 2022b). To further evaluate whether Mn₃O₄ nanorods are able to protect AC16 cardiomyocytes from H₂O₂-triggered apoptosis, we used a caspase-3 activity assay. As shown in Fig. 3, the activity of caspase-3 activity in the H₂O₂ group is markedly enhanced from 100 % to 333.12 ± 32.53 % in comparison with the control group, while Mn₃O₄ nanorods pretreatment reduced apoptosis induction on AC16 cells (216.56 ± 22.72 %). Indeed, Mn₃O₄ nanorods pretreatment led to a significant decrease in apoptosis rate (1.53-fold) compared to the H₂O₂ group (Fig. 3).

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Fig. 3

Caspase-3 activity assay in AC16 cell treated with H₂O₂ (0.5 mM) for 6 h, Mn₃O₄ nanorods for 24 h, or pretreated with Mn₃O₄ nanorods (5 µg/mL) for 18 h and then added by H₂O₂ (0.5 mM) for additional 6 h. **p < 0.01, ***p < 0.001.

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3.7 Mechanism of interaction of Mn₃O₄ nanorods with human hemoglobin

In the present study, we then analyzed the binding mode of the Mn₃O₄ nanorods to hemoglobin under simulated physiological conditions through intrinsic fluorescence measurements as well as CD study combined with molecular docking studies. Poor solubility of Mn₃O₄ nanorods in aqueous buffer solutions along with their heterogenous distribution disables the application of different types of calorimetry-based techniques for biomolecular interaction study. Then, we explored the consequences of Mn₃O₄ nanorod interaction and inspected their influence on the spatial and secondary structure of the protein by standard spectroscopic approaches. The purchased human hemoglobin was used to study the effects of prepared Mn₃O₄ nanorod on protein and we then compared our data with the literature, i.e., the binding affinity of the human hemoglobin–different nanoparticles.

3.7.1

3.7.1 Fluorescence quenching study

Quenching investigation of protein fluorescence induced by nanoparticles is known as a convenient approach for studying ligand-receptor interactions. The hemoglobin with a tetramer structure is composed of two αβ dimers. It has been shown that each dimer typically has three tryptophan (Trp) amino acid residues, namely α-Trp-14, β-Trp-15, and β-Trp-37. Moreover, five tyrosine (Tyr) amino acid residues are located in each αβ dimer: α-Tyr-24, α-Tyr-42, α-Tyr-140, β-Tyr-34, and β-Tyr-144 (Platanić Arizanović et al., 2023). Therefore, by excitation of the protein samples at 280 nm and the resultant changes in the intrinsic fluorescence of hemoglobin at 340 nm in the presence of the Mn₃O₄ nanorods may provide us with some useful detail regarding the local microenvironment of the Trp and Tyr moiety during binding (Cao et al., 2021, Li, Guo et al. 2022). Fig. 4 shows the typical quenching of hemoglobin fluorescence upon addition of increasing concentrations of the Mn₃O₄ nanorods. The optimum concentration of hemoglobin was fixed at 3 µM to reduce the auto-quenching of Trp/Tyr fluorescence by heme moieties (Hirsch and Nagel 1981, Platanić Arizanović et al., 2023). Human hemoglobin showed a strong fluorescence spectrum with a maximum emission at around 340 nm upon excitation at 280 nm, and Mn₃O₄ nanorods displayed a relatively low fluorescence intensity in comparison with the protein. Also, the probable fluorescence intensities of Mn₃O₄ nanorods were subtracted from the corresponding protein spectra. It was shown that Mn₃O₄ nanorods resulted in the fluorescence quenching of hemoglobin in a concentration-mediated manner at three temperatures of 298 K (Fig. 4A), 305 K (Fig. 4B), and 310 K (Fig. 4C), while no significant shift of the peak maximum was detected at all three temperatures. The data suggested that Mn₃O₄ nanorods interact with human hemoglobin and the fluorescence quenching could derive from a certain protein-nanorod complex formation (Li, Guo et al. 2022, Platanić Arizanović et al., 2023).

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Fig. 4

Intrinsic fluorescence spectroscopy analysis of hemoglobin (3 µM) either alone or with different concentrations of Mn₃O₄ nanorods (1–30  µM) at 298 K (A), 305 K (B), and 310 K (C). The excitation wavelength was fixed at 280 nm.

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3.7.2

3.7.2 Quenching mechanism

The Stern-Volmer Eq. (1) was used to determine the quenching mechanism of human hemoglobin induced by Mn₃O₄ nanorods as follows (Platanić Arizanović et al., 2023):

(1)

$$F_0/F=1+k_q\times {\tau }_0\left[Q\right]=1+K_{\mathit{SV}}\times \left[Q\right]$$ where F₀ and F are receptor fluorescence at 340 nm without and with Mn₃O₄ nanorods, respectively, k_q defines the quenching rate constant, τ₀ is the average lifetime of the hemoglobin (10⁻⁸), [Q] is the quencher (nanorod) concentration, and finally K_SV is the Stern-Volmer dynamic quenching constant, which can be determined from the slope of the linear F₀/F against [Q] plot. The Stern-Volmer plots of the human hemoglobin–Mn₃O₄ nanorod complex at different temperatures were shown to be almost linear for Mn₃O₄ nanorod concentrations up to 30 μM (Fig. 5A), suggesting that only one quenching mechanism (either static or dynamic) governs the protein quenching (Platanić Arizanović et al., 2023). In the static quenching mechanism, the formation of the ligand–protein system takes place in the ground state, while in the dynamic quenching mechanism the resultant complex is formed in the excited state of the fluorophore (van de Weert 2010). It was then deduced that K_SV values for the interaction of Mn₃O₄ nanorods and human hemoglobin were on the order of 10⁴ M⁻¹ (Table 1), revealing that there was a strong reduction in protein fluorescence with the addition of nanorods. Based on the data summarized in Table 1, the variations of the K_SV values are inversely proportional to temperature, characterizing the static quenching mechanism as a preferable process for the formation of Mn₃O₄ nanorods-hemoglobin complex (Ghalandari et al., 2022).

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Fig. 5

Stern-Volmer (A), modified stern-Volmer (B) and van’t Hoff (C) plots for the interaction of hemoglobin (3 µM) with different concentrations of Mn₃O₄ nanorods (1–30  µM).

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Table 1 Quenching parameters of the interaction of Mn₃O₄ nanorods with hemoglobin at 298 K, 305 K, and 310 K.

T (K)	KSV (M−1)	R2	kq (M−1 s−1)
298	4.02 × 104	0.98	4.02 × 1012
305	1.89 × 104	0.99	1.89 × 1012
310	1.30 × 104	0.96	1.30 × 1012

Also, the estimated values of k_q for the Mn₃O₄ nanorod binding to hemoglobin were found to be in the order of 10¹² M⁻¹ s⁻¹ (Table 1), much greater than the reported value of 2 × 10¹⁰ M⁻¹ s⁻¹, regarded as the basis of maximum associated constant in the dynamic quenching of macromolecules (Ware 1962). Therefore, this calculation further proved that the fluorescence quenching mechanism of hemoglobin is controlled by a static quenching process.

3.8 Molecular docking study

Many studies have used molecular docking to examine the atomic-level interactions between ligands/nanoparticles and proteins. The findings gained from molecular docking analysis can be used to learn more about the interaction forces and the different ways that ligands and proteins bind. In this study, the interaction of Mn₃O₄ nanorods with human hemoglobin was studied by molecular docking analysis. As in the TEM investigation, a heterogenous distribution of Mn₃O₄ nanorods was observed, we tried to design different kinds of Mn₃O₄ clusters showing different dimensions and explore their interaction with hemoglobin. First of all, we designed a Mn₃O₄ nanorod with a dimension of 1 nm diameter and 4 nm length and explored its interaction with hemoglobin. As shown in Fig. 6A, Mn₃O₄ nanorods interact with the region between α/β subunits (Fig. 6A(i)), while Lys 139 and Glu 131 have surrounded this cluster (Fig. 6A(ii), Table 4). Also, it was shown that these amino acid residues are in a distance in the range of 2.14–2.81 with oxygen atoms of ligands and can result in the formation of conventional hydrogen bonds and carbon-hydrogen bonds. The latter forces are regarded as covalent, which may result in a strong binding energy between Mn₃O₄ nanorods and hemoglobin (−15.41 kcal/mol, Table 4). The contribution of Lys 139 in the formation of hydrogen bonds is in line with experimental thermodynamic parameters. To further analyze the interaction of Mn₃O₄ nanorods with hemoglobin, different types of nanoclusters were designed. Therefore, the interactions of Mn₃O₄ nanorods with 3/4 length (Fig. 6B (i, ii)), ½ diameter, ¾ length (Fig. 6C (i, ii)), ½ diameter (Fig. 6D (i, ii)), ½ diameter, ½ length (Fig. 6E (i, ii)), ½ length (Fig. 6F (i, ii)), as well as spherical Mn₃O₄ clusters (2 nm) (Fig. 6G (i, ii)) with hemoglobin were assessed and the resultant data were summarized in Table 4. As summarized in Table 4, it can be observed that Mn₃O₄ nanorods with 1 nm diameter and 4 nm length showed the strongest binding affinity (−15.41 kcal/mol) with hemoglobin among all other types of clusters, followed by Mn₃O₄ nanorods with ¾ length (−9.39 kcal/mol), ½ diameter (−5.70 kcal/mol), ½ length (−4.79 kcal.mol), ½ diameter, ¾ length ½ (−4.18 kcal/mol), ½ diameter, ½ length (−1.13 kcal/mol), and spherical shape (2 nm, 29.56 kcal/mol). Therefore, it may be discussed that although all designed clusters have the same chemical composition, their binding energies can vary. Additionally, it was found that for all clusters the main dominant force upon the interaction of Mn₃O₄ nanorods with human hemoglobin was hydrogen bonding, which is in good agreement with experimental outcomes. Also, based on fluorescence spectroscopy data which determined that fluorescence quenching occurs in the presence of varying concentrations of Mn₃O₄ nanorods, we can claim that Tyr35 (interaction of ½ diameter/¾ length and ½ diameter Mn₃O₄ clusters with hemoglobin) and Trp37 (interaction of ¾ length Mn₃O₄ cluster with hemoglobin) are placed in the vicinity of binding site of Mn₃O₄ clusters-hemoglobin complex.

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Fig. 6

Molecular docking study of the interaction of Mn₃O₄ clusters with hemoglobin. The interaction of Mn₃O₄ nanorods with a dimension of 1 nm diameter and 4 nm length and hemoglobin (i), amino acid residues in the binding pocket (ii) (A). The interaction of Mn₃O₄ nanorod with 3/4 length of original cluster and hemoglobin (i), amino acid residues in the binding pocket (ii) (B). The interaction of Mn₃O₄ nanorods with a dimension of ½ diameter, ¾ length of original cluster and hemoglobin (i), amino acid residues in the binding pocket (ii) (C). The interaction of Mn₃O₄ nanorod with a dimension of ½ diameter of original cluster and hemoglobin (i), amino acid residues in the binding pocket (ii) (D). The interaction of Mn₃O₄ nanorods with a dimension of ½ diameter, ½ length of original cluster and hemoglobin (i), amino acid residues in the binding pocket (ii) (E). The interaction of Mn₃O₄ nanorod with a dimension of ½ length of original cluster and hemoglobin (i), amino acid residues in the binding pocket (ii) (F). The interaction of Mn₃O₄ nanoparticles with a spherical shape and a size of 2 nm and hemoglobin (i), amino acid residues in the binding pocket (ii) (G).

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Additionally, the solvent-accessible surface (SAS) of the ligand is often less than that of the free ligand when it interacts with proteins because a portion of the ligand that was solvent accessible in the free forms is buried during interaction. As shown in Fig. 7, the surface area of designed clusters is heavily correlated with their morphology, as Mn₃O₄ clusters with 1 nm diameter and 4 nm length as well as ½ diameter exhibited the highest levels of ASA among the designed clusters. It reveals that these clusters have failed to reach the core site of hemoglobin and preferentially bind to the surface amino acid residues.

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Fig. 7

Determination of solvent accessible surface (SAS) of Mn₃O₄ nanorod determined by molecular docking study of the interaction of Mn₃O₄ clusters with hemoglobin.

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3.9 Circular dichroism study

To obtain a further understanding of secondary structural changes of the hemoglobin in the presence of Mn₃O₄ nanorods, a CD study was carried out. This technique is a quantitative and sensitive approach studying the structure of proteins in an aqueous solution (Zeinabad et al., 2016). From Fig. 8, an intensive positive peak at 195 nm and 2 negative minima at 208 and 222 nm were observed, characterizing the presence of α-helical structure (Chakraborty et al., 2018). Thus, a thorough understanding of the secondary structure of hemoglobin was gained from far-UV CD analysis in the wavelength range of 190 to 250 nm. As shown in Fig. 8, the intensities of all bands at 195, 208 and 222 nm decrease upon the addition of Mn₃O₄ nanorods (30 µM). Indeed, in the presence of Mn₃O₄ nanorods (30 µM), the reduction in the ellipticity changes of the negative minima at 208 and 222 nm as well as the positive peak at 196 nm, provide us with some information regarding the secondary structural changes of hemoglobin in the presence of Mn₃O₄ nanorods. Quantitative analysis of the α-helix percentage in hemoglobin either alone or with Mn₃O₄ nanorods was determined by CDNN software. It was determined that the α-helix percentage of free hemoglobin was around ∼ 61.24 %, while this amount decreased to ∼ 58.77 % in the presence of Mn₃O₄ nanorods (30 µM). It has also been determined that gold nanoparticles after the interaction with hemoglobin resulted in a slight decrease in the α-helix percentage of protein (Chakraborty et al., 2018). In fact, the CD spectra of human hemoglobin at both minima, in the absence and presence of Mn₃O₄ nanorods altered slightly and seem to be comparable in shape. Thus, this observation revealed that the structure of hemoglobin remained predominantly α-helical even upon the interaction with the highest concentration of Mn₃O₄ nanorods (30 µM).

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Fig. 8

Circular dichroism (CD) spectroscopy study of hemoglobin (3 µM) either alone or with Mn₃O₄ nanorods (30  µM) at 298 K.

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Furthermore, it has been shown that ferric oxide nanoparticles (10–20 nm) did not induce any alteration in the secondary structure of hemoglobin even up to 40 M, evidenced by CD analysis (Zolghadri et al., 2010). However, it was found that the addition of silver nanoparticles (5–10 nm) with different concentrations of 18.7, 37.4, and 74.8 μM to hemoglobin solution resulted in a significant change in the secondary structure of the protein (Zolghadri et al., 2009), while green synthesized hexagonal silver nanoparticle did not induce a significant effect (Shahabadi et al., 2023). Additionally, Chetty et al. reported that cerium oxide nanoparticles (20.9 nm) with different concentrations of 25 μM and 150 μM upon the interaction with Hb led to a significant decrease in minima at 222 and 211 nm with around 20 and 50 % reduction of α-helices, respectively (Chetty and Singh 2020). Therefore, upon addition of Mn₃O₄ nanorods, the spectral shape of hemoglobin remains almost unchanged though a marginal variation in the amount of ellipticity, CD signal, was detected. This data indicated that although slight alterations had appeared in the secondary structure of hemoglobin as anticipated for the interaction of (nano)particles, the overall secondary structure of the protein was normally unchanged and its integrity was kept intact even after the interaction with a high concentration of Mn₃O₄ nanorods. Therefore, after the interaction of Mn₃O₄ nanorods with blood proteins like hemoglobin, we can deduce that no significant change in the secondary structural pattern of the proteins may occur.

All these findings stipulated that Mn₃O₄ nanorods may be employed as a promising co-antioxidant therapy.