β-Cyclodextrin-Stabilized Biosynthesis Nanozyme for Dual Enzyme Mimicking and Fenton Reaction with a High Potential Anticancer Agent.

The myth of inactivity of inorganic materials in a biological system breaks down by the discovery of nanozymes. From this time, the nanozyme has attracted huge attention for its high durability, cost-effective production, and easy storage over the natural enzyme. Moreover, the multienzyme-mimicking activity of nanozymes can regulate the level of reactive oxygen species (ROS) in an intercellular system. ROS can be generated by peroxidase (POD), oxidase (OD), and Fenton-like catalytic reaction by a nanozyme which kills the cancer cells by oxidative stress; therefore, it is important in CDT (chemo dynamic therapy). Our current study designed to investigate the enzyme mimicking behavior and anticancer ability of cerium-based nanomaterials because the cerium-based materials offer a high redox ability while maintaining nontoxicity and high stability. Our group synthesized CeZrO4 nanoparticles by a green method using β-cyclodextrin as a stabilizer and neem leaf extract as a reducing agent, exhibiting POD- and OD-like dual enzyme activities. The best enzyme catalytic activity is shown in pH = 4, indicating the high ROS generation in an acidic medium (tumor microenvironment) which is also supported by the Fenton-like behavior of CeZrO4 nanoparticles. Inspired by the high ROS generation in vitro method, we investigated the disruption of human kidney cells by this nanoparticle, successfully verified by the MTT assay. The harmful effect of ROS in a normal cell is also investigated by the in vitro MTT assay. The results suggested that the appreciable anticancer activity with minimal side effects by this synthesized nanomaterial.

Introduction

Natural biogenic enzymes have a high biocatalytic activity and are highly specific toward substrates and mediated a number of biological processes in the living system, so they have vast applications in medicinal science, sensing application, and bio-electro-catalysis.[1−4,30] However, they bear a certain limitation in applications due to the low operational stability, high expenses, and easy denaturation in a wide range of pH and temperatures.[5,6] Therefore, in terms of applicability under harsh conditions, a synthetic enzyme is needed. Yan and co-workers at first explored the magnetic iron oxide Fe3O4 nanoparticles that have a peroxidase (POD)-mimicking activity in 2007 which bankrupt the long-believed allegory of biological inactivity of inorganic nanomaterials.[7,8] From the time then, nanomaterial-analogue synthetic enzymes, frequently termed as “nanozymes”. Nanozymes exhibit the same bio-enzyme activity, including a certain merit, as their preparation is simple having low-cost and excellent stability in a wide range of pH and temperatures, which make them a valuable alternative of bio-enzymes. Recently, the nano-enzyme has been studied widely in various fields such as biosensing, biocatalyst, cancer therapy, nanomedicine, and environmental applications.[9−15] Among the semiconductor nanomaterials, Fe3O4 NPs, Co3O4 NPs, V2O5 NPs, and MnO2 NPs were studied for POD catalase, superoxide dismutase, and glucose POD-like property.[16−18] Various nanocomposites have been studied to boost their biocatalytic activity such as CuO/Pt NC, NiO2/C, MoO3/C nanorods, and CoO3/C nanocomposites.[19−23] Among the artificial nanozymes, POD surrounds a family of oxidoreductases which can possess the reduction of H2O2 with a real-time oxidation of organic substrates such as o-phenylenediamine (OPD), 3,3,5,5-tetramethylbenzidine (TMB), dopamine hydrochloride (DOPA), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and luminal.[23−27] Oxidase (OD) and POD nanozymes got much more attention in medicinal science recently because they are directly related with the ROS (reactive oxygen species) in synergistic cancer therapy. In principle, the nanozyme induces the generation of ROS in carcinoma cells which then disrupt the chemical redox state intercellular level and ruptures the cancer cells.[28−31] Normal CDT (chemo dynamic therapy) is occurred by the Fenton reaction catalyzed by intercellular H2O2 and nanomaterials which possess a redox pair (M/M) and produced highly reactive hydroxyl radicals, and having a powerful oxidation capability causes chronic oxidative damage in carcinoma cells.[31−35]

Cerium oxide (nanoceria) has gained enormous importance in recent years as nanocatalysts because of its exclusive physical and chemical properties.[36−38] Nanoceria has been extensively applied in a variety of fields, such as bioassays, catalysis, and antioxidant therapy. The CeO2 nanostructure shows a high catalytic activity in a range of applications owing to the existence of mixed valence states of Ce3+ and Ce4+, and also the presence of oxygen vacancies. The explanation to this catalytic action is that the redox couple can control between each state in a CeO2 ↔ CeO2– + x/2O2 (Ce4+ ↔ Ce3+) recycle system. The catalytic performance of nanoceria originates from the surface oxygen content, and increasing the Ce3+/(Ce3+ + Ce4+) ratio enhances the surface oxygen imperfection in the structure, leading to upgrading in catalytic properties.[39]

In this study, CeZrO4 nanocomposites were prepared by a green method using β-cyclodextrin with a small particle size, (approximately 10 nm) suitable for cell endocytosis. The prepared nanospheres have a highly porous-like nature and are found to mimic both POD- and OD-like activity in an acidic environment. The amount of endogenous H2O2 may increase by the OD-like activity in an acidic tumor microenvironment, and these endogenous H2O2 convert into highly reactive hydroxyl radicals by POD activity; moreover, cerium-based species possess high Fenton and Fenton-like catalytic activity, which can effectively convert the hydrogen peroxide into toxic hydroxyl radicals (•OH) in an acidic environment. Based on this hypothesis, human kidney cancer cell lines and noncancerous kidney cell line were used as cell models to investigate the in vitro anticancer effects and side effects of this protocol.

Result and Discussion

Characterization of Nanomaterials

Figure represents the systematic illustration of the synthesis of the CeZrO4 nanocomposite. The X-ray diffraction patterns of β-cyclodextrin-stabilized CeO2, ZrO2 are shown in Figure S1, and CeZrO4 are shown in Figure a and for CeO2, ZrO2. X-ray diffraction (XRD) curves of CeO2 and ZrO2 NPs reveal well-identified peaks that ideally match with the standard position of the reflection of pure CeO2 NP and two different phases of ZrO2 NP. Figure a shows all the diffraction peaks for CeZrO4 on the scale at 2θ = 29.27, 31.95, 46.23, 48.87, 56.01, and 76.31, which could be indexed to the (111), (011), (220), (331), and (133) facets of CeZrO4 with a cubic structure. All the peaks can be identified and assigned with reference to the JCPDS data (card No- 43-1002 for CeO2 and card No-81-1544 for t-ZrO2 and card No-81-1314 for m-ZrO2). The average crystalline size of CeZrO4 is calculated using Scherrer’s eq from the mean reflection of (111), (011), (220), and (133) planes was in the range 7.3 nm to 9.4 nm (shown in Table S1). The average crystalline sizes of CeO2 NPs and ZrO2 NPs are shown in Tables S2 and S3 in the Supporting Information.Where λ = the wavelength of the radiation (1.5406 A0), β = the full width at half-maximum intensity, and θ = the diffraction angle. The incorporation of ZrO2 in CeO2 lattices and formation of nanocomposites led to contract the lattices due to the replacement of cerium with zirconium of different sizes.[40−42]

Figure 1

Schematic representation for the preparation of green synthesized CeZrO4.

Figure 2

(a) XRD spectra of CeZrO4 nanoparticles, (b,c) SEM images of CeZrO4 nanoparticles with different resolutions, (d) SEM image of the CeO2 nanoparticle, (e) SEM image of the ZrO2 nanoparticle, and (f) size distribution of CeZrO4 nanoparticles.

Figure b,c represents the scanning electron microscopy (SEM) images of CeZrO4 nanocomposite at different resolutions, showing these as sponge-like shapes. Figure d showed the SEM image of CeO2, a rod-shape, and Figure e represented the SEM image of ZrO2, which is mostly irregular shapes. Meanwhile, it was found that the average particle size distribution of the CeZrO4 nanocatalyst was at∼14nm (shown in Figure f), which was closely similar in sizes measured from the XRD pattern using Scherrer’s formula. The size effect of CeZrO4 reflects its different activities compared with other nanoparticles.

In the energy-dispersive X-ray spectroscopy (EDAX) spectra, there is a uniform distribution of cerium, oxygen, and zirconium on the surface of the nanocatalyst shown in Figure a. In these signals, the peaks appeared in the areas 0–16 keV are directly related to the characteristic peak of cerium, zirconium, and oxygen atoms. Figure b represents the elemental distribution mapping of cerium, zirconium, and oxygen atoms. The red dot corresponds to the oxygen atom, and blue and green dots represent Ce and Zr atoms, respectively.

Figure 3

(a) EDAX spectrum of CeZrO4, (b) elemental mappings of cerium, zirconium, and oxygen atoms, (c) FTIR spectra of CeO2, ZrO2, and CeZrO4 nanoparticles, and (d) hydrodynamic particle size distribution of CeZrO4 nanoparticles.

Figure c represents the Fourier transform infrared (FTIR) spectra of CeO2, ZrO2, and CeZrO4 and are depicted as follows: in Figure c, the peaks appeared at 3432 cm–, indicating the presence of the O–H stretching frequency of water or hydroxyl groups. The bands appeared in the range 2923–2852 cm–1are due to the C–H bonds for the organic compounds. The frequency band at 2342 cm–1 may arise from the absorption of atmospheric CO2 on the metallic cations. The frequency at 1104 cm–1 indicates the C–O bending vibration present in organic compounds. The stretching frequencies in the region 725–576 cm–1indicated the formation of Zr–O and Ce–O bonds, confirming the incorporation of ZrO2 in CeO2 lattices.

Figure d shows the dynamic light scattering (DLS) spectra of CeZrO4, from which we revealed the average hydrodynamic particle size of the nanoparticles ∼15 nm. The hydrodynamic particle size of the CeZrO4 nanoparticles was higher than that obtained from the SEM analysis and XRD measurement. This is probably due to some extent of hydration of small size nanoparticles in the aqueous solution. This prominent nanocatalyst gives a better catalytic activity such as the Fenton-like reaction than other nanoparticles. The average hydrodynamic sizes of intrinsic ZrO2 and CeO2 have been found to be 13 and 24 nm, respectively, which are shown in Figures S2 and S3.

POD-Like Activity of CeO2, ZrO2, and CeZrO4 Nanomaterials

POD is a bio-enzyme of the oxidoreductases family which can catalyze the reduction of H2O2. Generally, the reduction of H2O2 is supervised by the change in the absorption profile due to color changes and the simultaneous oxidation of diverse organic (indicators) substrates such as TMB, DOPA, OPD, and ABTS. All these substrates lead to blue, pale pink, yellow, and green, respectively, in their oxidized form in the solution phase. Here, TMB and DOPA were selected as the colorimetric substrates. TMB and DOPA are oxidized to TMB-ox and dopamine-o quinone (amino chrome).[43,44] TMB-ox displays an absorption peak at 652 nm, and dopamine-o quinine exhibits the absorption peak of 470−490 nm broad peak in UV–visible spectroscopy. POD-like activities of prepared nanocomposites were demonstrated by catalyzing the oxidation of TMB and DOPA taking, as a POD substrate (indicators) in the presence of H2O2. Only TMB and DOPA did not show any coloration, but in the presence of CeZrO4 NPS, the solutions of TMB and DOPA were changed from colorless to blue and pale pink as shown in Figure a,c, with the maximum absorbance at around 652 and 490 nm recorded by UV–visible spectroscopy. The oxidizing ability of CeZrO4 is higher than those of CeO2 and ZrO2 nanoparticles, as shown in Figure a,c. The absorption peak catalyzed by CeZrO4 is slightly higher than catalyzed by CeO2, indicating the better POD activity of CeZrO4 shown than the other two. On the other hand, the peak intensity of ZrO2 is low, indicating that ZrO2 slowly oxidized the indicator TMB. The time-dependent nonenzymatic activity was checked only with TMB and the highest coloration observed after 15 min, and then, it was remaining constant, and later it changed to yellow coloration, which is shown in Figure b. Therefore, 15 min is the optimal time for the highest activity of the present system to appear. Further checking the applicability of a wide range of pH to POD activity of the as-prepared nanozyme, we conducted our studies with a different pH range from 1–7 with the substrate TMB only. The highest absorbance at 650 nm was observed at pH 4 shown in Figure d. Other pH-dependent catalytic activity is shown in Figure d. On increasing or decreasing the pH range, the absorption peak decreases simultaneously and transforms the color from blue to yellow at higher and lower pH due to the formation of yellow TMB diamine compound (TMBdi; ε450nm = 59 000 M–1 cm–1) through two-electron oxidation.[45] The reaction was started with the hydroxyl radicals (•OH) formation by the decomposition of H2O2 at the nanozyme interface demonstrated as an acceptable mechanism by which the substrates are oxidized to change its color and absorption peak profile.[46]

Figure 4

UV–vis spectra of the POD-like activity of CeZrO4 compared to model indicators including (a) TMB and (c) dopamine. (b) POD-like activity of CeZrO4 against different time intervals. (d) pH-dependent POD-like activity of CeZrO4 at a wavelength of 650 nm.

The efficiency of the POD-like enzyme activity and the binding nature of CeZrO4 NPs with substrates were further evaluated by steady-state kinetics, utilizing TMB and H2O2 as the enzymatic substrates of independent variables, and UV visible spectra of these corresponds are shown in Figure S4a,b. The POD-like enzyme tic activity exhibited by the CeZrO4 analogue follows the Michaelis–Menten model shown in Figure a,c for substrates TMB and H2O2, respectively. For both variables, the substrate concentration with reaction velocity can be fitted with double-reciprocal plots. All the reaction parameters including Km and Vmax are obtained from the Lineweaver–Burk plot for substrate TMB, maintaining the equation y = 0.54026 + 49.9108x, with R2 = 0.98661. Table displays the all-kinetic parameters including Michaelis constant (Km) and the maximum velocity reached by the reaction (Vmax).

Figure 5

Steady-state kinetic analysis of CeZrO4 as a POD mimetic. (a) Curve of velocity against the TMB concentration in the condition of 10 mM H2O2. (c) Curve of velocity against the H2O2 concentration under conditions of 0.3 mM TMB. (b,d) Double-reciprocal plots of (a,c), respectively. Condition: 250 μg·mL–1 and CeZrO4 in an acetate buffer (pH 5.0) at room temperature. Graphical representation of the POD-mimicking activity of CeZrO4 versus TMB (e).

Table 1

Tabular Format of Different Enzyme-Mimic Kinetic Parameters of CeZrONPs.

enzyme	enzyme kinetics	substrate	Km (mM)	Vmax (μM min–1)	R2 (value)
OD	LineweaverBurk	TMB	0.0543	1.223466	0.99432
POD	LineweaverBurk	H2O2	1.669765	1.068308	0.91226
POD	LineweaverBurk	TMB	0.09631	1.84245	0.97426
HRP	LineweaverBurk	H2O2	3.7	0.0871	NA
HRP	LineweaverBurk	TMB	0.434	0.1026	NA

The Vmax value is found to be 1.85 × 10–6 and the apparent Km value found with the substrate TMB is approximately 0.09237 mM for the POD enzyme, which is found to be much lower than the reported value of 0.434 mM for the HRP (horseradish peroxidase)[47] enzyme, depicted in Table , which indicates that our catalyst has an excellent binding capacity toward TMB than the normal biogenic enzyme HRP.

Moreover, the Km value for H2O2 is found to be somewhat higher than TMB, and the value is 1.669765 mM, and the kinetic parameters are calculated by the Lineweaver–Burk plot, maintaining the equation = 0.93606 + 1563.89502, R2 = 0.91222, as shown in Figure d. Next, the kinetic parameter Vmax was calculated as shown in Figure d, and the magnitude is found to be 1.0673 μM min–1. The Vmax value of CeZrO4 NPs was much higher than that of HRP enzymes, signifying that the CeZrO4 NPs have a good POD mimic activity. Figure e represents the systematic illustration of the POD-mimicking activity versus substrate TMB and H2O2, inset: the blue colored picture stands for the corresponding product formation.

OD-like Activity of CeO2, ZrO2, and CeZrO4 Nanomaterials

OD is an enzyme of the oxidoreductases family which catalyzes an oxidation reduction reaction, exclusively involving molecular dioxygen (O2) as the electron acceptor.[48] The reaction mechanism keeps on with the donation of a hydrogen atom, and the molecular oxygen gets reduced to hydrogen peroxide (H2O2) or water (H2O).[49] OD-like activities of CeZrO 4 were estimated by the colorimetric assay, performing the reaction between a chromogenic substrate TMB, dopamine, and catalyst without a supplementary oxidizing agent (H2O2). Colorless TMB and DOPA get oxidized to TMBox, a blue color oxidized product, and amino chrome, a pink color oxidized product, upon oxidation with the nanocatalyst, and adsorption bands appeared at 652 and 490 nm, respectively, to monitor the progress of the reaction by UV–visible spectroscopy. As shown in Figure b, TMB showed a blue coloration with the CeZrO4 nanocatalyst with a maximum absorbance appearing after 30 min, while no coloration is observed in the presence of TMB only. For comparison, same experiments were performed with other catalysts, CeO2 and ZrO2. A weak absorption band was observed for both CeO2 and ZrO2, indicating that these NPs slowly oxidized TMB and dopamine. The activity of CeO2 is higher than ZrO2, as shown in Figure a,c, respectively. Furthermore, we conducted our experiment in different pH values from pH 1 to 7 and optimized the pH condition for the best activity of the CeZrO4 catalyst, and the results are shown in Figure d. The nanocatalyst shows the best activity at pH 4, an optimal condition of the OD-like activity of the CeZrO4 catalyst. The absorption decreases at 652 nm; while increasing or decreasing the pH from optimal condition, the blue coloration vanishes above pH 7. A yellowish-green color was developed with the decrease in the pH equal to one.

Figure 6

UV–vis spectral monitoring of the OD-like activity of CeZrO4 compared to model indicators including (a) TMB and (c) dopamine. (b) OD-like activity of CeZrO4 against different time intervals. (d) pH-dependent OD-like activity of CeZrO4 at a wavelength of 650 nm.

In order to understand the binding efficiency and enzyme catalytic activity with the substrate TMB, the enzyme catalytic assets of OD mimicking CeZrO4 are studied by the enzyme kinetic method, and UV visible spectra are recorded with various concentrations of TMB, as shown in Figure S5. The typical Michaelis–Menten curves are plotted in Figure a. The two vital kinetic parameters, including maximum velocity (Vmax) and Michaelis–Menten constant (Km), are obtained by plotting the Lineweaver–Burk plot (Figure b) for substrate TMB, maintaining the equation y = 0.81735 + 44.6758x, R2 = 0.99432. The deceptive Km and Vmax values of the CeZrO4 OD nanozyme for TMB value are calculated to be 0.0543 mM, which is as low as others earlier studies.[50,51] Moreover, the Vmax value was found to be 1.224 μM min–1, which is significantly high, so the above kinetic studies approve that CeZrO4 exhibited good OD-like activity. Figure c represents the systematic illustration of the OD-mimicking activity versus substrate TMB, inset: the bluish green colored picture stands for the corresponding product formation.

Figure 7

Steady-state kinetic analysis of CeZrO4 as an OD mimetic. (a) Curve of velocity against the TMB concentration in Condition: 250 μg·mL–1 and CeZrO4 in an acetate buffer (pH 5.0) at room temperature. (b) Double-reciprocal plots of (a) graphical representation of the OD-mimicking activity of CeZrO4 versus TMB (c).

Fenton Reaction of CeO2, ZrO2, and CeZrO4 Nanomaterials

Subsequently, the •OH-generation by the decomposition of endogenous H2O2 is a crucial factor for CDT.[52] Therefore, the •OH-generating action of CeZrO4 was then evaluated by an in vitro method, where methylene blue (MB) taken as a model indicator which is degraded by newly generated •OH. As shown in Figure a, H2O2 alone negligibly degraded the MB, but catalyst with H2O2 together had a great impact on the degradation of MB. The absorption at 663 nm significantly decreases as measured in UV–visible spectroscopy, indicating the formation of •OH by the CeZrO4-mediated Fenton reaction. It was found that 87% MB degraded in only about 60 min, as shown in Figure b. Other catalysts also have shown that the Fenton reactions actively degrade MB as well, which are shown in Figure c. Nevertheless, the different scavengers have a significant effect on the degradation of MB. The active species trapping experiment was carried out to explore the reaction mechanism of the green-synthesized CeZrO4 nanocatalyst, as shown in Figure d. We have used isopropyl alcohol, benzoquinone, and N3– as a scavenger of hydroxyl radical (•OH) and superoxide radical (O2–•), respectively. The degradation efficiency greatly retarded in the presence of isopropanol compared to benzoquinone and azide ions. These results indicate that the •OH radicals were formed as reactive species in the degradation process and decrease the rate of reaction (shown in Figure S6).

Figure 8

(a) UV–vis spectral monitoring of degradation of MB by the CeZrO4-mediated Fenton-like reaction. (b) Impact of time on MB degradation by the CeZrO4 nanoparticle-mediated Fenton-like reaction. (c) Degradation % by different synthesized nanomaterials. (d) Influence of scavengers on the MB degradation by the CeZrO4-driven Fenton-like reaction.

Anticancer Effect of CeO2, ZrO2, and CeZrO4 Nanomaterials

From the earlier reports, it has been noticed that the nanosized materials, showing enzyme-like activities have a power to regulate the ROS levels and left a significant effect in biological organs.[53] Inspiring the excellent in-vitro ROS generation by both Fenton and enzyme mimicking of CeZrO4 nanoparticles, we conduct our study to evaluate their CDT effect. In vitro •OH radicals are generated in a larger amount from intercellular H2O2 in cancerous cells during CDT. It will be noticed that •OH is a stronger oxidant than H2O2E( = 2.80 V, E(H = 1.78 V, so it can cause severe oxidative damages in cancerous cells. Consequently, it performs as the main contributor to oxidative stress. Induction of oxidative stress by the nanozyme was measured by the fluorescence microscopy assay by utilizing the DCFH-DA probe. DCFH-DA is basically a nonfluorescent compound which is converted into fluorescent DCF and exhibited a strong green fluorescent, and the intracellular oxidative stress of a cancerous cell might be attributed to the production and accumulation of H2O2 in the cell, induced by the nanozyme which is then disrupting the H2O2 quantity in cell and initiated the generation of OH/O2 ROS by a Fenton-like reaction or POD-like activity.

Human kidney cancerous cell line (ACHN) and human kidney normal cell line (HEK-293) were chosen not only to see the effect of CeO2, ZrO2, and CeZrO4 at a biomolecular level but also on tissue specificity, as kidney cells gets exposed during every drug filtration. There was no significant effect on the growth of the HEK-293 cells in the presence CeO2, ZrO2, and CeZrO4 even at a higher dose of nanoparticles, as shown in Figure S7a, and the final OD found was somewhat lower for CeZrO4 than the other two, indicating that the Normal HEK cells are more viable in the presence of CeZrO4. In intra comparison, in between all three nanoparticles CeO2, ZrO2, and CeZrO4, the CeO2 nanoparticle was little effective as reported earlier Figure b. There was no toxicity in the chosen wide ranges of the complexes from 50–250 μg/mL even after repeating three times independently. In comparison the percentage viability of the ACHN cell line was measured by the change of OD at 570 nm as shown in Figurer S7b in the Supporting Information, and the bar diagram is depicted in Figure a in place of percentage cell toxicity. Surprisingly, we have found interesting results in the case of CeO2, ZrO2, and CeZrO4 and IC50 value (inhibitory concentration required for 50% toxicity) shown in Figure S8. After the drug treatment, the cell viability of the cancer cell line was proven to be concentration-dependent. No observable difference was observed in between 50–150 μg/ml concentrations of nanoparticles. In this experiment, CeO2 shows a higher toxicity than ZrO2 as published earlier by researchers. We have chosen kidney cancer cell lines (ACHN) for comparative studies of the effect of nanoparticles. Interestingly, we have found anticancer activity in CeO2, ZrO2, and CeZrO4 nanoparticles. It might be because of the mode of Fenton action of nanoparticles, and to prove it, we had calculated the IC50 value of all three nanoparticles and studied the production of hydrogen peroxide in the presence of IC50 values of CeO2, ZrO2, and CeZrO4. Intracellular ROS production was determined via H2DCFDA staining. In the results, there was no significant internal ROS production in control (blank) and ROS production was enhanced in the presence of H2O2 (as positive control), as shown in Figure a,b. The cells treated with IC50 values of CeO2, ZrO2, and CeZrO4 nanoparticles showed observable ROS production according to the concentration of nanoparticles Figure c–e. The intensity of fluorescence increased as per the concentration of ZrO2 and CeO2 nanoparticles, 1.228 and 0.559 μg/mL, respectively. We have found the higher amount of ROS formation and was clearly visible in cells with CeZrO4 nanoparticles with a concentration of 1.24 μg/mL. The cell toxicity in the MTT assay was also increased continuously by CeZrO4 nanoparticles in the cancer cell line, which resembles the fact that the complex is more capable of inducing cell toxicity in the cancer cell line. The IC50 values for each of the compound are calculated from the cell cytotoxicity graph and are represented in a separate table in Figure f.

Figure 9

Study of the anticancer potential of CeO2, ZrO2, and CeZrO4 nanoparticles through the MTT assay: (a) ACHN cell line (Human Embryonic Kidney cancerous cell line) was treated with different concentrations of CeO2, ZrO2, and CeZrO4, as given in Methods. The bar diagrams show percentage cell toxicity. (b) HEK-293 cell line (Human Embryonic Kidney normal cell line) was treated with different concentrations of CeO2, ZrO2, and CeZrO2, as given in Methods. The bar diagrams show percentage cell viability.

Figure 10

Fluorescence-based ROS production in the presence of IC50 values of CeO2, ZrO2, and CeZrO2 in the ACHN cell line. ACHN cell lines were treated with IC50 (Inhibitory Concentration 50) values of CeO2, ZrO2, and CeZrO4 with (a) blank (control), (b) H2O2, (c) CeO2-0.599 μg/mL (d), ZrO2-1.228 μg/mL (e) CeZrO4-1.24 μg/mL and (f) table shows IC50 values of the ACHN cell line.

All the nanoparticles were thoroughly studied for anticancer potential, and results were verified by the internal cellular stress. On the treated cancer cell line (ACHN), the nanoparticles may induce a certain cellular stress-like Fenton reaction and altered its response specifically in high dose exposure in the case of CeZrO4. The anticancer mechanism of CeZrO4 NPs is represented in Figure . The observed differences in cell toxicity indicate that there lies a significant difference in the sensitivity of cells toward the synthesized nanoparticles CeO2, ZrO2, and CeZrO4. The higher toxicity of the CeZrO4 nanoparticles clearly indicates their anticancer potency with sensitivity toward the normal cell line. However, many more studies in this aspect are required. These nanoparticles are nontoxic to normal cells, which suggests their tissue-specific cell-sensitive cytotoxic activity. On the basis of the above information, we can predict its safe application in cancer treatment. As predicted, ZrO2 shows more ROS production than CeO2 because of the higher dose of nanoparticles as per IC50 values. It is reported that, ZrO2 dissolution increases the intracellular [Zr4+] and is associated with high levels of ROS production. These oxygen radicals could be derived at the particle surface as well as from mitochondrial damage.[54] CeO2 NPs also showed higher fluorescence intensity due to more generation of ROS species.[55] However, the CeZrO4 nanoparticle complex formed by CeO2 and ZrO2 showed more ROS production due to which cell viability was significantly lost in treated cells. It could be a potential candidate for cancer therapy in the near future after more studies.

Figure 11

Graphical representation of the anticancer activity of synthesized CeZrO4.

Conclusion

In this work, an excellent CeZrO4 nanocomposite was synthesized via a green method using β-cyclodextrin as the stabilizer. The synthesized CeZrO4 nanocomposite and other NPs were characterized by XRD, EDX, SEM, DLS, and FTIR analysis. Owing to the contain mixed valence state of Ce, the composite materials offer several biomimetic catalytic activities including dual enzyme and OD- and POD-like activity and accelerated the Fenton-like reaction. The intrinsic nanozyme tic property is also shown by pure CeO2 and ZrO2, and synergistic behavior is observed in the composite. Highly reactive ROS generation in the intercellular level is catalyzed by OD, POD, and Fenton reaction and showed a significant in vitro anticancer effect while maintaining the minimum normal cell damage and red blood cell damage. Therefore, the present nanozyme showed the nanozyme tic anticancer effect with no side effect. We believe this new approach may become a potential material for biomedical science and promising candidates in organic- and electrocatalyst fields.

Materials and Methods

Materials

Ceric-ammonium nitrate, zirconium oxychloride octahydrate (ZrOCl2·8H2O, ≥99.5%), methylene blue (C16H18ClN3S.XH2O), β-cyclodextrin, dopamine hydrochloride, and TMB were purchased from Sigma-Aldrich. H2O2 (30 wt %), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Alfa-Asser. Fresh neem leaves were collected from the University of North Bengal Campus. All the chemicals are used in this work were analytical graded and used without further purification.

Neem Leaf Extract Preparation

To prepare a neem leaf extract, new and fresh leaves of “Azadirachta indica″ were collected from the University of North Bengal and washed several times under running tap water. After that, the leaves were cut into small pieces and again re-washed with deionized (DI) water to remove unnecessary particles, and small pieces of leaves are dried for 72 h at 60 °C temperature. After that, 10 mg of dried leaves were taken into 100 mL of DI water and heated at 80 °C with continuously stirred for 1 h. Subsequently, the homogeneous solution was allowed to cool at room temperature, followed by the filtration of the newly prepared extract by a Whatman-40 filter paper, and the filtered solution was stored in a refrigerator at 4 °C for further uses.

Preparation of Pure CeO2 NPs

We synthesized CeO2 as per the previously discussed method;[39] for the biosynthesis of β-cyclodextrin-capped CeO2 NPs, first 0.5 M ammonium ceric nitrate was dissolved in 150 mL of de-ionized water and stirred magnetically at 900 rpm for 1 h under ambient conditions for complete dissolution of the salts. After that, 10 mL of β-cyclodextrin solution added to this solution instantly changes the color of the solution to pink. After 30 min of stirring, 20 mL of freshly prepared neem leaf extracts was added to this solution and left the mixture for completing the reaction under stirring conditions. The color of the solution mixture changing from pink to brownish-yellow with a colloidal precipitate was observed, which indicated the formation of nanoparticles within the reaction mixture. Finally, the mixture was left at 85 °C in an air oven to obtain powder-like nanoparticles, followed by calcination in a muffle furnace at 700 °C

Preparation of Pure ZrO2 NPs

The same protocol was followed to synthesize the ZrO2NPs; at first, 100 mL of zirconium oxychloride (5 mM) was taken in a beaker and stirred for 30 min with the addition of β-cyclodextrin solution (10–5M) to obtain a homogeneous mixture. Subsequently, 10 mL of freshly prepared neem extracts was added drop wise, and the solution was stirred for another 30 min. The color of the solution changes to brown, indicating the formation of nanoparticles. Meanwhile the reaction was left for another 1 h to complete the reaction. Finally, the mixture was dried in an air oven and calcined at 700 °C in a muffle furnace.

Preparation of CeZrO4 Nanocomposites

CeZrO4 nanocomposites were synthesized via a facile green method using β-cyclodextrin as a stabilizer. For this purpose, 0.3 M ammonium ceric nitrate and 0.1 M zirconium oxychloride were dissolved in 200 mL of DI water. After complete dissolution, 20 mL of β-cyclodextrin (0.01 M) was added to the solution, and the color of the solution readily changes to pink. Subsequently, 10 mL of freshly prepared neem leaf extracts was added to this solution, and the solution was left for 1 h under the stirring condition to complete the reaction. After the mentioned time, the color of solution changes to brownish-yellow, indicating the formation of nanoparticles. Finally, the sample was dried by a heating mantle and calcined at 700 °C for further uses.

Characterization of Materials

The crystal structure of the prepared nanocomposites was analyzed by XRD at room temperature using an XPERT-PRO PW3071 diffractometer with Cu Kα (λ = 1.5418 Å) as target material using 40 kV accelerating voltage and 30 mA emission current. Morphologies and the average grain sizes of the nanoparticle atomic level dispersion were measured by SEM and elements by which the nanocomposites formed were analyzed by EDAX (JEOL JMS-5800) and elemental mapping studies. FTIR spectra were recorded at room temperature using a PerkinElmer Paragon 1000 FT-IR spectrometer (JEOL JMS-5800). The average hydrodynamic size was measured by DLS, Zetasizer nano instrument. The PL spectra are recorded in a JASCO FP 8500 spectrofluorometer instrument equipped with 150 W Xe source between 300 and 700 nm for different excitation wavelengths.

POD-like Activity

The POD-like activity assay was determined by using dopamine and TMB as enzymatic substrates at different pH values. Briefly, 160 μM TMB in dimethyl sulfoxide (DMSO) stock solution was taken into a 50 mL beaker containing 10 mL of NaAc/HAc buffer (pH 4.5–5), followed by the addition of 10 mM H2O2 and 250 μg/mL of nanocatalyst and then incubated for 15 min. After that, the absorbance of TMB was measured at a wavelength of 652 nm using UV–visible spectroscopy.

OD-like Activity

TMB and DOPA were used as an OD substrate to investigate the activities of the prepared nanozyme. First, we have taken 160 μM TMB in a DMSO stock solution in a 50 ml beaker containing 10 mL of NaAc/HAc buffer (pH 4.5–5), followed by the addition of 250 μg/mL of nanozyme and left the solution for 30 min. After that, the absorbance of TMB was measured at 652 nm using UV–visible spectroscopy. Furthermore, we study the effect of different parameters such as incubation time and pH on the catalytic reaction. The OD mimetic activity of the catalysts CeO2 NPs and CeZrO4 NPs were also evaluated separately.

Steady-State Kinetics

Steady-state kinetics of OD- and POD-like activity of CeZrO4 for TMB were also studied under similar reaction conditions by varying the final concentrations of TMB, which include 40, 80, 160, 320, and 640 μM while keeping the concentration of H2O2 constant. For evaluating the kinetic parameter of H2O2 as a substrate, the TMB concentration was maintained for 0.3 mM with varying the final concentrations of H2O2 to 1, 5, 10, 20, and 40 mM. All-important kinetics parameters, such as Km, Vmax, and specific, are calculated using four types of linear plot derived from the Michaelis–Menten equation.

Here, V represents the average rate of conversion of a substrate, Vmax is the maximum rate of conversion of a substrate, [S] stands for the substrate concentration, and Km is the Michaelis constant. Km is equal to the concentration of the substrate at which the rate of conversion becomes half of Vmax. The Michaelis constant Km represents the affinity (binding capacity) of an enzyme toward the substrate, and the low Km value indicated the high affinity of an enzyme for the substrate.

MTT Assay

ACHN (Human cancerous kidney cell line) was cultured in a 96-well micro titer plate at 37 °C in the presence of 5% carbon dioxide (CO2) at a density of 4 × 103 cells/well in 100 μL of Dulbecco’s modified Eagle medium Ham F-12 culture medium. After 24 h of incubation, drugs (CeO2, ZrO2, and CeZrO4) were added in each well at different concentrations (50, 100, 150, 200, and 250 μg/mL) in triplicate. After that, the micro titer plate was further incubated under the same experimental condition. Next day, the treated plate was withdrawn from the incubator and 10 μL (5 mg/mL) of MTT powder dissolved in 1× PBS was added in each well after discarding the previous culture media. Plates were then kept for 3 h in the abovementioned condition. Finally, 50 μL of isopropanol, a formazan solubilizer, was added to each well containing MTT solution and was shaken for about 5 min. At last, the absorbance was recorded at 620 nm in an ELISA reader. The percentage of cell toxicity was calculated as {(A – B)/A × 100}, where “A” is the mean optical density of control (untreated cells) and “B” is the mean optical density of treated cells with different drug concentrations.

Detection of Intracellular ROS Generation

For the detection of hydrogen peroxide in a cancer cell, H2DCFDA is used. A standard protocol[36] was performed with a minor alteration for the assessment of intracellular ROS. The oxidation of 2,7-dichlorofluorescein (H2DCF) to 2,7-dichlorodihydrofluorescein (H2DCFDA) was monitored for the determination of hydrogen peroxide (H2O2). The human kidney cancer cell line (ACHN) was cultured on a cover slip in 35 mm Petri dishes. Plates were incubated for 24 h with 5% CO2 in a N-biotech incubator. After 24 h of incubation, ACHN cells were treated with the previously determined IC50 concentration of each drug (IC50 was calculated via the cytotoxic experiment, i.e., MTT). The control plate was kept without any treatment. Then, cells were further incubated for 24 h at the same condition as mentioned above. Next day, the plates were withdrawn and washed twice with phosphate-buffered saline (PBS). H2O2 treatment was given to the positive control plate for 20 min. Then, 25 mM fluorescence dye 2,7-dichlorofluorescein diacetate was given to all the plates and further incubated for 30 min. After that, cells were washed with 1× PBS to remove the unbound dye, and glass slides were prepared by inverting the cover slips on the slide in a 20% glycerin solution. Fluorescence was captured under a light-emitting diode (LED)-based fluorescence microscope (Magnus MLXi). By using LED cassettes, samples were excited at 480 nm and using a long-pass filter emission.