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Peony-shaped zinc oxide nanoflower synthesized via hydrothermal route exhibits promising anticancer and anti-amyloid activity

BMC Pharmacology and Toxicology volume 25, Article number: 101 (2024) Cite this article

Abstract

Background

Cancer is the deadliest disease, and neurological disorders are also marked as slow progressive diseases, ultimately leading to death. Stopping two mouths with one morsel was the strategy that we used in this study.

Methods

We have synthesized peony-shaped zinc oxide nanoflowers (ZnO-NFs) and characterized them using various photophysical tools like UV-vis spectroscopy, zeta potential analysis, dynamic light scattering (DLS), FTIR, and scanning electron microscopy (SEM), and utilized these nanoflowers to monitor their anticancer and anti-amyloid activity. In vitro biocompatibility was assessed using fibroblasts and undifferentiated rat phaeochromocytoma cells, and in vivo, biocompatibility was estimated using haemolysis assay and zebrafish embryo development.

Results

The results demonstrated high biocompatibility of the as-synthesized ZnO-NFs up to a dose of 200 µg/ml. In vitro anticancer activity was evaluated using adherent (A375) and non-adherent (Dalton’s Lymphoma Ascites, DLA) cancer cell lines. The results indicated that the ZnO-NFs significantly killed the cancer cells in a dose-dependent way, showing an extraordinary effect on DLA cells. The anti-amyloid activity in vitro was explored using a spectrum of assays that were hallmarks in anti-amyloid studies like ThT fluorescence assay, DLS, turbidity assay, atomic force microscopy (AFM), and SEM analysis. Excellent anti-amyloid activity was observed in vitro at 50 µg/ml of ZnO-NFs.

Conclusion

We can conclude from the above results that the as-synthesized ZnO-NFs have a dual role as an anticancer as well as an anti-amyloid agent. In the future, animal models can be used to study the efficacy of the ZnO-NFs in cancer inhibition and amyloid degradation.

Graphical abstract

Peer Review reports

Introduction

The medical bionanotechnology field has expanded rapidly in the past few decades, bringing up several theranostic solutions for a spectrum of diseases [1]. Nanoparticles have an enormously high surface area to volume ratio that imparts an effective role at an ultra-low concentration compared to their bulk counterpart [2]. Metals and metal oxides are the most explored category of nanostructures that have been exploited towards targeted drug delivery [3, 4], biosensors designing [5], medical imaging, agriculture, and food [6], nano nutraceuticals, and anticancer activity. Cancer encompasses a variety of types depending on the site of occurrence, age of the patient, stage of the cancer, as well as metastatic status. Cancer is obviously one of the leading causes of death worldwide, and skin cancer also contributes hugely to the cancer death percentage [7]. On the other hand, ascitic tumors, which silently grow in the peritoneal cavity, are also another type of cancer, which is non-tumorigenic, and the ascites caused are named malignant ascites [8]. These two types of cancers have different natures and are challenging to treat with the same strategy. It becomes necessary to propose a treatment regime that can work efficiently for both types of cancer.

Type II diabetes and neurodegenerative disorders like Alzheimer’s disease (AD). Parkinson’s disease (PD), and prion disease possess a common attribute between them- the deposition of misfolded protein aggregates in different parts of the body [9]. In the case of type II insulin-dependent diabetes patients, the site of repeated insulin injection causes the development of a subcutaneous amyloid mass. On the other hand, in the case of the abovementioned neurodegenerative disorders, the misfolded proteins or peptides responsible are amyloid Aβ 42 for AD [10], α-synuclein for PD, and PrPSc for prion diseases. These misfolded proteins are insoluble, fibrillar with a predominant β-sheet-rich structure, and form amyloids that get deposited in the brain’s neuropil or above the neurons’ cell membranes and can cause neurofibrillary tangles or Lewy bodies. Thus, these diseases are commonly known as amyloidosis, and it has been observed that in the autopsy of the brain with AD, the level of metal ions like iron, aluminium, copper, and zinc is significantly higher than in normal individuals [11, 12]. Metal ions have been shown to play a vital role in the aggregation and degradation of these amyloids and in deciding the fate of the degraded amyloid fibrils [13]. It has been observed earlier that iron and copper enhance the formation of fibrillar structures, whereas zinc causes amorphous structure when co-incubated or sequentially added to Aβ 42 [14, 15]. Anticipation can be made from the above reports that zinc can play a pivotal role in changing the morphology of insoluble amyloid fibrils to soluble aggregates, adding value to the treatment strategy for amyloidosis.

The shape of nanoflowers and their surface functionalization can determine the fate of the cells they treat. In this study, a typical peony petal shape could elicit a different effect compared to the typical hexagonal petal structure of ZnO-NFs. Bare ZnO-NFs and albumin-coated ZnO-NFs have shown anticancer activity, as reported earlier [16, 17]. In a recent study, inorganic hybrid nanoflowers, which included ZnO-NFs impregnated with collagenase, were explored for their antiamyloid activity [18]. Keeping in mind the severity of both the diseases, cancer and amyloidosis, we attempted to synthesize a peony-shaped zinc oxide nanoflower (ZnO-NF), using the hydrothermal method, characterized them using different tools like UV-visible spectrophotometry, zeta potential, dynamic light scattering (DLS), and scanning electron microscopy (SEM).

Further, we explored its potential towards two types of cancer cell lines, skin cancer (A375 cells) and ascitic tumor cells (Dalton’s lymphoma ascites (DLA)). A step forward, the anti-amyloid potential of the as-synthesized ZnO-NFs was also explored using insulin amyloids (IA), a model amyloid used for exploring amyloidosis due to their structural similarity. Various tools, such as Thioflavin-T (ThT) fluorescence assay, DLS, and turbidity assay, were employed to evidence the IA degradation; atomic force microscopy (AFM) was used to visualize the degraded IA. The biocompatibility of the synthesized ZnO-NFs was monitored in vitro using normal fibroblast cells (Chinese hamster lung fibroblasts, V79) and undifferentiated rat phaeochromocytoma cells (PC12). In vivo, biocompatibility was assessed using hemocompatibility assay and zebrafish embryos. We speculate that the mode of administration of ZnO for cancer can be oral, topical, or intravenous. On the other hand, intranasal spray can be used for delivery to the brain, which can handle the limitation of drug delivery to the brain by circumventing the blood-brain barrier (BBB).

Materials and methods

Materials

Huminsulin 30/70 40 IU/ml was procured from Eli Lilly and Company (India) Limited), Thioflavin T (ThT) (Sigma-Aldrich, U.S.A.), Zinc acetate dihydrate, sodium hydroxide (NaOH), ethanol, Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin, Fetal Bovine Serum (FBS), antibiotics and antimycotic solution, sodium chloride (NaCl), Potassium bromide (KBr), dimethyl sulphoxide (DMSO), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue, acridine orange, ethidium bromide, from Hi-Media. DCFH-DA was purchased from Sigma Aldrich. Phosphate buffered saline (PBS) tablets were procured from Sigma Aldrich and reconstituted by dissolving one tablet in 200 ml of distilled water to yield 10 mM phosphate buffer solution (pH 7.4). Aqua regia was used to wash all the glassware, followed by double distilled water washing and drying in a hot air oven before use.

Synthesis of zinc oxide nanoflowers

ZnO NF was synthesized using the method described earlier [19], with slight modification. Zinc acetate dihydrate (0.2 M) was taken as a precursor and mixed with 1 M sodium hydroxide (NaOH) with a volume made up to 100 ml using distilled water. The solution was properly mixed using a magnetic stirrer for 1 h and was further taken in a three-necked refluxing bottle. The pH of the solution was made alkaline (pH = 12.5), and the solution was refluxed at 90 °C for 2 h. After the reflux was completed, a white-colored precipitate was observed in the walls of the refluxing flask, which was then scrapped off using a spatula and collected in a tube. This powder of ZnO-NF was then washed three times using methanol and kept for drying at room temperature in a dirt-free environment. The yield percentage was 86.4%. Post-drying, we could get the desired ZnO-NFs, which were subjected to characterization.

Characterization of the synthesized ZnO-NFs

UV-vis spectroscopy was used to analyze the absorption spectrum of the synthesized ZnO-NFs using a Shimadzu UV-1800 spectrophotometer. A solution of 0.1 mg/ml of ZnO-NFs was prepared in distilled water and sonicated in a water bath for 15 min, two times, to dissolve the nanoflowers and used for the absorption measurement. Since ZnO is sparingly soluble in water, we added 100 µl DMSO to solubilize the ZnO and then added 900 µl of PBS or distilled water (as necessary) to make a solution, as shown in Figure S1. The final concentration of DMSO was always less than 1% during the in vitro and in vivo studies. The same sample was diluted nearly 50X to measure the hydrodynamic diameter and zeta potential using Malvern zeta sizer. XRD analysis was done using the ZnO-NF powder using (XPERT-PRO), and the crystal size was calculated using the Debye–Scherrer equation [20]:

$$D = \frac{{0.9{\mkern 1mu} \lambda }}{{\upbeta \text{Cos}\uptheta }}$$

Where,

D denotes crystal size

λ denotes the X-ray wavelength

β denotes full width at half maximum (in radians)

θ denotes the diffraction angle (in radians)

The X-ray diffractometer contained CuKα as a source of radiation having wavelength 1.54 Å. The readings were taken in the 2θ range of 20.02°–89.98°, with a step size of 0.05°.

FTIR peaks were recorded using Bruker alpha IR spectrometer by preparing KBr pellets of ZnO-NF and recording the IR spectra in transmittance mode. The surface topology was visualized under SEM (FEI Quanta FEG 200-High Resolution Scanning Electron Microscope (SEM)) at SAIF, IIT, Madras.

In vitro biocompatibility and anticancer activity

Microscopic observation, MTT assay, and trypan blue assay

Whether or not the synthesized ZnO-NFs are biocompatible has to be monitored both in vitro and in vivo. Since our present investigation deals with anticancer and anti-amyloid activities, we selected normal fibroblasts and undifferentiated rat phaeochromocytoma cells to observe biocompatibility, respectively. MTT assay was used for the evaluation of in vitro biocompatibility using normal fibroblast cells (V79 cells) and undifferentiated rat phaeochromocytoma cells (PC12 cells). According to Deepika et al., the anticancer activity of skin cancer cell lines (A375 cells, adherent cancer cell lines) was also assessed using an MTT assay [21]. All the cells were maintained at 37 °C in a humidified atmosphere supplied with 5% CO2 in an Eppendorf CO2 incubator (CellXpert C170i-CO2 incubator, Eppendorf). The medium used for cell growth was DMEM, containing FBS (10%) and antibiotic-antimycotic solution (1%). Briefly, the V79 and A375 cells were seeded at a cell density of 5 × 104 cells/well in a 48-well plate and allowed to attach for 24 h. A stock solution of ZnO-NFs (1 mg/ml) was prepared in 10% DMSO and 90% complete DMEM, filtered with a 22-micron syringe filter under sterile conditions, and treatment of the cells was done with different doses of ZnO-NFs (25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml). Untreated cells were kept under the same condition and considered as control. 24 h post-treatment, MTT solution was added to each well (5 mg/ml, 50 µl) and incubated 4 h further in the CO2 incubator for the formazan crystal formation. The formazan crystals were solubilized using DMSO by repeated pipetting and mixing the solvent using a cell rocker. Ensuring the complete solubilization, the absorbance at 570 nm of the samples was recorded, and the cell viability percentage was estimated using the formula provided below:

$$\% \;{\text{cell viability}}=\left({\frac{{{\text{O}}.{\text{D}}.\;{\text{of sample}}}}{{{\text{O}}.{\text{D}}.\;{\text{of untreated control}}}}} \right) \times 100$$

The anticancer activity of DLA cells (ascitic tumor cells of suspended cell type), was done using MTT assay (25 µg/ml and 50 µg/ml) according to Deepika et al. [21], and trypan blue dye exclusion assay according to Strober [22]. DLA cells were procured from Amala Cancer Research Centre, Thrissur. The cells were of a suspended type and were taken in 15 ml sterile centrifuge tubes inside a BSL II biosafety cabinet, washed two times with sterile PBS (1X, pH = 7.4) by centrifuging at 1500 rpm for 5 min. Final washing was done with complete sterile DMEM, and the cells were suspended in 5 ml of fresh DMEM. 5 × 104 cells/well were seeded in two 48 well plates (one for MTT assay and another for trypan blue dye exclusion assay) and treated with different concentrations of ZnO-NFs. After 24 h, an MTT assay was done as described in the above section. For the trypan blue assay, the cells were mixed with 0.4% trypan blue dye under sterile conditions, stained for 10 min, loaded in a hemocytometer, and counted for cell viability. The dead cells were stained with the dye due to compromised cell membrane integrity, whereas the live cells with intact cell membranes did not take up the dye. The percentage of cell viability was calculated using the following formula.

$${\text{\% ~cell~viability}}=\left({\frac{{{\text{No}}.{\text{~of~unstained~cells}}}}{{{\text{Total~number~of~cells}}}}} \right) \times 100$$

Each experiment was done in triplicates.

Live dead assay using fluorescence microscopy

The live-dead assay was conducted according to Deepika et al., using a combination of the dual fluorescent dye, acridine orange, and ethidium bromide [21]. Briefly, the adherent cell lines, V79, PC12, and A375, were seeded over sterile coverslips kept inside 35 mm cell culture Petri dishes, and the cells were allowed to adhere for 24 h. ZnO-NFs were added at two concentrations, low (50 µg/ml) and high (200 µg/ml), and kept inside a CO2 incubator maintained at 37 ºC, with a humidified atmosphere for 24 h; untreated cells were kept as control. After the treatment, the culture medium was slowly aspirated without disturbing the cell layer and gently washed with PBS once to remove the residual medium. The dye mixture AO: EtBr = 3 µM: 1.9 µM, dissolved in PBS, was then added to the cells under sterile conditions and incubated at 37 ºC for 3 min in the dark, followed by excess dye removal using PBS and visualizing the cells under a fluorescent microscope (Olympus BX51 fluorescent Microscope) with appropriate filters. The live cells appeared green in color, whereas the dead cells appeared red. Randomly, five spots were photographed for each coverslip, and the cells were counted to calculate the cell viability using live dead assay by the formula given below:

$${\text{\% ~cell~viability}}=\left({\frac{{{\text{No}}.{\text{~of~green~cells}}}}{{{\text{Total~number~of~cells}}}}} \right) \times 100$$

The experiments were done in triplicates.

In vivo biocompatibility assay

Zebrafish embryo toxicity study

Transparent zebrafish embryos are robustly used to observe the developmental toxicity of nanoparticles [20]. The experiment was done after getting permission from the Institutional Animal Ethics Committee of Chettinad Academy of Research and Education (IAEC2/Proposal:162/A.Lr:124/Dt. 02.07.2024). We have assessed the in vivo toxicity of the as-synthesized ZnO-NFs where 30 embryos in each group (control, ZnO-NF-25 µg/ml, ZnO-NF-100 µg/ml, and ZnO-NF-200 µg/ml), was taken and monitored under a light microscope until they hatched according to Deepika et al. [21]. The cumulative hatchability was also calculated based on the data obtained according to the formula:

$$\begin{aligned}{\text{Cumulative~hatchability~}}\left({{\% }} \right)\\&=\frac{{{\text{Total~number~of~embryos~hatched}}}}{{{\text{Total~number~of~embryos~taken~initially}}}} \times {\text{~}}100\end{aligned}$$

Each experiment was done in triplicates.

Haemolysis assay

The hemolysis assay experiment was done using human blood after procurement of ethical clearance from the Institutional Human Ethical Committee (IHEC), Chettinad Academy of Research and Education (Ref No: IHEC-II/0655/24) on 26.04.2024. The methodology followed was strictly according to Harini et al. [23]. Briefly, human blood (3 ml) was drawn from a healthy male volunteer of age 21 years with informed consent in an EDTA-coated vacutainer. The blood was subjected to centrifugation at 1500 rpm for 15 min, which yielded an RBC pellet, and the pellet was washed three times with normal saline (0.9% NaCl). The RBC so obtained was then diluted 10 times using normal saline and divided into 7 tubes, each containing 100 µl of RBC and 900 µl of ZnO-NFs diluted at different concentrations (10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml) in normal saline. The positive control for RBC lysis was distilled water, and the negative control was normal saline. All the tubes were incubated at 37°C for 2 h and then subjected to centrifugation at 12,000 rpm for 1 min. Post centrifugation, an image was captured in a camera, and the supernatant was gently aspirated out in fresh tubes to record the absorbance at 541 nm (the absorption maxima of hemoglobin). The hemolysis percentage was calculated using the formula [23]:

$$\begin{aligned}{{\%} \text{~of~haemolysis}}\\&=\frac{{\left({{\text{OD~of~the~Sample~treated}} - {\text{OD~of~the~negative~control}}} \right)}}{{\left({{\text{OD~of~the~positive~control}} - {\text{OD~of~the~negative~control}}} \right)}}~ \times 100~\end{aligned}$$

Anti-amyloid activity

Insulin can form amyloids in vitro if kept under certain controlled conditions [24], and can act as a model amyloid for the study of amyloidosis [25]. In this study, we have incubated Huminsulin 30/70 40 IU/ml in a sealed centrifuge tube at 65 °C for 24 h for the in vitro amyloid formation.

Thioflavin T assay

Thioflavin T (ThT) is a dye that binds with amyloids and increases the fluorescence emission intensity at nearly 480 nm when excited at 440 nm and is used as a gold standard to detect amyloids. The absorption maxima (λmax) of ThT absorption has the shortest wavelength, 412 nm, and the longest wavelength, 450 nm, in an aqueous environment and in water being incorporated into the amyloid fibrils, respectively [26]. The λmax depends upon the microenvironment around the probe. In the present experiment, we used PBS, and the ThT showed a λmax at 412 nm. So, we have used this wavelength as the excitation wavelength in the fluorescence studies. The secondary structure of amyloids contains β-sheet rich regions, and the fluorescence enhancement mechanism after amyloid binding is due to the rotational immobilization of benzothiazole and aniline rings connector central C–C bond [27]. Moreover, ThT has been known to bind with the side chain channels that run along the amyloid fibril’s long axis [28]. ThT was dissolved in PBS at a concentration of 20 µM. 190 µl of IA with 10 µl of PBS (untreated control), 190 µl of IA with 10 µl of ZnO-NF (final concentration of ZnO-NFs-25 µg/ml, 50 µg/ml, 75 µg/ml, and 100 µg/ml) (sample), and 190 µl BSA (1 mg/ml) with10 µl of PBS (negative control) was incubated at 37° C for 24 h. The negative control was a non-amyloidogenic protein, BSA because it does not form amyloids after incubation at 65 °C for 24 h, and the same has been used previously [29]. After incubation, 10 µl of each sample was diluted in 3 ml distilled water with 10 µl of ThT, and the fluorescence intensity was measured at 412 nm excitation and emission from 440 to 600 nm. Only ThT fluorescence was also measured by taking 3 ml of distilled water and 10 µl of ThT, under the same measurement conditions. All fluorescence measurements were carried out using a Spectrofluorometer (Jasco-FP8300 Model).

Turbidity assay

Previously, it was shown that the size of the IA fibrils before and after degradation with any therapeutic agent can be assessed by measuring the solution absorbance at 600 nm [24, 30]. 190 µl of IA with 10 µl of PBS (untreated control) and 190 µl of IA with 10 µl of ZnO-NF (50 µg/ml) (sample) were incubated at 37° C for 24 h for amyloid degradation. Post incubation, 10 µl of each sample was diluted in 3 ml PBS, and the absorbance of the samples was measured at 600 nm.

Fibril size measurement using DLS

The size of the degraded IA fibrils after incubation with the as-synthesized ZnO-NFs was assessed using DLS, which gives the hydrodynamic diameter of the samples [24]. 190 µl of IA with 10 µl of PBS (untreated control) and 190 µl of IA with 10 µl of ZnO-NF (50 µg/ml) (sample) were incubated at 37° C for 24 h for amyloid degradation. Once the incubation was over, 10 µl of each sample was diluted in 1 ml PBS, and the hydrodynamic diameter was measured, indicating the size of the amyloid fibrils (degraded or undegraded).

Atomic force microscopy

The IA aggregation can be visualized if we can deploy AFM and check the z-height of the treated and untreated IA samples [25, 31, 32]. The z-height and surface roughness can indicate the amount of IA deposition in the sample [15]. We have analyzed our samples of IA and IA + ZnO-NF using AFM (WITec alpha300RA (WITec GmbH, Ulm, Germany) AFM, SNOM & RAMAN combined system) from SAIF, Mahatma Gandhi University, Kottayam, Kerala, India. 190 µl of IA with 10 µl of PBS (untreated control) and 190 µl of IA with 10 µl of ZnO-NF (50 µg/ml) (sample) were incubated at 37° C for 24 h for amyloid degradation. After the incubation, a drop of the sample was cast on a dirt and grease-free glass coverslip and dried in a closed box at room temperature. The surface was then analyzed using the Confocal Micro Raman spectrometer with AFM instrument.

Scanning electron microscopic studies

For scanning electron microscopy, the samples were prepared as follows: 190 µl of IA with 10 µl of PBS (untreated control) and 190 µl of IA with 10 µl of ZnO-NF (50 µg/ml) (sample) were incubated at 37° C for 24 h for amyloid degradation. After the incubation, a drop of the sample was cast on a dirt and grease-free glass coverslip and dried in a closed box at room temperature. The dried sample was analyzed using a Hi-Resolution Scanning Electron Microscope (HRSEM), Thermo Scientific Apreo S at SRM Institute, Kattankulathur, Chennai, with appropriate magnification.

ROS generation assay

The ROS generated by the ZnO-NFs in cancer cells were assessed using the procedure mentioned by Alarifi et al. and Heo et al., with slight modifications [33, 34]. 3 × 105 A375 cells per well were cultured in a six-well plate and incubated with complete DMEM (DMEM containing 10% FBS and 1% antibiotic solution) for 24 h for cell attachment. Treatment with ZnO-NFs at 50 µg/ml and 100 µg/ml was done, and the cells were further incubated for 24 h. The positive control was hydrogen peroxide (H2O2) (40 µg/ml). Post-treatment, the cells were washed with sterile PBS, and DCFH-DA (10 µM) solubilized in complete DMEM was added to each well, followed by 1 h incubation in the dark at 37 °C. The nonfluorescent DCFH-DA gets converted to fluorescent DCF in the presence of ROS. Thus, the amount of fluorescence obtained is directly proportional to the amount of ROS generated. After 1 h, the cells were lysed using DMSO and diluted in PBS. The fluorescence intensity emitted was measured using a spectrofluorimeter (Jasco Spectrofluorimeter FP8300, Tokyo, Japan). The excitation of the sample was done at 485 nm, and the emission was measured at 528 nm (the emission peak of the dye). The percentage of ROS generated was calculated by taking H2O2 as a positive control (100%). Untreated cells were also assessed for ROS generation and maintained under the same condition (control). The experiment was done in triplicate.

Statistical analysis

All the experiments were done in either triplicates or repeated three times. One-way ANOVA and Dunnett’s test were performed to analyze the data.

Results and discussion

Characterization of the as-synthesized ZnO-NFs

The characterization of synthesized ZnO-NFs was done to identify the absorption spectra, hydrodynamic diameter, stability, functional groups present, and surface topography using UV-visible spectrophotometer, dynamic light scattering, zeta potential, FTIR, and SEM analysis, respectively. The results are shown in Fig. 1, where it was evident that the absorption maximum of the synthesized ZnO-NF (Fig. 1a) was at 350 nm with a typical shoulder peak of ZnO nanoparticles [19, 29]. The hydrodynamic diameter was 609 nm (Fig. 1b), showing the flower shape can take a higher radius of water molecules around them while moving in Brownian motion inside water. The PDI was found to be 0.26, indicating monodispersity. The zeta potential was − 12 mV, which shows that the synthesized ZnO-NFs were relatively stable (Fig. 1c). Acetate ions stabilized the nanoflowers, so the surface charge was negative, and the magnitude of the zeta potential indicates that the structure was stable. Guidelines in drug delivery literature commonly classify nanoparticle dispersions based on their zeta potential values as follows: ±0–10 mV as highly unstable, ± 10–20 mV as relatively stable, ± 20–30 mV as moderately stable, and > ± 30 mV as highly stable [35, 36].

Fig. 1
figure 1

The (a) UV-visible spectrum (b) hydrodynamic diameter (c) zeta potential (d) FTIR and (e) XRD spectrum, and (f) SEM images of the synthesized ZnO-NFs (g) The HR-TEM images of the synthesized ZnO-NFs at 60,000 X magnification and (h) 1000000 X magnification (d spacing indicated by arrows)

Full size image

The FTIR peaks (Fig. 1d) correspond to different stretching, bending, and vibrations of bonds that supported the presence of ZnO in the sample. The samples were washed with methanol to remove any impurities and dried in a dirt-free atmosphere before proceeding to make the KBr pellet. Table 1 shows the different FTIR peaks obtained with their explanation. The peak at 834 cm-1 demonstrates the presence of Zn and O in the synthesized nanoflower, which is further confirmed by the XRD analysis showing the ZnO crystalline structure.

Table 1 The different FTIR peaks of the synthesized ZnO-NFs and the respective functional groups
Full size table

The XRD spectrum of the ZnO-NFs showed the characteristic peaks at the designated 2θ values (Fig. 1e). The peaks corroborated with the JCPDS pattern of ZnO (file no. (00-036-1451), and also with previously published reports [20]. This peak distribution confirmed the formation of ZnO nanoflowers, and the crystal size of the ZnO-NFs was found to be 18.19 nm with a lattice strain of 0.00634, calculated using Scherrer’s formula. The SEM images (Fig. 1f) of as-synthesized ZnO-NFs exhibited peony flower-like morphology with the protruded petals visible in the nanometer range. Figure 1g and h shows the HR-TEM images of the synthesized ZnO-NFs, indicating the flower petals as elongated structures originating from a core. At a higher magnification with a 5 nm scale bar, the distinct crystalline lattice planes of the sheets become clearly visible. Figure 1h highlights the characteristic d-spacing of approximately 0.2 nm, as indicated by the arrows. The d spacing information evidenced the crystalline structure of the nanostructure. The typical flower structure enhances the surface area hugely because each petal of the flower, being a part of the nanoparticle, provides additional surface [29]. Thus, from the above experiments, it could be concluded that we have successfully synthesized the ZnO-NFs.

In vitro biocompatibility of the ZnO-NFs

Any nanostructure for biomedical applications can be successfully implemented if it shows biocompatibility in a range of concentrations, both in vitro and in vivo. Normal lung fibroblasts (V79 cells) and undifferentiated rat phaeochromocytoma cells (PC12) were used to monitor the in vitro biocompatibility of the as-synthesized ZnO-NFs because our present study involved anticancer and anti-amyloid activity of the ZnO-NFs.

Fig. 2
figure 2

(a) The morphological observations under an inverted microscope (b) Live dead assay captured under a fluorescence microscope (c) MTT assay and (d) Cell viability percentage calculated using live-dead assay, of V79 cells after treatment with different doses of ZnO-NFs for 24 h. *** represents p < 0.001 compared to untreated control

Full size image

The in vitro biocompatibility was done using MTT assay, where the V79 cells were exposed to different doses of ZnO-NFs and kept under incubation for 24 h. After 24 h, the cells were observed using an inverted microscope, and images were captured randomly for a minimum of five spots in each treatment well. After image capturing, the cells were processed for MTT assay. The morphological observations (Fig. 2a) showed that the ZnO-NFs were non-toxic up to a dose of 100 µg/ml. At 200 µg/ml, there were a few cells visible to be dead and shrunken in morphology, whereas 400 µg/ml completely killed all the cells, making their morphology round. These results were also supported by the results of the MTT assay (Fig. 2c), where we could observe that up to a dose of 100 µg/ml, there was no significant difference in cell viability compared to the control cells, whereas 200 µg/ml showed 75% cell viability which rapidly reduced to 9% after treatment with 400 µg/ml of the ZnO-NF. The live dead assay also showed that up to 200 µg/ml, there were not many dead cells (red color stained) observed (Fig. 2b), which was also supported by the percentage of viable cells (Fig. 2d) scored after counting the live and dead cells under a fluorescent microscope. Obtaining these results, we restricted our further studies to a dose of 200 µg/ml.

In the case of PC12 cells, which can be taken as a model cell for any neurological studies, we could observe no morphology change after treatment with ZnO-NFs up to a dose of 200 µg/ml (Fig. 3a). Performance of live dead assay also confirmed that there was an absence of dead cells and the majority of live cells that appeared green in color under fluorescent microscope in untreated control as well as ZnO-NF treated PC12 cells at the dose of 50 µg/ml and 200 µg/ml (Fig. 3b). The MTT assay results (Fig. 3c) and cell counting by live-dead assay (Fig. 3d) also concluded that the as-synthesized ZnO-NFs could not induce any significant cell killing in the PC12 cells compared to the untreated control.

Fig. 3
figure 3

(a) The inverted microscopic images of PC12 cells (b) Live dead assay fluorescent microscopic image (c) MTT assay for cell viability, (d) Cell viability by Live dead assay of PC12 cells after treatment with different doses of ZnO-NFs for 24 h

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Anticancer activity using skin cancer cell line

The anticancer activity was governed for adherent (A375) as well as non-adherent (DLA) type of cancer cell line. Skin cancer cell line A375 was exposed to different doses of synthesized ZnO-NFs and was incubated for 24 h.

Fig. 4
figure 4

(a) The morphological observations under an inverted microscope (b) Live dead assay captured under a fluorescence microscope (c) MTT assay and (d) Cell viability percentage calculated using the live-dead assay of A375 cells after treatment with different doses of ZnO-NFs for 24 h. *** represents p < 0.001 compared to untreated control

Full size image

Post incubation, the A375 cells were observed for any morphological changes under inverted microscopy (Fig. 4a) followed by MTT assay (Fig. 4c). It was fascinating to see that at a dose of as low as 50 µg/ml, the morphology of the cells started to change from bipolar slender shape to round. This indicated that the synthesized ZnO-NFs could elicit the cancer cell-killing effect at a very low concentration. 200 µg/ml of ZnO-NFs made most of the cells round, and the cell numbers were also decreased. Further, 400 µg/ml could successfully kill all the A375 cells, evidenced by a total round morphology of the cells, which is a hallmark of dead cells. The morphological observation was also supported by MTT assay results, where a significant dose-dependent decrease in cell viability compared to untreated control was observed (Fig. 4c). A step forward, we conducted the Live dead assay for A375 cells after exposure to 50 µg/ml and 100 µg/ml of ZnO-NFs (Fig. 4b). The results showed that in control cells, all the cells were green in color, whereas after ZnO-NF treatment, the number of dead cells (red) increased. The increase was higher for 100 µg/ml ZnO-NF treatment. The number of live and dead cells was counted using a fluorescence microscope, and the cell viability was plotted (Fig. 4d). The results corroborated with the results of fluorescent microscopic studies and MTT assay. In our recent study, we have shown that ZnO nanosheets also exhibited anticancer activity against A549 and A375 cells [40].

Anticancer activity using DLA cancer cells

Dalton’s lymphoma ascites (DLA) can cause peritoneal cancerous tumors, which is a form of a liquid malignant tumor. We have obtained the cells from Amala Cancer Research Centre, Thrissur, and used them for our study. The cells were seeded in equal numbers in different wells of a multiwell plate, and treatment with different doses of ZnO-NF was done. 24 h after treatment, morphological observations were done under an inverted microscope followed by an MTT assay. The trypan blue dye exclusion assay was also done after 24 h of treatment with different doses of ZnO-NFs. Inverted microscopic images (Fig. 5a) revealed that in the control group, the cells were healthy with a perfect round morphology, typical for the suspended cell type. On the other hand, after treatment with ZnO-NFs, the cells were shrunken with a black-colored deposition in the periphery of the cell membrane, indicating damaged cells. The presence of giant cells was also observed in 50 µg/ml ZnO-NF treated cells indicating that the cells are targeted towards autophagy due to survival struggle [41]. These results were also in line with the MTT results (Fig. 5c), where the DLA cell viability was 65% and 39%, respectively, after 25 µg/ml and 50 µg/ml ZnO-NF treatment, and was significantly lower than untreated control. A step forward, the trypan blue results (Fig. 5b) also depicted that there was a rapid increase in the number of blue-colored cells after treatment with 50 µg/ml, 100 µg/ml, and 200 µg/ml of ZnO-NFs, indicating cell death. The trypan blue dye is taken up by dead cells, and they were counted in triplicate after loading in a hemocytometer, and the cell viability was plotted in Fig. 5d. The results showed that even as low as 25 µg/ml of ZnO-NF could elicit significant cell killing in DLA cells, whereas the same dose was benign for normal fibroblasts (V79) (Fig. 2). Thus, from the results of A375 and DLA cells, it can be concluded that the synthesized ZnO-NF specifically killed the cancer cells but did not affect the normal cells. This can be due to the leaky vasculature of cancer cells, which allows the nanoflowers to enter the cells and elicit cell killing, whereas the normal fibroblasts possessing intact cell membranes did not allow the nanoflowers to enter inside the cells. A similar response to silver nanoparticles and ZnO nanoparticles was observed earlier, where cancer cell-specific killing was performed [42,43,44,45]. Moreover, they may induce a local ROS load in the cytoplasm, thereby damaging the organelles and leading to cell death. ZnO also possesses the ability to induce apoptosis if treated at a particular dose [46].

Fig. 5
figure 5

(a) Morphological observation using an inverted microscope for DLA cells (b) Light microscopic image of trypan blue stained cells loaded in a hemocytometer (c) MTT assay demonstrating cell DLA viability (d) Percentage of DLA cell viability using trypan blue dye exclusion assay, after treatment with different concentrations of ZnO-NFs for 24 h. *** represents p < 0.001 compared to untreated control

Full size image

In vivo biocompatibility assay

In vivo, toxicities of the synthesized ZnO-NFs were conducted using hemocompatibility assay and zebrafish embryos. Direct interaction with RBCs can be observed using the hemolysis assay, whereas any developmental toxicity can be observed using transparent zebrafish embryos, where all the developmental stages can be visualized.

Fig. 6
figure 6

(a) Images of RBC lysis after treatment with different doses of ZnO-NFs. Negative control was 0.9% NaCl, and positive control was distilled water (b) The percentage of hemolysis plotted for different samples (c) Microscopic images, and (d) Cumulative hatchability of zebrafish embryos captured at different hpf

Full size image

The hemolysis assay demonstrated that there was no RBC lysis elicited by the ZnO-NFs up to a dose of 200 µg/ml as visualized in the tube images (Fig. 6a) and hemolysis percentage graph (Fig. 6b). Any agent that causes hemolysis below 5% is biocompatible, and, in this case, our synthesized ZnO-NF caused hemolysis below 4% at all the doses tested. Zebrafish embryos were treated with 50 µg/ml and 200 µg/ml of ZnO-NFs from 8 h post fertilization (hpf). The embryos were monitored till 120 hpf to score any developmental abnormality like pericardial edema, tail bent, yolk sac defect, etc., is visible or not. We could clearly see that there was no abnormality induced by ZnO-NFs at low and high doses (Fig. 6c), and there was no effect on the hatchability of the embryos (Fig. 6d). The cumulative hatchability was plotted against the progressive hpf, and 100% hatching was observed for control and ZnO-NF-treated embryos. These results ensured that our synthesized ZnO-NFs were highly biocompatible up to a dose of 200 µg/ml. Our previous study demonstrated that coating ZnO nanoparticles with natural polymer chitosan could protect the zebrafish embryos from bare ZnO-induced toxicity [20].

Anti-amyloid study

Identifying an agent that can break or dissociate amyloid aggregates can open an avenue for a spectrum of diseases like AD, PD, prion disease, as well as type II diabetes. Insulin amyloids (IA) were employed for this study because they resemble the structural similarity with other pathogenic amyloids containing more β-sheets. ZnO nanoparticles synthesized using clove and ginger have been shown to be exerting anti-inflammatory effects [47].

Fig. 7
figure 7

(a) The DLS peak (b) ThT fluorescence spectra at different concentrations, and (c) Turbidity assay graph of IA and IA + ZnO-NFs. (d) SEM image of IA, and (e) SEM image of IA + ZnO-NFs. AFM images of (f) IA and (g) IA + ZnO-NFs

Full size image

IA was pre-grown for 12 h at 65 ºC to form amyloids and further incubated at physiological temperature (37 ºC) with and without ZnO-NFs for 24 h (50 µg/ml). Post incubation, various analyses were done, and we can see from the hydrodynamic diameter measurements (Fig. 7a) that the IA fibril size was 1275 nm, which decreased to 342 nm after incubation with ZnO-NFs, evidencing IA degradation. Post incubation, we found that the nanoflowers and the undegraded IA were settled at the bottom of the tube, and the supernatant contained the degraded amyloids. We took the samples from the top layer and did the DLS experiment. Thioflavin T (ThT) is a dye that specifically binds to amyloids, gives enhanced fluorescence, and is used as a gold standard to detect the presence of amyloids. We can also quantitatively compare the amount of amyloids present in a sample before and after degradation using ThT assay. In this study, we have used BSA, a non-amyloidogenic protein, as a negative control and observed the IA degradation using ZnO-NFs (25 µg/ml, 50 µg/ml, 75 µg/ml, and 100 µg/ml) by ThT assay (Fig. 7b). The IA produced fluorescence after binding with ThT with high intensity (1958 a.u.), whereas ZnO-NFs treated IA reduced the ThT fluorescence emission (1627 a.u. for 25 µg/ml, 1343 a.u. for 50 µg/ml, 1245 a.u. for 75 µg/ml, and 1178 a.u. for 100 µg/ml), indicating degradation of amyloids in the 100 µg/ml treated sample up to 1.4-fold. On the other hand, BSA (703 a.u.) did not increase the fluorescence intensity much after binding with ThT (601 a.u.) because it does not contain any amyloids.

Turbidity of the amyloid sample also decreases if the amyloid gets degraded. In our study, we measured the turbidity of fully grown IA fibrils at 600 nm and the turbidity of the IA sample incubated with ZnO-NFs for 24 h. The turbidity data shows that there was a significant decrease (60% degradation) in the turbidity of IA when they were incubated with ZnO-NFs (Fig. 7c). The fibrillar morphology was observed using SEM (Fig. 7d), which clearly shows a branched IA fibril. Further, degradation of IA with ZnO-NF showed amorphous aggregates using SEM analysis (Fig. 7e). Further, degradation of IA with ZnO-NF showed a decrease in surface roughness (Fig. 7g) as well as a decline in z-height as observed by the surface topography captured using AFM, compared to IA (Fig. 7f).

The degradation may be induced by the petals of the ZnO-NF which had a high surface area to degrade the amyloids. In a previous study, it was observed that incubation with Zn causes the inhibition in fibril formation in vitro of Aβ 42 amyloids and gives rise to amorphous aggregates. Both Zn2+ caused a reduction in β-sheet content of Aβ 42 and increased random coil formation [14, 15]. Thus, the reduction in fibrillar morphology can be due to the presence of Zn in the NF structure which are in close vicinity of the IA. Many polymer-based nanoparticles have shown either an increase or decrease in amyloid fibrillation [48]. On the other hand, researchers have reported that bioinspired polymeric nanoparticles and nutraceutical-loaded polymeric nanoparticles could effectively inhibit the fibrillation of Aβ [49, 50]. Zinc oxide nanoparticles are known to exert their antioxidant and cytotoxic effects when synthesized using bioroute [51, 52].

ROS generation in A375 cells

The mechanism of cancer cell killing was demonstrated to be ROS generation, as evidenced by the DCF-DA reduction assay. The percentage of ROS generated was 100% for H2O2 (40 µg/ml) treatment, and in untreated control, the percentage was 27%. On the other hand, after treatment with 50 µg/ml and 100 µg/ml of the synthesized ZnO-NFs, the ROS generation was 58% and 67%, respectively (Fig. 8). This shows that one of the modes of cancer cell death was by generation of ROS. Due to the leaky nature of the cancer cell membrane, the synthesized nanoflowers could enter the cancer cells and generate ROS, which was not possible for normal cells with intact cell membranes.

Fig. 8
figure 8

The ROS generation percentage of untreated control A375 cells and A375 cells treated with ZnO-NFs (50 µg/ml and 100 µg/ml) and positive control H2O2 (40 µg/ml)

Full size image

Conclusion

Discussing the various life-threatening diseases, the few diseases that capture the top category are cancer and neurodegenerative disorders. A dual role of any therapeutic agent can pave the way for the judicial use of treatment modalities in handling these diseases. Our present study synthesized peony flower-shaped ZnO-NFs via the hydrothermal route and exploited the anticancer and anti-amyloid activities. The range of photophysical tools confirmed the nanoflowers’ synthesis, and their biocompatibility was explored in vitro using two types of cell lines- a normal fibroblast, V79, and an undifferentiated rat phaeochromocytoma cell, PC12. The magnificent biocompatibility in vitro encouraged us to explore the anticancer and anti-amyloid activities. The anticancer activity of these synthesized ZnO-NFs was exploited for adherent and non-adherent cancer cell lines. The dose-dependent decrease in cell viability for cancer cells was observed using the ZnO-NFs with a safe dose up to 200 µg/ml. One of the modes of cell death was found to be the generation of ROS in cancer cells. The results were more promising for the non-adherent cancer cell line, DLA, where the effect of killing was visible at a dose as low as 50 µg/ml. The in vivo biocompatibility of the as-synthesized ZnO-NFs was also documented using a hemolysis assay and zebrafish embryos, resulting in high biocompatibility up to a dose of 200 µg/ml. A step forward, we delved into the anti-amyloid activity of the synthesized ZnO-NFs. Our findings supported the dissociation of IA, as evidenced by the DLA assay, ThT fluorescence assay, turbidity assay, AFM, and SEM analysis. Altogether, our study could unveil the dual role of synthesized ZnO-NFs as an anticancer and anti-amyloid agent. Further studies are warranted to find out the efficacy of these ZnO-NFs in an in vivo model of cancer and amyloids.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ZnO-NFs:

Zinc oxide nanoflowers

DLS:

Dynamic light scattering

FTIR:

Fourier Transform Infrared Spectroscopy

SEM:

Scanning electron microscopy

DLA:

Dalton’s Lymphoma Ascites

AFM:

Atomic force microscopy

AD:

Alzheimer’s disease

PD:

Parkinson’s disease

ThT fluorescence assay:

Thioflavin-T fluorescence assay

BBB:

Blood brain barrier

IA:

Insulin amyloids

DMEM:

Dulbecco’s Modified Eagle’s Medium

FBS:

Fetal Bovine Serum

NaCl:

Sodium chloride

DMSO:

Dimethyl sulphoxide

KBr:

Potassium bromide

MTT-3:

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS:

Phosphate buffered saline

BSA:

Bovine serum albumin

NaOH:

Sodium hydroxide

EtBr:

Ethidium Bromide

AO:

Acridine orange

HRSEM:

Hi-Resolution Scanning Electron Microscope

XRD:

X-Ray diffraction

V79:

Chinese hamster lung fibroblasts

PC12:

Undifferentiated rat phaeochromocytoma cells

A375:

Skin cancer cell lines

A549:

Adenocarcinomic human alveolar basal epithelial cells

hpf:

Hours post fertilization

References

  1. Senthil R, Kavukcu SB, Vedakumari WS. Cellulose based biopolymer nanoscaffold: a possible biomedical applications. Int J Biol Macromol. 2023;246:125656.

    Article  CAS  PubMed  Google Scholar 

  2. Rochani A, Raveendran S. Theranostics: a new holistic Approach in Nanomedicine. Bionanotechnology in Cancer: Jenny Stanford Publishing; 2022. pp. 573–624.

    Google Scholar 

  3. Desai N, Momin M, Khan T, Gharat S, Ningthoujam RS, Omri A. Metallic nanoparticles as drug delivery system for the treatment of cancer. Expert Opin Drug Deliv. 2021;18(9):1261–90.

    Article  Google Scholar 

  4. Cherkasov VR, Mochalova EN, Babenyshev AV, Rozenberg JM, Sokolov IL, Nikitin MP. Antibody-directed metal-organic framework nanoparticles for targeted drug delivery. Acta Biomater. 2020;103:223–36.

    Article  CAS  PubMed  Google Scholar 

  5. Mercy DJ, Girigoswami K, Girigoswami A. A mini review on biosensor advancements-emphasis on quantum dots. Results Chem. 2023:101271.

  6. Thirumalai A, Girigoswami K, Harini K, Pallavi P, Gowtham P, Girigoswami A. A review of the current state of probiotic nanoencapsulation and its future prospects in biomedical applications. Biocatal Agric Biotechnol. 2024:103101.

  7. Hasan N, Nadaf A, Imran M, Jiba U, Sheikh A, Almalki WH, et al. Skin cancer: understanding the journey of transformation from conventional to advanced treatment approaches. Mol Cancer. 2023;22(1):168.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Sasikumar V, Govindan S, Rajendran G, Rajendran A, Ramani P, Pateiro M, et al. Protective effect of aqueous extract of Pleurotus eous against Dalton’s lymphoma ascites tumor in mice. J Agric Food Res. 2023;13:100638.

    CAS  Google Scholar 

  9. Metkar SK, Udayakumar S, Girigoswami A, Girigoswami K. Amyloidosis-history and development, emphasis on insulin and prion amyloids. Brain Disorders. 2023;13:100106.

    Article  Google Scholar 

  10. Marshall KE, Vadukul DM, Staras K, Serpell LC. Misfolded amyloid-β-42 impairs the endosomal–lysosomal pathway. Cell Mol Life Sci. 2020;77:5031–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lovell M, Robertson J, Teesdale W, Campbell J, Markesbery W. Copper, iron and zinc in Alzheimer’s disease senile plaques. J Neurol Sci. 1998;158(1):47–52.

    Article  CAS  PubMed  Google Scholar 

  12. Bagheri S, Squitti R, Haertlé T, Siotto M, Saboury AA. Role of copper in the onset of Alzheimer’s disease compared to other metals. Front Aging Neurosci. 2018;9:446.

    Article  PubMed  PubMed Central  Google Scholar 

  13. House E, Collingwood J, Khan A, Korchazkina O, Berthon G, Exley C. Aluminium, iron, zinc and copper influence the in vitro formation of amyloid fibrils of Aβ 42 in a manner which may have consequences for metal chelation therapy in Alzheimer’s disease. J Alzheimers Dis. 2004;6(3):291–301.

    Article  CAS  PubMed  Google Scholar 

  14. Ryu J, Girigoswami K, Ha C, Ku SH, Park CB. Influence of multiple metal ions on β-amyloid aggregation and dissociation on a solid surface. Biochemistry. 2008;47(19):5328–35.

    Article  CAS  PubMed  Google Scholar 

  15. Ha C, Ryu J, Park CB. Metal ions differentially influence the aggregation and deposition of Alzheimer’s β-amyloid on a solid template. Biochemistry. 2007;46(20):6118–25.

    Article  CAS  PubMed  Google Scholar 

  16. Paino IM, Gonçalves FJ, Souza FL, Zucolotto V. Zinc oxide flower-like nanostructures that exhibit enhanced toxicology effects in cancer cells. ACS Appl Mater Interfaces. 2016;8(48):32699–705.

    Article  CAS  PubMed  Google Scholar 

  17. Wang R, Zhang L, Razzaq A, Khan NU, Alfaifi MY, Elbehairi SEI, et al. Albumin-coated green-synthesized zinc oxide nanoflowers inhibit skin melanoma cells growth via intra-cellular oxidative stress. Int J Biol Macromol. 2024;263:130694.

    Article  CAS  PubMed  Google Scholar 

  18. Jamal HS, Raja R, Ahmed S, Yesiloz G, Ali SA. Immobilization of collagenase in inorganic hybrid nanoflowers with enhanced stability, proteolytic activity, and their anti-amyloid potential. Int J Biol Macromol. 2024:133114.

  19. Girigoswami A, Ramalakshmi M, Akhtar N, Metkar SK, Girigoswami K. ZnO Nanoflower petals mediated amyloid degradation-An in vitro electrokinetic potential approach. Mater Sci Engineering: C. 2019;101:169–78.

    Article  CAS  Google Scholar 

  20. Girigoswami K, Viswanathan M, Murugesan R, Girigoswami A. Studies on polymer-coated zinc oxide nanoparticles: UV-blocking efficacy and in vivo toxicity. Mater Sci Engineering: C. 2015;56:501–10.

    Article  CAS  Google Scholar 

  21. Deepika B, Pallavi P, Gowtham P, Girigoswami A, Girigoswami K. Anticancer potential of nanoformulated extract of Passiflora incarnata leaves. Biocatal Agric Biotechnol. 2024;57:103109.

    Article  CAS  Google Scholar 

  22. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015;111(1):A3. B. 1-A3. B.

  23. Harini K, Alomar SY, Vajagathali M, Manoharadas S, Thirumalai A, Girigoswami K, et al. Niosomal Bupropion: Exploring Therapeutic Front through Behav Profiling Pharmaceuticals. 2024;17(3):366.

    CAS  Google Scholar 

  24. Hsu R-L, Lee K-T, Wang J-H, Lee LY-L, Chen RP-Y. Amyloid-degrading ability of nattokinase from Bacillus subtilis natto. J Agric Food Chem. 2009;57(2):503–8.

    Article  CAS  PubMed  Google Scholar 

  25. Metkar SK, Girigoswami A, Murugesan R, Girigoswami K. Lumbrokinase for degradation and reduction of amyloid fibrils associated with amyloidosis. J Appl Biomed. 2017;15(2):96–104.

    Article  Google Scholar 

  26. Maskevich AA, Stsiapura VI, Kuzmitsky VA, Kuznetsova IM, Povarova OI, Uversky VN, et al. Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form. J Proteome Res. 2007;6(4):1392–401.

    Article  CAS  PubMed  Google Scholar 

  27. Voropai E, Samtsov M, Kaplevskii K, Maskevich AA, Stepuro V, Povarova OI, et al. Spectral properties of thioflavin T and its complexes with amyloid fibrils. J Appl Spectrosc. 2003;70:868–74.

    Article  CAS  Google Scholar 

  28. Krebs MR, Bromley EH, Donald AM. The binding of thioflavin-T to amyloid fibrils: localisation and implications. J Struct Biol. 2005;149(1):30–7.

    Article  CAS  PubMed  Google Scholar 

  29. Akhtar N, Metkar SK, Girigoswami A, Girigoswami K. ZnO nanoflower based sensitive nano-biosensor for amyloid detection. Mater Sci Engineering: C. 2017;78:960–8.

    Article  CAS  Google Scholar 

  30. Metkar SK, Girigoswami A, Vijayashree R, Girigoswami K. Attenuation of subcutaneous insulin induced amyloid mass in vivo using Lumbrokinase and serratiopeptidase. Int J Biol Macromol. 2020;163:128–34.

    Article  CAS  PubMed  Google Scholar 

  31. Girigoswami K, Ku SH, Ryu J, Park CB. A synthetic amyloid lawn system for high-throughput analysis of amyloid toxicity and drug screening. Biomaterials. 2008;29(18):2813–9.

    Article  CAS  PubMed  Google Scholar 

  32. Metkar SK, Girigoswami A, Murugesan R, Girigoswami K. In vitro and in vivo insulin amyloid degradation mediated by Serratiopeptidase. Mater Sci Engineering: C. 2017;70:728–35.

    Article  CAS  Google Scholar 

  33. Alarifi S, Ali D, Alkahtani S, Almeer RS. ROS-mediated apoptosis and genotoxicity induced by palladium nanoparticles in human skin malignant melanoma cells. Oxidative Med Cell Longev. 2017;2017(1):8439098.

    Article  Google Scholar 

  34. Heo J-R, Kim S-M, Hwang K-A, Kang J-H, Choi K-C. Resveratrol induced reactive oxygen species and endoplasmic reticulum stress-mediated apoptosis, and cell cycle arrest in the A375SM malignant melanoma cell line. Int J Mol Med. 2018;42(3):1427–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Patel VR, Agrawal Y. Nanosuspension: an approach to enhance solubility of drugs. J Adv Pharm Tech Res. 2011;2(2):81–7.

    Article  CAS  Google Scholar 

  36. Bhattacharjee S. DLS and zeta potential–what they are and what they are not? J Controlled Release. 2016;235:337–51.

    Article  CAS  Google Scholar 

  37. Bashir S, Awan MS, Farrukh MA, Naidu R, Khan SA, Rafique N, et al. In-vivo (albino mice) and in-vitro assimilation and toxicity of Zinc Oxide nanoparticles in Food materials. Int J Nanomed. 2022;17:4073.

    Article  CAS  Google Scholar 

  38. Kennepohl D, Farmer S, Reusch W. 12.9: infrared spectra of some common functional groups. LibreTexts: Chemistry. 2020.

    Google Scholar 

  39. Dulta K, Koşarsoy Ağçeli G, Chauhan P, Jasrotia R, Chauhan P. Ecofriendly synthesis of zinc oxide nanoparticles by Carica papaya leaf extract and their applications. J Cluster Sci. 2022:1–15.

  40. Bhuin A, Udayakumar S, Gopalarethinam J, Mukherjee D, Girigoswami K, Ponraj C, et al. Photocatalytic degradation of antibiotics and antimicrobial and anticancer activities of two-dimensional ZnO nanosheets. Sci Rep. 2024;14(1):10406.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yang D, Zhang M, Gan Y, Yang S, Wang J, Yu M, et al. Involvement of oxidative stress in ZnO NPs-induced apoptosis and autophagy of mouse GC-1 spg cells. Ecotoxicol Environ Saf. 2020;202:110960.

    Article  CAS  PubMed  Google Scholar 

  42. R SD, Girigoswami A, Meenakshi S, Deepika B, Harini K, Gowtham P, et al. Beneficial effects of bioinspired silver nanoparticles on zebrafish embryos including a gene expression study. ADMET DMPK. 2024;12(1):177–92.

    PubMed  PubMed Central  Google Scholar 

  43. Hanley C, Layne J, Punnoose A, Reddy K, Coombs I, Coombs A, et al. Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles. Nanotechnology. 2008;19(29):295103.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Rasmussen JW, Martinez E, Louka P, Wingett DG. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications. Expert Opin Drug Deliv. 2010;7(9):1063–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Akhtar MJ, Ahamed M, Kumar S, Khan MM, Ahmad J, Alrokayan SA. Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species. Int J Nanomed. 2012:845–57.

  46. Wang C, Hu X, Gao Y, Ji Y. ZnO nanoparticles treatment induces apoptosis by increasing intracellular ROS levels in LTEP-a‐2 cells. Biomed Res Int. 2015;2015(1):423287.

    PubMed  PubMed Central  Google Scholar 

  47. Selvaraj S, Chokkattu JJ, Shanmugam R, Neeharika S, Thangavelu L, Ramakrishnan M. Anti-inflammatory potential of a mouthwash formulated using clove and ginger mediated by zinc oxide nanoparticles: an in vitro study. World J Dentistry. 2023;14(5):394–401.

    Article  Google Scholar 

  48. Holubová M, Štěpánek P, Hrubý M. Polymer materials as promoters/inhibitors of amyloid fibril formation. Colloid Polym Sci. 2021;299:343–62.

    Article  Google Scholar 

  49. Joseph B, Thomas S, Sen N, Paschold A, Binder WH, Kumar S. Bioinspired synthetic polymers-based inhibitors of Alzheimer’s amyloid-β peptide aggregation. Polym Chem. 2023;14(4):392–411.

    Article  Google Scholar 

  50. Martano S, De Matteis V, Cascione M, Rinaldi R. Inorganic nanomaterials versus polymer-based nanoparticles for overcoming neurodegeneration. Nanomaterials. 2022;12(14):2337.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ashna Y, Roy A, Kumar R, Lakshmi T. Biosynthesis of zinc oxide nanoparticles using ginger oleoresin and its antioxidant and cytotoxic activity. Telematique. 2022:3981–90.

  52. Sariga Jayachandran DLT, Rajeshkumar Shanmugam DEP. Cytotoxic effects of Lemon Grass and Mint Formulation mediated Zinc Oxide nanoparticles against Lung Cancer Cell line. J Surv Fisheries Sci. 2023;10(1S):213–22.

    Google Scholar 

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Acknowledgements

The authors are grateful to Chettinad Academy of Research and Education for providing the infrastructure and facilities to conduct the research. B.D., S.U., G.J., and D.J.M., acknowledge Chettinad Academy of Research and Education for providing the PhD scholarship.

Funding

No funding was received to conduct the research work.

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Authors and Affiliations

  1. Medical Bionanotechnology, Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Chettinad Health City, Kelambakkam, Tamilnadu, 603103, India

    Agnishwar Girigoswami, Balasubramanian Deepika, Saranya Udayakumar, Gopalarethinam Janani, Devadass Jessy Mercy & Koyeli Girigoswami

  2. Centre for Global Health Research, Saveetha Medical College, Saveetha Institute of Medical and Technical Sciences, Thandalam, Chennai, 602101, India

    Koyeli Girigoswami

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Contributions

A.G., K.G.: Conceptualization, methodology, data curation, investigation, supervision, validation, writing the original draft, writing the final manuscript. B.D., S.U., G.J., D.J.M., Data curation, investigation.

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Correspondence to Koyeli Girigoswami.

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Ethical approval

The experiments on hemolysis assay were done by obtaining ethical clearance from the Institutional Human Ethical Committee (IHEC), Chettinad Academy of Research and Education (Ref No: IHEC-II/0655/24) on 26.04.2024. The experiments on zebrafish embryos were conducted by obtaining ethical clearance from the Institutional Animal Ethics Committee of Chettinad Academy of Research and Education (IAEC2/Proposal:162/A.Lr:124/Dt. 02.07.2024).

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Girigoswami, A., Deepika, B., Udayakumar, S. et al. Peony-shaped zinc oxide nanoflower synthesized via hydrothermal route exhibits promising anticancer and anti-amyloid activity. BMC Pharmacol Toxicol 25, 101 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40360-024-00830-x

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40360-024-00830-x

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BMC Pharmacology and Toxicology

ISSN: 2050-6511

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