Green synthesis and optimization of zinc oxide quantum dots using the Box–Behnken design, with anticancer activity against the MCF-7 cell line

A green strategy and cost-effective approach was adapted to prepare Zinc oxide quantum dots (ZnO QDs) for biomedical applications. The prepared ZnO QDs may hold great promise as sensing scanners for diagnostics and therapy, as demonstrated in our current study. Zinc sulfate, Azadirachta indica, Aloe vera gel and Catharanthus roseus leaves extract were used to synthesize a novel natural Zinc oxide bionanocomposite (ZnO BC) and used as a precursor to prepare ZnO QDs by microwave-assisted technique. The ZnO BC was characterized by SEM–EDX, FT-IR, XRD, Zeta potential, and particle size analysis. The optical properties of QDs were investigated using UV and PL spectrophotometers. Experimental factors like the concentrations of ZnO Nps, C. roseus, and Aloe vera were evaluated using Box–Behnken design (BBD). MTT and hemolysis assay was performed using ZnO BC and ZnO QDs. Maximum absorbance was observed at optimized values of 0.5% ZnO Nps, 1 g A. vera gel, and 0.5 ml C. roseus leaf extract of ZnO QDs against BBD. There was a decreased viability rate, ranging from 60 to 15% for 0.5 mg/ml ZnO BC and 45 to 5% for 5 mg/ml ZnO QDs which revealed a tenfold decrease in cell viability with less concentration scale for 5 mg/ml of ZnO QDs when compared with that of 0.5 mg/ml ZnO BC. Also, the hemolysis test shows that the hemolysis ratio was below 0.5%, indicating non-hemolysis of ZnO QDs. Cellular morphology by results was supported by phase-contrast microscopy images. Good biocompatibility and high anticancer activity were noticed for ZnO QDs when compared to ZnO BC and provide versatile applications in the field of nano-biomedicine.


Introduction
Semiconductor carbon quantum dots of low dimensional (< 10 nm) have got notable consideration for possible use in medical applications such as diagnosis and treatment where larger nanoparticles (> 10 nm) are ineffective [1]. Quantum dots (QDs) have gained widespread attention due to their advantageous properties such as low poisonousness, small sizes, water solvency, biocompatibility, and exceptional photoluminescence [2]. Quantum dots (QDs) is a family of carbonaceous nanostructures and one of the emerging nanomaterials [3]. QDs are characterized as nanoparticles with molecule size more modest than it excite on Bohr radius which is around < 20 nm [4]. Additionally, QDs can be simply synthesized with low-cost methods that put aside complex, toxic, time-consuming and high-cost synthesis methods [5]. A Different expansive range of applications like solar cells, chemo-sensor, drug delivery, biosensor, cell imaging and catalysis [6][7][8][9][10][11] have been incorporated using carbon quantum dots (CQDs) because of their distinct physical and chemical properties like water solubility, brilliant fluorescence, non-toxicity and biocompatibility. Then again, the capability of utilizing CQDs in different applications expands the need to synthesize them on an industrial scale which is harmless to the ecosystem and green cycles. To avoid the issues of the chemical method of nanoparticle preparation an eco-friendly approach was adopted using plant material which is a green strategy [12]. Heaps of literature surveys have demonstrated benefits in green strategies which are natural, cost-effective, environmentally friendly, and simple to increase much of the time, the shortfall of compound chemicals, poisonous foreign substances and utilizing low-valued precursors [13][14][15]. Other than these, there are numerous possible effective molecules in the plant interceded synthesis of nanoparticles which is appropriate for a wide scope of biomedical applications [12].
A couple of designed strategies of QDs have been accounted. For example, pyrolysis, aqueous carbonization strategy, microwave radiation, laser removal, curve release, and plasma treatment [16][17][18][19][20][21]. Among them, microwave strategy is considered as a fundamental and green engineered approach due to its possible interaction, utilization of fluid medium and arrangement of QDs with better quantum yield. Also, microwave radiation was thought to be the best, cheapest, and quickest way to create fluorescent QDs, on the grounds that it does not use a lot of equipment, is not difficult to perform and saves time. The microwave-assisted heating method does not require any firm reaction conditions and is easy to operate compared to other synthetic methods. It is a one-step method of preparation and stabilization of QDs. Therefore, making it an economically and environmentally friendly method that could be used in the large-scale production of fluorescent QDs [22].
Metal and metal oxide nanomaterials have demonstrated critical restorative effects in scientific progress [23] because these nanoparticles have a high surface-to-volume ratio, which allows for better interaction with cancer cells and microscopic organisms [24]. Apart from the huge excitation on restricting energy, ZnO is a major semiconductor material with a direct wide bandgap and high directivity at 25 °C. The way that ZnO has solid pharmacological properties which increases its utilization in medical services regions, for example, anticancer, antimicrobial, and cancer prevention agents [25]. So, preparation of ZnO nano-material using metals like Zinc can be added with the plant-based synthesis of ZnO quantum dots and would pave a good way to fight against cancer in the forthcoming generations.
Among different cancers, breast cancer is one of the most widely recognized cancer disease in females in the entire world (WCR, 2008). 23% of all recently occurring cancers in women known as breast cancer has been observed worldwide, and 13.7% has been addressed as death due to breast cancer in both genders [26]. Medication, both optional and necessary, is currently thought to play an emerging role in cancer prevention. Plant-based materials from nature are inside simple reach and are an amazingly encouraging technique for chemo-prevention to impede the advancement of cancer in humans [27]. As an outcome, semiconductor nanoparticles/quantum dots (QDs) of low-dimensional have obtained incredible consideration in cancer treatment using nanotechnology [28].
Optimization of various factors over evaluating independent variable and responses relationship is given by Response surface methodology (RSM) [29,30]. This technique has been successfully applied in a variety of biotechnology fields, as well as some new studies for the green synthesis of carbon quantum dots [31,32]. RSM was used for the optimization of variables for the biological synthesis of ZnO QDs.
An enormous number of late written works uncovered that thoughtful and simple fabrications of QDs for various cancer investigations and also other biological applications have taken serious efforts. Zavareh et al. for example, reported that carbon quantum dots made of Chitosan were used as an anticancer medication to deliver 5-fluorouracil [9], Li et al. reported that a carbon quantum dot was used for combined photodynamic-chemotherapy examination of cancer cells [33], Ganesan Muthusankar et al. formed a productive probe for the simultaneous determination of anticancer and antibiotic drugs using nitrogen-doped carbon QDs implanted CO 3 O 4 with multiwall carbon nanotubes. [34]. Cutrim et al. prepared and characterized nanoconjugate-based QDs and an in vitro anticancer performance was checked [35]. ZnO QDs were prepared for the evaluation of breast cancer, was reported by Zahra Fakhroueian et al. [36]. Also, a chitosan-encapsulated ZnO QDs was designed for in vitro drug delivery response by Yuan et al. [37]. Accordingly, these research works got the impact of the greenway of QDs synthesis with compound interactions of useful hetero-atom doping, a gathering of an antecedent, which gives the way to the solid arrangement of quantum yield. Nature reliably gets additional thought from material scientists considering its unlimited focal point for making novel materials with less biological effect. To enrich biomass firm QDs, alternate bio-waste/plant materials were used as regular forerunners to set up the quantum dot material [38,39]. Because of their exceptional fluorescence and identifying properties, biomass-derived QDs have received a lot of attention from scientists as a candidate material for semiconductor quantum dots and other fluorescent regular components.
Catharanthus roseus is additionally notable as Madagascar; C. roseus is a type of blossoming plant having a place with the family Apocynaceae. C. roseus originated on the Indian Ocean Island of Madagascar, and it is now widely distributed in Australia, China, South America, Indonesia, and North America. The plant's leaves are abundant in monomeric indole alkaloids such as vindoline and catharanthine, which have remarkable therapeutic properties such as anticancer, antibacterial, antifungal, antimicrobial, cancer prevention agent, wound healing, and antiviral activities. Likewise, plant leaf concentrates are a rich source of chemically significant terpenoid, indole alkaloids, and it is currently used in the semi-union of two anticancer medications (vinorelbine and vinflunine) in pharmaceutical companies [40,41]. Thus, it is profoundly alluring to set up a proficient QDs from C. roseus leaves without the guide of solid corrosive dissolvable, surface passivation reagent, as well as a complicated post-treatment procedure [38].
By taking into consideration of the above facts, a novel method that combines ZnO BC derived ZnO QDs was synthesized using extract of C. roseus and Aloe vera gel, which was then converted to QDs and assessment of their biological activities are reported. C. roseus leaves were selected since it contains vinblastine and vincristine. Chemical constituents of C. roseus are expected to play an important role in the development of ZnO QDs. Interestingly, we exhibit the anticancer activity of C. roseus-derived ZnO QDs which are represented in Scheme 1. The connection between the biological activity of cancer cells in anticancer activity and hemolytic activity of low-dimensional greensynthesized QDs are the major highlights of this work. The results of commercial products and control samples were compared. We have primarily engaged one-pot approach to prepare QDs without any passivation agent by a simple microwave-assisted technique which is a green synthetic and eco-friendly approach applying C. roseus leaves as carbon origin. Critically, our outcomes uncovered that C. roseusbased QDs have minuscule molecule size, low harmfulness, solid fluorescence and biocompatibility, which have been utilized in cell imaging of MCF-7 cell line. C. roseus-based ZnO QDs in recent research have great likely application for biomedical and environmental pollution.

Reagents and chemicals
C. roseus (Vinca rosea) leaves were collected from nearby local farms in Tiruvannamalai; Neem leaves and Aloe vera were gathered from Arunai Engineering college campus, Tiruvannamalai. Other chemicals like pharmaceutical grade Zinc Sulfate (ZnSO 4 ) were purchased from Hi-tech enterprise Pvt Ltd, Chennai. Sodium hydroxide and Glacial acetic acid were purchased from Qualiens Chennai, India.

Synthesis of ZnO Nps using Neem (Azadirachta indica) leaf extract
Neem leaves of around 30 g were completely washed using distilled water and were dried and crushed. 1 L distilled water was added to this and bubbled for around 15

Preparation of Bionanocomposite (ZnO BC) using C. roseus extract
Prior to preparation, the gathered C. roseus leaves, which are high in carbon source, were thoroughly washed in deionized water, hacked into small pieces, and dried at room temperature. 50 ml of deionized water was mixed with 0.5 g of C. roseus leaves and bubbled for 15 min. From then on, it was permitted to reach room temperature. The pale yellowish arrangement was obtained and separated using a 0.45 μm layer channel to remove unreacted natural moieties and unwanted large particles [38]. 0.5% ZnO Nps were dispersed in 1 g of Aloe vera gel for 2-3 h and it was sonicated for 5 min at 50 °C. After that, 0.5 ml of C. roseus leaf extract was added dropwise and stirred for 24 h to achieve a homogeneous mixture. At last, the precipitate was dried at 50 °C overnight in the oven. A brownish-black powder of ZnO BC was formed and used as a natural carbon source for the synthesis of ZnO QDs.

Biogenic formation of ZnO QDs from ZnO BC (C. roseus extract)
A one-pot synthesis of microwave-assisted procedure was chosen to blend the QDs from the C. roseus leaves Scheme 1 Schematic representation of the synthesis of ZnO QDs from ZnO BC extract prepared from ZnO BC as a natural carbon source. 20 ml of 5 mg/ml ZnO QDs was prepared using 0.5 mg/ ml ZnO BC and suspended in distilled water of 30 ml, kept under magnetic stirrer for 10 min. The ZnO BC suspension was then exposed to microwave for 15 min and then at 8000 rpm, it was centrifuged for 10 min to assist ZnO QDs synthesis. The supernatant was collected and observed under UV light trans-illumination for fluorescence property and stored for further applications. This entire process for the synthesis of ZnO QDs is also shown in scheme 1.

Characterization studies
The UV absorption (Perkin Elmer Lambda 9) was carried out to find the excitation wavelength of QDs. To characterize the optical and semiconductor properties of fluorescence quantum dots, photoluminescence spectra (HORIBA) have been performed. Zeta potential and Particle size (HORIBA) estimation tests were carried out using a nano-material vs. pure ZnO. The surface morphology and elemental analysis of ZnO BC and pure ZnO were examined using a scanning electron microscope (SEM; JEOL JSM 6360) coupled with an energy-dispersive X-ray (Oxford Instruments, INCApentaFETx3). To investigate the effect of organic species in plant extract on ZnO BC and pure ZnO, the FT-IR spectrum (IR prestige 21, Shimadzu) was used. X-ray diffraction (XRD) was used to examine the crystalline nature of the as-prepared ZnO BC and pure ZnO (Bruker D8 Advance).

Morphological analysis in MCF-7 cell line
Inverted phase-contrast microscope at 20X magnification (OLYMPUS LS CKX 31) was used to see the macroscopic and microscopic changes that occurred in MCF-7 cells which were exposed to different doses of 500-25 µg/ml of ZnO BC (0.5 mg/ml) and 3.125-50 µg/ml of ZnO QDs (5 mg/ml).

Hemocompatibility assay
To determine the toxic effect of ZnO QDs with red blood cells (RBC), hemolytic assay was carried out using a whole blood sample. Fresh blood was collected using a disposable syringe and stored in a centrifuge tube containing EDTA to prevent the blood from clotting. The blood sample was diluted using 1 × PBS buffer in equal volume [46]. To 0.5 ml of blood, 5 mg/ml ZnO QDs of the prepared sample was added in different concentrations 50-250 μl which an increment of 50 μl in an Eppendorf tube, 3-h incubation time was given with agitation for a few minutes after every 1 h. Then, the sample was centrifuged for 10 min at 1600 rpm and then removed the supernatant and the optical density was checked at 540 nm.

Statistical analysis for anticancer activity
Analysis of Variance (ANOVA) and Tukey's test were applied to know the high range measurable domain of difference between mean values of cell viability of different cell lines treated with ZnO QDs vs. ZnO BC. All tests were carried out in triplicate, and the results were reported as mean ± SD [47]. Hence, from the results, statistical significance with p values < 0.05 was contemplated.

Data analysis of Box-Behnken design for ZnO QDs
The Box-Behnken design was used in this study to estimate the synergy of the factors for a concentration of ZnO Nps (X1), A. vera (X2), and C. roseus (X3) on the absorbance response of ZnO QDs. The range and levels of the variables were observed. For the prepared design, 17 runs were performed for the desired factors and levels in this study. Design Expert-13 software was used to analyze the variables of response surfaces inside the experimental scope. As a result, three additional confirmation experiments were carried out to validate the statistical experimental statistics. Finally, UV-Vis and PL examinations were performed in derived optimum conditions after ZnO QDs biosynthesis to demonstrate the reasonableness of the BBD.

Visual observation of ZnO QDs
With the developing interest in green synthesis and to restricting the utilization of toxic chemicals and nanoparticles of heavy metals, the improvement of organic, biomimetic, and biochemical methodologies for forming nanoparticles was wanted. Green strategies for nanoparticles synthesis are more invaluable than physicochemical techniques because of their eco-friendly and non-toxic nature. Figure 1 shows the visual observation of prepared ZnO QDs from C. roseus leaf extract by a microwaveassisted method. The commercial ZnO and synthesized ZnO were used as a control sample in order to prove the fluorescence property emitted by ZnO QDs on exposure to UV light. The presence of a good fluorescence effect was noticed in ZnO QDs (Fig. 1c) prepared from ZnO BC alone when compared to control samples a) commercial ZnO and b) synthesized ZnO. Apart from this, the prepared ZnO BC and ZnO QDs were characterized using XRD, FT-IR, SEM-EDX, UV spectroscopy, PL spectroscopy, particle size and Zeta potential analysis.

UV and photoluminescence spectrum
UV spectroscopy was finished to break down the optical properties of processed ZnO QDs (bare) including ZnO QDs (C. roseus) and shown in Fig. 2a. The UV range revealed a change in intensities from 270 and 360 nm, confirming the π-π* and n-π* electronic changes caused by the presence of C-O and C-C functional groups in ZnO QDs (C.roseus) [38]. To impress the properties of Photoluminescence of QDs exhaustively, an excitation frequency was adjusted from 270 to 350 nm with a period of 10 nm. The most extreme outflow of 500 nm and 550 nm was seen at an excitation frequency of 330 nm for ZnO QDs (bare) and ZnO QDs of C. roseus (displayed in Fig. 2b). This wavelength confirms that the ZnO QDs prepared out of C. roseus leaf extract showed high intensity when compared to that with ZnO QDs (bare). It was seen that the extract of natural material acts as penetrating and reducing agents to synthesize quantum dots. The presence of greenish-blue color was seen with the naked eye due to the presence of high intensity which confirms the presence of good optical property [48].

X-ray diffraction (XRD) analysis
The XRD pattern of pure ZnO and ZnO BC is shown in Fig. 3. According to the reference data for ZnO wurtzite in JCPDS no. 36-1451, the hexagonal ZnO wurtzite structure is well indexed by all of the diffraction peaks of ZnO BC and pure ZnO. When compared to pure ZnO (Fig. 3a), highly intense peaks were observed at the ZnO lattice structures of ZnO BC (Fig. 3b). This is due to the formation of hydrogen bonds by carbohydrates present in phytochemicals of C. roseus, A. vera gel, and Neem extract present in ZnO BC. The high crystalline strength was confirmed by the presence of a high intense peak of pure ZnO ( where θ is the Bragg's angle, K = 0.89 (a constant), β denotes the full width at half maximum (FWHM) of the selected XRD peak and λ (0.15406 nm) is the X-ray wavelength. The fact that ZnO BC has a smaller crystalline size than pure ZnO confirms that the natural agents combined well and functioned as natural precursors during the synthesis of ZnO BC.

Fourier transform infrared (FT-IR) spectral analysis
The explained FT-IR spectra peaks are shown in Fig. 4 as evidence of the synthetic effectiveness of pure ZnO and ZnO BC. The expansive peaks of ZnO BC around 3495 cm −1 are attributed to the extension of -OH, which confirms alcoholic, phenolic, and carboxylic structures, and the peak at 2230-2018 cm −1 was compared to C≡N, which is due to the amides present in natural precursors of ZnO BC (C. roseus, A. vera gel, and Neem extract). ZnO BC's aliphatic and aromatic C-H stretches are at 2359 cm −1 . Furthermore, the peaks at 1620 cm −1 and 1101 cm −1 identified its C=O and C-O groups. Also, the peaks at 1142 cm −1 and 628 cm −1 confirm the C=C and C-C bond due to the presence of polyphenols in the phytochemicals of C. roseus extract, indicating that the synthesized ZnO BC acts as a carbon rich source in the preparation of Quantum dots [12,38]. The presence of zinc and oxygen is marked by the appearance of ZnO stretching at 3500 cm −1 in the pure ZnO's XRD pattern. The presence of a ZnO bond has been confirmed by the presence of peaks at 1200 cm −1 for both pure ZnO and ZnO BC, indicating the presence of zinc and oxygen. Other than ZnO bonds, no other peaks have formed for the pure ZnO pattern. This demonstrates that zinc oxide was well combined with natural precursors (C. roseus extract, A. vera gel, and Neem extract) acted as capping and reducing agents to form zinc oxide bionanocomposite.

Scanning electron microscopy and energy-dispersive X-ray analysis
The morphological components of ZnO BC and pure ZnO observed under SEM revealed particle sizes of approximately 23 nm and 36 nm, respectively. Because of the reduction of precursor salts by natural C. roseus extract, the state of ZnO BC was observed to be spherical, crystalline and homogeneous (Fig. 5a). Because of the presence of zinc oxide ions, pure ZnO was found to be amorphous in nature (Fig. 5c). As a result, C. roseus, A. vera gel, and Neem extract responded as reducing and capping agents in ZnO BC, and the results were consistent with the XRD patterns of ZnO BC and pure ZnO. The EDX spectra revealed that the zinc and oxygen components in ZnO BC were combined at 12.81% and 50.67%, respectively (Fig. 5b). The carbon peak in the spectrum represents the adsorbed constituents of the leaf extracts [49]. Since, carbon is the primary chemical constituent in the chemical structures of both C. roseus and A. Vera leaves. The EDX spectrum confirms the presence of ZnO in ZnO BC by showing particle formation via bioreduction of plant leaf extracts. In addition, as shown in Fig. 5d, the EDX spectra of pure ZnO revealed the presence of only zinc and oxygen, indicating that it behaves as a metallic precursor in the preparation of nanocomposites. Furthermore, the EDX results agree with the FT-IR spectral analysis of pure ZnO and ZnO BC.

Particle size distribution and Zeta potential measurements
Particle size distribution and Zeta potential (Zetasizer NanoZS, Malvern Instruments, UK) were used to measure and compare the stability of ZnO QDs, ZnO BC, and pure ZnO. The measurements were obtained by averaging three repeated measurements. The particle size distributions of ZnO QDs, ZnO BC, and pure ZnO were generally narrow, with mean particle sizes of 16.2 nm, 22.7 nm, and 36.9 nm respectively, as shown in Fig. 6a1, a2, and a3. This demonstrates that the obtained ZnO QDs and ZnO BC are smaller in size than pure ZnO [50]. Surface charges of about − 2.5 mV, − 9.7 mV, and − 12.4 mV were observed for the ZnO QDs, ZnO BC, and pure ZnO respectively, indicating the presence of ZnO QDs formation and blending of Zinc sulfate, A. vera, C. roseus and Neem leaf extract in ZnO BC. This had an effect on the size and surface charges of ZnO QDs and ZnO BC. The stability of quantum dots and Bionanocomposites was suggested, and the value of zeta potential for ZnO QDs is within the stable low range when compared to ZnO BC and pure ZnO [49]. As a result of the residual surfactants, ZnO QDs may accumulate and act as more stable components.

Optimization and confirmation experiments of ZnO QDs using RSM
Optimization of variables concentration for the synthesis of ZnO QDs was achieved through Box-Behnken design (BBD) of experiments. BBD was particularly preferred for accurate estimates, low-cost and high-quality products development with specific desired features demanded in research.
The data of absorbance of ZnO QDs were analyzed using analysis of variance (ANOVA), regression coefficients, and regression equation using Design Expert-13 software. The absorbance as a contemporary function of the concentration of ZnO Nps, A. vera, and C. roseus is represented in the polynomial equation shown below in Eq. (2) We can learn from the analysis that the model's coefficient of determination (R 2 ) is 0.9337, indicating that the model is significant. The model F value is 10.95, indicating that the model is suitable for addressing the genuine connections among the selected factors. Because the P values are less than 0.05, the independent factors A, B, and C, as well as the quadratic term of AB, AC, BC, and A 2 , B 2 , C 2 , have a large effect on the absorbance of the obtained ZnO QDs. The 2D form plots and the 3D response surfaces of the reactions using Eq. (2) for the QDs absorbance are displayed in Fig. 7. To demonstrate the intuitive effects of autonomous factors on responses, one variable was kept constant while the other two shifted within a specific range. The shapes of response surfaces and contour plots revealed the extent of interaction between various factors and their nature [29]. Less conspicuous or negligible connections are shown by circular contour plots, whereas relatively notable interactions are generally shown by elliptical shape plots (Fig. 8) illustrating that the concentration of ZnO Nps, A. vera, and C. roseus significantly influences the absorbance of the as-prepared ZnO QDs, while the circular nature in Fig. 8b demonstrates that the absorbance is not affected by the concentration value of precursor solution significantly. Graphical optimizations by Design-Expert software provide optimum conditions for the microwave method. Thinking about the two expenses and effectiveness, the optimum operating parameters were observed to be A = 1 ml, B = 1 g, and C = 0.5 ml. That is, the concentration of ZnO Nps, A. vera, and C. roseus values was fixed at 1 ml, 1 g, and 0.5 ml, separately. Affirmation tests were conducted in three replicates under these conditions. The observed mean absorbance was found to be generally consistent with the expected qualities. The QDs obtained had a high absorbance up to 330 nm, which corresponds to the UV properties of the synthesized ZnO QDs.

MTT assay
MTT assay was used to compare the cytotoxicity of ZnO QDs to ZnO BC. A control (untreated cells) has been established for the comparison of treated cells. In our test, the control shows 100 percent feasibility with the cell lines that were developed to evaluate anticancer activity [50]. The breast cancer cell line was treated against 5 mg/ml ZnO QDs and 0.5 mg/ml ZnO BC of varying concentrations ranging from 3.125 to 50 µg/ml and 25 to 500 µg/ml, respectively. It was inspected from the test that there was decreased viability rate, ranging from 60 to 15% for 0.5 mg/ml ZnO BC and 45 to 5% for 5 mg/ml ZnO QDs which revealed a tenfold decrease in cell viability with less concentration scale for 5 mg/ml of ZnO QDs when compared with that of 0.5 mg/ ml ZnO BC. Phase-contrast microscopy images were shown in Fig. 9a and b which also distinctly supports the percentage viability of cells for ZnO BC and ZnO QDs. Therefore, the prepared ZnO QDs exhibit more biocompatibility when compared to ZnO BC [44]. The ZnO QDs-treated cells showed more strain with that of ZnO BC due to the reduced particle size and presence of green materials (C. roseus and A. vera) act as a rich carbon source for the synthesis of ZnO QDs. These carbon sources are in turn responsible for the anticancer activity and due to its penetrating property against breast cancer cells it can be used as an efficient anticancer drug.

Hemocompatibility
Hemolytic assay of ZnO QDs was checked to test its blood compatibility. From the quantitative assessment of Hemoglobin, furthermore, from the actual perception, the test was directed to assess the toxicity of synthesized 5 mg/ ml ZnO QDs at various concentrations, i.e., 50, 100, 150, 200, and 250 μg/ml, it is prominent that the OD value for hemolysis was significantly higher with 250 μg/ml, while just slight hemolysis was seen with 50 μg/ml rather than the negative control ( Supplementary Fig. 1). The positive control caused the most hemolysis. The statistical significance using ANOVA was obtained as p < 0.05 and the Hemolysis tests were conducted in triplicate and results was obtained for mean vs. standard deviation as shown in Fig. 10. According to ASTM (American Society for Testing and Materials Designation), which is standard practice for assessing the hemolytic properties of materials. This indicates that hemolysis of less than 2% is considered non-hemolytic, 2-5% is considered somewhat hemolytic, and > 5% is considered hemolytic [42,50]. As a result, the results of 5 mg/ml ZnO QDs have met the material standard of Hemolysis.

Cytotoxicity mechanism of ZnO QDs and ZnO BC on MCF-7 cells
The ZnO QDs-treated cells showed more strain with that of ZnO BC due to the reduced particle size and when these highly crystalline nanomaterials are in direct contact with cancer cells can induce physical damage to the outer cell membrane because of the negative charge present on the outer orbital of ZnO BC and ZnO QDs nanomaterials which are good acceptors and cause damage to the cancer cells. The cell damage caused by ZnO QDs is substantially higher than that of ZnO BC due to reduced particle size, concentration and surface penetration property of fluorescence ZnO Nps (ml) and C. roseus (ml), c A. vera (g) and C. roseus (ml) using BBD of RSM for investigation of interacted parameters belonging to the synthesis of ZnO QDs quantum dots [51]. Therefore, strong interaction is due to charge transfer between the low concentration of ZnO QDs and cancer cell membrane cause cell damage these are proven by MTT assay images of phase-contrast microscopy of MCF-7 cells treated. Also, these cell membranes cause reactive oxygen species (ROS) when they are in direct contact with the nanocomposite and QDs. This causes disintegration into the cells due to the cytotoxic nature of carbon rich ZnO nanomaterials. They penetrate through the mitochondria of the cancer cells and destruct the DNA strains in the nuclei [52]. Zn 2+ ion release from ZnO QDs and ZnO BC can be considered as one of the main mechanisms involved the cytotoxicity of cancer cells [53]. Also, natural agents (Azadirachta indica, Aloe vera gel, and Catharanthus roseus) present in green-synthesized ZnO nanomaterials consist of various anticancer agents as phytochemicals like flavonoids, Limonoids, Quercetin, Nimbin, Lectins, alkaloids, Terpenoids, Phenols, and Tannins; these act as an antioxidant agent and has the characteristic to control or stop the free radical activity against cancer cells [54][55][56]. High uptake of these phytochemicals present in green-synthesized ZnO nanomaterials on cancer cells is possible due to the C. roseus which is a rich source of polyphenols which acts as binding agent to the surface of cancer cells. Therefore, Fig. 9 a, b Cell viability and MTT assay images using phase contrast microscopy of MCF-7 cells treated against ZnO BC and ZnO QDs of concentration ranges:9a-ZnO BC (i) Control (ii) 500 µg/ml (iii) 250 µg/ml (iv) 100 µg/ml (v) 50 µg/ml (vi) 25 µg/ml; 9b-ZnO QDs (i) control (ii) 50 µg/ml (iii) 25 µg/ml (iv)12.5 µg/ml (v) 6.25 µg/ml (vi) 3.125 µg/ml, respectively. All tests were carried out in triplicate, and the results are shown as mean vs. standard deviation as shown in graph (a, b). As a result of the findings, statistical significance was determined using p values < 0.05 cellular uptake of ZnO QDs plays a vital role in cancer cell destruction faster because of its high fluorescence property and low particle size compared with that of ZnO BC [57,58]. Understanding the cytotoxicity of ZnO nanomaterials is critical for the development of relatively hygienic biomaterials for use in the treatment of diseases such as cancer. This cytotoxicity mechanism is sketched in Fig. 11.

Conclusion
We, for the first time, have prepared and reported ZnO QDs derived from bionanocomposite ZnO BC by adapting microwave-assisted method without using any toxic chemicals. Also, ZnO QDs were optimized using BBD of RSM to obtain the optimum reaction conditions of concentration value of ZnO Nps, A. vera, and C. roseus. Under the optimal conditions, the concentration value of ZnO Nps, A. vera, and C. roseus values was fixed at 0.5%, 1 g, and 0.5 ml, respectively. The obtained ZnO QDs have a high absorbance at 330 nm which was in contrast to that of characterization results obtained. The low concentration range of ZnO QDs revealed favorable cytotoxicity when compared to ZnO BC. From these results, it can be concluded that due to the surface penetration property of ZnO QDs (C. roseus and A. vera), it could act as a new type of fluorescence probe that may act as a nano-carrier and can be known to the nanomedical worldwide market after finishing its clinical preliminary and provide promising potential in the field of breast cancer treatment research.