One-pot biosynthesis of CdS quantum dots through in vitro regeneration of hairy roots of Rhaphanus sativus L. And their apoptosis effect on MCF-7 and AGS cancerous human cell lines

Development of green based synthesis of nanoparticles has been regarded as a novel and safe alternative method compared to conventional methods. Semiconductor cadmium sulfide quantum dots (CdS QDs) possess unique biological and medical applications includes labeling cells, diagnosing of diseases and imaging intercellular events. The present paper reports the biosynthesis of CdS QDs through aqueous extracts of the regenerated hairy roots of Rhaphanus sativus L. as the organic source for both reducing and stabilizing of Cd and S precursor ions. The characterization of synthesized QDs showed maximum absorbance peak of 460 nm and fluorescence spectrum of cadmium sulfide at 530 nm. The results of Transmission Electron Microscope (TEM) and EDS analysis demonstrated that the particles were morphologically spherical with size distribution between 2–7 nm and confirmed presence of CdS QDs. Fourier transform infrared spectroscopy (FT-IR) also showed the active presence of aromatic, amino, and carboxyl groups on the surface of quantum dots. Cytotoxicity effect of the synthesized CdS QDs on two cell lines include MCF-7 breast cancer and AGS gastric cancer were assayed through MTT assay. The results showed significant inhibitory effects of synthesized QDs on treated cells in a dose dependent manner. It was also concluded that CdS QDs had more apoptosis effect on MCF-7 cells rather than AGS cell lines. The obtained results clearly illustrated that the synthesis of CdS quantum dots with standard features would be possible through cost-effective, reliable, environmentally friendly and less toxic alternative method compared to chemical and physical processes and the MTT toxicity assay also illustrated the significant apoptotic effects of synthesized CdS QDs on carcinogenesis.


Introduction
Recently, many attempts have been made to discover and introduce novel therapeutic agents to target different cancers [1][2][3]. Nanomaterials are a novel group of materials with diameter between 1-100 nm that could present in different shapes and proportions. Given their shapes, they are classified into Zero-dimensional, onedimensional, and two-dimensional [4]. Recently, some of advanced types of metallic nanomaterials have been applied in pharmaceutical applications such as anti-cancer [5][6][7][8], antimicrobial [9,10] effects. QDs are considered as semi-conductive zero-dimensional nanocrystals with sizes in range of 1-20 nm. These materials are characterized by a nanoparticle core consisting of hundreds or thousands of elements in group II and VI Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
(Cadmium, Techtentium, Zinc, Selenium) or III (Tantalum) and V (Indium) [11,12]. The photophysical properties of quantum dots is dependent to their particle size, therefore, controlling the particles growth is crucially important. The quantum dots with larger size create shorter band gap and the more spectral power distribution. Accordingly, the color distribution in various wavelengths can be created by variations in nanoparticle size [13,14]. Among them, Cadmium sulfide QDs (CdS QDs) displays upper direct band gap of 2.4 eV and a small Excitation Bohr Radius of 2.4 nm [15,16]. They are also characterized by their high quantum function, large molar extinction coefficients, narrow/wide absorption, balanced photoluminescence (PL), near infrared (NIR) UV spectrum and high efficiencies compared with other fluorescence probes [17]. These features have made them interesting for their possible application in industry such as in light emitting diodes (LEDs), solar cells, lasers, absorption filters, piezoelectric converters, and biological sensors [18][19][20][21][22][23][24][25][26][27][28][29][30].
Commonly, chemical synthesis of quantum dots produces a measure of toxic components which preclude them for biological applications. It has been found in recent decade that biological systems including bacteria [31], yeast [32] and fungi [33], could revitalize and convert metallic ions into metallic nanoparticles by their proteins and metabolites. These studies showed that plant synthesis of quantum dots is very considerable due to their low costs, short production time, safety and large production amounts. Plant metabolites like terpenoids, polyphenols, alkaloids, phenolic acids, and proteins play a vital role in revitalizing metallic ions [34]. The reduction of metallic ions and producing nanoparticles are dependent to a variety of factors. In additions to the presence of biologically active molecules, the factors include pH level of reaction mixture, incubation temperature, reaction time, concentration of salts and ionic electrochemical potential are determining factors during QDs biosynthesis [35]. So, production of QDs through green synthesis could be consider as a novel and safe alternative approach through biological processes. One popular method in such synthesis was exploiting of plant organs, tissues, or cells [36]. Among them hairy roots were preferred over other plant organs due to their high capacity for accumulation of heavy metals [37] and it was regarded as a successful method in producing important secondary metabolites that could contribute to nanoparticle formation [38]. The induction of hairy roots would be possible by infecting the plant tissue with Agrobacterium rhizogenes bacterium. In this way, the resultant transformed roots are genetically stable with quick growth pattern in hormone-free environment. Rhaphanus sativus L. is an annual plant of Brassicaceae family that has high food value because of high content of anti-oxidants, glucozinoids, isothiocyanate, diet dissolved fibers and vitamins B and C [39]. It also has 4-(methylthio)-3-butenyl isothiocyanate, allyl isothiocayanate, benzyl isothiocyanate and phenethyl isothiocyanate as well as flavonoids such as kaempherol glycosides, peroxidases and antioxidants [40]. These plants derived substances could play an important role for reduction and following green synthesis of metal ions present in their growth medium. Peptides and proteins also have functional groups in their lateral chains that facilitate the attachment of metal ions and may facilitate nanoparticles formation [41]. In the present paper, a novel and facile one-pot approach was introduced for biological synthesis of cadmium sulfide via hairy roots of Rhaphanus sativus L. As illustrated in scheme 1 (A) the Rhaphanus sativus L plant explant was infected by Agrobacterium rhizogenes and finally resulted to hairy root induction. Next for synthesis process, first, the medium containing hairy root extract was prepared and Na 2 S and CdSO 4 precursor salts were added to the medium respectively under different pH condition (scheme 1(B)). Finally, the structure, morphology and luminescence of the synthesized CdS QDs were characterized. The toxicity effect of these green synthesized CdS QDs were analyzed through determination of their apoptosis effect on two human cancer cell lines. The study provides an economical and safe approach for synthesis of fluorescent CdS QDs with distinguished properties and well compatible with chemical synthesized approaches and the clinical effect of synthesized CdS QDs were also determined through MTT assay for their further biological application.

Materials and methodology
Preparation of hairy roots Green synthesis of QDs was done via producing hairy roots of Rhaphanus sativus L according to previous report [42]. The induction of hairy roots was performed in a liquid hormone free MS medium using Agrobacterium rhizogenes incubating at 28°C for 15 days. After the set period, the grown hairy roots were washed with sterile distilled water in order to remove any possible impurity. Following hairy roots were cut into small pieces. 100 ml of sterile distilled water was added to 10 g of hairy roots of Rhaphanus sativus L. The samples were placed in shakers with 110 rpm at 60°C. A paper filter was used to separate the small particles. The resultant extract was centrifuged at 12 000 rpm for 15 min. Then pH level of liquid extract of Rhaphanus sativus L was adjusted in range of 3-11 using 1% H 2 So 4 compound and 0.5 M of NaOH. In order to remove tissue residues, aqueous extract of hairy roots was filtered through a 0.2 micron filter in a sterilized laminar hood and kept at room temperature for biosynthesis of CdS QDs.

Biological synthesis of quantum dots of Cadmium sulfide
For biological synthesis of QDs, 30 ml of the hairy roots extract was added to a 100 ml Erlenmeyer flask which was put in a stirrer with 250 rpm at 28°C. Then, 2 ml of CdSO 4 0.025 M was syringed into the extract. The solution was put in a stirrer with 250 rpm at 28°C in a dark setting for 3 days. Following 500 μl of Na 2 S 0.5 M was added into the solution (solution color turned to yellowish). Then the solution was put in a stirrer with 250 rpm at 28°C in a dark condition. After 4 days, the solution was centrifuged at 5000 rpm for 10 min and the upper phase was used throughout the experiment. In addition, salt-free extract was used as control solution.

Characterization of synthesized CdS QDs
Synthesized CdS QDs were analyzed for their various features includes absorption spectrum, emission spectrum, morphology, crystallographic structure, presence of functional groups and average size of synthesized particles which determined by UV-vis spectrophotometer (PerkinElmer LAMBDA 950), Fluorescence spectrophotometry (PerkinElmer LS 55), Field emission scanning electron microscopy (FESEM), X'Pert PRO MPD x-ray diffraction, Fourier-transform infrared spectroscopy (FTIR) (IF505 manufactured by BRUKER) and Zetasizer Nano Series model of Dynamic Light Scattering (DLS) instrument by Malvern Corporation, respectively.

Cell culture
The human cell lines including MCF-7 breast cancer cells and AGS gastric cancer cells were cultured in DMEM medium supplemented with 10% FBS and 60 mg ml −1 penicillin and 100 mg ml −1 streptomycin antibiotics, in a humidified 5% CO 2 atmosphere at 37°C for 72 h (medium refreshed after 48 h) to ensure the cell growth.

MTT assay
The cytotoxic effects of CdS QDs on human cancer cells were determined using colorimetric MTT assay. The initial density of 1×10 4 cells per well was chosen to culture were grown in 96-well plates containing 100 ml DMEM medium with 10% FBS for 24 h. For determining of cytotoxicity effect, different concentration of CdS QDs was prepared upon addition of distilled water to the stock solution with serial dilution method. The increasing concentration of CdS QDs solution (3.25, 6.25, 12.5, 25, 50, 100 mg ml −1 ) were added to the wells. As control, the same concentrations of plant extract were used to compare with obtained results. After incubation of plates for 24 h, the medium was removed and replaced by fresh medium and incubated for additional 24 h. Then wells were filled with 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg ml −1 in phosphate buffered saline (PBS), pH 7.4) and incubated in a humidified atmosphere for 4 h at 37°C. After the incubation, the containing media were removed and 100 ml of 99.9% dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. Absorbance was immediately determined at 570 nm (reference wavelength 630 nm) using a microplate reader (infinite NanoQuant M 200), Tecan (Zurich, Switzerland). Treated cells in CdS QDs containing medium were compared to control cells in blank plant extract medium (Cell viability=A treated /A control ×100) and expressed as the mean±SD of triplicates [43,44].

Results and discussion
Characterization of synthesized CdS QDs The particle size distribution histogram determined from FESEM image is shown in figure 1(a). It was found that quantum dots of cadmium sulfide synthesized by aqueous extract of hairy roots of radish (at pH 6) were well dispersed with spherical morphology with average size of 2-7 nm. DLS analysis was also used in order to determine the QDs size more accurately. The particle size histogram of synthesized QDs in figure 1(b) showed narrow size distribution having 2-7 nm in diameter. For further confirmation, TEM analysis was also performed and TEM image ( figure 1(C)) verified the size of QDs around 3 nm. In order to obtain further insight to the features of the CdS QDs, EDX analysis was also performed. According to EDX analysis the presence of Cd, S, Na and K ions in the reaction was confirmed (figure 2). The weight percentage composition of synthesized CdS QDs were 13.87, 8.58 for Cd and S elements respectively. Presence of Cd, S and Na ions are due to CdSo 4 and Na 2 S salt precursors and presence of K ions was attributed to either the water or plant cell tissue. UV-visible spectrophotometry UV visible analysis is critical in investing the behavior of nanocrystalline semiconductors. The absorption wavelength range for both crude extract of hairy roots and synthesized quantum dots was measured at 200-600 nm. As it was illustrated in figure 1 the absorption spectrum of hairy roots extract show absorption band around 260 nm while synthesized QDs solution show absorption band between 350-550 nm with the maximum wavelength occurred in 460 nm. It was concluded that peak at 460 nm correspond to the QDs nanoparticles with a diameter 4-6 nm. In similar studies aqueous extract of hairy roots of Linaria maroccana L [38] and Fusarium oxysporum [34] were used for synthesis of cadmium sulfide quantum dots and the wavelength range of 450-462 nm determined as the absorption spectrum. The aqueous solution of QDs exhibited bright yellow color as demonstrated in inset of figure 3.

Luminescence of CdS QDs
Luminescence property is considered as the most significant and applicable feature of QDs due to its broad application in many studies. Fluorescence emission spectra of QDs were analyzed at different excitation wavelengths ranging from 300 to 600 nm. The highest emission intensity at 520 nm was observed for excitation at 370 nm. Figure 2 shows the luminescence spectrum for crude sample compared to synthesized CdS QDs after excitation in 370 nm wavelength. As it is shown the fluorescence emission of synthesized QDs observed at 520 nm with red shift compared to control sample at 370 nm wavelength. The aqueous solution of CdS QDs exhibited bright green luminescence compared to control sample under UV light as illustrated inset of figure 4. It was also reported previously that luminescence band was found at 425-500 nm in a study which applied aqueous extract of hairy roots of Linaria maroccana L [38] and R.palustris [45] for the synthesis of cadmium sulfide quantum dots. Absorption and luminescence of semiconductor nanoparticles depend on type, size, and surface properties and is affected by the reciprocal impact between nanoparticles surface and the environment and the reciprocal impact among the nanoparticles themselves [46]. The colloidal solution storage of CdS QDs was extremely stable and did not show aggregation even after three months.  Fourier transform infrared spectroscopy (FTIR) FTIR analysis was performed to study the mechanism of CdS QDs formation and also determine the presence of functional group on the surface of QDs. FTIR spectrometry shows that absorption vector in a 1634.32 cm −1 point belongs to Amide compounds (N-H) of the polypeptides or proteins while 2069.29 cm −1 is the absorption frequency of (C-H) asymmetric groups and 3436.05 cm −1 belongs to hydroxyl groups (OH) ( figure 6). Similar results also reported previously on the synthesis of CdS QDs synthesis by F.oxysporium [33]. FTIR spectrum revealed the presence of hydrophilic functional groups over the surface of QDs leading to their excellent water solubility. Also, presence of proteins, functional groups and carbohydrates play crucial roles in bio functionalization and further application of QDs nanoparticles in biosynthesis.

Optimization of synthesis procedure
In order to evaluate the effect of pH on biosynthesis of cadmium sulfide QDs, the reaction was performed at different pH using aqueous extract of hairy roots of radish. It was found that maximum fluorescence intensity of synthesized quantum dots was progressively enhanced with an increase in the hydroxyl ion concentration. Comparing the pH values showed approximately 2-fold increase in the fluorescence intensity of CdS QDs at pH=11 compared to that at pH=3 ( figure 7(A)). It was also found that pH level of aqueous extract plays a major part in formation of nanoparticles with different fluorescence intensity correspond to their sizes and may indicating the growth of CdS QDs during the biochemical process [48]. Any variation in natural pH levels of the aqueous extract affects the attachment or recovery of metallic cations and anions during the synthesis. It was assumed that separating a proton from ugenol OH group would lead to resonance and, through further oxidation and this construct may recovers metallic cations and consequently, nanoparticles are formed.
The optimum temperature for synthesis of QDs were determined through incubation of reaction at varying temperature. As it was shown in figure 7(B) the highest intensity of synthesized QDs was at 28°C, and this temperature was selected as optimum point. Another determining factor for the synthesis of CdS QDs is the reaction time. In order to determine the optimum period, the fluorescence emission of synthesized QDs were monitored during the reaction. The obtained results showed the maximum intensity was obtained after 4 days and the fluorescence emission remained stable in next days (figure 7(C)). So, the 4 days reaction time was selected as the optimum reaction time.
Cytotoxicity of synthesized CdS QDs MCF-7 and AGS cancerous cell lines were treated with concentration series of CdS QDs (0-100 mg ml −1 ) and cell viability was determined. The results of MTT assay showed the dose dependent decrease in cell viability of treated cells with CdS QDs solution compared to treated cells with blank plant extract as a control. As shown in figure 8, the amount of cell viability decreased upon addition of higher concentration of QDs in both cell lines. Although this effect was more significant for MCF-7 cells. Viability of MCF-7 cells inhibited significantly at 12.5 mg ml −1 and reached its highest effectiveness (20%) at maximum concentration of CdS QDs ( figure 8(A)). On the other side CdS QDs induced significant apoptosis on AGS cells at 25 mg ml −1 and its maximum concentration led to 50% viability of treated cells ( figure 8(B)). Plant hairy root extract alone could not play significant role for inhibition of both cell lines (figures 8(C), (D)). So, it was concluded that the MCF-7 cells showed more sensitivity to toxicity of CdS QDs compared to AGS cells. Also, in order to confirm the apoptosis effect of CdS QDs on MCF-7 cells, microscopic images were shown in figures 8(a)-(c) that illustrated after incubation of cells with 3 doses of incubated synthesized CdS QDs at 100, 50 and 25 mg ml −1 respectively, resulted to cell apoptosis. The plant extract (figure 8(d)) and control (figure 8(e)) couldn't induce any apoptosis effects on MCF-7 cells.

Conclusion
Remarkable properties of QDs in industries and medicine, made them as interesting targets. They are usually produced by costly chemical methods which leave a measure of toxic agents on nanoparticles that might cause problems in later applications. Recently, plant extracts have been introduced as convenient alternatives in producing metallic nanoparticles. In the present experiment regenerated induced hairy roots were used in biosynthesis of CdS QDs, since they are capable of quick controlled growth in lab setting and have superior property rather than the original plant in aspect of secondary metabolites content. The UV-Vis spectrophotometry and luminescence were used in determining optical properties of synthesized CdS QDs. The absorption and fluorescence emission spectrum of synthesized QDs were in 460 nm and 520 nm wavelengths respectively. Morphologically spherical and structurally crystalline, the synthesized particles showed acceptable stability due to presence of proteins and functional groups. According to TEM results QDs were found to be mostly 2-10 nm. The apoptosis effect of synthesized CdS QDs were investigated on MCF-7 and AGS cell lines. Obtained results showed both cells experienced decreased viability over increased concentration of applied QDs samples and the MCF-7 cells were more sensitive to applied CdS QDs. According to these results, aqueous extract of hairy roots of radish (Rhaphanus sativus L.) is a potential factory for producing CdS QDs and their biological inhibitory effects on cancer cells which was directly dependent to QDs presented their possible therapeutic application.