Dietary flavone Chrysin ( 5 , 7-Dihydroxyflavone ChR ) functionalized highly-stable metal nanoformulations for improved anticancer applications

Nanomaterials of noble metals with unique size, shape and composition receives much attention owing to their versatile functionality in personalized cancer nanomedicine. Chrysin (ChR), a natural anticancer bioflavonoid, emerged as a potential drug therapy for almost all types of cancer, however it has poor solubility and bioavailability. Herein, we report a new approach to formulate biofunctionalized metallic silver (ChR–AgNPs) and gold (ChR–AuNPs) nanoparticles using ChR as a direct bioreductant and capping agent. Size and dispersity of nanoparticles (NPs) were controlled through fixing different reaction conditions such as the temperature, pH, concentration of metal ion, stoichiometric proportion of the reaction mixture and incubation time based on their optical properties and SPR effect in UV-visible spectroscopy. The role of hydroxyl and carbonyl groups in functionalizing the metal ions with ChR was confirmed with Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. It was also substantiated that the oxygen group from ChR donates electrons to metal ion and results in complexation; ionic Ag+ and Au3+ were reduced to Ag0 and Au0 nano-forms. The physiochemical state of obtained NPs was characterized through different exclusive instrumentation, which shows the presence of highly-stable, spherical, crystalline ChR–AgNPs and ChR–AuNPs with an average size of 14 ± 6 nm and 6 ± 2 nm respectively. In vitro anticancer results revealed that the formulated metallic NPs exhibit enhanced cytotoxicity over ChR in the treatment of two different breast carcinoma cell lines (MDA-MB-231 and MDA-MB-468). Furthermore, it was evident that the NPs cause cell death via the induction of apoptosis. A hemolysis assay with human erythrocytes demonstrates good blood biocompatibility of the NPs. Thus, the ChR functionalized metal NPs can be potentially employed as a combinational drug-nano platform for breast cancer therapy.

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Introduction
Nano-biotechnology is constantly fast growing research field of the present time which combines biotechnology, nanotechnology, material science, chemical processing and system engineering.It actively engaged in tailoring of new functional nanomaterials with 10 −9 meter dimension for various industrial and biomedical applications.Interestingly, these dynamic nanomaterials acquire inimitable size, shape and surface properties comparatively to their macroscale counterparts. 1Recently, a huge attention was given for green synthesis of metal nanoparticles through exploiting different biological entities like bacteria 2 , fungi 3 , actinomyctes 4 , yeast 5 , algae 6 and plants. 7By virtue of their facile, eco-friendly and cost-effective schemes biogenesis was fixed to be most preferred route among other conventional methods. 8Many studies have demonstrated that the active metabolites such as polyphenols 9 , reducing sugars 10 and proteins 11,12 were found to play a key role in reduction and stabilization of nanoparticles (NPs).Interaction of biomolecules with nanomaterials will offer new improved end products.Although, biogenic nanomaterials were found to be an effective contrivance, the actual reduction mechanism and getting narrow size distribution were yet to be clarified.Over the past decade cancer continues to be a huge burden for mankind, despite the existence of diverse therapeutic strategies.Especially, breast cancer is the most common malignancy among women, incidence rate varied between countries with respect to geography, economic background, life style, age, stage at presentation and biological characteristics. 13Occurrence of newer breast cancer cases will get two fold increases by the end of 2020. 14Early detection dihydroxyflavone), is a natural bioactive dietary flavone abundantly found in honey, passion flowers, Oroxylum indicum and propolis.It owns stupendous medicinal properties such as anticancer, anti-inflammatory, antioxidant, hepatoprotective, antimicrobial and anti-diabetic effects.Also, it is now well established that ChR inhibits cancer cell growth through induction of apoptosis, cell cycle arrest, inhibition of angiogenesis, invasion and metastasis without adverse side effects to normal cells. 17While ChR was proposed to be an extraordinary chemotherapeutic agent, it always suffers with lot of efficacy limitations like poor solubility and bioavailability. 18An ideal nanoparticulate system of noble metals, polymers and other inorganic materials can improve target-specificity, porosity, solubility and increased bioavailability of various chemotherapeutic agents.19   The large surface area to volume ratio and different structural properties of nano-drugs would target tumour sites by a passive process.Also, arrangement of leaky vasculature and poor lymphatic drainage of cancer cells will leads to enhanced permeability and retention (EPR) effect.Materials at nano scale level can easily pass through the cellular barriers and strongly interacts with functional biomolecules.20   Ultimately, it will offer many biomedical insights for clinical level applications.
Noble metal NPs such as gold, silver, copper, platinum, palladium, iron, zinc and titanium have gained colossal attention due to their indispensable applications in drug delivery 21 , imaging 22 , Surface-Enhanced Raman Scattering (SERS) detection 23 , antioxidant 24 , anti-inflammatory 25 , bactericidal 26 and cancer theranostics. 27Metal-flavonoid complexes have elicited great interest in recent years for their potential therapeutic applications.In an attempt to further improve the anticancer efficacy of ChR, we used this flavone as instant reductant to generate functionalized highly-stable AgNPs and AuNPs.Most importantly it reveals the reduction mechanism and kinetics of biogenesis of nanomaterials (Scheme 1).On the other hand, in vitro anticancer studies demonstrate the enhanced chemotherapeutic potential of formulated NPs for breast cancer therapy.Influence of these parameters on size, shape and yield of the NPs were studied preliminarily through SPR absorbance spectra in UV-Visible spectrometer.

Characterization of ChR-AgNPs and ChR-AuNPs
Reduction of AgNO 3 and HAucl 4 were monitored by UV-Visible spectrophotometer based on SPR of all the reaction parameters.Before, high throughput characterization the colloidal NPs suspensions were purified by dialysis using a cellulose tube (MWcutoff 12 400 D) against 1 L of deionized water for 24 h at 30° C to remove excess metal ion and unreacted ChR.To perform UV-Vis, a small aliquot of sample was diluted with distilled water and absorbance maxima were scanned by Perkin-Elmer Lambda 2 UV198 UV-Visible Spectrophotometer, at the wavelength of 300-700 nm.FTIR spectroscopic measurements was carried out to study the surface chemistry of NPs, samples were mixed with KBr powder and pelletized after drying the transmittance were recorded using JASCO 460 PLUS FTIR spectrometer (Wavelength range between 4000 cm -1 to 400 cm -1 ).X-ray diffraction (XRD) was performed to determine the dimension of synthesized NPs with h, k, l values.The diffraction pattern was obtained with conditions at 40 kV and 30 mA in Cu, Ka radiation and mean particles size (L) (PAN analytical X pert PRO Model) of NPs were calculated using following Debye-Scherrrer's equation.L = 0.9λ/β cos h θ Where, λ is the wavelength of X-ray, β is full width and half maximum and θ is the Bragg's angle.The surface oxidation state and presence of element in the sample were studied using XPS.It was carried out using an omicron ESCA spectrometer with monochromatized Al Kα radiation.Morphology of nanoparticles was studied using the images Please do not adjust margins Please do not adjust margins obtained with high resolution transmission electron microscope (HRTEM).To perform TEM analysis, purified NPs solution were allowed for sonication for 10-20min, a drop of this solution was used to make a thin layer on copper coated grid and allowed to dry.The micrographs were taken at different magnification using JEOL JEM 2100 HRTEM operating at 100Kev.Energy-dispersive X-ray spectroscopy (EDS or EDX or EDAX) is an analytical technique used for elemental analysis or chemical characterization of sample.EDX spectra and selected area diffraction (SEAD) pattern of NPs were obtained along with HRTEM (JEOL JEM 2100) analysis.Size distribution and surface charge of synthesized NPs were measured using dynamic light scattering (DLS) and zeta potential analyzer (Malvern Zetasizer, Nano-ZS90).Concentrations of formulated NPs were measured with inductively coupled plasma atomic emission spectroscopy (ICP-OES Perkin-Elmer Optima 5300 DV model).

Analysis of Apoptosis (TUNEL assay)
The level of apoptosis induced by ChR and formulated NPs were identified via a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) staining using the in Situ Cell Death Detection Kit-Fluorescein (Roche Molecular Biochemicals, Chemicon Int., Temecula, CA, USA) as per manufacturer's instructions.Briefly, cells were grown on poly-L-lysine-coated glass cover slips and treated with ChR, ChR-AgNPs and ChR-AuNPs for 48 hours.Subsequently, the medium was removed and slides were washed three times with PBS (pH 7.4), fixed with 4% paraformaldehyde in PBS (pH 7.4) and permeabilized with 0.1% Triton X-100.Aliquots (50 mL) of the reaction mixtures were applied to cover slides and placed in a humidified incubator at 37°C for 60 minutes.After incubation, cells were washed with PBS, air dried and mounted on slides.Finally, the slides were examined by epi-fluorescence microscopy.

Hemocompatibility assay
Hemocompatibility assay was performed as per the earlier report 30

Result and Discussion
Generation of metallic Ag and Au nanostructures by reduction of AgNO 3 and HAucl 4 with chrysin was initially confirmed through the formation of yellowish brown and ruby red colour respectively.Fig. 1a1  Please do not adjust margins Please do not adjust margins to fabricate AgNPs and AuNPs without addition of any other toxic chemical ingredient.

Size controlled synthesis of ChR-AgNPs and ChR-AuNPs
As mentioned earlier, after blending ChR with metal ions the reaction mixture exhibits variations in colour due to excitation of Surface Plasmon Resonance (SPR), formation of yellowish brown colour and ruby red colour indicates the synthesis of ChR-AgNPs and ChR-AuNPs.The optimal reaction mixture to synthesis monodispersed stable nanomaterials was fixed as 0.5 mM ChR and 1mM metal ions in 1:2 ratio (Fig. S1a, b and c & Fig. S2a, b and c Supporting Information).NPs generated at this stochiometric proportion generates intense SPR spectra which clearly denotes the presence of smaller nanoparticles, whereas the other precursor concentration produces broad and weaker SPR peak due to increased polydispersity. 33As per, LaMer model it was envisaged that formation of NPs could only happen when the precursor concentration is within a suitable range for nucleation.However, this range might vary amongst different biomass-assisted synthesis approaches.Dubey et al., 2010 observed that NPs synthesized at higher metal ion concentration were larger and red shift occurred in SPR spectra as an indicative of polydispersity.

34
Likewise, physiological parameters such as temperature and pH mainly influence the synthesis of NPs.Increasing the temperature (37°C to 100°C) reflects in the nucleation growth of NPs.Synthesis was found higher at 90°C when compared to other temperature, ChR-AgNPs and ChR-AuNPs synthesized at 90°C shows SPR peak at 420 nm & 530 nm (Figure S1d & Figure S2d Supporting Information).Above 90°C the SPR peak was observed at lower wavelength regions due to reduced size range of NPs and they were found unstable.It was believed that lesser synthesis of NPs occurred at lower temperature because of the plasmon band was not accompanied with a significant increase in intensity at lower temperature.

35
Increasing the temperature reflects in SPR peak, at higher temperature rate of NPs synthesis reaching maximal point which shows intense absorbance peak relatively to the size of NPs.
36 pH of the reaction medium plays a crucial role in metal ion reduction, from our results it was inferred that, in alkaline pH-9 (Fig. S1e Supporting Information) the synthesis of ChR-AgNPs was found higher.In alkaline pH, the aggregation of NPs was believed to be favoured over the nucleation to form narrow size nanoparticles. 37On the other hand, stable ChR-AuNPs were formed at acidic pH-6 (Fig. S2e Supporting Information), it generates an intense SPR peak at 530 nm.Usually, biosorption mechanism of Au is ionic rather than covalent due to the dependence of Au binding with pH.At lower pH, gold is present in solution in anionic form (AuCl 4 ) and the functional groups of active biocompounds on the biomass surface such as hydroxyl groups tend to undergo protonation and become positively charged.The overall positively charged surface could promote the interaction between protonated functional groups and the negatively charged (AuCl 4 ) through electrostatic attraction or electrovalent bond. 38As a result, biosorption was preferred over bioreduction of Au ions.The bioreduction of gold could occur through the oxidation of hydroxyl to carbonyl groups.The pH of the colloid is lowered in most cases after the synthesis completed.Our study, the complete reduction of metal ion was noticed after 60min incubation time, yield of NPs was direct proportion with reaction time.Similarly, Dwivedi et al., 2010 reported that the peak absorbance was sharper when the contact time was increased at 2h time duration. 39As compared to previous reports our synthesis method requires much shorter time which can be useful for easy scaling up process.

Characterization of ChR-AgNPs and ChR-AuNPs
Generally, noble metal NPs were known to exhibit SPR phenomenon where conducting electrons of metals oscillate collectively in resonance with certain wavelengths upon interaction with an electromagnetic field.These SPR band highly depends on type, size, shape of the NPs and surrounding environment. 40UV-visible spectroscopy displays intense SPR peak at 420 nm for ChR-AgNPs and 530 nm for ChR-AuNPs (Fig. 1c & d) respectively, the synthesized NPs were stable at room temperature even after 45 days.The IR spectrum of free ChR manifests prominent transmittance located at 3080, 3009, 2945, 1655 and 1610 cm−1 (Fig. 2).The strong bands between 3500-3000 cm−1 corresponds to the O-H stretch whereas 1655 and 1610 cm−1 aƩributes C=O and C=C respectively.The characteristic IR transmittance of synthesized NPs clearly implicates that functional groups of ChR actively participates in coordination with metal ions.Variations in the intensity of transmittance at 3500-3000 cm-1 noticed in formulated ChR-NPs complex which clearly depicts the symmetrical and asymmetrical stretching modes of O-H undergoes changes after the reduction.This difference in intensity suggests the loss of one O-H group during the coordination to the metal ions. 41A strong band at about 1655 cm-1 detected in the transmittance of ligand is assigned to C=O which was shifted to 1638 cm-1 in the spectra of ChR-NPs complex which denotes that functionalization of ChR on metal NPs occurs through the C=O oxygen atom.

42
XRD pattern of ChR-AgNPs and ChR-AuNPs were interpreted with JCPDS intensitives, after reduction the diffraction peaks at 2θ = 38.03•,46.18• and 63.43• were indexed as (1 1 1), (2 0 0) and (2 2 0) planes of a faced centered cubic (fcc) lattice of silver (JCPDS, file no.04-0783) (Fig. 3a1).The XRD patterns displayed here are consistent with earlier reports. 43Likewise, for AuNPs the XRD peak corresponding to four peaks (JCPDS, No. 89−3722) at (38.17˚), (44.36˚) and (64.65˚) (Fig. 3b1) which are found to be an identical with those reported for the standard gold metal (Au˚). 44The mean size of ChR-AgNPs and ChR-AuNPs was calculated using Debye-Scherrer's equation by determining the width of (1 1 1) peak and found to be 15 and 8 nm respectively which is fairly in agreement with the HRTEM measurement.Fig. 3a2 & b2 displays the XPS general scan spectrum of ChR-AgNPs and ChR-AuNPs which shows the presence of strong C1s, O1s, Ag3d and Au4d core levels accordingly.The strong Please do not adjust margins Please do not adjust margins signal of Ag 3d (370 eV) and Au4f (87.5 eV) indicates the presence of Ag and Au metal.The C 1s peak observed at a binding energy of ~285 eV serves as a reference to correct the binding energy NPs.shift and it also stems from ChR to coordinate.The spectrum also consists of O (~531 eV) elements in their respective binding energy positions due to the interaction of ChR with synthesized NPs.Thus, it was concluded from XPS measurements that the metal ions were reduced to nano metallic form, and the NPs were capped by ChR. 24,42HRTEM micrographs display the fine configuration of uniform size spherical ChR-AgNPs with mean size of 14±6 nm (Figure 4a).Interestingly, Au colloid displays both spherical and oval-shaped NPs with an average size of 6±2 nm (Fig. 5a).It was also found that both ChR-AgNPs and ChR-AuNPs having a thin layer of ChR coating on its surface, particles were well dispersed and stable for long period of time.There is no direct contact of particles were noticed, it's mainly due to the presence capping agent.45   Higher magnification TEM micrographs exposes excellent crystallinity of NPs, the distance of 0.23 nm between lattice planes is in agreement with the (1 1 1) lattice spacing of face centered cubic (fcc) Ag (d111 = 0.2359 nm) and Au (0.235 nm).Crystalline nature of the NPs was further evidenced by the SAED pattern (Fig. 3(d)).Clear lattice fringes in high-resolution TEM image and the typical SAED pattern (Fig. 4b & 5b) with bright circular rings corresponds to (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) planes indicates that the synthesized NPs are highly crystalline.

42
EADX spectra display a strong metal peak for Ag and Au, the presence of copper is due to copper grid used for the HRTEManalysis (Fig. S3a & b).The hydrodynamic diameter of ChR-Ag and AuNPs were evaluated by DLS which confirms the particle size distribution respectively (Fig. 4c & 5c), their corresponding zeta potential value is suggesting high stability of NPs (Fig. S4a  & b).The large negative potential value could be due to the capping agent, which generate repulsive forces between the NPs.

46
ICP-OES results specifies the concentration of synthesized NPs were quantified to be 65.89 & 103.3 mg/L of ChR-AgNPs and ChR-AuNPs in that order.It denotes that more than 80% of the metal ions have been reduced to nano scale values.

Analysis of cell viability (MTT assay)
Cell viability assay clearly explains the cellular response to a toxicant, in our study synthesized NPs exhibits higher anticancer activity than ChR.There was a dose-dependent cellular toxicity was observed in ChR (0, 5, 10, 20, 25, 50, 75, 100, 150 and 200 μg/ml), synthesized ChR-AgNPs and ChR-AuNPs (0, 5, 10, 20, 30, 40, and 50 μg/ml) treated MDA-MB-468 and MDA-MB-231 breast cancer cell lines.ChR-AgNPs gives more cytotoxic effect (IC50-15 μg/ml & 12 μg/ml) followed by ChR-AuNPs (IC50-19 μg/ml & 21 μg/ml) (Fig. 6 a1 & b1) and ChR (IC50-72 μg/ml & 35 μg/ml) (Figure 6 a2 & b2) against treated MDA-MB-468 and MDA-MB-231 breast cancer cell lines.Especially, the cytotoxicity effect of formulated NPs were much stronger than free ChR, depicts the improved anticancer efficacy of ChR after getting functionalized with Ag and Au nanostructures.The NPs size, shape, surface area and surface functionalization are major factors that influence biokinetics and toxicity. 47It should be mentioned that the concentration of synthesized NPs used in this case was very less when compared to ChR.This decrease in cell viability with increase in NPs concentration, suggests that more number of NPs could accumulate inside cells resulting in enhanced stress, ultimately leading to cell death.These results clearly specify the enhanced effectiveness of the ChR functionalized NPs against cancer cells.It was demonstrated in our earlier study that AgNPs synthesized using phamolocolgically important Dendrophthoe falcata with a size range of 5-45 nm has shown enhanced cytotoxicity against human breast carcinoma cells (MCF-7) compared to the aqueous plant extract. 37Similarly, Selim and Hendi (2012) reported the toxic responses of AuNPs to human breast epithelial MCF-7 cells, they noticed the cytotoxicity effect of AuNPs on MCF-7 cells and apoptotic response in dose depended manner.

Analysis of apoptosis (TUNEL assay)
Apoptosis is the key event in cancer therapy that can be measured with the activation caspase-cascade, chromatin aggregation, partition of cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria, and nuclear material. 49We performed TUNEL staining to identify apoptotic cell death induced by ChR, synthesized ChR-AgNPs and ChR-AuNPs.TUNEL-positive nuclei were found throughout the photomicrographs of the treated groups but few in untreated controls, and numbers of positive nuclei increased with treatment time (Fig. 7a &b).Previous references have showed that the possible mechanism involved in the AgNPs induced cellular toxicity begin with the cellular uptake of inorganic nanoparticles through clathrin-dependent endocytosis and macropinocytosis.
50 Inorganic NPs profoundly interact with cells and intracellular macromolecules like proteins and DNA. 51Cellular uptake of NPs leads to generation of reactive oxygen species which provoke oxidative stress.It is clearly evidenced that synthesized NPs (both Ag and Au) induces cell damage through loss of cell membrane integrity, oxidative stress and apoptosis.Several factors influences toxicity of NPs such as dose, time and size of the particles and it was found that biogenic AgNPs and AuNPs shows does and time dependent toxicity against HeLa cells.

Hemocompitibility assay
Hemocompitibility of ChR, synthesized ChR-AgNPs and ChR-AuNPs were assessed upon measuring the damage to human RBCs.Our result shows that synthesized NPs exhibits comparatively lower red haemoglobin release than ChR.Fig. 8 a & b are photographs of the RBCs exposed to ChR-AgNPs, ChR-AuNPs and free ChR at different concentrations.As shown in Fig. 8, compared to positive control and ChR the haemolytic activity of NPs was less considerable, implying its safe nature in application.The mechanisms of direct haemolytic activity for different toxic agents were found to be non-specific.Especially, the plant derived xenobiotic compounds such as phenols, are capable of promoting haemolysis through oxidation of haemoglobin, forming metahemoglobin. 56Our data highly corroborate with Ruden et al 57 who reported that silver nanoparticles did not show haemolytic activity against erythrocytes even at the higher concentrations (up to 1024 µg/mL).In another report, AuNPs synthesized using Z. officinale extract have shown high level compatibility with the blood cells which do not initiates any aggregation of cells and also the NPs do not seem to activate the platelets.It was inferred that surface passivation of nanomaterials with different bio agents will improve their biocompatibility.

Diagram 1 Scheme 1
Scheme 1 Illustration of reduction and functionalization of AgNPs and AuNPs using Chrysin (ChR) as direct bioreductant

Fig. 7
Fig. 7 Induction of apoptosis by formulated ChR-AgNPs, ChR-AuNPs and free ChR were measured through TUNNEL assay in treated (a) MDA-MB-468 and (b) MDA-MB-231 breast carcinoma cell lines.Epi-fluorescence microscopic image shows apoptotic cells (TUNELpositive nuclei) at different incubation time intervals (24 h and 48h).

Size controlled synthesis of ChR-AgNPs and ChR-AuNPs
Heat inactivated fetal calf serum (FBS), minimum essential medium (MEM), glutamine, EDTA and trypsin were purchased from Sigma-Aldrich (St. Louis, USA).All glasswares were washed with distilled water followed by acetone and dried in oven before use.Breast cancer cell lines MDA-MB-231 and MDA-MB-468 were obtained from National Centre for Cell Science (NCCS), Pune, India.