Nano-magnetite supported N-heterocyclic carbene-gold complex with pendant ferrocenyl group as a multi-modal anti-breast cancer agent

A novel nano-magnetite supported N-heterocyclic carbene-gold complex with pendant ferrocenyl group has been prepared by using a multi-step procedure. The complex displayed considerable anticancer activity against the MCF-7 breast cancer cell line with IC50 = 32.38 ± 0.31 µg/mL as compared with standard drug 5-fluorouracil (IC50 = 25.29 ± 0.11 µg/mL) in the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The in-ovo anti-angiogenic activity of the complex using chicken chorioallantoic membrane (CAM) assay showed significant decrease in secondary blood vessels indicating that it is an efficient angiogenesis inhibitor. The magnetic hyperthermia studies of the complex revealed a significant specific absorption rate (SAR) value of 246.974 W g−1 with therapeutic temperature of 46.8°C.


Introduction
Cancer is a leading cause of death, accounting for nearly 10 million deaths in 2020 [1].Breast cancer accounts for second leading cause of cancer-related deaths in females worldwide.Breast cancer is invasive with a significant degree of inflammation and potential to metastasize [2].The worldwide incidences of breast cancers are increasing at an alarming rate since last decade.Traditional therapeutic approaches for breast cancer are based on the expression status of the hormonal receptors such as estrogen and progesterone receptors (ER and PR) and human epidermal growth factor receptor 2 (HER2) [3].The triple-negative breast cancers (TNBC) lack hormonal receptors and are more difficult to treat.The basic cancer treatment modalities include a variety of treatments such as surgery, chemotherapy, radiotherapy and hormonal therapy [4].Many of these therapies are painful, involve high-cost treatment and sometimes cause severe complications [5].To alleviate these problems, development of a target-based strategy with multi-modal therapeutic action is highly warranted [6].
N-Heterocyclic carbenes (NHCs) are driving much attention in the cancer research due to their fascinating properties such as ease of preparation, unique architecture, high stability in physiological fluids, a wide choice and fine tuning of wingtip substituents in order to control lipophilicity and favourable physico-chemical properties [7].NHCs are regarded as workhorses of medicinal chemistry especially for developing new metal-based complexes for anticancer therapy as they act as

Materials and methods
All reactions were carried out under air atmosphere in dried glassware.A Perkin-Elmer Spectrometer (Model no.783USA) was used to get FT-IR spectra of compounds in the range of 400-4000 cm −1 using KBr pellets to confirm the material and surface functionalization.The weight loss curves were obtained by heating the sample using instrument TA SDT Q600 V20.9 Build 20 from 25 o C to 1000°C.The elemental composition was analyzed by Perkin-Elmer 2400, Series II, CHNS/O analyser.Phase identification and structural analysis of synthesized complex were studied using X-ray diffractometer RIGAKU Mini flex 600 (XRD) in the 2θ range from 20° to 80°.The morphology of samples was examined using a Philips CM 200 model TEM, with an operating voltage of 20-200 kV.The surface morphology of nanoparticles was examined by JFOL JSM-IT200 model (SEM).Magnetization measurements were performed on Lakeshore magnetometer, USA, Model 7410 (VSM).Inductions heating of all samples were performed in a plastic microcentrifuge tube (1.5 mL) with a 6 cm diameter coil (4 turns).Nano-magnetite (1), silicacoated nano-magnetite (2) and 3-chlopropyl modified silica-coated nano-magnetite (4) were prepared following literature procedure [19][20][21].All other chemicals were obtained from local suppliers and used without further purification.

Preparation of cell culture
The proliferation studies of nano-magnetite (1), [Nano-magnetite-FemTriaz]Cl (6) and nano-magnetite-FemTriaz-NHC-Au complex (7) were accomplished by MTT assay using human breast cancer MCF-7 cells.At first, cancer cells were allowed to grow in an incubator (Thermo Scientific BB150) under optimal conditions.When they reached 70-80% confluence, they were employed for cell plating purposes.After aspirating the old media from the cell plate, the cells were washed three times with sterile phosphate-buffered saline (PBS) at pH 7.4.Then, PBS was discarded by washing and trypsin was added in such way that it was evenly distributed onto cell surfaces.After adding trypsin, cells were incubated for 1 min under 5% CO 2 at 37°C.The cell flasks were then tapped to promote their cell segregation which was monitored by inverted microscope.Trypsin was inactivated by 5 mL freshly prepared 10% fetal bovine serum (FBS) media.The grown cells were counted to dilute to 2.5 × 10 5 cells/mL and were inoculated into wells (100 μL cells/well) to incubate at 37°C under 5% CO 2 [22].

MTT Assay
After incubating inoculated cancer cells overnight, cell attachment was assured; after diluting the nano-magnetite (1), [Nano-magnetite-FemTriaz] Cl (6) and nano-magnetite-FemTriaz-NHC-Au complex (7) with media as per desired concentration, the plates were again incubated for 72 h while keeping 1 3 Nano-magnetite supported N-heterocyclic carbene-gold… temperature and CO 2 atmosphere unchanged.A 20 μL of MTT reagent, namely [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], was added in individual well and they were again incubated for 4 h.The wells for formazan crystal formation were observed under the microscope.The yellowish MTT was reduced to dark colored formazan by viable cells only.After removing the medium completely.50 μL of DMSO was added and they were again incubated for 5 min under CO 2 environment.Finally, triplicate samples were analyzed by measuring the absorbance of each sample by a microplate reader (Benesphera E21) at a wavelength of 550 nm.

In-ovo angiogenic assay (Yolk sac membrane assay)
In-ovo angiogenic assay was performed to evaluate the effect of nano-magnetite-FemTriaz-NHC-Au complex (7) along with corresponding bulk compounds on angiogenesis pattern of chick yolk sac membrane.The dose for assay was kept in accordance with in-vitro anticancer activity assay.Yolk sac membrane (YSM) Assay was performed according to Datar et al. with slight modification [23].For this, zero hour fertilized eggs were collected from central egg hatching centre, Kolhapur.After wiping the eggs with 70% alcohol, good quality eggs were incubated at 37 o C with 60-70% relative humidity.Fertilized chick eggs were treated with disc diffused with 7 and assessed for antiangiogenic activity on 3rd, 6th and 8th day post incubation.Chorioallantoic membrane area was observed and photographed with the help of stereomicroscope.The inhibitory effect of 7 on angiogenesis in chorioallantoic membrane was evaluated and assessed according to the scoring system followed in several studies [24,25].According to angiogenic scoring system, the change in the density of the capillaries around the disc was evaluated in the form of score on the basis of which the extent of anti-antigenicity is evaluated.Anti-angiogenic scores (average score) of 7 were calculated by using following formula.
Thermal gravimetric analysis (TGA) of bare nano-magnetite (1) and nanomagnetite-FemTriaz-NHC-Au complex (7) was carried out at heating rate of at 10°C min −1 under nitrogen and the weight loss was measured from ambient temperature to 1000°C.The thermograms of 1 and 7 are given in Fig. 2 and 3.The first weight loss of 5.27 and 5.15% observed in thermograms of 1 and 7 temperatures up to 129.6 and 104.9°C, respectively, are due to desorption of water molecules from the surface.TGA curve of 1 displayed no significant weight loss from 130-800 °C solely indicating the presence of iron oxides molecules (Fig. 2).The combined weight loss of 13.64% at temperature range 104.9-625.5°Cfor 7 was attributed to decomposition of organic ligand, i.e., FemTriaz and organic component on the surface of nano-magnetite.Finally, the large residual weight of 74.85% is due to the formation of silica, gold and other metallic oxides which possess high thermal stability (Fig. 3) [30].
The EDX analysis of nano-magnetite-FemTriaz-NHC-Au complex (7) indicated C, O, Fe, N, Cl, Si and Au as the major elements.It is revealed that Fe and O which are constituent elements of core of nano-magnetite are contributing major percentage of 7 whereas nitrogen, chlorine, silica and gold are present in minor amounts.The EDX analysis revealed Au loading of 0.075 mmol per gram of 7 (Fig. 4).
The crystallite size was calculated by using Debye-Scherrer equation on the basis of most intense peak (3 1 1) at 2θ value 35.54° and found to be 52.1 nm.From the XRD analysis, it is evident that crystallographic structure of 1 is retained in 7 even after multi-step functionalization.
The morphology of nano-magnetite-FemTriaz-NHC-Au complex (7) was investigated using transmission electron microscopy (TEM).The TEM images (Fig. 6) indicate that nanoparticles of 7 are entirely composed of crystals with a relatively uniform, spherical shape and non-smooth surface.Nano-magnetite particles were clearly seen as dark regions surrounded by gray shells in the micrograph (Fig. 6a,  b) [32].A fine observation of Fig. 6c indicates average nanoparticle size of 7 to be 12 nm with a lattice fringe width distance of d (311) = 2.6 Å, which reveals the formation of perfect crystal structure.Selected area electron diffraction (SAED) pattern of 7 exhibited four strong diffraction rings with bright dotted pattern of crystallographic planes (3 1 1), (4 0 0), (3 3 1) and (4 2 2), respectively, which persuades polycrystalline nature (Fig. 6d).
The morphology of nano-magnetite-FemTriaz-NHC-Au complex (7) was studied using scanning electron microscopy (SEM).The SEM images of 7 demonstrate irregular morphology randomly distributed as shown in (Fig. 7a-d).Elemental mapping images show the presence of C, N, O, Si, Fe, Cl and Au which are uniformly distributed in the structure (Fig. 8).Furthermore, an overlay map image confirms the presence of elements such as C, N, O, Si, Fe, Cl and Au in the structure of nanomagnetite-FemTriaz-NHC-Au complex (7) (Fig. 9).
The magnetic character of the bare nano-magnetite (1) and nano-magnetite-Fem-Triaz-NHC-Au complex (7) was investigated by measuring magnetization as a function of field and temperature using vibrating sample magnetometer (VSM) (Fig. 10).The magnetization (ms) of 1 was found to be 62 emug −1 and decreases to 27 emug −1 for 7.The decrease in ms value for 7 is attributed to successful covalent functionalization of 1 [33].Although ms value of 7 is comparatively less as compared to 1, it is sufficient to show anticipated hyperthermia activity.

Anticancer activity
Nano-magnetite (1), [Nano-magnetite-FemTriaz]Cl (6) and nano-magnetite-Fem-Triaz-NHC-Au complex (7) were screened for in-vitro anticancer activity against human breast cancer MCF-7 cells using MTT assay.The cell lines were incubated with nanoparticles of 1, 6 and 7 for 48 h with concentration of 20, 40, 60, 80 and 100 µg/mL in the 5% CO 2 atmosphere.The results of MTT assay are summarized in Table 1.The IC 50 value and the % of inhibition are correlated to each other in a dose-response curve, which is employed to determine the effectiveness of a test sample that inhibits a biological target.The results revealed that 1, 6 and 7 displayed considerable anticancer activity against human breast cancer   However, as the concentration of the 1, 6 and 7 increased, the percentage of inhibition gradually approached and potentially surpassed that of 5-fluorouracil.
The significant anticancer activity of 7 is attributed to synergistic result of steric and electronic effects displayed by bioactive triazole-based NHC ligand with bulky ferrocenyl substituent at wingtip position and gold metal center.It is well accepted that NHC ligands centered with gold can induce apoptosis in several cancer cell lines via inhibition of selenoenzyme and thioredoxin reductase (Trxr) [34].In addition, gold-based NHCs are also popular to cause fragmentation of DNA and RNA strands via inhibition of topoisomerase.Ferrocene being a redox active molecule acts as reactive oxidative species (ROS) production promoter leading to the elevated oxidative stress and mitochondrial arrest.Thus, the significantly higher anticancer activity of 7 is attributed to synergistic effect of NHC ligand architecture, gold metal and pendant ferrocenyl group.

Angiogenesis study (CAM assay)
Angiogenesis is the process of development of new blood vessels from the existing vasculature.Anti-angiogenic drugs are therapeutics that prevent tumors from developing their own blood vessels.This may slow or potentially stop the growth of the malignancy via inhibition of angiogenesis [36].Vascular endothelial growth factor (VEGF) is an angiogenic factor which is highly potent and selective.VEGF was initially discovered for its capacity to induce vascular permeability and encourage endothelial cell development, but it has since proven to be an essential requirement Nano-magnetite supported N-heterocyclic carbene-gold… for tumor formation [37].One of the most promising approaches in medicine is the loading and delivery of diverse natural and synthetic anti-angiogenic compounds via nanotechnology.Targeted therapy with nanocarriers allows drug-loaded nanostructures to reach more easily to the desired areas in the body (cells, tissues and organs) and allows a more controlled drug release profile.Furthermore, depending on their nano-size design, nanoparticles may exhibit anti-angiogenic properties [38].
In light of this, nano-magnetite-FemTriaz-NHC-Au complex (7) was evaluated for angiogenesis on fertilized chick eggs employing an in-ovo CAM assay.The eggs treated with 7 were assessed for anti-angiogenic activities on 3rd, 6th and 8th day post-incubation and anti-angiogenic effect was observed (Table 3 and Fig. 11).The  results revealed that 7 displayed anti-angiogenic activity on yolk sac membrane vasculature with anti-angiogenic score ranging from 0.2-1.23.Based on these results, we conclude that 7 exhibits significant anti-angiogenic activity on the yolk sac membrane vasculature, as shown in Fig. 12.It is worth mentioning that at a concentration of 5 mg/mL, the density of secondary blood vessels decreases significantly.Nano-magnetite supported N-heterocyclic carbene-gold…

Magnetic hyperthermia study
In order to evaluate hyperthermia potential, the induction heating properties of nano-magnetite (1), [Nano-magnetite-FemTriaz]Cl (6) and of nano-magnetite-FemTriaz-NHC-Au complex (7) were determined at fixed magnetic field 335.2 Oe at frequency 277 kHz for 10 min run with sample concentrations of 5 mg/mL prepared in distilled water as shown in Table 4. From the nature of temperature kinetic bars, it is observed that 1, 6 and 7 attains the temperature of 72.2°C, 51.4°C and 46.8°C, respectively, within short time as shown in Fig. 13.The therapeutic temperature (ΔT max ) obtained for 7 is 46.8 °C which is sufficient to induce hyperthermia temperature for cancer treatment.It is noteworthy to mention that temperature of 42 o C-48°C is considered as the most effective for hyperthermia therapy.The decrease in hyperthermia temperature from 1 to 7 is due to the surface functionalization.The fall in heating rates could be further attributed to a thermal effect due  Cl (6) and nano-magnetite-FemTriaz-NHC-Au complex (7) at concentration of 5 mg/mL at fixed applied magnetic field 335.2 kHz as a function of variable field to magnetic degradation of the particles.Further, detailed investigation of effect of magnetic field variations on 7 has been performed by using magnetic fields 251.4 Oe and 335.2 Oe (300 A and 400 A) at frequency of 277 kHz for 10 min run with concentration of 5 mg/mL prepared in distilled water (Table 5).From the nature of temperature kinetic bars, it is clear that 7 attained the desired hyperthermia temperature of 42.2 o C and 46.8°C for the concentration of 5 mg/mL (Fig. 14).

Induction heating studies (hyperthermia therapy)
In order to use nano-magnetite-FemTriaz-NHC-Au complex (7) as a heating mediator in magnetic fluid hyperthermia therapy for cancer, nanoparticles should produce maximum rise in temperature at the lowest concentration at physiological safe range of applied external AC magnetic field and frequency.Actual rise in temperature within span of 10 min for the samples is measured at fixed frequency (277 kHz) and nano-magnetite concentration (5 mg/mL −1 ) at varying magnetic field, 251.4 and 335.2 Oe (Fig. 14).The heat generated by the nano-magnetites is determined by the specific absorption rate (SAR) and expressed in W g -1 .SAR value of nano-magnetite (1),  Nano-magnetite supported N-heterocyclic carbene-gold… [Nano-magnetite-FemTriaz]Cl (6) and of nano-magnetite-FemTriaz-NHC-Au complex (7) were calculated by using the equation [39] The value of SAR depends on the size and concentration of particles, suspension medium, the magnitude and frequency of an external alternating magnetic field.The magnetic induction heating is mainly caused by the hysteresis and residual loss in an AC magnetic field.It is observed from Fig. 14 that the SAR values decrease from 1 to 7 and found to be 623.7 W g −1 , 336.2 W g −1 , 246.9 W g −1 , respectively, at fixed magnetic field 335.2 Oe (400 A).
The decrease in SAR values from 1 to 7 is attributed to the surface functionalization of 1.The highest SAR value of 246.9 W g −1 is good enough to meet the requirements of hyperthermia therapy.

Conclusions
In the present investigation, nano-magnetite-FemTriaz-NHC-Au complex (7) has been successfully synthesized.The structural, magnetic, anti-angiogenic, cytotoxic and anticancer properties of 7 were studied.The data of XRD and TEM confirm that resulting powder is a single-phase inverse spinel structure of crystalline nanomagnetite and spherical in shape with an average size about 12 nm.The MTT assay revealed that 7 displayed significant in-vitro anticancer activity against human breast MCF-7 cell line with IC 50 value 32.38 ± 0.31 µg/mL.The complex 7 displayed strong anti-angiogenic activity with the score 1.23 against yolk sac membrane vasculature.The effects of induction heating of 7 at concentration 5 mg/mL under an external alternating magnetic field of varying strength 335.2 Oe was studied.Temperatures necessary for the hyperthermia treatment were obtained for the complex 7 and found to be maximum of heating temperature ΔT max = 46.8°C.The highest 246.974W g −1 SAR value for 7 was obtained at an applied field of 335.2 Oe (400 A) at 277 kHz with a particle concentration of 5 mg/mL.In conclusion, the nano-magnetite supported N-heterocyclic carbon-gold complex with pendant ferrocenyl group synthesized in the present work is a multi-modal anticancer agent owing to anti-breast cancer, anti-angiogenic and hyperthermia potential.

Table 2
Anticancer activity of reported complexes against Human breast Cancer cells (MCF-7)