NIR light active ternary modified ZnO nanocomposites for combined cancer therapy.

Recent developments in nanomedicine for cancer therapy enable nanoparticles for tumour specific therapeutics. Certain nanoparticles with their inherent physical/chemical properties can themselves act as drugs. Also they can be designed to respond to either tumor microenvironment or externally applied physical stimuli such as temperature, light, magnetic field, and ultrasound for tumor-targeted and enhanced anticancer efficacy. In this study, a simple design of cost-effective ternary modified zinc oxide nanocomposites possessing near-infrared (NIR) absorbance were synthesized using simple, fast, thermal decomposition route with hydrazine precursors. The in vitro cytotoxicity of these nanocomposites studied on human breast cancer cells (MCF-7) and the human embryonic kidney normal cells (HEK 293) by MTT assay show that they are highly selective and are dose dependent against both the cell lines. The developed nanocomposites can be used for combined photothermal (PTT) and photo dynamic (PDT) cancer therapy.

Introduction

Today cancer is one of the leading causes of death worldwide, which is mainly treated with chemo therapy, radiation, and surgery. While chemotherapy and radiotherapy suffer from severe toxic side effects to normal tissues, surgery in most occasions could not remove all cancer cells in the human body. Nanotechnology and nanomedicine can offer a more targeted approach in the treatment of cancer. Nanoparticles with their small size preferentially enter into the leaky vascular tumour tissues, and are then retained in the tumour bed, by a process recognized as the enhanced permeation and retention (EPR) effect [1]. In this regard, metal oxide semiconductor nanoparticles is of interest, as they can participate in cellular redox reactions and have photocatalytic activity [2]. Zinc oxide (ZnO) belonging to a group of metal oxides is a wide band-gap semiconductor which absorbs in the UV region. ZnO NPs are biocompatible having antibacterial properties and show inherent differential toxicity against rapidly dividing cancer cells [3, 4, 5]. Generation of intracellular reactive oxygen species (ROS) is a major cytotoxic mechanism of ZnO NPs leading to cell death [6, 7]. For nanoscale ZnO large numbers of valence band holes and/or conduction band electrons are available on the surface due to crystal defects. These electron-hole pairs get trapped by dissolved oxygen in the aqueous solution inside the cell resulting in the production reactive of oxygen species (ROS). Generally cancer cells themselves produce more ROS than normal cells owing to their faster metabolic rate. Therefore, exogenous ROS-generating agents like ZnO NPs produces more ROS in cancer cells than normal cells resulting in huge oxidative stress in the cancer cell and eventually killing the cell [8, 9]. The selective toxicity of ZnO NPs can further be improved by external stimulation like photo activation.

Phototherapeutics, involving photodynamic therapy (PDT) and photothermal therapy (PTT) using nanomaterials have received attention, due to their selective and localized therapeutic effect by light irradiation [10]. PDT, a minimally invasive method for treating cancer, uses photosensitizing agents which upon exposure to cancer cells can produce toxic reactive oxygen species (ROS) such as hydroxyl radical, hydrogen peroxide, and superoxide under light irradiation leading to cancer cell death [11]. PTT is also a minimally invasive therapeutic strategy, where light irradiation is converted by photothermal agents to heat, leading to thermal ablation of cancer cells with subsequent death of the malignant cells [12]. The photothermal agents for PTT should have absorption in the near infrared region (NIR) (700–2500 nm) which is a transparency window for biological tissues. Dual-modal phototherapeutics that combine PDT and PTT can have synergistic effects which enhance the therapeutic efficacy compared to PDT or PTT alone [13, 14]. Photoabsorbing agents with improved efficiency of reactive oxygen species (ROS) generation, high absorption, photostability and near-infrared (NIR) light absorption are necessary for dual-modal phototherapeutics to treat diseases. Metal based nanoscale materials exhibit higher absorption and have high photostability [15].

Upon UV irradiation ZnO nanoparticles produces more electrons and holes which increases their ability to generate ROS which could further enhance the growth inhibition of cancer cells. Thus ZnO nanoparticles could play an important role in the PDT. Instead of dangerous ultraviolet (UV) radiation light, NIR is a very good choice for PDT, which not only can afford deeper penetration depths, but also impose lower photo-toxicity on normal cells and tissues. ZnO NPs are also known as one of photothermal (PTT) agents for cancer treatment [16, 17]. Zinc oxide nanoparticles with their ability to produce ROS, if modified to absorb NIR light instead of UV, it could be used for a dual PDT and PTT therapy and thus enhance its inherent toxicity and selectivity towards cancer cells.

Doping is mainly developed, with the aim of shifting light absorption towards higher wavelength region and simultaneous doping (co-doping) of two or three transition metals or metal ions could successfully modify the electronic structures of ZnO and shift its absorption edge to a lower energy [18]. In this study we have prepared (Ag,Cu,Co) co-doped ZnO nanostructures from thermal decomposition of novel precursors (Ag,Cu,Co) co-doped zinc laurate hydrazinate which were prepared by coprecipitation method [19]. Dopants are so choosen that they are biologically active and can tune electronic and optical property [6, 20, 21, 22]. The carboxylic acid is lauric acid which is a medium-length long-chain fatty acid having anti microbial properties [23]. Thermal decomposition of solid solution precursor metal carboxylate hydrazinate [24] has been used to prepare fine-particle mixed metal ferrites [25] cobaltites [26] and yttria [27]. These precursors once ignited, decompose in the presence of air auto catalytically, with the evolution of N2, H2 and CO2 to yield fine particle oxides. Hydrazine by virtue of its coordinating ability with transition metal can act as a neutral, monodentate, bidentate and bridged bidentate ligand with metal carboxylates. The heat required for the formation of nanosized particles is provided by the combustion of hydrazine and carbonaceous mass of the carboxylate precursor. The synthesis is inexpensive, simple, fast, and yields high purity products in a single step. In this paper the preparation, characterization, optical properties of (Ag,Cu,Co) co-doped zinc oxide nanocomposites as well as the cytotoxic response in human breast cancer (MCF-7) cells and the human embryonic kidney normal cells (HEK 293) are investigated.

Experimental

Preparation of the precursor (Ag,Cu,Co) co-doped zinc laurate hydrazinate

The precursor was prepared by coprecipitation method. First the ethonolic solution (50ml) of lauric acid (1.020g, 0.51x10−2mol) was stirred with hydrazine hydrate N2H4_H2O (99–100%) (4 ml, 8 x10−2mol). To this, a freshly prepared aqueous solution (50 ml) of zinc nitrate hexahydrate (1.23 g, 0.4x10- 2mol) mixed with silver nitrate (0.1g, 0.05x10−2mol), copper nitrate hexahydrate (0.05g, 0.01x10−2mol) and cobalt nitrate hexahydrate (0.1g, 0.05x10−2mol) was added dropwise with constant stirring. The precursor formed immediately, was kept aside for an hour for digestion, filtered, washed with water and alcohol followed by diethylether and air-dried. All reagents used for the reaction are of analytical grade.

Preparation of AgCuCoZnO nanocomposites

Thermal decomposition of the precursor led to the formation of oxide nanoparticles. The dried precursor was transferred to silica crucible and heated to red hot condition in an ordinary atmosphere. The precursor got ignited, underwent autocatalytic decomposition using atmospheric oxygen resulting in the formation of fine oxide products. The obtained nanoparticles were further heated at 400֠ C to remove any residual carbon formed during the decomposition.

(Ag,Cu,Co) co-doped ZnO nanocomposites prepared, hereafter referred to as AgCuCoZnO nanocomposites.

Quantitative methods

The hydrazine content in the precursor was chemically determined volumetrically using 0.025 M KIO3 as titrant under Andrew's condition [28]. Elemental analysis of C, H and N were performed on a Elementar Vario EL III analyzer. Actual doping concentration was quantified with inductively coupled plasma atomic emission spectroscopy (ICP-AES) using Thermo Electron IRIS INTREPID II XSP DUO Flexible axial and radial view instrument, with high concentration capabilities, after digesting the precursor in HNO3, made to 50ml, filtered and analysed.

Sample characterization

All reagents for the synthesis were obtained commercially and used as received. The infrared spectrum of the solid precursor sample was recorded using KBr pellets in the range of 4000–400 cm−1 on Thermo Nicolet, Avatar 370-FT-IR spectrometer. EDX energy-dispersive X-ray spectroscopy was performed by SIGMA HV – Carl Zeiss with Bruker Quantax 200 – Z10 EDS Detector. The simultaneous TG-DTA experiment was carried out in Perkin Elmer, Diamond TG/DTA thermal analyzers. Thermal analysis was carried out in nitrogen atmosphere at the heating rate of 10֩ C per minute using 4.365mg of the sample. Platinum cups were used as sample holders and alumina as reference. The temperature range was ambient to 700֠ C. XRD pattern was recorded using Bruker AXS D8 Advance X-ray diffractometer using CuK_ radiation (1.5406 A°) at 40 kV and 40 mA. Scanning electron microscopy (SEM) was performed with a JEOL Model JSM - 6390LVmicroscope. The morphology of the synthesized NPs was characterized using transmission electron microscopy (TEM) Jeol/JEM 2100 model operating with an accelerating voltage of 200 kV. UV-Vis-NIR absorption spectra was recorded at room temperature on Perkin Elmer lambda 950 UV-VIS-NIR instrument with spectral range of 175–3300 nm having Deuterium lamp (UV region)and Tungsten- Halogen lamp (VIS-NIR region)as source, PMT (UV-VIS) and Peltier cooled PbS (NIR) as Detectors.

In vitro cytotoxicity

Cell lines and cell culture

The human breast adenocarcinoma cell line (MCF7) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 370֩ C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. The human embryonic kidney cell line (HEK 293) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 370֩ C 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Estimation of cytotoxicity: cell treatment procedure

The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1 × 105 cells/ml. One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370֩ C, 5% CO2, 95% air and 100% relative humidity. After 24h the cells were treated with serial concentrations of the test samples. They were initially dispersed by sonication in phosphate buffered saline (PBS) and an aliquot of the sample solution was diluted to twice the desired final maximum test concentration with serum free medium. Additional four serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 μl of these different sample dilutions were added to the appropriate wells already containing 100 μl of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 370֩ C, 5% CO2, 95% air and 100% relative humidity. The medium without samples were served as control and triplicate was maintained for all concentrations.

MTT assay

[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT)] is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells. After 48 h of incubation, 15μl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370֠C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100μl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula, % Cell Inhibition = 100-{Abs (sample)/Abs (control) x100}.Nonlinear regression graph was plotted between % Cell inhibition and Log concentration and IC50 was determined using Graph Pad Prism software.

Results and discussion

Characterization of the precursor

Chemical analysis

The percentage of hydrazine, carbon, nitrogen, hydrogen and metals present in the precursors were analysed and given in Table 1.

Table 1

Chemical analysis of precursor.

Analysis		AgCuCo co-doped Zinc laurate Hydrazinate
		obs	cal
Hydrazine %		16.88	17.57
Elemental Analysis %	C	37.75	37.16
	H	9.00	8.80
	N	14.68	15.89
Metal %	Zn	20.87	21.70
	Ag	2.80	2.55
	Cu	0.83	0.74
	Co	1.54	1.34

Thermal analysis

As can be observed from Fig. 1, the precursor loses weight in two steps. From TG curve the first step is the loss of surface water molecule at a temperature range between100֩ C and 110֩ C. The corresponding peak in DTA curve is observed as an endotherm at 108֩ C. The major weight loss of 68% on the TG curve from 320֩ C to 420֩ C is attributed to the second step involving simultaneous dehydrazination and decarboxylation of the precursor. This is indicated by a sharp endothermic peak in DTA curve at 413֩ C which gives AgCuCoZnO as the final residue, which is stable over 700֩ C without any further weight loss. The thermal analysis data are given in Table 2.

Fig. 1

Thermogravimetric-differential thermal analysis (TG-DTA) curve of precursor.

Table 2

Thermal analysis of precursor.

Precursor	TG temp range °c	Mass loss %	DTA Peak temp °c	Decomposition product
(AgCuCo)co-doped Zinc laurate hydrazinate	100–110	2.31%	108 (-)	Loss of one water molecule
	320–420	68.0%	413 (-)	Loss of two N2H4 molecule & decarboxylation

The analytical data of the precursor obtained from chemical (Table 1) and thermal (Table 2) analysis are found to be in good agreement with the values calculated, considering the molecular stoichiometric composition as Ag0.1Cu0.05Co0.1Zn(C12H23O2)(N2H4)2 for precursor (Ag,Cu,Co) codoped Zinc laurate hydrazinate.

FT-IR analysis

The FT-IR spectra of the precursor shown in Fig. 2 have three bands in the region (3248–3356) cm−1, which are characteristic of N–H stretching frequencies. The N–N stretching frequency is observed at 972 cm−1which confirms the presence of hydrazine as bidentate bridging ligand [29]. The asymmetric stretching frequencies of the carboxylate group in the precursor are observed at 1604 cm−1 and 1543cm−1. The symmetric stretching frequencies of the carboxylate group in the precursor are observed at 1442 cm−1 and 1404 cm−1. The (asym–sym) separation of 162 cm−1 and 139 cm−1 indicate the bidentate bridging nature of carboxylate group. These results support the formation of (AgCuCo) codoped Zn(lau)(N2H4)2 precursor. The infrared spectra analysis data are given in Table3.

Fig. 2

FT-IR analysis of precursor.

Table 3

FT-IR analysis of precursor.

Precursor	νO-H cm−1	νN-H cm-1	νasyo=c=o cm−1	νsyo=c=o cm-1	Δν cm-1	νN-N cm-1
(Ag,Cu,Co)co-doped Zinc laurate hydrazinate	---	3248–3356	16041543	14421404	162139	972

Characterization of the synthesized AgCuCoZnO nanocomposites

From the FT-IR spectra in Fig. 3, we observe peak only at 490cm−1 for AgCuCoZnO nanocomposites, which corresponds to the metal-oxygen (Zn–O) stretching vibration. Peaks corresponding to organic species and hydrazine are completely removed. This confirms that after thermal decomposition only the metal oxide exist.

Fig. 3

FT-IR spectra of AgCuCoZnO nanocomposites.

Energy dispersive X-ray analysis (EDX)

The EDX analysis shown in Fig. 4 exhibit the presence of metals in AgCuCoZnO nanocomposites and it is clear that there is incorporation of all metal dopants and no other elemental impurities present in the synthesized nanostructures.

Fig. 4

EDS of AgCuCoZnO nanocomposites.

ICP-AES analysis

The compositions of nanocomposites were analysed using ICP-AES and given in Table 4. The estimated values of metals agreed with the expected values. This result confirms the absence of loss of elements during thermal decomposition.

Table 4

Composition of AgCuCoZnO nanocomposites obtained from ICP-AES analysis.

Nanocomposite	Zn%		Ag%		Cu%		Co%
	Obs	Cal	Obs	Cal	Obs	Cal	Obs	Cal
AgCuCoZnO	60.01	60.62	7.44	8.14	2.11	2.41	4.00	4.48

X-ray diffraction pattern (XRD)

A typical XRD pattern of AgCuCoZnO nanocomposites is shown in Fig. 5, which shows planes corresponding to both wurtzite phase of ZnO and face-centered cubic (fcc) phase of metallic Ag. The peaks at 31.70°, 34.39°, 36.19°,47.51°, 56.55°, 62.86°, 67.93°, 69.03° can respectively be indexed to (100), (002), (101), (102), (110), (103), (112) and (201) planes of hexagonal wurtzite ZnO structure (JCPDS No36-1451). The three diffraction peaks observed at 38.05°, 44.22° and 77.49° correspond respectively to (111), (200) and (311) planes of the face-centered cubic structure of Ag (JCPDS No 04–0783). The results suggest that silver is not getting incorporated into the zinc oxide lattice but rather forms on the surface of zinc oxide as metallic silver [30]. No obvious intensity peak of metallic Co, Cu or related compounds is detected under the sensitivity of an X-ray diffractometer, which rules out the possibility of any secondary phase. Thus the dopants (Co and Cu) are well doped in the ZnO lattice. The average particle size is calculated using Debye-Scherrer formula, D = Kλ/β cosθ, where D is crystallite size (nm), θ is Bragg diffraction angle, λ is the source wavelength (1.54) and β is the width of the XRD peak at half maximum height. The calculated average particle size of AgCuCoZnO nanocomposites is found to be around 16nm.

Fig. 5

X-ray diffraction pattern of AgCuCoZnO nanocomposites.

SEM and TEM analysis

The SEM image shows the external morphology of nanoparticles. From Fig. 6 AgCuCoZnO nanocomposites exhibit that, the majority of particles are of polygonal shape. From TEM micrographs in Fig. 7 the average diameter is calculated, from measuring over 100 particles in random fields of TEM view. The average TEM diameter of AgCuCoZnO nanocomposites is between 10 to 20 nm, supporting the XRD data. The selected area electron diffraction (SAED) pattern of AgCuCoZnO nanocomposites have sharp spots indicative of polycrystalline nature with symmetrical orientation as shown in Fig. 8.

Fig. 6

SEM image of AgCuCoZnO nanocomposites.

Fig. 7

TEM image of AgCuCoZnO nanocomposites.

Fig. 8

SAED pattern of AgCuCoZnO nanocomposites.

UV-VISIBLE-NIR absorption spectra

ZnO NPs respond only to UV light. Absorption spectra for AgCuCoZnO nanocomposites present a significant absorbance in the visible and NIR region as shown in Fig. 9. X-Ray diffraction reveals the presence of metallic Ag on the surface of ZnO lattice. The Metal dopants (Co and Cu) can narrow the band gap of ZnO NPs by introducing new energy levels between VB and CB and reduce the carrier recombination centers, thereby achieving the enhancement of visible-light absorption. Metallic Ag which has strong electron accepting ability suggest strong interfacial electronic coupling between Ag and CuCoZnO and shows surface plasmon resonance. The surface plasmon absorption maximum red shifts to the NIR region when Ag nanoparticles form assemblies or aggregates [31].

Fig. 9

UV-VISIBLE-NIR absorption spectra of AgCuCoZnO nanocomposites.

In vitro cytotoxicity AgCuCoZnO nanocomposites

The synthesized AgCuCoZnO nanocomposites were tested for their potent cytotoxic activity against human breast cancer cells (MCF-7) and human embryonic kidney normal cell line (HEK 293) using MTT assay. MCF-7 and HEK 293 cell viability were evaluated after 48 h exposure to AgCuCoZnO nanocomposites of various concentrations ranging from (6.25 μg/ml to 50 μg/ml). The living cells, convert the MTT assay to an insoluble purple formazan. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using micro plate reader. The % cell viability of the nanocomposites towards cancer and normal cell lines were determined using the following formula, % Cell viability = {Abs (sample)/Abs (control) x100} and shown in Fig. 10.

Fig. 10

The percentage viability of MCF7 cancer cell lines and HEK 293 normal cell lines using MTT assay after 48 h exposure to AgCuCoZnO (6.25–50 μg/mL). Data represented are mean ± standard deviation of three identical experiments made in triplicate.

Evaluation of IC50 value

Fig. 11 shows the Nonlinear regression graph plotted between % Cell inhibition and Log concentration. IC50 was determined using Graph Pad Prism software. Here we present novel findings that cancerous cells are markedly more susceptible (1.8 times) to AgCuCoZnO nanocomposites mediated toxicity than their normal counterparts (the IC50 value 22 μg/ml for cancer cells and 40 μg/ml for normal cells). At lower concentrations AgCuCoZnO nanocomposite are less toxic to normal cells. It is necessary to reduce IC50 to nanomolar concentrations, so that it is non toxic to normal cells. The interesting fact at this juncture is AgCuCoZnO nanocomposites absorb in near-infrared (NIR) region. NIR absorption is critical for both photo dynamic therapy and photo thermal therapy because of minimal light absorption by water and hemoglobin and the resulting optimal tissue penetration of the light in this region. AgCuCoZnO nanocomposites on irradiating with NIR light may produce greater level of ROS and can heat up the cancer tissues. The inherent toxicity of AgCuCoZnO nanocomposites along with combined therapy of PDT and PTT may reduce the IC50 to nanomolar concentrations and further improve the cancer cell toxicity. Thus these AgCuCoZnO nanocomposites can act as a potential candidate for developing anti-cancer therapeutics. ROS generation is proposed as one of the key cytotoxic mechanism [7, 8] for ZnO nanoparticles. The mechanism of cytotoxicity of AgCuCoZnO nanocomposites is not fully understood.

Fig. 11

Cytotoxicity of AgCuCoZnO against (a) cancer cell line (MCF 7) IC50 value 22 μg/ml and (b) normal kidney (HEK 293) cell line IC 50 value 40 μg/ml.

Conclusion

The synthesis of AgCuCoZnO nanocomposites were successfully achieved by a simple thermal decomposition method. Primarily, the composition, morphology and optical properties were determined. The cytotoxicity of the AgCuCoZnO nanostructures against human breast cancer cells (MCF-7) and the human embryonic kidney normal cells (HEK 293) in vitro were studied. We present the finding that cancerous cells are markedly more susceptible than normal cells. As the nanocomposites have strong absorption in the visible and NIR region, they can be used as photosensitizers for PDT/PTT. The selective cytotoxicity of the nanocomposites against cancer cells can be improved by irradiating with visible or NIR light. Thus these novel nanocomposites are promising material for cancer therapy. Further research on anticancer activity of AgCuCoZnO nanocomposites in different types of cancer cells is warranted. A great amount of work is still needed to develop practical applications of these novel nanomaterials.

Declarations

Author contribution statement

K Vasuki, R Manimekalai: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.