Ag/AgFeO2: An Outstanding Magnetically Responsive Photocatalyst for HeLa Cell Eradication.

A superfast, room-temperature, one-step carrier-solvent-assisted interfacial reaction process was developed to prepare Ag/AgFeO2 composite nanocrystals (NCs) of less than 10 nm in size within a 1 min reaction time. These composite NCs were with a direct energy band gap of 2.0 eV and were paramagnetic, making them suitable for optical activation and magnetic manipulation. These composite NCs, applied as a photocatalyst for the treatment of HeLa cells, achieved a significant reduction of 74% in cell viability within 30 min. These Ag/AgFeO2 composite NCs proved to be a promising magnetically guidable photocatalyst for cancer cell treatment.

Introduction

Cancer is one of the leading causes of mortality as well as morbidity worldwide. Current medical treatments for cancers include surgery, radiotherapy, chemotherapy, hormone therapy as well as stem cell transplant. Unfortunately, these treatment options often bring side effects to patients. Some side effects can even take over the patient’s daily life. Therefore, developing a new treatment option that minimizes the side effect is urgently needed.

Photocatalysts, capable of producing highly oxidative photoinduced holes and highly reductive photoinduced electrons upon absorption of optical energies, have been intensively applied in pollutant degradation, air purification, and bacteria eradication.[1−7] The highly reactive oxygen species (ROS) generated from the reactions between the photoinduced charge carriers, electrons and holes, and the oxygen and water molecules adsorbed on the surfaces of the photocatalyst play the key role in achieving the intended functionality of the above-mentioned applications. The ROS, in fact, exist in normal cells, the so-called intracellular ROS, and act as an intracellular signaling messenger, involved in the regulation of cell proliferation, metabolic alterations, and angiogenesis.[8] A suitable level of ROS is necessary for cell survival, while excessive amounts of ROS can trigger cell apoptosis through damaging the membrane and interior of the cells. Over the past few years, applications of photocatalysts in cancer cell treatment grab the attention of researchers in the area and emerge as a promising alternative to the traditional cancer treatment options that suffer from severe side effects.[9−13]

In recent years, spinel-structured materials, such as Fe3O4, ZnFe2O4, and SnFe2O4, have been demonstrated as a class of outstanding photocatalysts for pollutant degradation.[3,14−19] Silver ferrite, AgFeO2, a much less studied spinel-structured photocatalyst, is expected to exhibit decent photocatalytic activities and is thus a promising candidate as a photocatalyst for cancer cell treatment. In addition, silver particles are widely used in medical applications because of their superior performances in preventing microbial infections.[20] Silver particles are also found to exhibit excellent performances in wound healing, diagnosis, and pharmacological treatments.[21] Silver however is comparatively more expensive, making the treatments that involve silver particles economically less advantageous.[22] To reduce the usage of silver, compositing a minor amount of Ag with a major constituent of AgFeO2 may prove a promising effective photocatalyst option for cancer cell treatment.

In this study, a superfast, room-temperature, one-step carrier-solvent-assisted interfacial reaction process was developed to prepare Ag/AgFeO2 composite nanocrystals (NCs). This newly developed photocatalyst possessed a narrow energy band gap of 2.0 eV, making it suitable for light-triggered photocatalytic functions, and was paramagnetic, feasible for magnetic guiding and targeting when in use for practical cancer treatments.

HeLa cells, one of the most recalcitrant cancer cells, have often been taken as the model cancer cells for the development of cancer cell treatments. Previously developed photocatalysts for treating the HeLa cells are mainly based on TiO2, the most well-known and studied photocatalyst, mostly metal-decorated or metal-doped.[23−32] The synthetic methods for these and other photocatalysts often involve complicated procedures,[23−27,29−38] long preparation times,[23,26,27,29,31−37] and/or high temperatures/pressures.[10,23,26−29,31,32,34,37,38] Furthermore, the HeLa cell treatment utilizing the above-mentioned photocatalysts often require high photocatalyst dosages (>100 μg/mL)[23,27,28,30,31,34,35,38] and long treatment times (>100 min).[23,28,31,38] In this study, we developed a simple one-step carrier-solvent-assisted interfacial reaction process that can produce Ag/AgFeO2 composite NCs of less than 10 nm in size at room temperature under ambient pressure with only 1 min reaction time. The Ag/AgFeO2 composite NCs achieved a significant reduction of 74% in cell viability toward the HeLa cells at a photocatalyst dosage of 40 μg/mL and 30 min treatment time. Most importantly, the Ag/AgFeO2 composite NCs, unlike previously developed photocatalysts, are magnetically responsive and thus feasible for magnetic guiding and targeting when in practical use. The present Ag/AgFeO2 composite NCs prove to be a promising magnetically guidable photocatalyst for cancer cell treatment.

Results and Discussion

Characterizations of the Photocatalyst, Ag/AgFeO2

The product photocatalyst first characterized its crystalline composition with the X-ray diffraction (XRD) measurement. As evident from Figure , the product photocatalyst was composed of two crystalline components, Ag (JCPDS 04-0783) and AgFeO2 (JCPDS 70-1452), without the presence of silver oxides and iron oxides. Five major diffraction peaks located at the 2θ values of 28.4, 34.8, 61.0, 68.5, and 72.7° were identified for AgFeO2 and two minor ones located at 38.1 and 77.4° were attributed to Ag. It can be concluded from the comparison of the diffraction peak intensities that AgFeO2 was the major component of the photocatalyst with Ag as the minor one. Furthermore, the grain size of AgFeO2 can be estimated to be around 6.7 nm with the Scherrer equation based on the full width at half-maximum value of the most pronounced diffraction peak located at 34.8°. The photocatalyst, termed Ag/AgFeO2, was paramagnetic and can be collected with an externally applied magnetic field. The photograph shown as the inset of Figure demonstrates the excellent magnetic collectability of the Ag/AgFeO2 NCs. All Ag/AgFeO2 NCs were securely adhered to the tube wall with a magnet. This magnetic responsiveness confirms that AgFeO2 is the major constituent of the photocatalyst because Ag is not magnetically responsive. The magnetic responsiveness of the photocatalyst is advantageous when the photocatalyst is applied for cancer cell treatment because magnetic guiding and targeting are possible.

Figure 1

XRD pattern of the product photocatalyst. Inset: Magnetic collection of Ag/AgFeO2 NCs.

The morphology, size, and crystalline structure of the Ag/AgFeO2 NCs were examined by using a high-resolution transmission electron microscope. Figure a shows a typical high-resolution transmission electron microscopy (HRTEM) image of the Ag/AgFeO2 NCs at low magnification. It is to be stressed that because of the paramagnetism of the sample, the NCs drifted around under the bombardment of the high-energy electron beam, and it was thus difficult to obtain sharp TEM images of the sample. When examined carefully, one can identify the NCs and estimate their sizes.

Figure 2

(A,B) HRTEM images of Ag/AgFeO2 NCs.

The NC size distribution thus determined based on 250 NCs is presented in Figure S1, which shows most of the sizes of the NCs fall in the range of 6–7 nm. This crystal size is in good agreement with the grain size, 6.7 nm, estimated from the XRD pattern. Figure b shows another HRTEM image, from which the interlayer distances of 0.26, 0.13, 0.31, and 0.24 nm can be determined, matching well with the d-spacing of the (1 0 1), (2 0 1), and (0 0 4) lattice planes of AgFeO2 and the (1 1 1) lattice planes of Ag, respectively. Furthermore, the inset shows a single NC with the lattice planes of both Ag and AgFeO2 identified, confirming the composite structure of the Ag/AgFeO2 NC.

The composition of the Ag/AgFeO2 NCs was determined with EDX spectroscopy. The atomic ratio of Ag versus Fe was determined to be around 1.1:1, with Ag slightly higher than Fe, consistent with the composition of major AgFeO2 combined with minor Ag. Note that the signal of Pt identified in the spectrum came from the pretreatment of the sample with Pt to enhance the electrical conductivity of the sample. Furthermore, the elemental EDX mapping was conducted to further study the composition of the Ag/AgFeO2 NCs. The mapping of Ag (green) and Fe (red) was shown in the inset of Figure . Evidently, both Ag and Fe were uniformly distributed throughout the sample, implying a uniform coupling of Ag and AgFeO2 instead of separate phases of Ag and AgFeO2.

Figure 3

Energy-dispersive X-ray (EDX) spectrum of Ag/AgFeO2 NCs. Inset: Area mapping of Ag and Fe elements.

The energy band gap of the Ag/AgFeO2 NCs was determined with the UV–vis absorption spectrum as shown in Figure . The onset absorption wavelength was determined to be 620 nm, corresponding to an energy band gap of 2.0 eV. The energy band gap can be more accurately determined by converting the UV–vis spectrum into the corresponding Tauc plot, the inset of Figure , from which a value of 2.0 eV was obtained for the energy band gap, in excellent agreement with that determined from the onset absorption wavelength. The Ag/AgFeO2 NCs, with an energy band gap much smaller than 3.1 eV, can thus be effectively triggered with UV light to generate photoinduced electrons and holes, from which hydroxyl radicals, a powerful ROS, are produced for the eradication of cancer cells.

Figure 4

Ultraviolet–visible (UV–vis) absorption spectrum of Ag/AgFeO2 NCs. Inset: Corresponding Tauc plot.

Cell Treatment Efficacy of Ag/AgFeO2 NCs

The Ag/AgFeO2 NCs were applied as a photocatalyst in cancer cell treatment, taking HeLa cells as a model. The efficacy of the Ag/AgFeO2 NCs on the eradication of HeLa cells was evaluated with the cell viability and cell morphology upon treating HeLa cells with the NCs under UV irradiation. The cell viability and cell morphology data were collected at three different stages: before treatment, after treatment, and 24 h of regrowth after treatment. The comparison between the results of cell viability and cell morphology before and after the treatment gives immediate indication on the performance of the Ag/AgFeO2 treatment. As to the 24 h regrowth after treatment, its purpose is to study the recovery of the cells from photodamage, a further confirmation of the cell apoptosis.

Light irradiation, concentration of the photocatalyst, and treatment time are three main parameters to be investigated. We designed four sets of experiments, as summarized in Table S1, to study the efficacy of the Ag/AgFeO2 treatment. Experiment 1, without the application of both light irradiation and photocatalysts, served as a control to highlight the effects of light irradiation and photocatalysts. When conducting the experiments, the cells were transferred from a CO2-guarded incubator to a biosafety cabinet for sample preparation, and the subsequent treatment was carried out in a UV transilluminator where no controlled level of CO2 was provided. Therefore, experiment 1 also served the purpose of investigating the effect of ambient conditions on the cell viability and cell morphology within the experimental time frame. Meanwhile, experiments 2 and 3, with the application of only light irradiation or photocatalysts, respectively, were designed to investigate the effects of plain light irradiation or photocatalysts on the test cells. Experiment 4, with the application of both light irradiation and photocatalysts, served as the main experiment to evaluate the efficacy of the Ag/AgFeO2 NCs on HeLa cell eradication. The cell viability was determined as the percentage of the number of living cells against total cell number. In this study, trypan blue, a blue color dye, was used to quantify the cell viability. Living cells, protected by cell walls, can resist the penetration of trypan blue and remain unstained, whereas dead cells, with the cell walls damaged, can be penetrated by trypan blue and appeared blue under an inverted microscope. Figure S2 illustrates the difference between unstained living cells and trypan blue stained dead cells under an inverted microscope. Cell counts were then carried out on the total number of the stained and unstained cells to quantify the cell viability.

The cell viability data obtained from experiments 1–3 at three stages with treatment times up to 30 min were compiled in Figure S3 as histogram plots. Both mean values and standard deviations (based on the three experiments) were indicated in the histograms. Evidently, no significant cell eradication was achieved with all three sets of experiments. A slight reduction in the cell viability, around 4%, was within the experimental errors. The results of experiment 1 confirm that HeLa cells are strong enough to survive in the ambient condition. The results of experiments 2 and 3 indicate that neither light irradiation nor photocatalysts alone can do harm to HeLa cells.

Figure shows the cell viability results for experiment 4 conducted at three increasing concentrations of Ag/AgFeO2 NCs. The results confirm that the Ag/AgFeO2 NCs under UV light irradiation can effect cell eradication. The Ag/AgFeO2 NCs, with an energy band gap of 2.0 eV, were able to absorb UV light to generate photoinduced electrons and holes, from which hydroxyl radicals, a powerful ROS, were subsequently created to attack the cells. Several points can be made from Figure . First, the cell viability decreased with increasing treatment time. This is expected because ROS generation continued with the increasing treatment time to effect an increasing extent of cell eradication. Second, the cell viability generally dropped further after 24 h of regrowth. This implies that the cell eradication continued during the regrowth period. Third, the cell eradication performance depends critically on the concentration of the Ag/AgFeO2 NCs, with an optimal concentration of 40 μg/mL, achieving a significant reduction in the cell viability of 74% before and after the treatment. The reduction in cell viability achieved with the Ag/AgFeO2 concentrations of 20 and 60 μg/mL at the end of 30 min treatment was only 20 and 13%, respectively. This drastic effect of the photocatalyst concentration may be explained as follows. Theoretically, the more the photocatalyst exists in the system, the more the ROS is generated for cell eradication. The generation of ROS however occurs at the particle surface of the photocatalyst. These photocatalyst NCs aggregate to a certain extent in the culture medium, thus reducing the effective exposed surfaces for the ROS generation. Figure shows the size distributions of the Ag/AgFeO2 NC suspensions of 20, 40, and 60 μg/mL determined with the dynamic light scattering spectroscopy. The polydispersity indices of the 20, 40, and 60 μg/mL Ag/AgFeO2 NC suspensions were 0.363, 0.407, and 0.603, respectively. It is evident that the Ag/AgFeO2 NCs are prone to aggregation because of their large surface-to-volume ratios,[39] and the Ag/AgFeO2 NCs aggregated to some extent forming larger-sized particle clusters. The extent of aggregation becomes more severe as the Ag/AgFeO2 NC concentration increases, with the size distribution shifting toward the larger size side. The competition between the amount of the Ag/AgFeO2 NCs present in the system and the aggregation extent experienced by the Ag/AgFeO2 NCs leads to the existence of an optimum Ag/AgFeO2 NC concentration of 40 μg/mL. This phenomenon explains why the reduction in cell viability was the lowest at 60 μg/mL and the highest at 40 μg/mL.

Figure 5

Cell viability vs treatment time for experiment 4 at photocatalyst concentrations of (A) 20, (B) 40, and (C) 60 μg/mL. Red denotes before treatment, blue denotes after treatment, and green denotes 24 h regrowth after treatment.

Figure 6

Dynamic light scattering size distributions of Ag/AgFeO2 NC suspensions of 20, 40, and 60 μg/mL.

In addition to cell viability measurements, the treatment outcome can also be characterized with observation of the cell morphology at different treatment stages. The cell morphology was recorded with an inverted microscope at the three treatment stages: before treatment, after treatment, and 24 h regrowth after the treatment. For this morphological study, cell confluence of more than 95% was ensured before the treatment. Figure S4 shows typical microscopic images for the HeLa cell samples with confluency much less and slightly greater than 95%. Cell samples with confluency much less than 95% can be observed with cells separated from one another. Dead cells shrink and detach from the walls, giving reduced confluency. Therefore, the microscopic images offer a qualitative visualizable measure of the cell treatment outcome.

Figure S5 shows the morphology of the test cells at the three treatment stages at the end of the 30 min treatment for experiment 1. Regardless of the treatment stage, all three images exhibited high confluency close to 100%, in consistent with the cell viability data. The HeLa cells survive well in ambient conditions. For experiment 4, Figure shows the corresponding cell images at the three treatment stages for photocatalyst concentrations of 20, 40, and 60 μg/mL. The trends in confluency reduction match very well with those observed in the cell viability. The reduction in confluency was the most pronounced for the case of 40 μg/mL, only limited for the case of 20 μg/mL, and the least for the case of 60 μg/mL after treatment. The black objects observed in the images are large aggregated Ag/AgFeO2 particles.

Figure 7

Morphology of the HeLa cells (A) before treatment, (B) after treatment, and (C) 24 h regrowth after treatment, with UV exposure time of 30 min and 20 μg/mL of Ag/AgFeO2. (Experiment 4) (D–F) For the case of 40 μg/mL and (G–I) for the case of 60 μg/mL.

The production of hydroxyl radicals during the photocatalytic treatment is responsible for destroying the cell membrane and subsequent apoptosis of the cells. It is thus interesting to investigate the correlation between the production of the hydroxyl radicals and the cell apoptosis. A photoluminescence (PL)-based technique was developed for the quantification of hydroxyl radicals.[40,41] Coumarin is used as a probe molecule to detect the formation of hydroxyl radicals because it reacts with the hydroxyl radicals to form a highly fluorescent 7-hydroxycoumarin molecule, which emitted PL light at 456 nm with an excitation wavelength of 332 nm. The higher the concentration of the hydroxyl radical, the higher the intensity of the PL. Figure shows the PL spectra recorded for characterizing the hydroxyl radical concentration generated from the Ag/AgFeO2 NC suspensions of 20, 40, and 60 μg/mL at illumination times of 0, 15, and 30 min, respectively. It is evident that the hydroxyl radical concentration increases with the increasing reaction time, and the Ag/AgFeO2 suspension of 40 μg/mL gives the highest hydroxyl radical concentration, with that of 20 coming next and 60 the last, in good agreement with the cell apoptosis results obtained.

Figure 8

PL spectra of Ag/AgFeO2 NC suspensions of 20, 40, and 60 μg/mL at illumination times of 0, 15, and 30 min, respectively.

Conclusions

Ag/AgFeO2 composite NCs of less than 10 nm in size were prepared with a one-step carrier-solvent-assisted interfacial reaction process. The process proceeded at room temperature under ambient pressure and produced the composite NCs in one step within 1 min. The main constituent of the composite NC, AgFeO2, is a paramagnetic narrow band gap semiconductor with a direct energy band gap of 2.0 eV, suitable for UV light activation and magnetic manipulation. These composite NCs were applied as a photocatalyst for the treatment of the HeLa cells, one of the most recalcitrant cancer cells. The presence of UV light or photocatalyst alone did no harm to the cancer cells. However, the irradiation of the photocatalyst with UV light achieved a significant reduction of 74% in cell viability within a 30 min treatment time. The Ag/AgFeO2 composite NCs were thus proved to be a promising magnetically guidable photocatalyst for the treatment of the HeLa cells. The HeLa cell eradication effectiveness of the present photocatalyst was compared with those of the previously reported works in Table S2. The Ag/AgFeO2 composite NCs appear to be a superior choice considering the low catalyst concentration and short treatment time involved, not to mention the potential magnetically guidable capability.

Experimental Section

Materials

Materials used in the present study included AgNO3 (Acros, 99%), Fe(NO3)3·9H2O (J. T. Baker, 98%), ethanol (Sigma-Aldrich, 99%), chloroform (Sigma-Aldrich, 99%), aqueous NaOH solution, 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich), 90% ethanol solution, fetal bovine serum (FBS) (Biowest), HeLa cell line, penicillin (Millipore), phosphate-buffered saline (Amresco), RPMI 1640 medium (Matrioux (M) SDN BHD), and sodium hydrogen carbonate (BioWhittaker). All chemicals were used as received without further purification.

Preparation of Ag/AgFeO2 Composite NCs

The Ag/AgFeO2 composite NCs were prepared with a one-step carrier-solvent-assisted interfacial reaction process.[39] Briefly, 0.1 g of AgNO3 and 0.4756 g of Fe(NO3)3·9H2O were dissolved in 22.5 mL of ethanol to serve as a precursor solution. An amount of 3.75 mL precursor solution was added into chloroform of equal volume. An amount of 7.5 mL aqueous NaOH solution (0.8 M), lighter than and immiscible with chloroform, was then added to the above solution to generate an interface between the aqueous and chloroform domains. Ethanol, miscible with both water and chloroform but having higher affinity toward water, served as the carrier solvent, carrying the precursor from the lower chloroform domain to the upper aqueous domain to react for the formation of Ag/AgFeO2 composite NCs through sol–gel reactions. The reaction time was controlled to be 1 min.

Cancer Cell Culture and Viability Assay

The HeLa cells were cultured in RPMI 1640 medium (pH 7) supplemented by 9.9% FBS and 0.99% penicillin in a humidified incubator under 5% CO2 atmosphere at 37 °C. Here, penicillin was added to reduce the possibility of microbial contamination in the cell culture, whereas FBS served as the nutrient source for the cancer cells. The cell confluence, observed with an inverted microscope at a magnification of 10×, was controlled to be over 95% before any photocatalytic treatments, and cell viability tests can be performed.

The cell viability was determined with a hemocytometer using the trypan blue staining method to differentiate the living from the dead cells. The cell membrane of a living cell is impermeable to trypan blue, whereas the compromised cell membrane of a dead cell allows permeation of trypan blue, from which the cell viability can be readily determined with an inverted microscope at a magnification of 10×. The cell samples were first detached from the cell culture flask wall with an appropriate amount of 0.25% trypsin–EDTA solution to form the cell suspension. The cell suspension was then mixed with an equal volume of trypan blue for the possible staining for microscopic observation.

Cancer Cell Treatment with a Photocatalyst

Prior to the photocatalytic treatment, the cell sample was examined under an inverted microscope to ensure sufficient confluence. For the photocatalytic treatment experiment, the old culture medium was replaced with a new culture medium containing the Ag/AgFeO2 NCs of a desired concentration and incubated for 48 h. The culture was then irradiated with UV light provided by a UV transilluminator (Gel Logic 112, Carestream) at room temperature for a desired time duration before the cell viability determination. After the photocatalytic treatment, the photocatalyst-containing medium was replaced with fresh culture medium, and the treated cell sample was incubated for another 24 h to investigate the regrowth of the survivor cells. All experiments were conducted in triplicate.

The effects of two treatment parameters, the concentration of the Ag/AgFeO2 NCs and the duration of light irradiation, were investigated. The concentration of the Ag/AgFeO2 NCs was increased from 0 to 60 μg/mL at a 20 μg/mL interval, whereas the duration of light irradiation was increased from 0 to 30 min at a 5 min interval. Four groups of experimental setting, summarized in Table S1, were compared to reveal the detailed functionality of the Ag/AgFeO2 NCs on killing the HeLa cells. The first group was without the presence of a photocatalyst and light irradiation. The purpose of this group was to investigate how strong the HeLa cells were at ambient conditions away from their comfort zone, 37 °C and 5% CO2 atmosphere. The second group was treated with the photocatalyst without light irradiation, whereas the third group was irradiated with light in the absence of a photocatalyst. The fourth group had both photocatalyst and light irradiation in action. From the comparison of the last three groups, one can reveal the roles played by the photocatalyst and light irradiation in HeLa cell eradication.

Measurement of Hydroxyl Radicals

Typically, 1 mL of 10–3 M coumarin aqueous solution was added into 2 mL of Ag/AgFeO2 photocatalyst suspensions of 20, 40, and 60 μg/mL, and the PL intensity at 456 nm was recorded at a 5 min interval for 30 min under light illumination.

Characterizations

The crystalline structure of the photocatalyst was characterized with an X-ray diffractometer (MAC Science, MXP18, Cu Kα). The morphology and crystalline structure of the photocatalyst were observed with a high-resolution transmission electron microscope (JEOL, JEM-3000F, 300 kV). The band gap of the photocatalyst was determined with UV–vis spectroscopy (Hitachi, U-2800). EDX spectroscopy (Oxford 6587, Oxford Instruments) was conducted to determine the elemental composition of the photocatalyst. The size distribution of the NC suspension was determined with dynamic light scattering spectroscopy (Malvern ZEN1600), in which DI water was used as the solvent. The PL spectra were recorded with a fluorescence spectrometer (Hitachi F-4500). The cell images were observed and taken using an inverted microscope (Olympus CKX41).