|Year : 2020 | Volume
| Issue : 2 | Page : 45-51
Near-infrared-responsive silver-capped magnetic nanoclusters for cancer therapy
Amit Tewari, Ruby Gupta, Deepika Sharma
Institute of Nano Science and Technology, Habitat Centre, Mohali, Punjab, India
|Date of Submission||02-May-2020|
|Date of Acceptance||09-May-2020|
|Date of Web Publication||23-Jun-2020|
Dr. Deepika Sharma
Institute of Nano Science and Technology, Habitat Centre, Mohali, Punjab
Source of Support: None, Conflict of Interest: None
Aim: Near-infrared (NIR)-based photothermal therapy (PTT) has been proposed as a prospective adjuvant to traditional chemotherapy. The present work aims to study the impact of silver-coated magnetic nanoparticles as a PTT agent against multiple cancer cell lines. Materials and Methods: Silver-coated magnetic nanoclusters (Ag-MNCs) were synthesized by a modified method and characterized using X-ray diffraction, transmission electron microscopy, Fourier-transform infrared spectroscopy, and ultraviolet–visible absorption spectra. Its effect as an agent for NIR-based PTT was assessed on four different human cell lines, namely glioblastoma cell line U-87 MG, osteosarcoma MG-63, lung carcinoma A549, and triple-negative breast cancer cell line MDA-MB-231 by irradiation with 750 nm NIR laser for 10 min. Cellular damage was assessed in terms of MTT and cell cycle analysis and visualized by confocal microscopy. Results: The Ag-MNCs were successfully generated and exhibited excellent hyperthermic rise when exposed to NIR laser. A reduction of more than 60% of the cells was observed in the MTT assay. Confocal microscopy also confirmed significant nuclear damage to cells exposed to PTT in the presence of Ag-MNCs. Conclusion: Our results confirm that the Ag-MNCs have an excellent hyperthermic profile and as the test results indicate that it can be utilized as an agent for NIR-based PTT against various types of cancer cells.
Keywords: Human cell lines, magnetic nanoclusters, near infrared, photothermal therapy, silver
|How to cite this article:|
Tewari A, Gupta R, Sharma D. Near-infrared-responsive silver-capped magnetic nanoclusters for cancer therapy. J Radiat Cancer Res 2020;11:45-51
|How to cite this URL:|
Tewari A, Gupta R, Sharma D. Near-infrared-responsive silver-capped magnetic nanoclusters for cancer therapy. J Radiat Cancer Res [serial online] 2020 [cited 2020 Jul 14];11:45-51. Available from: http://www.journalrcr.org/text.asp?2020/11/2/45/287447
Amit Tewari & Ruby Gupta: Both the authors have equal
| Introduction|| |
Cancer continues to be one of the leading causes of death worldwide. It is well established that cancer is a complex disease caused by a combination of genetic factors, environmental factors, carcinogen exposure, lifestyle, and work habits. Several hallmarks and common features of cancer such as unlimited replication potential, evading apoptosis, and dysregulation in various cellular pathways have also been proposed. Current treatment modalities generally focus on chemotherapy and radiotherapy; however, the low selectivity of these methods implies that normal healthy cells are affected as a by-product of the targeting of cancerous cells. Hence, the current research has focused on alternative methods of cancer treatment and exploiting advances made in nanotechnology to solve these problems.,, Treatment modalities based on metallic particles, especially magnetic nanoparticles, have attracted a lot of attention because of their uniform distribution size, biocompatibility, high magnetization values, enhanced targeting abilities, and minimum invasiveness.,,, Noble metal-coated metallic nanoparticles have grabbed special attention because of their antibacterial nature, biocompatibility, and potential to be exploited for photothermal therapy (PTT) and optical imaging.,,, PTT which involves the production of heat in response to absorption of near-infrared (NIR) light has been the focus of recent research because it is an excellent adjuvant to traditional chemotherapy and is especially desirable because of its noninvasiveness and ability to be directed when combined with magnetic nanoparticles.,,,,, A sustained increase in the tissue temperature from 37°C to 42°C–45°C without changing the physiological microenvironment has been shown to cause tumor cell death by activating various cell degradation mechanisms., PTT utilizing near-infrared (NIR) absorption is particularly useful because of the low scattering and minimum absorption by blood and other surrounding soft tissue of the target cancer cells, hence minimizing any unwanted side effects.,,
In this work, we aim to engineer double dopant iron-based magnetic nanoclusters (MNCs) coated with silver (Ag-MNCs) as an agent for NIR-based PTT. The MNCs are then tested for its NIR-based PTT response, cytotoxic effects on various cell lines, and the results were analyzed by cell cycle analysis and modifications in nuclear morphology. To the best of our knowledge, the particle synthesis is novel and has not been explored for PTT thus far.
| Material and Methods|| |
All the chemicals and reagents used in the study were of analytical grade and purchased from Sigma-Aldrich unless otherwise specified.
Synthesis and characterization of photothermal agents
Synthesis of Fe3O4 nanoparticles
The Fe3O4 nanoparticles synthesized earlier in the laboratory were used as seeds to generate nanoclusters for application in PTT. To do so, 20 mg of Fe3O4 nanoparticles was re-suspended in 11.5 mL water under sonication. After complete dispersal, mixture of 450 mg of FeCl2.4H2O, 40 mg MnCl2, and 10 mg ZnCl2 salts were added to the Fe3O4 solution. The mixture was sonicated for another 30 min. After incubation, 500 mg of citric acid was added to form stable dispersions of magnetic nanoparticles and stirred for 2 h. Then, the reaction mixture was reduced by adding 2.5 mL of ammonium hydroxide (NH4 OH) under vigorous stirring. The reaction was air stirred for 10 min, after which the mixture was added into a Teflon vessel and kept at 150°C for 4 h. The reaction was then cooled to room temperature (RT) and washed with water several times.
Synthesis of Fe3O4 core – Ag shell particles
The MNCs were coated using a protocol suggested by Mandal et al. The synthesized Fe3O4 nanoparticles to be used as core material and anhydrous silver nitrate (AgNO3) were taken together in a 1:1 molar ratio. Glucose was then added to reduce the silver ion to its metallic state that would ultimately enable it to form a capping layer onto the surface area of core Fe3O4 nanoparticles. The reaction mixture was sonicated for 15 min. The reaction was further carried out by heating in a water bath with slow stirring for an hour. A change in color from black to a brownish shade was used as a visual indicator of the progress of the reaction. The nanoparticles (Ag-MNCs) were then separated by centrifugation yielding a clear supernatant.
Characterization of nanoparticles
Bruker D8 Advance X-ray diffraction (XRD) system was used to evaluate the crystallite size and structure of the nanoparticles using Cu Kα radiation source from 20 to 80° (2θ). A JEOL JEM-2100 transmission electron microscope (TEM) operating at an acceleration voltage of 200 kV was used to evaluate the particle size and size distribution profile of the nanomaterials. The mean diameter of the particle was calculated using the Scherrer equation:
Where θ is the angle at which the reference peak occurs, θ is the X-ray wavelength, b is the FWHM of the XRD peak, and K is the shape factor.
Energy dispersive X-ray (EDX) spectroscopy equipped with scanning electron microscopy was used to study the elemental distribution in the MNCs.
FTIR measurements using a Bruker Vertex 70 FTIR spectrophotometer were done to confirm silver coating onto the surface of core Fe3O4 MNCs. A spectral range from 4000 to 400 cm−1 was investigated. The signal was obtained by averaging 64 scans at a resolution of 4 cm−1. Ultraviolet–visible (UV–Vis) spectra of the MNCs were recorded using a UV-2600 UV − Vis spectrophotometer (Shimadzu, Japan).
Photothermal hyperthermia measurements
Laser hyperthermia was conducted by a NIR continuous laser at 750 nm (laser components). The aqueous dispersion of Ag-MNCs (0–5 mg/mL) was exposed to the laser for 10 min. A digital thermometer inserted into the wells was used to measure the temperature rise.
Four human origin cell lines, namely glioblastoma cell line U-87 MG, osteosarcoma MG-63, lung carcinoma A549, and triple-negative breast cancer cell line MDA-MB-231 were purchased from NCCS, Pune (India), and maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) and ×100 antibiotic-antimycotic solution (HiMedia) at 37°C with 5% CO2 in a humidified atmosphere. All the experiments were performed in triplicates.
MTT assay to measure cell viability
The biocompatibility of the synthesized nanomaterial was investigated on a normal murine fibroblasts cell line (L929) in a concentration range of 0–1000 μg/mL using MTT assay. All cancer cell lines used were then treated with a biocompatible concentration of 250 μg/mL of Ag-MNCs, as determined by the biocompatibility assay on L929 (data not shown). After 6 h of incubation, the cells were exposed to PTT for 10 min. After exposure, the cells were incubated back at 37°C to recover from the heat stress. The treatment controls were established as follows: (a) untreated cells; (b) cells treated with NIR alone, and (c) cells treated with Ag-MNCs alone. For all the samples, viability was determined 24 h after the treatment using the conventional MTT assay by applying the given formula:
Cell Viability (%) = (At÷ Ac) × 100
Where At is the average absorbance of test wells and Ac is the average absorbance of control wells. All experiments were performed in triplicates.
Cell cycle analysis
The effect of the treatment on various cancer cell lines was investigated using cell cycle analysis by the protocol suggested by Thinon et al. Cells were harvested and washed with phosphate-buffered saline (PBS) after the appropriate treatments. About 70% ice-cold ethanol was used to re-disperse and fix the obtained cell pellets overnight at a temperature of −20°C. Following this, the cells were then washed with PBS and stained using 50 μg/mL freshly prepared solution of propidium iodide containing 10 μg/mL RNase. Immediate analysis using flow cytometer (BD Biosciences, UK) was carried out after incubation at 37°C for 30 min.
Study of nuclear morphology
The effect of treatments on cellular nucleus was analyzed by staining cells with 5 μg/mL solution of Hoechst-33342 in PBS for 10 min at RT. Following incubation, cells were washed and observed under Zeiss LSM 880 confocal microscope (Carl Zeiss, Thornwood, New York).
| Results and Discussion|| |
Characterization of magnetic nanoclusters
The XRD spectra confirmed the crystallite structure and composition of the synthesized MNCs as magnetite (Fe3O4). As represented in [Figure 1]A, the diffraction peaks centered at 2θ angles of 30.24, 35.41, 42.72, 56.37, and 62.32 can be indexed from (220), (311), (400), (511), and (440) faces of the inverse spinel iron oxide, respectively [Figure 1]A: a], while the additional diffraction peaks observed in XRD spectra of Ag-MNCs centered at 2θ angles of 38.13, 44.24, and 64.24 came from the (111), (200), and (220) faces of face-centered cubic silver, respectively [Figure 1]A: b]. The presence of these additional diffraction peaks in Ag-MNCs confirms the presence of Ag in the MNCs, indicating the successful generation of Ag-capped MNCs as Ag-Fe3O4(Ag-MNCs). The crystallite size of individual iron oxide grain for MNCs as calculated by the Scherrer formula was determined to be ~7 nm, while that of Ag-MNCs was 6 nm. Furthermore, the XRD peak analysis indicated formation of single-phase cubic structures with no extra peaks indicating the absence of any trace of impurity in the nanomaterial.
|Figure 1: (A) X-ray diffraction spectra; (B) FTIR spectra; (C) TEM image analysis of MNCs, and (D) Silver-coated magnetic nanoclusters; (E) EDX elemental spectra of MNCs and (F) Silver-coated magnetic nanoclusters; (G) UV spectra of analysis of MNCs and (H) Silver-coated magnetic nanoclusters|
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FTIR measurements were further performed to confirm the silver coating onto the surface layer of MNCs [Figure 1]B. The observed intense bands were compared with standard values to identify the functional groups. FTIR spectrum shows absorption bands at 3460, 3020, 2965, 1737, 1368, 1222, and 560 cm−1 indicating the presence of Ag as a capping agent onto the surface layer of MNCs. The bands at 3460 cm−1 in the spectra correspond to O-H stretching vibration indicating the presence of alcoholic groups. Bands at 3020 and 2965 cm−1 region correspond to C-H stretching. The band at 1743 cm−1 corresponds to non-conjugated C–C stretching. The band at 1368 cm − 1 exemplifies the N=O symmetry stretching typical of the nitro compound. The band at 1222 cm−1 corresponds to C-N stretching of amines. These functional groups have a role in capping of Ag-MNCs as previously reported in studies., The MNCs (both bare and Ag coated) also exhibited the characteristic peak of Fe-O bond vibrations at 560 cm−1 region.
[Figure 1]C and [Figure 1]D represents the TEM image analysis of the synthesized MNCs and Ag-MNCs, respectively. For Ag-MNCs [Figure 1]D, Ag is in dark contrast, while lighter structures correspond to magnetite MNCs. EDX spectra were further extracted to determine the elemental distribution, namely iron (Fe), manganese (Mn), zinc (Zn), oxygen (O), and carbon (C) from the MNCs [Figure 1]E and additionally silver (Ag) in Ag-MNCs [Figure 1]F. As seen in the table listing elemental composition [Figure 1]F, the presence of Ag further confirms the successful coating onto the surface layer of MNCs. Moreover, the table also confirms the successful doping of magnetite MNCs with both Mn and Zn [Figure 1]E and [Figure 1]F.
It was observed that solution of silver nitrate turned the solution of MNCs from black to light brown indicating the formation of Ag-MNCs. The UV-Vis absorption spectra of both MNCs and Ag-MNCs were further recorded at a wavelength range of 300–800 nm, as shown in [Figure 1]G and [Figure 1]H, respectively. A single strong and broad surface plasmon resonance peak was observed at 405 nm that confirmed the synthesis of Ag-MNCs [Figure 1]H. The absorption spectra of MNCs indicated the particles to be NIR active and were thus evaluated for their role in PTT.,
Photothermal hyperthermia measurements
The silver coating onto the surface layer of MNCs dramatically increased the photothermal response of the MNCs as compared to the uncoated MNCs and Fe-MNCs [Figure 2]A. Further, concentration-dependent (0.5–5 mg/mL) temperature rise of Ag-MNCs was tested on laser irradiation [Figure 2]B. With increasing MNC concentration, the total degree rise in temperature was observed to increase. At the highest concentration tested (5 mg/mL), PTT resulted in approximately 14°C rise in temperature, while at the lowest concentration (0.5 mg/mL), a rise of upto 7°C was observed. The results indicate that the Ag-capped MNCs were able to attain the desired hyperthermic window (ΔT = 5°C–8°C) at all the concentrations tested indicating it be a potential theranostic candidate for cancer therapy.,
|Figure 2: Photothermal hyperthermia measurements of (a) Different nanosystems synthesized; (b) Silver-coated magnetic nanoclusters at different concentrations. The grey-2shaded region represents the desired hyperthermic window (ΔT = 5°C–8°C)|
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Effect on cell viability
To access the effect of PTT on viability of cancer cells, conventional MTT assay was employed. The effect was investigated on four different cancer cell lines, namely glioma (U87-MG), bone (MG-63), lung (A549), and breast (MDA-MBA-231) [Figure 3]. The results clearly show that for all the cancer cells tested, a very slight reduction in cell viability is observed when cells are exposed to radiation alone. Further, on treatment with Ag-MNCs, a greater reduction in cell viability was observed indicating the cytotoxic potential of the magnetic nanomaterial. However, maximum reduction in cell viability was observed on laser irradiation of cancer cells in the presence of Ag-MNCs. All of the cell lines experienced a more than 60% reduction in cell viability when exposed to dual treatment with NIR and Ag-MNCs. After PTT in the presence of Ag-MNCs, ~34% cells were observed viable for U87-MG, ~35% cells were observed viable for MG-63, ~15% cells were observed viable for A549, and ~12% cells were observed viable for MDA-MBA-231 cell lines. The results indicate that the synthesized nanomaterial is an effective agent with potential to be used in NIR-mediated PTT against various types of cancers. Studies have reported effective cancer cell death on exposure to temperature at 40°C–60°C for several minutes.,, In accordance with our findings, Liu et al. have also reported the application of Ag-capped Fe3O4 nanoparticles for reduction of ovarian cancer cell line viability to ~25% on irradiation with NIR laser (808 nm) for 10 min. Thus, demonstrating that photothermal effect of Ag-capped MNPs can be employed for tumor regression by NIR laser irradiation.
|Figure 3: MTT assay on various cell lines for (1) Control cells (untreated), (2) Control cells treated with NIR alone, (3) Cells treated with silver-coated magnetic nanoclusters, and (4) Cells treated with NIR in the presence of silver-coated magnetic nanoclusters|
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Cell cycle analysis
Cell cycle analysis was further done to evaluate the possible DNA damage after the appropriate treatments on all the four cancer cell lines by subjecting cells to flow cytometry. On DNA damage, cells are known to accumulate in G1 phase, DNA synthesis phase (S), or in G2/M phase of the cell cycle. [Figure 4] represents the results obtained from cell cycle analysis. For all the cell lines studied, arrest in G2/M phase of the cell cycle was observed after treatment with NIR laser for 10 min in the presence of Ag-MNCs. A slight decrease in percentage of cells in S-phase of the cell cycle was also observed in comparison to the controls taken. The results obtained are in accordance with various other studies which have reported arrest of cells in G2/M phase of the cell cycle after treatment with other Ag-capped nanomaterials.,
|Figure 4: Cell cycle analysis for (a) U87-MG, (b) MG-63, (c) A549, and (d) MDA-MBA-231 cell lines for (1) Control cells (untreated), (2) Control cells treated with NIR alone, (3) Cells treated with silver-coated magnetic nanoclusters, and (4) Cells treated with NIR in the presence of silver-coated magnetic nanoclusters. The bar graphs represent the total population of cells in different phases of cell cycle after appropriate treatments of each respective cell lines|
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Effect on nucleus morphology of cancer cells
Confocal microscopy clearly reveals deterioration of the cellular structure when exposed to PTT for all cell lines investigated. As seen in [Figure 5], exposure of cancer cells to NIR alone did not have any significant effect on the nuclear morphology of cells, whereas after treatment with Ag-MNCs, changes in cellular nuclei were evident. Consistent with cell viability test results, maximum deterioration of nuclear morphology was observed in the treatment Group 4 (NIR in the presence of Ag-MNCs).
|Figure 5: Hoechst staining to determine effect of treatments on cellular nucleus|
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The significant reduction observed in viability of cancer cells after NIR exposure in the presence of Ag-MNCs [Figure 3] can be attributed to the observed morphological alterations in the cellular nuclei [Figure 5]. Various studies have reported such morphological alterations in nuclei to be conducive to apoptotic cell death.,,,
| Conclusion|| |
We have successfully shown the production of silver-coated MNCs (Ag-MNCs) as potential candidates for NIR-based PTT of multiple cancers. The Ag-MNCs exhibited a significant rise in temperature when exposed to NIR laser of wavelength 750 nm. The MNCs demonstrated excellent results in terms of a decrease in cell viability, along with arrest in G2/M phase of the cell cycle on NIR exposure. Moreover, significant alterations in cellular nuclei were also evident that ultimately resulted in the death of cancer cells. In conclusion, the results substantiate the potential of Ag-MNCs to be employed as effective phototheranostic agents for cancer therapy.
Authors dedicate this work to all the front line warriors from India, who are fighting against the COVID-19 pandemic.
Financial support and sponsorship
This work was supported by DST-SERB project under [grant ECR/2017/000049].
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]