DOI:
10.1039/C3RA45338C
(Paper)
RSC Adv., 2014,
4, 5819-5825
Gold–chitin–manganese dioxide ternary composite nanogels for radio frequency assisted cancer therapy
Received
25th September 2013
, Accepted 1st November 2013
First published on 1st November 2013
Abstract
Gold nanoparticles (Au-NPs) based chitin-MnO2 ternary composite nanogels (ACM-TNGs) were prepared by the regeneration of chitin along with MnO2 nanorods (5–20 nm) and the incorporation of 10 nm sized Au-NPs to make “ACM-TNGs”. They were characterized with FT-IR, TG, and UV spectroscopy. The SEM showed spindle shaped 200 nm sized chitin–MnO2 nanogels (chitin–MnO2 NGs), whereas ACM-TNGs had spindle sizes of 220 nm. The ACM-TNGs were compatible up to 1 mg mL−1 and showed uptake into L929, HDF, MG63, T47D and A375 cell lines without affecting the cellular morphology. ACM-TNGs showed conductivity, and heating under a radio frequency (RF) source at 100 W for 2 min. They also showed the ability to kill breast cancer cells under RF radiation at 100 W for 2 min, when compared with the chitin–MnO2 NGs. The RF assisted ablation of breast cancer cells was confirmed by a live/dead assay. These results suggests that ACM-TNGs could be useful for the RF assisted cancer cells ablation with minimal toxicity compared with MnO2 nanorods.
Introduction
Metal oxides like TiO2, ZnO and their composites have attracted interest in radio frequency assisted (RF) cancer therapy, which has been used in the biomedical field for the past couple of years.1 However, the current treatment modality lacks a good treatment regimen, as it is completely invasive, non-specific and can result in unwanted side effects to the patient.2 During the past ten years, using advanced nanotechnology, researchers have developed different methods to address these issues, in which gold nanoparticle (Au-NP) based nanosystems are important to produce the RF assisted ablation of cancer cells at the in vitro level.3 Similarly, iron oxide nanoparticles also showed RF assisted heating.4,5 However, the toxicity associated with these metallic nanoparticles has to be reduced by appropriate chemical or physical modification with polymers. Nanogels (NGs) can reduce toxicity associated with metallic ions by trapping them within their multilayered polymeric chains.6
“Composite nanogels” are a new research area which can address the various problems associated with an ordinary NG system.7 In metallic nanoparticle based composite NG systems, the metallic components can be coated with a polymeric NG system to avoid leaching of the metal ions into the surrounding body tissues, reducing the toxicity.8 Chitin based NG systems are interesting in terms of their functionality via –NHCOCH3, –NH2 and –OH groups, which can easily be modified with active ligands. Chitin NGs are cytocompatible, hemocompatible and degrade slowly, which makes them suitable for various biomedical applications viz. drug delivery and imaging.9–12 Au-NPs, on the other hand, are best known for their cytocompatibility and RF heatability.13 However, the RF heating capability of Au-NPs is dependent on the way they are made. In general, ion free methodologies are essential for RF heating associated applications. The ionic content associated with Au-NPs could cause unwanted heat effects. Therefore ion free methodologies would give RF heating only from the Au-NPs. However, since it is essential to maintain the heat effects in order to kill cancer cells, a molecular adjuvant would be beneficial for the extra heat effects from Au-NPs.
Manganese dioxide (MnO2) is a transition metal oxide with excellent physical and chemical properties.14 It has many polymorphic forms, such as α, β, γ and δ types which show distinctive properties and are widely used in many areas such as catalysis, ion-sieves, biosensing, energy storage etc.15 Due to its exceptional chemical stability, high sensitivity, low cost, abundance and good biocompatibility, it has been also widely used in bioengineering applications.16 α-MnO2 is known for its excellent wave absorption performance in lower frequency bands which extends its RF applications.17 It is also known that its permeability is almost unity, and the real part and the imaginary part are nearly 1 and 0, respectively.18 However, its permittivity (∼22 F m−1) is much greater than its permeability indicating that it is capable of absorbing frequencies of 2–18 GHz.18 Moreover, its temperature can increase from 298 to 1378 K in 100 s at the speed of 10.80 K s−1 under electromagnetic wave irradiation, so it can be used as a promising adjuvant nanomaterial along with Au-NPs for RF related cancer research.18
Therefore combining gold, chitin and MnO2 into a ternary NGs system would be ideal for the improved RF assisted ablation of cancer cells. Thus we focused on the preparation, characterization, cytocompatibility, cellular localization and RF assisted heating of ACM-TNGs at the in vitro level.
Experimental
(a) Materials
α-Chitin with a molecular weight of 150 kDa and a degree of acetylation of 72.4% was purchased from Koyo chemical Co., Ltd., Japan. CaCl2 and methanol were purchased from Qualigens, India. Chloroauric acid and stains were purchased from Sigma Aldrich, Bangalore. The cell lines were provided by the National Center for Cell Sciences (NCCS), Pune, India.
(b) Preparation of MnO2 nanorods
The chemicals used in the present study were of analytical grade obtained from Sigma Aldrich. The synthesis and growth mechanism of MnO2 nanorods by the hydrothermal method have been discussed in detail in our previous work.14 To describe briefly, 0.1 M MnSO4 and 0.1 M KMnO4 were dissolved in 30 mL distilled water. To this, H2SO4 (60% concentration) was added under vigorous stirring conditions for 30 min, maintaining a pH between 4 and 5. The prepared solution was transferred to a teflon-coated hydrothermal container and autoclaved at 150 °C for 30 min. The obtained precipitate was thoroughly washed using distilled water and centrifuged to obtain a pellet and dried at 60 °C.
(c) Preparation of Au-NPs
The Au-NPs were prepared according to the existing literature19 with slight modifications to avoid a high ionic concentration in the Au-NPs suspension. 300 μL 0.l M HAuCl4·6H2O was treated with 10 mL 0.20% starch solution, then 200 μL 0.l M D-glucose was added and the reaction mixture was heated to 50 °C. The pH was adjusted with a 1% tris buffer solution. A deep wine red coloration after 5 min indicated the formation of 10 nm sized Au-NPs.
(d) Preparation of gold-based chitin–MnO2 nanorod ternary composite nanogels (ACM-TNGs)
For the preparation of ACM-TNGs, 5 mL chitin solution (which was dissolved in a stock solution of 800 g CaCl2–1.2 L methanol mixture) with a 12.5 mg mL−1 concentration of chitin was mixed with 400 μL MnO2 (containing 1 mg of MnO2 nanorods). This solution was further mixed with 5 mL methanol solution to regenerate chitin NGs over MnO2 nanorods (chitin–MnO2). This was then probe sonicated at 50 amperes for about 2 min. These steps were repeated for a further 3 times, washed with distilled water and centrifuged at 20
000 rpm to obtain a pellet. This was then redispersed with distilled water and 400 μL colloidal Au-NPs (containing 0.5 mg mL−1 Au-NPs) was added to the chitin–MnO2. The whole solution was incubated for 1 h to obtain the final ACM-TNGs.
(e) Phase and morphological analysis
Morphology and phase analyses of the synthesized nanorods were performed using transmission electron microscopy-selective area energy dispersive X-ray (TEM, Model: JEOL, JEM-2100F) and X-ray diffraction analyses (XRD, X'Pert PRO Analytical), respectively. Image J software was used to determine the diameter size distribution of the nanowires. Scanning electrochemical microscopy (SECM, Nanotech, Munich) using a Pt probe (diameter 25 μm2) and 0.1 M KOH as a conducting medium was used for mapping the current across the surface.
(f) Labelling rhodamine-123 with ACM-TNGs (rhod-123-ACM-TNGs)
The rhodamine-123 (excitation and emission wavelength of 511 and 536 nm) dye20 labelled ACM-TNGs were synthesized by incorporating rhodamine-123 dye after the final washing of ACM-TNGs. Briefly, 2 mL ACM-TNGs were incubated with 40 μL of rhodamine-123 dye for 12 h with continuous stirring. Finally, the un-bound dye was removed by centrifuging the rhodamine-123-labelled ACM-TNGs at 5000 rpm for 5 min. The resulting pellet was re-suspended in 2 mL millipore water for further studies.
(g) Cell culture
L929 (mouse embryonic fibroblast cells), HDF (Human Dermal fibroblast cells), A375 (Melanoma cells, NCCS Pune), T47D (Human ductal carcinoma cells) and MG63 (Human osteosarcoma cells) were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS). The cells were incubated with 5% CO2. After reaching confluency, the cells were detached from the flask with trypsin–EDTA. The cell suspension was centrifuged at 3000 rpm for 3 min and then re-suspended in the growth medium for further studies.
(h) Cytocompatibility studies of ACM-TNGs
For compatibility experiments, the cells were cultured in 10% MEM and seeded on a 96 well plate with a density of 10
000 cells per cm2. MTT assay was performed to evaluate the cytocompatibility of the prepared ACM-TNGs. Three different concentrations of the ACM-TNGs and dye labelled ACM-TNGs (20, 100 and 500 μg mL−1) were prepared by dilution with the media. After reaching 90% confluency, the cells were washed with PBS buffer and different concentrations of the ACM-TNGs were added. Cells in media alone, devoid of ACM-TNGs, acted as a negative control and wells treated with Triton X-100 acted as a positive control for a period of 24 h. 5 mg of MTT was dissolved in 1 mL of PBS and filter sterilized. 10 μL of the MTT solution was further diluted to 100 μL with 90 μL of serum-free phenol red free medium. The cells were incubated with 100 μL of the above solution for 4 h to form formazan crystals by mitochondrial dehydrogenase. 100 μL of the solubilisation buffer (10% Triton X-100, 0.1 N HCl and isopropanol) was added to each well and incubated at room temperature for 1 h to dissolve the formazan crystals. The optical density of the solution was measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate reader (Bio-Tek Power Wave XS). Triplicate samples were analyzed for each experiment.
(i) Cell uptake studies
Acid etched cover slips kept in 24 well plates were cultured in 10% MEM and loaded with L929, HDF, MG63, A375 and T47D cells with a seeding density of 5000 cells per cover slip and incubated for 24 h for the cells to attach to the well. After the 24 h incubation, the media was removed and the wells were carefully washed with PBS buffer. Then the ACM-TNGs at a concentration of 0.2 mg mL−1 were added along with the media in triplicate to the wells and incubated for 4 h. Thereafter the media with the sample were removed and the cover slips were processed for fluorescent microscopy according to the existing protocols.
(j) Anti-actin–DAPI staining for cellular localization of rhod-123–ACM-TNGs
Tracking of rhodamine-123 (green emission) labelled ACM-TNGs (rhod-123–ACM-TNGs) by DAPI–anti-actin staining was carried out on L929, HDF, MG63, A375 and T47D cells. The cells were cultured in 10% MEM and grown on cover slips in 24 well plates with a seeding density of 5 × 104 per well for 24 h. The rhod-123–ACM-TNGs were treated with cells with a concentration of 0.2 mg mL−1. After 24 h incubation, the cells on the cover slips were washed with PBS after removing the media from the wells and fixed with 5% paraformaldehyde for 20 min, followed by a PBS wash. Then, the cells were permeabilized with 0.5% Triton for 5 min, Triton was neutralized with 1% FBS in PBS and the cells were washed with PBS. Next, actin dye was added to the cells according to the manufacturers’ protocol. After 1 h incubation with anti-actin stain, 100 μL DAPI (1
:
15 ratio in PBS) was added to each of the wells. The stained samples were dried for 24 h. Thereafter, the samples were fixed on slides using DPX as a mountant. The samples were analyzed using fluorescent microscopy.
(k) Radio frequency heating of ACM-TNGs
The RF exposure system operated at a frequency of 13.56 MHz with an adjustable power output between ∼10 W and 1000 W. The RF generator/power amplifier (COMDEL, CX1250S/A, cooled RF generator) was connected through a type-N cable to a variable matching network (COMDEL/MATCH PRO CPMX 2500, CODEL INC), which matched the impedance of the power amplifier signal to the water cooled, solenoid antenna. To expose the ACM-TNGs, 4 mL of the sample with the required concentration was placed into a 35 × 10 mm Petri dish and positioned on a glass Petriplate very close to the outer edge of the coil. Each sample was exposed to the RF signal at an amplifier setting of 100 W for 2 min. The temperature change of the solution was measured over time with a digital thermometer.
(l) RF ablation of cancer cells in vitro
RF assisted ablation of cancer cells was carried out after treating 1 mg mL−1 ACM-TNGs with T47D cells for 24 h. The un-bound ACM-TNGs were removed by PBS washings, followed by deionized water treatment for the experiment. The T47D cells, after ACMT-TNG treatment, were kept in the RF incubator for 2 min at 100 Watts to obtain a maximum temperature range of ∼70 °C. The treated cells were then tested for viability with MTT and a live/dead assay kit.
(m) Statistics
The experiments were carried out in triplicate and values are expressed as mean ± standard deviation (SD). A Student's t-test was conducted to determine the significance. A probability level of p < 0.05 was considered to be statistically significant.
Results and discussion
The prepared ACM-TNGs were highly stable with a zeta potential of +24 mV, whereas the control chitin NGs and chitin–MnO2 NGs had a zeta potential of +44.25 and +40.34, respectively. The expected structural composition of ACM-TNGs is shown in Fig. 1. Chitin is enriched with functionalities such as NH2, –OH, and –NHCOCH3, which can readily interact with the MnO2 nanorods to form chitin–MnO2 NGs. The Au-NPs were added to the chitin–MnO2 NGs (Fig. 2a) and bound to them via strong hydrogen bonding provided by the chitin–MnO2 NGs. Both chitin–MnO2 and ACM-TNGs are highly water dispersible as shown in Fig. 2b and c. The % MnO2 nanorods in the ternary composite NGs are 27% Mn and 56% for the Au nanoparticles, which has been confirmed by the EDS (Fig. 2e) based elemental tracing.
 |
| Fig. 1 The expected structure of the ternary composite nanogels based on chitin–MnO2 nanorods and Au-NPs. | |
 |
| Fig. 2 (a) Preparative method for the ACM-TNGs. (b) Control chitin NGs; (c) chitin–MnO2 NGs and (d) stable ACM-TNGs, (e) rhod-123–ACM-TNGs after centrifugation at 10 000 rpm for 5 min and (f) its supernatant; (g) energy dispersive spectral data for ACM-TNGs displaying the % content of Au and MnO2 in the final nanosuspension of ACM-TNGs. | |
Since MnO2 nanorods have a small particle size, the chitin NGs can easily roll over them and form a stable interaction via hydrogen bonding or metal–ligand interaction. Au-NPs, on the other hand, can become entrapped in the polymer interlocks in chitin–MnO2 NGs.
The rhod-123 dye labeling can effectively occur through the hydrogen bonding between the –NH2 from rhod-123 and the –OH/–NHCOCH3 functionalities of ACM-TNGs. The hydrogen bonding interaction in the polymer is generally a strong interaction, since it has a chain structure compared to a single molecule. Thus the bound dye would not leach once it enters the cellular compartments. The dye-labeling helps in recognition of cellular localized ACM-TNGs.21
Fig. 3a and b are the representative TEM images showing the morphology of hydrothermally synthesized MnO2 nanorods. High-resolution (HR)-TEM imaging showed the interplanar spacings to be ∼0.24 nm along the growth direction and ∼0.31 nm along the diameter. This was confirmed by Fast Fourier Transform (FFT) analysis (see inset Fig. 3b).
 |
| Fig. 3 (a) TEM for control MnO2 nanorods; (b) its high resolution image; (c) TEM image for the Au-NPs; (d) SEM image of chitin–MnO2 and (e) SEM image of ACM-TNGs. | |
Fig. 3c shows the uniformly distributed 10 nm sized Au-NPs. The chitin–MnO2 NGs and ACM-TNGs also showed the same type of structure as the MnO2 nanorods (Fig. 3d and e). The chitin–MnO2 NGs showed a size of 200 nm, whereas the ACM-TNGs showed a particle size of 220 nm.
The broad absorption band centered at around 3400–3500 cm−1 may be due to the –OH stretching vibration. It was clear that ACM-TNGs give only the split transmittance peaks of chitin at 1660 and 1638 cm−1 corresponding to the amide I region and the transmittance peak at 1560 cm−1 corresponding to the amide II region. This revealed that the MnO2 was covered by the chitin NGs. The enhanced peak observed at 980 cm−1 could be due to the presence of the starch coated Au-NPs in the ACM-TNGs (Fig. 4a). The rhod-123 labeling was confirmed by the peak broadening at 3450 cm−1 as shown in Fig. 4a. This could be due to the hydrogen bonding interactions viz. the –NH2 functionalities of rhod-123 with either the –OH or –NHCOCH3 of the chitin component in the ACM-TNGs. The MnO2 and Au-NPs absorptions were observed in the final ACM-TNGs, which showed that they have retained their properties even after the combination with chitin NGs (Fig. 4b).
 |
| Fig. 4 (a) FTIR analysis for control chitin, control CNGs, control MnO2, chitin–MnO2, Au-NPs, ACM-TNGs and rhod-123-ACM-TNGs; (b) UV spectral analysis for control CNGs, chitin–MnO2, Au-NPs and ACM-TNGs. | |
SECM was used to investigate the localized charge transfer mechanisms across the composite–electrolyte interfaces. Fig. 5a and b show the spatial mapping of the surface current on the composite (chitin–MnO2 and ACM-TNGs) by keeping the distance between the probe and the conducting surface constant. It was seen that for a scanned area of 100 μm2, the topography established by the current peaks in ACM-TNGs was more pronounced on a given current scale, than that for the chitin–MnO2 NGs. This high conductivity could be due to the presence of Au-NPs in the ACM-TNGs, whereas the MnO2 could enhance only minimal conductivity in the chitin–MnO2 NGs.
 |
| Fig. 5 SECM analyses for (a) chitin–MnO2 and (b) ACM-TNGs. | |
The cytocompatibility tests were analyzed by a MTT assay on the ACM-TNGs. The MTT assay showed that both chitin–MnO2 NGs and ACM-TNGs are cytocompatible with L929, HDF, MG63, T47D and A375 cells (Fig. 6a and b). The ACM-TNGs are cytocompatible even up to the higher concentration of 1 mg mL−1. Fig. 7 shows the compatibility of chitin–MnO2 NGs and ACM-TNGs with HDF cells, demonstrating that the morphology has been retained even after treatment for 24 h. The cellular localization studies on L929, HDF, A375, MG63, and T47D cells revealed that the rhodamine-123-labelled ACM-TNGs were taken up without disturbing the cell morphology (Fig. 8).
 |
| Fig. 6 Cytocompatibility with (a) chitin–MnO2 NGs and (b) ACM-TNGs on L929, HDF, T47D, MG63 and A375 cell lines after 24 hours of exposure (* represents n = 3, p < 0.5). | |
 |
| Fig. 7 Cytocompatibility assessments by anti-actin and DAPI staining of the cytoskeleton and nucleus respectively on HDF cells after treatment with chitin–MnO2 and ACM-TNGs for 24 hours. | |
 |
| Fig. 8 Cellular localization studies of rhod–ACM-TNGs on L929, HDF, MG63, A375 and T47D cells after 24 h (anti-actin stains the cytoskeleton (red emission); DAPI stains the nucleus (blue emission) and rhodamine-123 has green/greenish yellow color respectively). | |
The up scaled versions of the fluorescent images showed fluorescence of rhodamine-123 (Fig. 8) even after 24 h of exposure. The higher magnified images showed a clear uptake of the rhodamine-123 labelled ACM-TNGs inside the cells. The higher uptake in the cancer cells of rhodamine-123 labelled ACM-TNGs could be due to their +ve surface charge, meaning that they could be more readily taken up by the relatively negatively charged cancer cells than the normal fibroblast HDF and L929 cells.
It is known that RF waves can interact with metallic nanoparticles to generate heat energy.22–27 The 220 nm sized ACM-TNGs were exposed to the RF signal and significant heating was observed with an increase of 45 °C for 1 mg mL−1 concentration of ACM-TNGs (where the Au concentration was 0.5 and MnO2 concentration was 0.2 mg mL−1 respectively) (Fig. 9). To make sure the heating was only via the presence of MnO2 and Au-NPs in the ACM-TNGs, deionized H2O was tested as a negative control. The DI H2O exhibited negligible heating of ∼1 °C. It was clear that the better RF heating at 100 W was dependent on the presence of Au-NPs in the ACM-TNGs. The deionized water was used as a control in the RF experiment to make sure that the heating was occurring only through the Au and MnO2 content. Heat dissipation from the ACM-TNGs could be caused by the delay in the relaxation of the magnetic moment either through the rotation within the particle (Néel) or the rotation of the particle itself (Brownian) when they are exposed to an AC magnetic field with magnetic field reversal times shorter than the magnetic relaxation times of the particles.22 The applied RF dependent heating efficiency of the ACM-TNGs would be beneficial for their use as magnetic hyperthermia of cancer cells, and magnetic nanoparticle assisted drug delivery to many disease sites24–27 (Fig. 10).
 |
| Fig. 9 RF assisted heating of control chitin–MnO2 and ACM-TNGs at different powers with 0.5 mg Au, 1 mg chitin and 0.27 mg mL−1 MnO2 nanorods in the ACM-TNGs; DI water and T47D cells in DI water were used as controls (* represents n = 3, p < 0.5). | |
 |
| Fig. 10 Effective RF assisted ablation of breast cancer cells by ACM-TNGs compared to the chitin–MnO2 at 100 watts for 2 min. (a) MTT assay on RF treated and untreated ACM-TNGs at 100 watts/2 min; (b) optical micrographs for the same and (c) live/dead assay shows the complete destruction of the breast cancer cells after ACM-TNG treatment at 100 watts/2 min RF exposure (* represents n = 3, p < 0.5). | |
Conclusions
In summary, we have succeeded in synthesizing gold based chitin–MnO2 ternary composite nanogels (ACM-TNGs) by regeneration chemistry. The 220 nm sized ACM-TNGs showed cytocompatibility with L929, HDF, A375, MG63 and T47D cells. Further, these were labelled with rhod-123 to track their cellular localization. The ACM-TNGs showed applied power dependent RF heating, which could be useful for magnetic hyperthermia for cancer therapy. The effect of RF assisted ablation of cancer cells on T47D cells was shown to be successful with the addition of Au-NPs, in comparison with the control chitin–MnO2 NGs. In short, ACM-TNGs are cytocompatible, RF heatable and can also be used for cancer cells tracking, which makes them ideal RF assisted hyperthermic agents for cancer therapy.
Acknowledgements
The Department of Biotechnology (DBT), Government of India supported this work; under a center grant of the Nanoscience and Nanotechnology Initiative program (Ref. no. BT/PR10850/NNT/28/127/2008). N. Sanoj Rejinold acknowledges the Council of Scientific and Industrial Research (CSIR) for the financial support through Senior Research Fellowship (SRF Award no: 9/963 (0017)2K11-EMR-I). The authors are thankful to Mr Sajin. P. Ravi for his help in SEM studies.
Notes and references
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