Article pubs.acs.org/molecularpharmaceutics
Enhanced Microwave Hyperthermia of Cancer Cells with Fullerene Mingrui Sun,†,∇ Asimina Kiourti,‡,∇ Hai Wang,†,§ Shuting Zhao,† Gang Zhao,∥ Xiongbin Lu,⊥ John L. Volakis,‡ and Xiaoming He*,†,§,# †
Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, United States ElectroScience Laboratory, Department of Electrical and Computer Engineering, The Ohio State University, Columbus, Ohio 43212, United States § Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States ∥ Centre for Biomedical Engineering, Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China ⊥ Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States # Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, United States ‡
ABSTRACT: Hyperthermia generated with various energy sources including microwave has been widely studied for cancer treatment. However, the potential damage due to nontargeted heating of normal tissue is a major hurdle to its widespread application. Fullerene is a potential agent for improving cancer therapy with microwave hyperthermia but is limited by its poor solubility in water for biomedical applications. Here we report a combination therapy for enhanced cancer cell destruction by combining microwave heating with C60-PCNPs consisting of fullerene (C60) encapsulated in Pluronic F127-chitosan nanoparticles (PCNPs) with high water solubility. A cell culture dish integrated with an antenna was fabricated to generate microwave (2.7 GHz) for heating PC-3 human prostate cancer cells either with or without the C60PCNPs. The cell viability data show that the C60-PCNPs alone have minimal cytotoxicity. The combination of microwave heating and C60-PCNPs is significantly more effective than the microwave heating alone in killing the cancer cells (7.5 versus 42.2% cell survival). Moreover, the combination of microwave heating and C60-PCNPs is significantly more destructive to the cancer cells than the combination of simple water-bath heating (with a similar thermal history to microwave heating) and C60PCNPs (7.5 versus 32.5% survival) because the C60 in the many nanoparticles taken up by the cells can absorb the microwave energy and convert it into heat to enhance heating inside the cells under microwave irradiation. These data suggest the great potential of targeted heating via fullerene for enhanced cancer treatment by microwave hyperthermia. KEYWORDS: fullerene, microwave, hyperthermia, Pluronic F127, chitosan
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INTRODUCTION Hyperthermia has been widely studied for cancer treatment by heating to destroy tumor with various energy sources including ultrasound, laser, radiofrequency (RF), and microwave.1−5 Although localized heating can be achieved by designing the antennas to control the distribution of microwave field, the nontargeted nature of microwave heating based on the absorption of microwave energy by tissue water could cause significant damage to the normal tissue adjacent to tumor.6−9 To address this issue, nanoparticles have been explored to enhance the outcome of microwave hyperthermia. For instance, several studies have demonstrated that carbon nanotubes (CNTs) can absorb and convert the microwave energy into heat for microwave hyperthermia of cancer cells.10−12 The C60 fullerene is a promising candidate for cancer therapy and known for its photodynamic effect to produce the singlet oxygen species under visible and near-infrared light illumination.13−18 However, it is still not widely used probably due to its poor solubility in not only water but also commonly © XXXX American Chemical Society
used organic solvents (e.g., dimethyl sulfoxide or DMSO). Therefore, studies have been conducted to improve its water solubility for biomedical applications. For example, Detrembleur et al. synthesized a well-defined PVOH/C60 nanohybrid, and Iwamoto et al. prepared a C60-NVP copolymer, both of which have high water solubility.13,14 Liu et al. synthesized a PEG-conjugated fullerene with Gd 3+ for photodynamic therapy.16 Fan et al. reported a study using water-dispersible fullerene aggregates with anticancer drug to achieve chemotherapy and photodynamic therapy under blue light irradiation.17 However, these studies chemically modified the fullerene to synthesize the water-soluble nanoparticles, which may alter the physical and chemical properties of fullerene and Special Issue: Physics-Inspired Micro/Nanotherapeutics Received: December 26, 2015 Revised: May 10, 2016 Accepted: May 19, 2016
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DOI: 10.1021/acs.molpharmaceut.5b00984 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Figure 1. Microwave dish used in this study: (a) an overview of the design including the cover, bottom, and antenna; and (b) the side, bottom, and top views of the antenna.
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its interactions with the biological systems.19−21 Moreover, fullerene also has been demonstrated to absorb the energy carried in microwave field and convert it into heat.22 This makes fullerene a promising agent for enhancing microwave hyperthermia of cancer cells, which has not been explored in the literature. In this study, we developed a novel method of using Pluronic F127-chitosan nanoparticles (PCNPs) to encapsulate free fullerene (C60) without any chemical modification to synthesize the fullerene-encapsulated Pluronic F127-chitosan nanoparticles (C60-PCNPs). The resultant C60-PCNPs are soluble in cell culture medium and can be taken up by PC-3 human prostate cancer cells. Moreover, a cell culture dish equipped with an antenna was used to generate microwave for studying the effect of the combination of microwave and the C60-PCNPs on the cancer cells. Our data suggest the great potential of using fullerene to achieve specific heating of cancer cells for enhanced cancer treatment by microwave hyperthermia.
EXPERIMENTAL SECTION
Fabrication of Cell Culture Dish Integrated with Microwave Antenna (Microwave Dish). The configuration of the microwave dish is shown in Figure 1. It consists of a standard 60 mm (outer diameter) cell culture Petri dish covered with copper tapes on its outer surface and integrated with a microwave antenna (Figure 1a). The inner diameter of the Petri dish is 54 mm and the height is 13.5 mm. The antenna (Figure 1b) consists of a feed, a copper ground plane, a Rogers TMM4 ceramic substrate, and a copper patch whose detailed geometry was reported elsewhere.23,24 The antenna is fed by a 50 Ω coaxial cable connected to a 2.7 GHz signal generator (Anritsu 68369A/NV) with a power amplifier (Mini-Circuits ZHL-42), which was used to generate a power of 1.26 W for all the microwave heating experiments in this study. This antenna was designed to homogeneously heat ∼3 mL of cell culture medium in the dish with a resonance frequency of ∼2.7 GHz. In this study, the microwave dish was enclosed in Styrofoam during microwave heating to reduce the heat loss. A type-K precision-fine-wire thermocouple (Omega) and an Extech TM100 thermometer with 0.1 °C resolution were used to B
DOI: 10.1021/acs.molpharmaceut.5b00984 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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curcumin and fullerene in the PCNPs, for which 5 mg of PCNPs was dissolved in 2 mL of DI water, and both 1 mg of fullerene and 1 mg of curcumin were dissolved in 1 mL of toluene. The two samples were then mixed together. The mixture was processed to obtain dry fullerene and curcumin encapsulated Pluronic F127-chitosan nanoparticles (C60-CPCNPs) in the same way as that for obtaining the C60-PCNPs. The uptake of the C60-C-PCNPs by PC-3 cells was imaged using an Olympus FV1000 confocal microscope. For this study, glass coverslips (12 mm) were dipped into 1 mg/mL type I collagen solution in phosphate buffered saline (PBS) and dried to obtain the collagen-coated glass coverslips. PC-3 cancer cells were then cultured with the glass coverslips in a 60 mm Petri dish overnight for the cells to attach on the surface of the glass coverslips.25 Afterward, the cells on the coverslips were incubated with 3 mg/mL C60-C-PCNPs dissolved in cell culture medium for 2.5 h at 37 °C. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Finally, the glass coverslips with attached PC-3 cells were mounted on glass slides using antifade mounting medium (Vector Laboratories, Burlingame, CA, USA) for confocal imaging. To quantify cellular uptake of the C60-PCNPs, 1 × 106 of PC-3 cancer cells were cultured with the collagen-coated glass coverslips in 5 mL cell culture medium in a 60 mm Petri dish overnight for the cells to attach on the surface of the glass coverslips (some cells attached to the bottom surface of the dish). Afterward, the cells on the coverslips were incubated with 3 mg/mL C60-PCNPs dissolved in cell culture medium for 2.5 h at 37 °C. Cells cultured in medium without the C60-PCNPs were also studied as control. The cells were then washed three times with cell culture medium. Finally, the glass coverslips with attached PC-3 cells were immersed into 1 mL of toluene and treated with a Branson (Danbury, CT, USA) ultrasonic cleaner for 30 min to lyse the cells and transfer to the fullerene taken up by the cells into toluene. After centrifuging the resultant samples at 751 g, the absorbance spectra of the toluene supernatants were measured using a Beckman Coulter DU 800 spectrophotometer. The concentration of fullerene inside the PC-3 cancer cells with the C60-PCNPs treatment was estimated by interpolation based on the peak value at 334 nm for fullerene in the absorbance spectra of the toluene supernatants. Heating of Cells with Microwave and Hot Water Bath. The cell samples with the C60-PCNPs treatment were prepared as follows: a total of 6 × 104 PC-3 cancer cells in 300 μL of medium were cultured at the central region of the 60 mm Petri dish for 1 day; the cell culture medium was replaced with 3 mL of culture medium containing 3 mg/mL C60PCNPs; the cells were incubated with the nanoparticlecontaining medium for 2.5 h at 37 °C; the culture medium with the C60-PCNPs was removed and the cells washed 3 times using cell culture medium without any nanoparticles; and 3 mL of cell culture medium (without any nanoparticles) was added to the cells in the 60 mm Petri dish for further study. For microwave heating, the Petri dish was covered with copper tape and the cell sample heated inside the coppercovered dish by microwave for 22 min when the temperature of the medium at the edge of the dish reached 43 °C. The cell sample was then passively cooled down at room temperature, the copper tape removed, and the cells further incubated for 48 h at 37 °C in humidified air with 5% CO2 before measuring the cell viability.
measure and record the temperature of the cell-culture medium in the dish. Synthesis of Pluronic F127-Chitosan Nanoparticles (PCNPs). The synthesis of the PCNPs was detailed elsewhere.25 Briefly, Pluronic F127 was activated by adding dropwise 26 mM Pluronic F127 in benzene into 160 mM 4nitrophenyl chloroformate (4-NPC) in benzene with a volume ratio of 1:1 and stirring the mixture for 3 h in N2 atmosphere at room temperature. The activated Pluronic F127 was precipitated and filtered using excess (ice-cold) diethyl ether for three times. At last, the polymer was dried under vacuum overnight. To synthesize the PCNPs, a total of 5 mL of the activated Pluronic F127 (300 mg/mL) in dichloromethane was added drop by drop into 5 mL of chitosan solution (15 mg/ mL) in deionized (DI) water at pH 10 under the sonication at 16% of maximum amplitude for 3 min using a Branson 450 digital sonifier (Danbury, CT, USA). The resultant emulsion was rotary-evaporated to remove dichloromethane, dialyzed against DI water overnight using a 50 kDa Spectra/Por dialysis tube, and further dialyzed against DI water for 3 h using a 1000 kDa Spectra/Por dialysis tube. Finally, the sample was freezedried for 48 h to obtain the dry PCNPs. Synthesis of the Fullerene (C60) Encapsulated PCNPs (C60-PCNPs). To synthesize the C60-PCNPs, 5 mg of PCNPs was dissolved in 2 mL of DI water, and 1 mg of fullerene was dissolved in 1 mL of toluene. The two solutions were mixed together, and the mixture was shaken using a vortex mixer (Fisher Scientific) at the amplitude 7 for 3 h. The mixture was then rotary-evaporated to remove toluene. Finally, the product was filtered using a 450 nm filter and freeze-dried overnight to obtain dry C60-PCNPs. Characterization of Nanoparticles. The size of all nanoparticles in aqueous solution was measured using a Brookhaven (Holtsville, NY, USA) 90 Plus/BI-MAS dynamic light scattering (DLS) instrument, for which the nanoparticles were dissolved in DI water at 1 mg/mL. The morphology of all nanoparticles was assessed using scanning electron microscopy (SEM). The dry samples were put on a freshly cleaved mica grid and a thin film of Au sputtered onto the samples. An FEI (Hillsboro, OR, USA) NOVA nano400 scanning electron microscope was then used to image the nanoparticles. Successful encapsulation of fullerene in the C60-PCNPs was studied/confirmed using a Beckman Coulter DU 800 spectrophotometer. Various samples (1 mg/mL of C60PCNPs dissolved in DI water or toluene, 1 mg/mL of PCNPs dissolved in DI water or toluene, together with DI water and toluene) were measured. In addition, fullerene dissolved in toluene with various concentrations (0.005 mg/mL to 0.03 mg/mL) were studied. The loading content of fullerene in the C60-PCNPs was calculated by interpolation based on the peak value (at 334 nm) in the absorbance spectra of 1 mg/mL C60-PCNPs in toluene and the various toluene solutions of fullerene. Cell Culture. PC-3 human prostate cancer cells were cultured in 75 cm2 T-flasks at 37 °C in humidified air with 5% CO2. The cell culture medium was the F-12K base medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The medium was refreshed every 2 days. Characterization of Cellular Uptake of Nanoparticles. Curcumin was used to show the uptake of the C60-PCNPs by the PC-3 cancer cells. This was done by encapsulating both C
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Figure 2. Visual and quantitative analysis of fullerene (C60) encapsulated in Pluronic F127-chitosan nanoparticles (PCNPs): (a) typical pictures and the corresponding absorbance spectra of seven samples (1−7) together with a picture of fullerene in DI water (8); and (b) the absorbance spectra of different concentrations of fullerene dissolved in toluene. The peak at 334 nm is due to fullerene.
Statistical Analysis. All data were reported as mean ± standard deviation from at least three independent experiments at different days. Student’s two-tailed t test assuming equal variance was calculated using the SAS (Cary, NC, USA) JMP 10 to assess statistical significance (p < 0.05).
For water-bath heating, cell samples in the Petri dish were put into a 1000 mL beaker containing 200 mL of DI water. The Petri dish floats on the DI water in the beaker due to buoyancy. The beaker was then heated on a VWR hot plate, which was experimented to create the same thermal history in the culture medium of the Petri dish as that in the center of the microwave dish for microwave heating. After heating, the Petri dish was taken out of the beaker and passively cooled down at room temperature. The cell samples were then incubated for 48 h at 37 °C in humidified air with 5% CO2 before measuring cell viability. Measurement of Cell Viability. The Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies) was utilized to measure the cell viability in this study, for which the medium of the samples after 48 h of culture was replaced with 2 mL of fresh cell culture medium. A total of 100 μL of CCK-8 was then added to the fresh culture medium and the samples incubated at 37 °C for 4 h. Finally, the cell culture medium with CCK-8 was tested using a PerkinElmer 2030 mutilabel reader VICTOR X3 to obtain the cell viability according to the manufacturer’s instruction.
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RESULTS AND DISCUSSION Quantification of the Encapsulation of Fullerene in the C60-PCNPs. Figure 2a shows the appearances and absorbance spectra of fullerene (C60), PCNPs, and C60PCNPs dissolved in the colorless solvents (i.e., DI water (6) or toluene (7)). The solution of PCNPs in DI water (5) appears faint yellow, while the solution of C60-PCNPs dissolved in DI water is dark yellow (1). This suggests the presence of fullerene in the C60-PCNPs that can be dissolved in water because free fullerene forms aggregates in water and would not change the color of the sample (8). The solution of fullerene dissolved in toluene is purple (3), and the toluene solution of PCNPs is faint yellow (4), while the solution of C60-PCNPs dissolved in toluene is light pink (2). This light pink is probably a result of the combination of the faint yellow of PCNPs and purple of D
DOI: 10.1021/acs.molpharmaceut.5b00984 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Molecular Pharmaceutics fullerene in toluene because both the PCNPs and C60 fullerene could dissolve in toluene. This suggests that fullerene is physically encapsulated in the PCNPs without any strong chemical bond formation. Moreover, the homogeneous appearance of the C60-PCNPs in water suggests that the C60 was encapsulated inside the nanoparticle core consisting of the more hydrophobic block of poly(propylene glycol) (PPG) in Pluronic F127.25−27 Otherwise, the C60-PCNPs would form aggregate in DI water similarly to pure fullerene (8). The absorbance spectra of these samples (except the suspension of fullerene in DI water) further confirm the presence of fullerene in the C60-PCNPs as indicated by the fullerene peak at ∼334 nm for the two samples (1 and 2) with the C60-PCNPs. As shown in Figure 2b, the absorbance peak at 334 nm of fullerene dissolved in toluene is linearly correlated to the concentration of fullerene. Based on this relationship and the absorbance peak values at ∼334 nm of 1 mg/mL C60PCNPs in toluene and 1 mg/mL PCNPs in toluene, the loading content of fullerene in the C60-PCNPs is determined to be 23.0 ± 0.4 μg/mg. Characterization of the Size and Morphology of the C60-PCNPs. The size of the C60-PCNPs and PCNPs in aqueous solution determined using DLS is shown in Figure 3a. The diameter of the C60-PCNPs is ∼342 nm at room temperature, which is similar to that of the empty PCNPs (∼350 nm). However, the diameter of the C60-PCNPs stays almost the same as the temperature increases to 37 °C, while the diameter of the empty PCNPs decreases to ∼20 nm on average at 37 °C. The latter is consistent with that reported
previously for the nanoparticles made of Pluronic F127 and chitosan due to the thermal responsiveness of the Pluronic F127 polymer.25−27 These results suggest that fullerene encapsulated in the PCNPs changes the thermal responsiveness of the PCNPs. Figure 3b shows the SEM images of the dry PCNPs and C60-PCNPs. The dry PCNPs (∼400 nm) appear larger than the dry C60-PCNPs (∼200 nm). These data suggest that the presence of the highly hydrophobic fullerene in the C60-PCNPs can induce shrinking/collapse of the polymeric components of the nanoparticles possibly around the fullerene matrix in the nanoparticles during drying. Cellular Uptake of the C60-PCNPs. To evaluate cellular uptake of the C60-PCNPs, C60-C-PCNPs were synthesized by using PCNPs to encapsulate both fullerene and curcumin. Curcumin is chosen because it has poor water solubility so that it can be coencapsulated with fullerene in the PCNPs and its fluorescence property can be used to visualize cellular uptake of the nanoparticles using confocal fluorescence microscopy.25 Figure 4a shows the size distribution of the C60-C-PCNPs dissolved in DI water at both 22 and 37 °C. The C60-C-PCNPs are similar to the C60-PCNPs in size at both temperatures. Since materials on the surface of the two nanoparticles are also the same (i.e., chitosan cross-linked poly(ethylene glycol)),25−27 the C60-C-PCNPs can be used as a surrogate to study cellular uptake of the C60-PCNPs. Figure 4b shows typical differential interference contrast (DIC) images, fluorescence images of curcumin (excitation at 488 nm and emission at 520 nm), and their merged images for the control and C60-C-PCNP treated groups. There is no green fluorescence in the cells without C60-C-PCNP treatment and the fluorescence in the cells with C60-C-PCNP treatment is evident, which shows that the PC-3 cancer cells can take up the C60-C-PCNPs. As discussed above, the ability of PC-3 cells to take up the C60-C-PCNPs indicates that they can also take up the C60-PCNPs because these two types of nanoparticles have similar size and surface property. Furthermore, the concentration of fullerene inside the PC-3 cells treated with C60-PCNPs was quantified. The absorbance spectra of toluene supernatants from the PC-3 cells with and without the treatment of C60-PCNPs are shown in Figure 4c. There is one peak at 334 nm in the spectrum of toluene supernatant for PC-3 cells treated with C60-PCNPs, while the peak at 334 nm is not observable in the spectrum for control PC-3 cells without C60-PCNPs treatment. This peak at 334 nm is the peak of fullerene dissolved in toluene and was used together with the data shown in Figure 2b to determine the concentration of fullerene in the toluene supernatants. The concentration of fullerene inside PC-3 cells (CC60_cell) with the C60-PCNPs treatment was calculated as follows: CC 60_cell =
CC 60_tolueneVtoluene NcellVcell
where CC60_toluene is the concentration of fullerene in the toluene supernatant; Vtoluene is the volume of toluene supernatant, which is 1 mL; Ncell is the number of PC-3 cells, which was counted as (9.0 ± 0.3) × 105 after detaching the cells with trypsin; and Vcell is the volume of PC-3 cancer cells, which can be calculated using the diameter of the cells (18 ± 5 μm, measured with 400 PC-3 cells). Based on the absorbance of the peak at 334 nm in the spectra of PC-3 cells with C60-PCNPs treatment subtracted with the absorbance value at 334 nm of PC-3 cells without C60-PCNPs, the concentration of the
Figure 3. Characterization of the C60-PCNPs: (a) the size distribution of the PCNPs and C60-PCNPs in aqueous solution at 22 and 37 °C; and (b) morphology of dry PCNPs and C60-PCNPs. E
DOI: 10.1021/acs.molpharmaceut.5b00984 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Figure 4. Characterization of cellular uptake of the C60-PCNPs: (a) the size distribution of PCNPs coencapsulated with C60 and curcumin (C60-CPCNPs) at 22 and 37 °C; (b) confocal fluorescence images of PC-3 cancer cells without and with C60-C-PCNPs treatment; and (c) the absorbance spectra showing the uptake of C60-PCNPs by PC-3 cells, indicated by the peak at 334 nm of fullerene for the cells treated with C60-PCNPs but not the control cells without C60-PCNPs treatment. For the fluorescence imaging, the excitation wavelength is 488 nm and the emission wavelength is 520 nm. DIC: differential interference contrast. Scale bar: 20 μm.
significantly increases the temperature to 55.5 ± 1.8 °C from 50.5 ± 1.5 °C for medium alone with 22 min of heating. The temperature in medium with the intracellular concentration of fullerene is significantly higher than that of medium alone after 5 min heating and can be higher than 43 °C after only 10 min compared to 14 min for heating medium alone. It is worth noting that we cultured the cells only at the center (∼2 cm in diameter versus 6 cm in diameter for the dish) of the microwave dish. Therefore, the thermal history of the cell culture medium alone at the center of the microwave dish represents extracellular heating and cooling, while the thermal history of cell culture medium with 19 mg/mL C60-PCNPs at the center of the microwave dish represents intracellular heating and cooling.
fullerene in the toluene supernatant was determined to be 1.20 ± 0.04 μg/mL. Finally, CC60_cell was calculated as 436 ± 13 μg/ mL, which means the concentration of C60-PCNPs is 19.0 ± 0.6 mg/mL because the loading content of fullerene in the C60-PCNPs is 23.0 ± 0.4 μg/mg. Enhancement of Microwave Hyperthermia with the C60-PCNPs. Figure 5a shows the thermal histories in the cell culture medium with 19 mg/mL C60-PCNPs, the medium with 19 mg/mL PCNPs, and the medium alone at the center of the microwave dish during microwave heating and the subsequent phase of passively cooling down at room temperature, in the same way as we conducted further cell viability studies. Empty PCNPs have no significant effect on the thermal history in the medium. Importantly, adding the intracellular concentration of fullerene encapsulated in the C60-PCNPs into the medium F
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microwave irradiation via mathematical modeling, although it is beyond the scope of this work. To evaluate the effect of the intracellular C60-PCNPs on microwave hyperthermia of the PC-3 cancer cells, we studied a total of six groups: control (cells cultured in medium without any treatment), C60-PCNPs treatment, water-bath heating without C60-PCNPs treatment, water-bath heating with C60PCNPs treatment, microwave heating without C60-PCNPs treatment, and microwave heating with C60-PCNPs treatment. The corresponding cell viability (after two-day culture) data are shown in Figure 5c. The viability of cells treated with C60PCNPs in the absence of any heating is high, indicating minimal cytotoxicity of the C60-PCNPs. Moreover, the microwave heating with intracellular C60-PCNPs is significantly more effective than the microwave heating alone in killing the cancer cells (7.5 versus 42.2% in cell viability). This indicates that it is the thermal effect of the fullerene (encapsulated in the C60-PCNPs taken up by the PC-3 cancer cells) in the microwave field that enhances the destruction of the cancer cells in the microwave dish because the PCNPs have minimal microwave-induced thermal effect according to Figure 5a and previous studies.22 The cell viability after the water bath heating without C60PCNPs and microwave heating without C60-PCNPs is not significantly different, suggesting that microwave does not damage the cells via a mechanism other than heating the medium since the thermal history in medium for the two heating methods are similar (Figure 5b). Moreover, the cell viability after the microwave heating with C60-PCNPs is significantly lower than that of water bath heating with C60PCNPs (7.5 versus 32.5%), again suggesting the interaction between microwave and the fullerene in the intracellular C60PCNPs significantly enhances the cell killing effect of microwave (because the empty PCNPs have minimal microwave-induced thermal effect.). These data suggest that the direct cell damage by microwave per se is negligible, and the damage to cells during both water-bath heating and microwave irradiation is mainly a result of thermally induced cell injury. These aforementioned cell viability data also suggest that the thermal effect of fullerene in cells under microwave irradiation can be used to achieve much enhanced killing of cancer cells. This is understandable with the thermal histories shown in Figure 5a: the presence of 436 ± 13 μg/mL intracellular fullerene (corresponding to 19 mg/mL C60-PCNPs) could result in significantly higher temperature inside cells than the extracellular medium without fullerene, and the difference is ∼5 °C (55.5 ± 1.8 °C versus 50.5 ± 1.5 °C) at the end of heating. Such an increase in hyperthermic temperature (i.e., ≥43 °C) has been shown to be capable of inducing significantly more injury to cancer cells according to the hyperthermic literature because the rate of cell injury is strongly dependent on the hyperthermic temperature.28−31 According to the confocal fluorescence image shown in Figure 4b, the C60-PCNPs disperse all over the cytoplasm. Therefore, the collective thermal effect of all fullerene inside the PC-3 cells under microwave heating should be the main mechanism of cell injury according to the aforementioned thermal data and analysis. Nonetheless, it is also possible to locally disrupt the cell membrane or other subcellular organelles by the C60-PCNPs during microwave heating if many of the C60-PCNPs are close to the membrane or subcellular organelles, particularly when short-pulsed (on the order of milliseconds or shorter) energy source is applied at a
Figure 5. Enhanced cancer cell destruction of microwave hyperthermia with fullerene: (a) the thermal histories in the cell culture medium with 19 mg/mL C60-PCNPs, the medium with 19 mg/mL PCNPs, and the medium alone at the center of the microwave dish during microwave heating and the subsequent phase of passively cooling down at room temperature, in the same way as we conducted further cell viability studies; (b) the thermal histories in the medium at the center of the dish with microwave versus water-bath heating; and (c) viability of cancer cells 2 days after various treatment groups. The C60-PCNPs were taken up inside the cells and there were no C60PCNPs outside the cells in the cell culture medium during either microwave or water bath heating. *p < 0.05.
Figure 5b shows a comparison of the thermal histories in the cell culture medium at the center of the dish during the microwave versus water-bath heating processes. There is no significant difference between the two thermal histories when the temperature is above 43 °C, the critical temperature above which thermal injury to cells is believed to occur.5,28 The only difference between these two heating treatments is that for the microwave heating treatment, the PC-3 cells receive not only heat from cell culture medium but also heat via fullerene from the electromagnetic field of microwave directly if there are C60PCNPs in the cells. Our studies show that fullerene is capable of more efficiently absorbing/converting microwave energy into heat than either the cell culture medium22 or the medium with PCNPs (Figure 5a). Further study is warranted to quantify the specific absorption rate of the nanoparticles and use it for predicting the heat generation around the nanoparticles under G
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Molecular Pharmaceutics high power according to previous analyses.32,33 However, in this study, microwave with a low power (1.26 W) was applied for a long time (22 min). Therefore, the effect of disrupting the cell membrane or other subcellular organelles by the C60PCNPs under the condition of microwave heating in this study should be minimal. Lastly, our nanoparticles can be taken up by cancer cells (Figure 4b) and should be beneficial for in vivo applications to enhance microwave hyperthermia via intratumoral injection if it is a viable option. However, their size should be further reduced to less than ∼100 nm in order to utilize the effect of the enhanced permeability and retention (EPR) of the tumor vasculature for passively targeting the tumor in vivo via systemic delivery (e.g., intravenous injection).18,34 In summary, we developed C60-PCNPs by encapsulating free fullerene in Pluronic F127-chitosan nanoparticles to improve the solubility and cellular uptake of fullerene with minimal cytotoxicity. A microwave dish was fabricated and used to test the capability of fullerene in enhancing microwave hyperthermia for cancer treatment. Our data show the great promise of achieving targeted/localized microwave heating via fullerene taken up by cancer cells for enhancing the outcome of microwave hyperthermia.
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AUTHOR INFORMATION
Corresponding Author
*Address: Department of Biomedical Engineering, The Ohio State University, 308 BRT, 473 West 12th Avenue, Columbus, Ohio 43210, United States. Phone: (614) 247-8759. Fax: (614) 292-7301. E-mail:
[email protected]. Author Contributions ∇
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by an American Cancer Society (ACS) Research Scholar Grant (No. 120936-RSG-11-109-01CDD) to X.H. The authors would like to thank Wei Rao for her technical help.
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