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Biocompatible chitosan-carbon nanocage hybrids for sustained drug release and highly efficient laser and microwave co-irradiation induced cancer therapy
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Biocompatible Chitosan-Carbon Nanocage Hybrids for Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy Yuliang Guo , Yang Chen , Pomchol Han , Yuxiong Liu , Wenhao Li , Fangliang Zhu , Kai Fu , Maoquan Chu PII: DOI: Reference:
S1742-7061(19)30839-6 https://doi.org/10.1016/j.actbio.2019.12.010 ACTBIO 6493
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Acta Biomaterialia
Received date: Revised date: Accepted date:
13 August 2019 9 December 2019 10 December 2019
Please cite this article as: Yuliang Guo , Yang Chen , Pomchol Han , Yuxiong Liu , Wenhao Li , Fangliang Zhu , Kai Fu , Maoquan Chu , Biocompatible Chitosan-Carbon Nanocage Hybrids for Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.010
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Biocompatible
Chitosan-Carbon
Nanocage
Hybrids
for
Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy
Yuliang Guo a,1, Yang Chen a,b,c,1, Pomchol Han a, Yuxiong Liu a, Wenhao Li a, Fangliang Zhu a
, Kai Fu a, Maoquan Chu a,*
a
Rehabilitation department at Shanghai Putuo District People's Hospital, School of Life
Sciences and Technology, Tongji University, Shanghai 200092, P. R. China. b
Institute of Biophysics, Chinese Academy of Science, Beijing 100101, P. R. China.
c
University of Chinese Academy of Sciences, Beijing 100049, P. R. China.
1
Yuliang Guo and Yang Chen contributed equally to this work.
* Corresponding author. Tel: 86 21 65988653. E-mail: [email protected] (M. Chu). Graphical abstract
1
ABSTRACT Graphitic carbon nanocages (GCNCs) are unique graphene-based nanomaterials that can be used for cancer photothermal therapy (PTT). However, low toxicity GCNC-based organic/inorganic hybrid biomaterials for microwave irradiation assisted PTT have not yet been reported. In the present study, chitosan (CS)-coated GCNCs (CS-GCNCs) loaded with 5-fluorouracil (5Fu) were used for cancer therapy when activated by 808-nm laser and microwave co-irradiation. The cytotoxicity of GCNCs was significantly reduced after coating with CS. For example, fewer cell-cycle defects were caused by CS-GCNCs in comparison with non-coated GCNCs. The release rate of 5Fu from CS-GCNCs was significantly slower than that of 5Fu from GCNCs, providing sustained release. The release rate could be accelerated by 808-nm laser and microwave co-irradiation. The 5Fu in CS-GCNCs retained high cancer cell killing bioactivity by enhancing the caspase-3 expression level. The cancer cell killing and tumor inhibition efficiencies of the 5Fu-loaded nanomaterials increased significantly under 808-nm laser and microwave co-irradiation. The strong cell killing and tumor ablation activities were due to the synergy of the enhanced GCNC thermal effect caused by laser and microwave co-irradiation and the chemotherapy of 5Fu. Our research opens a door for the development of drug-loaded GCNC-based nano-biomaterials for chemo-photothermal synergistic therapy with the assistance of microwave irradiation. KEYWORDS. Graphitic carbon nanocages; low cytotoxicity; sustained drug release; laser microwave co-irradiation; cancer therapy
2
Statement of Significance Graphitic carbon nanocages (GCNCs) are graphene-based nanomaterials that can be used for both drug loading and cancer photothermal therapy (PTT). Herein, we showed that chitosan (CS)-GCNCs hybrid biomaterials had very low cytotoxicity, high ability for loading drug, and exhibited sustained drug release. In particular, although low-power microwaves alone are unable to trigger cancer cell damage by GCNCs, the cell killing and mouse tumor inhibition efficiencies were significantly improved by near-infrared (NIR) laser and microwave co-irradiation compared with laser-triggered PTT alone. This combined use of laser and microwave co-irradiation promises essential therapeutic modality and opens a new avenue for PTT.
3
1. Introduction Although cancer therapies have greatly improved in recent decades, cancer remains a serious disease that causes many deaths worldwide [1]. Any approach that has the potential to improve the efficiency of cancer therapy is therefore welcome. Nanotechnology shows significant potential to improve cancer treatment through highly selective targeting of cancer cells, which reduces side effects compared with traditional methods. Among
various
nano-biomaterials
developed
to
date,
carbon-based
nanomaterials—which include graphene-based nanomaterials [2], nanodiamond [3], carbon dots [4], and activated carbon [5]—are among the most extensively studied. These nanomaterials show lower toxicity towards cells and animals than nanomaterials containing heavy metals such as semiconductor quantum dots. Graphitic carbon nanocages (GCNCs)—unique graphene-based nanomaterials—are hollow porous nanoparticles with a large internal cage with a diameter of a few tens of nanometers and a thin graphitic shell with a thickness of several nanometers [6,7]. Because of their unique geometric structure, GCNCs exhibit good physical and chemical properties and have been extensively investigated for various applications [8–11] such as supercapacitors [11]. In the present study GCNCs are used for near-infrared (NIR) laser-induced cancer therapy with the assistance of microwave irradiation (Fig. 1). The current strategy—the combination
of
laser
and
microwave
irradiation
to
improve
the
efficiency
of
nanoparticle-mediated cancer phototherapy—has not previously been reported. Microwave irradiation was introduced during NIR laser-induced PTT as microwaves exhibit strong tissue penetration, high heat generation rate, and homogeneity of heating. These 4
advantages make microwave heating markedly superior to using infrared lamps. In this approach, microwave irradiation is used to raise the initial temperature of the mouse (particularly the tumor) before low-power (0.25 W/cm2) NIR laser irradiation, which is different to nanomaterial-mediated microwave thermal therapy [12–15]. In recent research, microwave-sensitive nanomaterials with high microwave–thermal conversions have been used [12–15] where the microwave probe must be placed very close to the tumor (e.g., 1 cm away from the tumor of a fixed tumor-bearing mouse [15]), which may not be convenient for the clinical treatment of tumors in deep tissue. In the present study, these limitations are overcome as the distance between the microwave probe and mouse tumor reaches over 3 cm. Although increasing the laser power density and concentration of PTT agents may improve the PTT effect, low laser power [16] and a low PTT agent concentration are safer for clinical application. To improve their biocompatibility and cancer therapy efficiency, the GCNCs were encapsulated in chitosan (CS) polymer nanoparticles and loaded with 5-fluorouracil (5Fu). The 5Fu-loaded CS-GCNCs nanosystem exhibits sustained drug release, a strong ability to disturb the cell cycle and upregulate the expression of apoptosis-related proteins, and a strong ability to inhibit mouse tumor growth through the synergistic effect of chemotherapy and photothermal therapy (PTT) upon NIR laser irradiation. GCNCs were coated with CS because CS is a natural polymer with desirable properties such as nontoxicity, biodegradability, immunogenicity, bioadhesivity, high charge density, and low cost [17], and has been widely used as a sustained-release or responsive-release drug carrier [18–20]. The results presented in this work will facilitate the application of CS-GCNCs in biomedicine and provide a strategy for nanomaterial-mediated cancer therapy using the combination of laser and microwave irradiation. 5
Fig. 1. 5Fu-loaded chitosan-coated graphitic carbon nanocages for chemo-photothermal therapy with the assistance of microwave irradiation.
2. Experimental section 2.1. Cell line, animals, and materials The sources of the human nasopharyngeal carcinoma (CNE) cell line, BALB/c nude mice, and all reagents, including the 5Fu-loaded CS-GCNCs (abbreviated to CS-GCNCs(5Fu) in the following sections) developed by our research group are listed in the Supporting Information. All animal experiments were performed in accordance with the University of Tongji Institutional Animal Care and Use Committee Guidelines. The chitosan (CS) used in this study was analytical grade (deacetylation degree: 80%–95%, viscosity: 50–800 mPa·s). The detailed information of all reagents is shown in the Supporting Information.
2.2. Microwave-induced heating of nanoparticles in aqueous solution
6
CS-GCNC aqueous suspensions (GCNC: 0.01, 0.2, or 0.5 mg/mL, 200 μL for each sample) were placed in small glass tubes. The suspensions [temperature: 25.0±0.2 °C] were exposed to microwave irradiation (SPW-1 Microwave Therapy Instrument, Shengpu Medical Equipment Technology, Xuzhou, China) for 0–10 min. The power of the microwaves was set as 2, 5, or 10 W, and the frequency was maintained at 2450 MHz. The distance between the microwave probe and aqueous suspensions was 1 cm. A Ti29 thermal imager (Fluke Corporation, Everett, WA, USA) was used to measure the sample temperature. Distilled water and water containing 2% polyvinylpyrrolidone (PVP) (200 μL) was irradiated under the same conditions as a control. To measure the heat generated by GCNCs under microwave irradiation, GCNCs were dispersed in distilled water containing 2% (PVP). Each experiment was conducted three times (note: as GCNC is poorly dispersed in distilled water, 2% PVP was added in order to achieve uniform dispersion for the measurements; CS-GCNCs exhibited good stability in both distilled water and buffer solution, therefore no PVP was used to disperse CS-coated GCNCs for experiments).
2.3. Heating of the nanoparticle aqueous suspensions irradiated by both NIR laser and microwave CS-GCNC aqueous suspensions (0.2 mg/mL, 200 μL for each sample) at room temperature were placed in glass tubes and then exposed to microwave irradiation. When an initial suspension temperature of 28, 31, or 34 °C was achieved and maintained, the suspensions were simultaneously irradiated with an 808-nm laser (0.25 W/cm2) (Shanghai Inter-Diff Optoelectronics Tech. Co., Ltd., Shanghai, China) and the same microwaves for 20 min. Each experiment was conducted three times.
7
2.4. Analysis of cell cycle, caspase-3, and viability of nanoparticle-treated cells 2.4.1 Measurement of cell cycle distribution. CNE cells were incubated with culture medium-dispersed samples (GCNCs, CS-GCNCs, CS-GCNCs(5Fu), and free 5Fu) in a six-well plate in a CO2 incubator at 37 °C for 24 or 48 h. After incubation the cells were collected, fixed for 2 h with 70% ethanol, and then stained with propidium iodide. The cell fluorescence intensity (wavelength: 488 nm) was measured using a flow cytometer (BD FACSVerse, USA). Each experiment was conducted five times. 2.4.2 Caspase-3 analysis. CNE cells were treated with CS-GCNCs(5Fu) or 5Fu in a six-well plate for 24 or 48 h. The cells were collected, and caspase-3 levels were analyzed using a Caspase 3 Activity Assay Kit according to the provided protocol (Beyotime Biotechnology, Shanghai, China). Each experiment was conducted five times. 2.4.3 Cell viability assay. CellTiter-Glo™ reagent was used to detect the viabilities of CNE cells treated with CS-GCNCs. The viabilities of cells incubated with GCNCs, CS-GCNCs(5Fu), chitosan nanoparticles, and free 5Fu were also determined.
2.5. Apoptosis and viability of nanoparticle-treated cells upon microwave irradiation or microwave and NIR laser co-irradiation Serum-free medium-dispersed samples were added to cells in a 96-well plate. The cells at room temperature were irradiated with 5 W microwaves until the temperature of the medium was maintained at ~28 °C. The cells were then continuously exposed to the microwave irradiation for a further 20 min. In another group, cells incubated with the same samples were pre-irradiated with microwaves until the temperature of the medium was maintained at ~28 °C; the cells were then simultaneously exposed to laser and microwave irradiation for 20 min. The samples were pre-incubated with cells for 24 h and then treated with 8
microwaves or laser and microwaves using the same methods. The distance between the microwave probe and cells was kept to ~1 cm. After irradiation, cell apoptosis and viability were detected using the methods described above. The samples included CS-GCNCs (0.8 mg/mL), 5Fu (0.17 mg/mL), and CS-GCNCs(5Fu) (0.8 mg/mL). Each experiment was conducted five times. (Note: for cell and animal experiments, as the stability of GCNCs in HEPES buffer was better than that in distilled water, HEPES buffer was used to disperse the GCNCs, instead of distilled water containing 2% PVP, to remove the effect of PVP on the biological experiments).
2.6. Animal experiments To prepare the tumor-bearing mice, nude mice were subcutaneously injected with CNE cells. Six groups of mice with the same tumor size (~4 mm) were then used for the in vivo therapy experiments. The samples were intratumorally injected into the mice and the tumors were treated with 808-nm laser irradiation, microwave irradiation (3W), or laser and microwave co-irradiation. The distance between the microwave probe and tumor was fixed at ~3 cm (as we combined laser irradiation with microwave irradiation, it was necessary to leave a space so that the tumors could be easily exposed to the laser light. This distance (~3 cm) or greater distance is suitable for laser/microwave co-irradiation, and may also be appropriate for the clinical treatment of tumors). Laser and microwave irradiation was repeated once daily until the tumors were not detected, at which point the mice were no longer treated with the laser or microwave irradiation. The detailed animal experimental methods are shown in Fig. S1. Other experimental methods are described in detail in the Supporting Information.
9
2.7. Statistical analysis A non-parametric test (Mann–Whitney) and Student t-test were used for statistical analysis. Comparisons between results of in vitro experiments were performed using a non-parametric test (Mann–Whitney). The Student t-test was used to compare the means of two samples. P<0.05, P<0.01, and P<0.001 indicated statistically significant, highly statistically significant, and very highly statistically significant, respectively.
3. Results 3.1 Properties of GCNC and CS-GCNC nanoparticles The GCNCs were hollow in structure (Fig. 2a). In our previous work, we showed that the cage structure may be graphitic because the Raman spectrum of the GCNCs contained two prominent bands (D and G bands) consistent with graphite [21]. Here we further demonstrated using EDS that the GCNCs were primarily composed of C with a small amount of O (Fig. 2b). In the XRD pattern a strong peak at 2θ = 26.0° and two weak peaks at 2θ = 42.4° and 44.3° are assigned to the (002), (100), and (101) diffraction planes of the hexagonal structure of graphite (JCPDS Card no.41-1487), respectively (Fig. 2c). The graphitic nature of the GCNCs was further confirmed by the FT-IR spectrum (Fig. 2d). The FT-IR spectrum showed a prominent peak at around 1620 cm−1, as well as several weak peaks at 1542, 1510, and 1450 cm −1, which can be assigned to C=C stretching modes, attributed to polycyclic aromatic hydrocarbons; the two peaks at around 1384 and 1317 cm−1, are attributed to the C-O stretching mode of the carboxyl group; and several weak peaks, for example at 1725, 1708, and 1695 cm−1, are attributed to the C=O stretching mode of the carboxyl group; the strong peaks at around 3440 and 1110 cm−1 are due to the stretching and bending vibrations of O-H, respectively; the peaks at around 3033, 2917, and 2846 10
cm−1 are due to the stretching vibration of C-H, and several peaks at low wavenumber such as 825, 777, and 617 cm−1 are attributed to the bending vibrations of C-H in aromatic hydrocarbons. These findings indicated that the as-synthesized hollow nanoparticles are graphitic structures containing carboxyl and hydroxyl groups. A ‘water-in-oil’ microemulsion system was used to fabricate CS-GCNCs and CS nanoparticles. After addition of sodium tripolyphosphate (TPP), CS in the ‘water-in-oil’ microemulsion system assembled into nanoparticles (Fig. 2e). When TPP was added to a mixture of CS and GCNCs in a microemulsion, CS-GCNCs nanocomposites formed, and few free GCNCs could be observed (Fig. 2f). Although the diameter of an individual CS-GCNC was approximately 600 nm, the average hydrodynamic size of the CS-GCNCs was 960.8 ± 184.6 nm (Fig. 2g). In most cases, the hydrodynamic size measured using laser scattering is larger than the size of the same nanoparticles measured by TEM as the firmly aggregated and loosely bound particles are all detected as one particle. Although these large-sized CS-GCNCs were not uniform in size and shape, these CS-GCNCs could be used for cancer therapy as these particles were injected directly into mouse tumors to inhibit tumor growth. Submicrometer- or micrometer-sized particles with not uniform sizes for intratumoral injection for cancer therapy have been reported in both basic research [22,23] and clinical applications [24]. This administration route is safer for patients and animals than intravenous injection. The suspension stability of GCNCs in aqueous solution was markedly improved after the GCNCs were coated with CS, which was confirmed by images of the samples (Fig. 2h) and optical absorption changes of the sample supernatant over time (Fig. 2i). These results indicate that most of the GCNCs were coated or hybridized with CS. In this study, the nanoparticles were intratumorally injected into mouse tumors for in vivo therapy. The size of the particles was appropriate for tumor therapy as it does not affect the injection management and also 11
prevents the release of particles from the tumor tissue.
Fig. 2. Characterization of GCNC and CS-GCNC nanoparticles. (a) HRTEM image, (b) EDS spectrum, (c) XRD pattern, and (d) FT-IR spectrum of GCNCs. TEM images of (e) CS and (f) CS-GCNC nanoparticles. (g) Hydrodynamic size of CS-GCNCs. (h) Suspension stability of GCNCs and CS-GCNCs in HEPES buffer solution. (i) Relative optical absorption at 808 nm of the supernatant of CS-GCNCs in HEPES buffer solution with time. (j) Absorption spectra of GCNCs, CS nanoparticles, and CS-GCNCs. (k) The relationship between absorbance at 808 nm and the concentrations of GCNCs and CS-GCNCs. Photostability of (l) GCNCs and (m) CS-GCNCs (CNC concentration: 0.1 mg/mL) in water over six 808-nm laser irradiation on/off cycles. For the measurement of absorption and photothermal conversion in aqueous solution, GCNCs were dispersed in water containing 2% PVP.
3.2. Absorption coefficient, photothermal conversion, and photostability of GCNCs and CS-GCNCs at 808 nm The absorption coefficient of the GCNCs at 808 nm was 0.83±0.06 L/g·cm. After the 12
GCNCs were incorporated into a CS polymer shell, the resulting CS-GCNCs nanoparticles exhibited similar absorbance to that of the GCNCs between ~300 and 1100 nm (Fig. 2j). The absorption intensities of different concentration CS-GCNC suspensions at 808 nm were consistently higher than those of GCNCs under the same conditions (Fig. 2k). This is because the absorption of the CS-GCNCs had contributions from both the GCNCs and CS. The absorption coefficient of CS-GCNCs at 808 nm was 0.88±0.08 L/g·cm, which was slightly higher than that of the GCNCs (P>0.05), which indicates that CS-GCNCs may have potential for use as photothermal agents. Aqueous suspensions of both GCNC and CS-GCNC nanoparticles exhibited rapid photothermal conversion upon laser irradiation (Fig. 2l, 2m). The photothermal conversion efficiency of CS-GCNCs (23.7%) was similar to that of the GCNCs (23.9%), and other nanoparticles such as prophyrin immobilized nanographene oxide (19.9%) [25] and gold nanorods (22.3%) [26]. The GCNCs exhibited good photostability under 808-nm laser irradiation both before and after coating with CS (Fig. 2l, 2m), which is advantageous for long-term cancer PTT.
3.3. Thermal conversion of GCNCs and CS-GCNCs treated with microwaves or microwave and 808-nm laser co-irradiation. When an aqueous dispersion of CS-GCNCs (200 μL) was exposed to microwave irradiation (the microwave probe was placed 1 cm away from the sample), the temperature of the aqueous suspension increased rapidly with prolonged exposure time and when the microwave power was increased from 2 to 10 W (2450 MHz) (Fig. 3a and b). The temperature increase profiles of distilled water alone (200 μL) under the same microwave irradiation conditions were similar to those of water-dispersed CS-GCNCs. In addition, the 13
temperature of CS-GCNC aqueous suspensions under microwave irradiation did not increase markedly upon raising the concentration of CS-GCNCs (GCNC concentration was increased from 0.01 to 0.5 mg/mL), as shown in Fig. 3c and d. These results indicate that CS-GCNCs
are
not
microwave-absorbing
materials.
GCNCs
are
also
not
microwave-sensitive materials (Fig. S2). To investigate the contribution of the heat generated by microwave irradiation to the thermal conversion of laser-triggered nanoparticles, CS-GCNC suspensions were irradiated with microwaves until they first reached a particular temperature (e.g. 28, 31, or 34°C). This temperature did not change perceptibly during the subsequent ~20 min of microwave irradiation under the same conditions. The results showed that when a CS-GCNC aqueous suspension (0.1 mg/mL) was exposed to microwave irradiation until the temperature of the suspension increased and remained at a certain level; and then the suspension was co-irradiated with the laser and microwaves (Fig. 3e), the sample was heated more quickly than in the case of a suspension exposed to the laser irradiation alone (Fig. 3f and g). For example, the difference in initial temperature between the groups exposed to the laser only and the laser and microwaves was ~6.0 °C, whereas the difference in final temperature between these two groups was ~10.5 °C. The results described above indicate that although microwave irradiation at the investigated power does not efficiently trigger heat generation by GCNCs or CS-GCNCs, there may be a combined effect of interaction between the photothermal effect and microwave thermal effect. That is, the NIR-absorbing materials may be simultaneously exposed to both microwave and NIR laser irradiation to enhance the final temperature of the materials. This is important for cancer therapy because both microwave and laser irradiation were applied at low power, therefore only the tumor tissue containing the NIR-absorbing 14
material should be damaged by the enhanced thermal effect realized under the co-irradiation of a NIR laser and microwaves.
Fig. 3. Temperature changes of CS-GCNCs in aqueous solution upon laser irradiation, microwave irradiation, and laser and microwave co-irradiation. The temperature increase of CS-GCNC aqueous suspensions and distilled water upon microwave irradiation at (a, b) different microwave powers (GCNCs: 0.2 mg/mL) and (c, d) different GCNC concentrations (microwave power: 10 W). (e) Schematic illustration of the method of laser and microwave co-irradiation. (f, g) Photothermal conversion of distilled water-dispersed CS-GCNCs upon laser, and laser and microwave (3 W) co-irradiation, and controls. (a, c, f) Representative infrared thermal images. (b, d, g) Temperature intensities obtained from the images in a, c, and f, respectively. For the groups exposed to laser and microwave co-irradiation, the CS-GCNC suspensions or mouse tumors containing CS-GCNCs were first exposed to microwave irradiation until the temperature increased to 28 or 31 °C (for aqueous suspensions) or 38 °C (for tumors) and then maintained at these temperatures, followed by 20 min of laser exposure. During the laser exposure, CS-GCNC suspensions and mouse tumors were continuously irradiated with the microwaves. L, MW, and IT are laser, 15
microwave, and initial temperature, respectively. 3.4. Cytotoxicity of GCNCs and CS-GCNCs 3.4.1 Cell cycles of cancer cells treated with nanoparticles. The cell cycles of CNE cells treated with CS-GCNCs (0.1 mg/mL), even for 48 h, did not significantly change compared with those of the untreated cells (control) (P>0.05) (Fig. 4a, 4b). However, when the cells were treated with GCNCs (0.1 mg/mL) without CS coating for 48 h, the G0/G1 and G2/M phases significantly increased and decreased, respectively, compared with the control (P<0.01 for G0/G1 and P<0.001 for G2/M, compared with the control) (Fig. S3). These results imply that the toxicity of GCNCs was lowered by CS coating. 3.4.2 Cell viabilities of cancer cells treated with nanoparticles. As shown in Fig. 4c, the CNE cells after incubation with low-concentration GCNCs (e.g., 0.01 mg/mL) for 48 or 72 h exhibited high viability. However, the viabilities of cells incubated with the GCNCs at higher concentration (e.g., 0.1 mg/mL) decreased to (81.9±7.5)% and (77.1±4.4)% after 48 and 72 h of incubation, respectively. Interestingly, when CS-GCNCs containing the same amount of GCNCs as described above were added to the cells and incubated for 48 and 72 h, the cell viabilities ranged from (96.3±4.3)% to (100.4±4.7)%. These results further indicate that the CS polymer coating reduced the toxicity of the GCNCs. The CS coating is therefore necessary to improve the biocompatibility of GCNCs.
16
Fig. 4. The cell cycle and viability of CNE cells treated with CS-GCNCs. (a, b) The effect of nanoparticles on cell cycle distribution. Cells were incubated with CS-GCNCs (0.1 mg/mL) for 24 or 48 h or left untreated. (c) Viabilities of cells after incubation with GCNCs and CS-GCNCs for 48 or 72 h. Observed significance levels were ***P<0.001, **P<0.01, and *P<0.05.
3.5. Drug release and bioactivity of 5Fu loaded CS-GCNCs 5Fu has been widely used for clinical cancer chemotherapy. In a previous paper [27], we calculated that the loading efficiency of 5Fu was (22.4±2.0)%, i.e., 22.4±2.0 mg of 5Fu was loaded per 100 mg of CS-GCNCs. As shown in Fig. 5a, 5Fu was slowly released from 5Fu-GCNCs at room temperature. After 96 h of dialysis, only 74.13±2.8% of 5Fu was released. 5Fu-CS nanoparticles also exhibited sustained drug release and CS-GCNCs further delayed the drug release. After 96 h of dialysis at room temperature, only (74.1±2.8)% and (31.1 ±0.8)% of 5Fu was released from the 5Fu-GCNCs and CS-GCNCs(5Fu), respectively. At 37 °C, the release of 5Fu from CS-GCNCs(5Fu) increased slightly to ~33.6±0.6% after 96 h of dialysis (Fig. 5b). The release rate of 5Fu from the CS-GCNCs(5Fu) could be accelerated by NIR laser irradiation or laser and microwave co-irradiation. As shown in Fig. 5c, when CS-GCNCs(5Fu) 17
suspended in aqueous solution were exposed to an 808 nm laser for 20 min, the amount of 5Fu released from the nanoparticles [(10.1 ± 0.8)%] was 2.11 times higher than that without irradiation [(4.8 ± 0.6)%]. The microwave irradiation may also have contributed to increasing the 5Fu release rate, that is: under 3 W microwave irradiation, the release rate of 5Fu from the nanoparticles with and without laser irradiation increased slightly to [(10.5 ± 0.6%)] and [(5.1 ± 1.0%)], however neither showed a significant difference (P>0.05) compared with the release rates without microwave irradiation (Fig. 5c). The NIR/microwave-enhanced drug release is related to the increased temperature of the drug-loaded nanoparticles. The aqueous suspension of nanoparticles can be easily heated by NIR laser or microwave irradiation. The higher the temperature, the faster the molecular thermal motion. Therefore, the release rate of 5Fu from CS-GCNCs(5Fu) at an initial temperature of 37 °C with or without laser or microwave irradiation must be faster than that from particles with an initial temperature of ~25 °C. The drug release profile of CS-GCNCs(5Fu) described above is beneficial for long-term dual-modal cancer therapy. That is: when CS-GCNCs(5Fu) in tumor tissue were irradiated with an NIR laser or laser and microwaves, tumors were synchronously attacked by the photothermal effect of the GCNCs and the stimulated release of the 5Fu chemotherapeutic from the nanocarriers; in the absence of irradiation, however, only a small amount of drug was released. As depicted in Fig. 5d–g and h, the populations of free 5Fu (0.17 mg/mL) treated CNE cells in the sub-G0 phase after incubation for 24 or 48 h were significantly enhanced compared with those of the cells without 5Fu treatment in the same phase (P<0.01), which indicates that 5Fu has high toxicity to cells. The cells were arrested in the G0/G1 and S phases after the cells were incubated with CS-GCNCs(5Fu) (containing 0.17 mg/mL of 5Fu) 18
or 5Fu for 24 or 48 h. Cell-cycle defects in free 5Fu-treated cells were more serious than those in CS-GCNCs(5Fu)-treated cells, which may be related to the incomplete release of 5Fu from the CS-GCNCs nanoparticles.
Fig. 5. Release rate of 5Fu from CS-GCNCs and the cytotoxicity of CS-GCNCs(5Fu) and controls. (a) Comparison of the release rates of 5Fu from GCNCs, CS-GCNCs, and CS nanoparticles at room temperature (25 °C). (b) Comparison of the release rates of 5FU from CS-GCNCs at 25 °C and 37°C. (c) Laser, microwave, and both induced 5FU release from the CS-GCNCs. For the cytotoxicity study the cells were treated with CS-GCNCs(5Fu) or free 5Fu (CS-GCNCs: 0.8 mg/mL, 5Fu: 0.17 mg/mL) for 24 or 48 h. (d–g) Representative images and (h) histograms of cell cycle distributions. (i) Activities of caspase-3 extracted from the cells. (j) Cell viabilities. Observed significance levels were ***P and #P<0.001, **P<0.01, and *P<0.05. L and MW are laser and microwave, respectively. 19
The expression level of caspase-3 in CNE cells significantly increased after the cells were treated with 5Fu (Fig. 5i). The level of caspase-3 after 48 h of incubation was significantly higher than that after 24 h of incubation. Caspase-3 is an apoptosis-related cysteine peptidase; the higher the caspase-3 level, the greater the cell apoptosis. The cell viability decreased from 100% to (85.7±3.0)% and (63.4±5.8)% after exposure to 5Fu for 24 and 48 h, respectively (Fig. 5j). CS-GCNCs(5Fu) (GCNCs: 0.2 mg/mL, 5Fu: 0.17 mg/mL) also induced significant CNE cell apoptosis by enhancing the caspase-3 expression level, and significantly lowered cell viability (Fig. 5j). The expression of caspase-3 in the 5Fu-loaded nanoparticle group was less than that in the 5Fu group, which may be a result of the slow release of 5Fu from the nanoparticles. However, 5Fu still exhibited high bioactivity leading to damage of cancer cells after incorporation into CS-GCNCs.
3.6. Ability of nanoparticles to kill cancer cells under laser and microwave co-irradiation Both qualitative and quantitative analyses showed that the cell viabilities were close to 100% when the cells were treated with CS (0.6 mg/mL) or CS-GCNCs (0.8 mg/mL) for 20 min (Fig. 6). However, as shown in Fig. 6a, many apoptotic or dead cells could be observed after the cells incubated with CS-GCNCs were exposed to the 808-nm laser for 20 min. The strong cancer cell killing ability of the CS-GCNCs and laser combination was also confirmed by quantitative analyses [cell viability: (52.4±3.9)%] (Fig. 6b). The main phototoxic mechanism of the CS-GCNCs was their photothermal effect because no toxic reactive oxygen species (ROS) were produced by the laser-irradiated GCNCs (Fig. S4). The low number of red fluorescent cells in Fig. 6a shows that the CS nanoparticles were unable to damage cells with or without laser irradiation. As shown in Fig. 6b, the viabilities of cells 20
(initial temperature: ~25 °C) treated with CS or CS-GCNCs after microwave exposure at 5 W exceeded 98%, which showed no significant difference to cells incubated with HEPES buffer exposed to the same microwave irradiation protocol [cell viability: (100.0±1.8)%]. This is because the materials barely absorb microwave energy and the applied microwave irradiation was only at a power of 5 W. However, after the cells incubated with CS-GCNCs were exposed to microwave irradiation to achieve a final culture medium temperature of ~28 °C, and then co-irradiated with microwaves and laser for 20 minutes, the cell viabilities significantly decreased compared with those of the cases where laser irradiation alone was used (P<0.001). For example, in the group with CS-GCNCs and laser exposure, the cell viability was (52.4±3.9)%, whereas for the group with CS-GCNCs and laser and microwave co-irradiation, the cell viability was only (39.4±3.5)%. The strong cell-killing ability of CS-GCNCs under laser and microwave co-irradiation was also confirmed by qualitative measurements (Fig. 6a). These results imply that laser and microwave co-irradiation is beneficial for cancer PTT, as high efficiency PTT may be achieved using low concentration PTT agents and/or a low power density laser.
Fig. 6. The cell killing efficiency of CS-GCNCs upon laser irradiation, microwave irradiation, or laser and microwave co-irradiation and controls. (a) Qualitative measurements. (b) Quantitative measurements. Observed significance levels were ***P and #P<0.001. In which, #
indicates differences between the corresponding results with and without laser or laser and
microwave co-irradiation. L and MW are laser and microwave, respectively. 21
To demonstrate that thermal stimulation and the toxicity of 5Fu exhibit a synergistic cell-killing effect, CNE cancer cells were incubated with CS-GCNCs(5Fu) for a longer time before irradiation so that the cytotoxicity of 5Fu could be observed. After the cells were treated with CS-GCNCs(5Fu) (GCNCs: 0.2 mg/mL) or free 5Fu (0.17 mg/mL) for 24 h (at this time point 30±2.3% of 5Fu was released from CS-GCNCs(5Fu)), the percentages of cell apoptosis were (15.6±0.9)% and (18.7±1.6)%, respectively (Fig. 7a,7b). The percentage of cell apoptosis increased significantly to (67.6±2.7)% after the cells were treated with CS-GCNCs(5Fu) in the same manner as described above and exposed to the laser for 20 min. A similar result was observed in the CS-GCNCs group. This is because the cytotoxicities of both 5Fu and GCNCs are time dependent. A cell viability assay further confirmed the synergistic cell-killing effect of thermal-chemotherapy in the CS-GCNCs(5Fu) group under laser irradiation (Fig. 7c). Importantly, when CS-GCNCs and CS-GCNCs(5Fu) were pre-incubated with cells for 24 h, NIR laser and microwave co-irradiation further improved the cell-killing efficiency of CS-GCNCs and CS-GCNCs(5Fu) (Fig. 7c). For example, after CS-GCNCs(5Fu) were incubated with cells for 24 h and then the cells were exposed to microwave and laser irradiation for 20 min, the cell viability decreased to just (19.2±1.0)%. In contrast, 5Fu and nanomaterials exposed to the microwaves alone did not significantly accelerate cell death (Fig. 7c).
22
Fig. 7. Effects of pre-incubation, laser irradiation, microwave irradiation, and laser and microwave co-irradiation of CS-GCNCs(5Fu), CS-GCNCs, or free 5Fu on CNE cell viability. CNE cells were treated with the samples for 24 h before laser and/or microwave irradiation (5 W, 2450 MHz). (a) Dot plot representation of dual staining of CNE cells with annexin FITC-A and propidium iodide and (b) Cell apoptosis percentages obtained by flow cytometry. (c) Cell viabilities. L and MW are laser and microwave, respectively.
3.7. In vivo cancer therapy In a previous paper [27], we showed that the growth of mouse CNE tumors was significantly inhibited by laser light-triggered CS-GCNCs and CS-GCNCs(5Fu). For nanoparticle-mediated PTT, enhancing local temperature can markedly accelerate the inhibition of tumor growth [27]. Because microwave irradiation can substantially increase the final temperature of laser-irradiated mouse tumors containing CS-GCNCs, we explored 808-nm laser and microwave (3 W, 2450 MHz) co-irradiation for CS-GCNCs-mediated cancer therapy. For a mouse tumor injected directly with CS-GCNCs, microwave irradiation 23
alone (the “CS-GCNCs+MW” group, note: MW is microwave) did not efficiently inhibit tumor growth because the average tumor growth rate was similar to those of tumors in the “HEPES+MW” and “HEPES, no irradiation” groups (Fig. 8a, 8b), and the tumor sizes and average weights in the “CS-GCNCs+MW” group 19 days post-injection were also not significantly different to those in the “HEPES+MW” group (Fig. 8d,8e). This is because CS-GCNCs are not microwave-sensitive materials and the microwave irradiation was at a power of just 3 W. This may be beneficial for clinical applications because such microwave irradiation should not harm normal tissue. The growth of tumors injected with 5Fu and exposed to microwave irradiation (the “5Fu+MW” group) was similar to that of 5Fu-injected tumors that were not subjected to further irradiation [27], which implies that 5Fu at the selected dose did not generate heat under microwave irradiation. The tumor growth (Fig. 8a, 8c) and the sizes and average weights of tumors 19 days post-injection (Fig. 8b, 8e) in the “5Fu+MW” and “CS-GCNCs(5Fu)+MW” groups further indicate that low power microwaves alone did not accelerate damage to tumor tissue by 5Fu or CS-GCNCs. However, when mice were intratumorally injected with CS-GCNCs and the tumors were co-irradiated with microwaves and laser (that is, the “CS-GCNCs+MW+L” group, note: L is laser), the tumor growth was significantly slower than that in the “CS-GCNCs+L” group (Fig. 8a, 8d). Tumors (n=4) were no longer detected after 7 days of co-irradiation and only skin tissue was resected after treatment (Fig. 8b), which was further confirmed by the histological images as only skin cells were detected at the tumor site (Fig. 8f). The tumors (n=4) in the “CS-GCNCs+L” group were not detected after 9 days of laser irradiation. The tumor size in the “CS-GCNCs+MW+L” group was significantly reduced compared with that in the “CS-GCNCs+L” group after 3 days of irradiation (P<0.001) (Fig. 8d). We measured the 24
photothermal conversion of CS-GCNCs in mouse tumors upon laser and microwave irradiation. The results showed that the temperature of the tumor injected with HEPES-dispersed CS-GCNCs increased from normal body temperature to 49.5±0.3 °C after 20 min of co-irradiation, which was significantly higher than the temperature of the tumor injected with HEPES buffer alone and exposed to the same irradiation (Fig. S11); and also higher than the temperature of the tumor injected with CS-GCNCs and exposed to laser irradiation alone (47.0±0.1°C) [27]. These observations indicate that the efficiency of CS-GCNCs-mediated cancer PTT can be obviously improved with the assistance of microwave
irradiation.
Tumors
treated
with
“CS-GCNCs(5Fu)+MW+L”
and
“CS-GCNCs(5Fu)+L” were no longer detected after 4 and 6 days of irradiation, respectively. Only skin was resected at the original tumor sites in the “CS-GCNCs(5Fu)+MW+L” group after treatment (Fig. 8b,8e). These results further demonstrated that co-irradiation with microwaves and laser could reduce the tumor treatment time compared with laser irradiation alone, which is meaningful for clinical use. In addition, the tumors treated with the “CS-GCNCs(5Fu)+MW+L” combination also disappeared more quickly than those treated with “CS-GCNCs+MW+L”. As shown in Fig. 8f, some shrunken cancer cells appeared in the tumor of the “5Fu+MW” group 19 days post-injection, which is similar to the results obtained for the “CS-GCNCs(5Fu)+MW” group. At this time point, the tumor tissues in the “HEPES+MW” and “CS-GCNCs+MW” groups were not noticeably damaged. These results further indicate that microwave irradiation alone is safe for cells and tissues, but the assistance of microwave irradiation is important to improve the nanoparticle-mediated cancer PTT effect. The synergistic effect of chemotherapy and the enhanced thermal effect caused by laser and microwave co-irradiation resulted in the strongest tumor ablation activity. 25
Fig. 8. Tumor growth inhibition by CS-GCNCs, 5Fu, and CS-GCNCs(5Fu) under laser irradiation, microwave irradiation, or laser and microwave co-irradiation. (a) Representative photographs of mice (all photographs are shown in Fig. S6–10). The mouse photographs for the “CS-GCNCs+L”, “CS-GCNCs(5Fu)+L”, and HEPEs groups are not shown here because they were reported in our recent work [27]. (b) Morphologies of tumors or skin. (c, d) Growth rates of tumors. (e–f) Tumors and skin (for the tumors that were not detected) at the original tumor site resected from mice 19 days post-injection. (e) Tumor weights and (f) histological images of the tumors or skin. Significance level observed was *P<0.05. L and MW are laser and microwave, respectively. To conveniently observe the differences in tumor growth between groups with laser irradiation and laser and microwave co-irradiation, the growth curves for the “CS-GCNCs+L”, “CS-GCNCs(5Fu)+L”, and HEPES groups, which were reported in our recent work [27], are also shown in this figure.
4. Conclusions We synthesized GCNCs and CS-GCNCs and showed that the changes in cell cycle distribution of nasopharyngeal carcinoma cells influenced by CS-GCNCs were suppressed 26
compared with those induced by the GCNCs without CS coating, and the final toxicity of CS-GCNCs to cells was significantly reduced compared with that of the GCNCs. CS-GCNCs(5Fu) exhibited sustained drug release and NIR laser irradiation or laser and microwave co-irradiation accelerated the drug release. Compared with that of free 5Fu, CS-GCNCs(5Fu) maintained high bioactivity, altering the cell cycle, improving apoptosis proteinase caspase-3 expression, and inducing cell death. The cell viability significantly decreased after photothermal ablation by CS-GCNCs or combined treatment with CS-GCNCs and 5Fu under NIR laser exposure. We combined microwave and photothermal irradiation to significantly enhance the cancer therapy efficiency of CS-GCNCs and CS-GCNCs(5Fu). The combination effect of microwave thermal and photothermal stimuli led to effective CS-GCNCs cell-killing action and inhibited mouse tumor growth. Because the microwave and laser irradiation used in this study were safe for cells when used alone, and owing to the good biocompatibility, high efficacy in cancer therapy, and simple synthesis of CS-GCNCs; the nanosystems (i.e., CS-GCNCs and 5Fu-loaded CS-GCNCs) based on graphitic nanocages possess considerable promise for clinical cancer therapy through enhanced PTT and chemo-thermal synergistic therapy triggered by the combination of NIR laser and microwaves.
Competing Interests The authors declared that no competing interests exist.
Acknowledgements This study was financially supported by the National Natural Science Foundation of China (31370961, 31570960), and the Science and Technology Innovation Foundation of 27
Shanghai (13NM1402000).
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Biocompatible Chitosan-Carbon Nanocage Hybrids for Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy Yuliang Guo , Yang Chen , Pomchol Han , Yuxiong Liu , Wenhao Li , Fangliang Zhu , Kai Fu , Maoquan Chu PII: DOI: Reference:
S1742-7061(19)30839-6 https://doi.org/10.1016/j.actbio.2019.12.010 ACTBIO 6493
To appear in:
Acta Biomaterialia
Received date: Revised date: Accepted date:
13 August 2019 9 December 2019 10 December 2019
Please cite this article as: Yuliang Guo , Yang Chen , Pomchol Han , Yuxiong Liu , Wenhao Li , Fangliang Zhu , Kai Fu , Maoquan Chu , Biocompatible Chitosan-Carbon Nanocage Hybrids for Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.010
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Biocompatible
Chitosan-Carbon
Nanocage
Hybrids
for
Sustained Drug Release and Highly Efficient Laser and Microwave Co-irradiation Induced Cancer Therapy
Yuliang Guo a,1, Yang Chen a,b,c,1, Pomchol Han a, Yuxiong Liu a, Wenhao Li a, Fangliang Zhu a
, Kai Fu a, Maoquan Chu a,*
a
Rehabilitation department at Shanghai Putuo District People's Hospital, School of Life
Sciences and Technology, Tongji University, Shanghai 200092, P. R. China. b
Institute of Biophysics, Chinese Academy of Science, Beijing 100101, P. R. China.
c
University of Chinese Academy of Sciences, Beijing 100049, P. R. China.
1
Yuliang Guo and Yang Chen contributed equally to this work.
* Corresponding author. Tel: 86 21 65988653. E-mail: [email protected] (M. Chu). Graphical abstract
1
ABSTRACT Graphitic carbon nanocages (GCNCs) are unique graphene-based nanomaterials that can be used for cancer photothermal therapy (PTT). However, low toxicity GCNC-based organic/inorganic hybrid biomaterials for microwave irradiation assisted PTT have not yet been reported. In the present study, chitosan (CS)-coated GCNCs (CS-GCNCs) loaded with 5-fluorouracil (5Fu) were used for cancer therapy when activated by 808-nm laser and microwave co-irradiation. The cytotoxicity of GCNCs was significantly reduced after coating with CS. For example, fewer cell-cycle defects were caused by CS-GCNCs in comparison with non-coated GCNCs. The release rate of 5Fu from CS-GCNCs was significantly slower than that of 5Fu from GCNCs, providing sustained release. The release rate could be accelerated by 808-nm laser and microwave co-irradiation. The 5Fu in CS-GCNCs retained high cancer cell killing bioactivity by enhancing the caspase-3 expression level. The cancer cell killing and tumor inhibition efficiencies of the 5Fu-loaded nanomaterials increased significantly under 808-nm laser and microwave co-irradiation. The strong cell killing and tumor ablation activities were due to the synergy of the enhanced GCNC thermal effect caused by laser and microwave co-irradiation and the chemotherapy of 5Fu. Our research opens a door for the development of drug-loaded GCNC-based nano-biomaterials for chemo-photothermal synergistic therapy with the assistance of microwave irradiation. KEYWORDS. Graphitic carbon nanocages; low cytotoxicity; sustained drug release; laser microwave co-irradiation; cancer therapy
2
Statement of Significance Graphitic carbon nanocages (GCNCs) are graphene-based nanomaterials that can be used for both drug loading and cancer photothermal therapy (PTT). Herein, we showed that chitosan (CS)-GCNCs hybrid biomaterials had very low cytotoxicity, high ability for loading drug, and exhibited sustained drug release. In particular, although low-power microwaves alone are unable to trigger cancer cell damage by GCNCs, the cell killing and mouse tumor inhibition efficiencies were significantly improved by near-infrared (NIR) laser and microwave co-irradiation compared with laser-triggered PTT alone. This combined use of laser and microwave co-irradiation promises essential therapeutic modality and opens a new avenue for PTT.
3
1. Introduction Although cancer therapies have greatly improved in recent decades, cancer remains a serious disease that causes many deaths worldwide [1]. Any approach that has the potential to improve the efficiency of cancer therapy is therefore welcome. Nanotechnology shows significant potential to improve cancer treatment through highly selective targeting of cancer cells, which reduces side effects compared with traditional methods. Among
various
nano-biomaterials
developed
to
date,
carbon-based
nanomaterials—which include graphene-based nanomaterials [2], nanodiamond [3], carbon dots [4], and activated carbon [5]—are among the most extensively studied. These nanomaterials show lower toxicity towards cells and animals than nanomaterials containing heavy metals such as semiconductor quantum dots. Graphitic carbon nanocages (GCNCs)—unique graphene-based nanomaterials—are hollow porous nanoparticles with a large internal cage with a diameter of a few tens of nanometers and a thin graphitic shell with a thickness of several nanometers [6,7]. Because of their unique geometric structure, GCNCs exhibit good physical and chemical properties and have been extensively investigated for various applications [8–11] such as supercapacitors [11]. In the present study GCNCs are used for near-infrared (NIR) laser-induced cancer therapy with the assistance of microwave irradiation (Fig. 1). The current strategy—the combination
of
laser
and
microwave
irradiation
to
improve
the
efficiency
of
nanoparticle-mediated cancer phototherapy—has not previously been reported. Microwave irradiation was introduced during NIR laser-induced PTT as microwaves exhibit strong tissue penetration, high heat generation rate, and homogeneity of heating. These 4
advantages make microwave heating markedly superior to using infrared lamps. In this approach, microwave irradiation is used to raise the initial temperature of the mouse (particularly the tumor) before low-power (0.25 W/cm2) NIR laser irradiation, which is different to nanomaterial-mediated microwave thermal therapy [12–15]. In recent research, microwave-sensitive nanomaterials with high microwave–thermal conversions have been used [12–15] where the microwave probe must be placed very close to the tumor (e.g., 1 cm away from the tumor of a fixed tumor-bearing mouse [15]), which may not be convenient for the clinical treatment of tumors in deep tissue. In the present study, these limitations are overcome as the distance between the microwave probe and mouse tumor reaches over 3 cm. Although increasing the laser power density and concentration of PTT agents may improve the PTT effect, low laser power [16] and a low PTT agent concentration are safer for clinical application. To improve their biocompatibility and cancer therapy efficiency, the GCNCs were encapsulated in chitosan (CS) polymer nanoparticles and loaded with 5-fluorouracil (5Fu). The 5Fu-loaded CS-GCNCs nanosystem exhibits sustained drug release, a strong ability to disturb the cell cycle and upregulate the expression of apoptosis-related proteins, and a strong ability to inhibit mouse tumor growth through the synergistic effect of chemotherapy and photothermal therapy (PTT) upon NIR laser irradiation. GCNCs were coated with CS because CS is a natural polymer with desirable properties such as nontoxicity, biodegradability, immunogenicity, bioadhesivity, high charge density, and low cost [17], and has been widely used as a sustained-release or responsive-release drug carrier [18–20]. The results presented in this work will facilitate the application of CS-GCNCs in biomedicine and provide a strategy for nanomaterial-mediated cancer therapy using the combination of laser and microwave irradiation. 5
Fig. 1. 5Fu-loaded chitosan-coated graphitic carbon nanocages for chemo-photothermal therapy with the assistance of microwave irradiation.
2. Experimental section 2.1. Cell line, animals, and materials The sources of the human nasopharyngeal carcinoma (CNE) cell line, BALB/c nude mice, and all reagents, including the 5Fu-loaded CS-GCNCs (abbreviated to CS-GCNCs(5Fu) in the following sections) developed by our research group are listed in the Supporting Information. All animal experiments were performed in accordance with the University of Tongji Institutional Animal Care and Use Committee Guidelines. The chitosan (CS) used in this study was analytical grade (deacetylation degree: 80%–95%, viscosity: 50–800 mPa·s). The detailed information of all reagents is shown in the Supporting Information.
2.2. Microwave-induced heating of nanoparticles in aqueous solution
6
CS-GCNC aqueous suspensions (GCNC: 0.01, 0.2, or 0.5 mg/mL, 200 μL for each sample) were placed in small glass tubes. The suspensions [temperature: 25.0±0.2 °C] were exposed to microwave irradiation (SPW-1 Microwave Therapy Instrument, Shengpu Medical Equipment Technology, Xuzhou, China) for 0–10 min. The power of the microwaves was set as 2, 5, or 10 W, and the frequency was maintained at 2450 MHz. The distance between the microwave probe and aqueous suspensions was 1 cm. A Ti29 thermal imager (Fluke Corporation, Everett, WA, USA) was used to measure the sample temperature. Distilled water and water containing 2% polyvinylpyrrolidone (PVP) (200 μL) was irradiated under the same conditions as a control. To measure the heat generated by GCNCs under microwave irradiation, GCNCs were dispersed in distilled water containing 2% (PVP). Each experiment was conducted three times (note: as GCNC is poorly dispersed in distilled water, 2% PVP was added in order to achieve uniform dispersion for the measurements; CS-GCNCs exhibited good stability in both distilled water and buffer solution, therefore no PVP was used to disperse CS-coated GCNCs for experiments).
2.3. Heating of the nanoparticle aqueous suspensions irradiated by both NIR laser and microwave CS-GCNC aqueous suspensions (0.2 mg/mL, 200 μL for each sample) at room temperature were placed in glass tubes and then exposed to microwave irradiation. When an initial suspension temperature of 28, 31, or 34 °C was achieved and maintained, the suspensions were simultaneously irradiated with an 808-nm laser (0.25 W/cm2) (Shanghai Inter-Diff Optoelectronics Tech. Co., Ltd., Shanghai, China) and the same microwaves for 20 min. Each experiment was conducted three times.
7
2.4. Analysis of cell cycle, caspase-3, and viability of nanoparticle-treated cells 2.4.1 Measurement of cell cycle distribution. CNE cells were incubated with culture medium-dispersed samples (GCNCs, CS-GCNCs, CS-GCNCs(5Fu), and free 5Fu) in a six-well plate in a CO2 incubator at 37 °C for 24 or 48 h. After incubation the cells were collected, fixed for 2 h with 70% ethanol, and then stained with propidium iodide. The cell fluorescence intensity (wavelength: 488 nm) was measured using a flow cytometer (BD FACSVerse, USA). Each experiment was conducted five times. 2.4.2 Caspase-3 analysis. CNE cells were treated with CS-GCNCs(5Fu) or 5Fu in a six-well plate for 24 or 48 h. The cells were collected, and caspase-3 levels were analyzed using a Caspase 3 Activity Assay Kit according to the provided protocol (Beyotime Biotechnology, Shanghai, China). Each experiment was conducted five times. 2.4.3 Cell viability assay. CellTiter-Glo™ reagent was used to detect the viabilities of CNE cells treated with CS-GCNCs. The viabilities of cells incubated with GCNCs, CS-GCNCs(5Fu), chitosan nanoparticles, and free 5Fu were also determined.
2.5. Apoptosis and viability of nanoparticle-treated cells upon microwave irradiation or microwave and NIR laser co-irradiation Serum-free medium-dispersed samples were added to cells in a 96-well plate. The cells at room temperature were irradiated with 5 W microwaves until the temperature of the medium was maintained at ~28 °C. The cells were then continuously exposed to the microwave irradiation for a further 20 min. In another group, cells incubated with the same samples were pre-irradiated with microwaves until the temperature of the medium was maintained at ~28 °C; the cells were then simultaneously exposed to laser and microwave irradiation for 20 min. The samples were pre-incubated with cells for 24 h and then treated with 8
microwaves or laser and microwaves using the same methods. The distance between the microwave probe and cells was kept to ~1 cm. After irradiation, cell apoptosis and viability were detected using the methods described above. The samples included CS-GCNCs (0.8 mg/mL), 5Fu (0.17 mg/mL), and CS-GCNCs(5Fu) (0.8 mg/mL). Each experiment was conducted five times. (Note: for cell and animal experiments, as the stability of GCNCs in HEPES buffer was better than that in distilled water, HEPES buffer was used to disperse the GCNCs, instead of distilled water containing 2% PVP, to remove the effect of PVP on the biological experiments).
2.6. Animal experiments To prepare the tumor-bearing mice, nude mice were subcutaneously injected with CNE cells. Six groups of mice with the same tumor size (~4 mm) were then used for the in vivo therapy experiments. The samples were intratumorally injected into the mice and the tumors were treated with 808-nm laser irradiation, microwave irradiation (3W), or laser and microwave co-irradiation. The distance between the microwave probe and tumor was fixed at ~3 cm (as we combined laser irradiation with microwave irradiation, it was necessary to leave a space so that the tumors could be easily exposed to the laser light. This distance (~3 cm) or greater distance is suitable for laser/microwave co-irradiation, and may also be appropriate for the clinical treatment of tumors). Laser and microwave irradiation was repeated once daily until the tumors were not detected, at which point the mice were no longer treated with the laser or microwave irradiation. The detailed animal experimental methods are shown in Fig. S1. Other experimental methods are described in detail in the Supporting Information.
9
2.7. Statistical analysis A non-parametric test (Mann–Whitney) and Student t-test were used for statistical analysis. Comparisons between results of in vitro experiments were performed using a non-parametric test (Mann–Whitney). The Student t-test was used to compare the means of two samples. P<0.05, P<0.01, and P<0.001 indicated statistically significant, highly statistically significant, and very highly statistically significant, respectively.
3. Results 3.1 Properties of GCNC and CS-GCNC nanoparticles The GCNCs were hollow in structure (Fig. 2a). In our previous work, we showed that the cage structure may be graphitic because the Raman spectrum of the GCNCs contained two prominent bands (D and G bands) consistent with graphite [21]. Here we further demonstrated using EDS that the GCNCs were primarily composed of C with a small amount of O (Fig. 2b). In the XRD pattern a strong peak at 2θ = 26.0° and two weak peaks at 2θ = 42.4° and 44.3° are assigned to the (002), (100), and (101) diffraction planes of the hexagonal structure of graphite (JCPDS Card no.41-1487), respectively (Fig. 2c). The graphitic nature of the GCNCs was further confirmed by the FT-IR spectrum (Fig. 2d). The FT-IR spectrum showed a prominent peak at around 1620 cm−1, as well as several weak peaks at 1542, 1510, and 1450 cm −1, which can be assigned to C=C stretching modes, attributed to polycyclic aromatic hydrocarbons; the two peaks at around 1384 and 1317 cm−1, are attributed to the C-O stretching mode of the carboxyl group; and several weak peaks, for example at 1725, 1708, and 1695 cm−1, are attributed to the C=O stretching mode of the carboxyl group; the strong peaks at around 3440 and 1110 cm−1 are due to the stretching and bending vibrations of O-H, respectively; the peaks at around 3033, 2917, and 2846 10
cm−1 are due to the stretching vibration of C-H, and several peaks at low wavenumber such as 825, 777, and 617 cm−1 are attributed to the bending vibrations of C-H in aromatic hydrocarbons. These findings indicated that the as-synthesized hollow nanoparticles are graphitic structures containing carboxyl and hydroxyl groups. A ‘water-in-oil’ microemulsion system was used to fabricate CS-GCNCs and CS nanoparticles. After addition of sodium tripolyphosphate (TPP), CS in the ‘water-in-oil’ microemulsion system assembled into nanoparticles (Fig. 2e). When TPP was added to a mixture of CS and GCNCs in a microemulsion, CS-GCNCs nanocomposites formed, and few free GCNCs could be observed (Fig. 2f). Although the diameter of an individual CS-GCNC was approximately 600 nm, the average hydrodynamic size of the CS-GCNCs was 960.8 ± 184.6 nm (Fig. 2g). In most cases, the hydrodynamic size measured using laser scattering is larger than the size of the same nanoparticles measured by TEM as the firmly aggregated and loosely bound particles are all detected as one particle. Although these large-sized CS-GCNCs were not uniform in size and shape, these CS-GCNCs could be used for cancer therapy as these particles were injected directly into mouse tumors to inhibit tumor growth. Submicrometer- or micrometer-sized particles with not uniform sizes for intratumoral injection for cancer therapy have been reported in both basic research [22,23] and clinical applications [24]. This administration route is safer for patients and animals than intravenous injection. The suspension stability of GCNCs in aqueous solution was markedly improved after the GCNCs were coated with CS, which was confirmed by images of the samples (Fig. 2h) and optical absorption changes of the sample supernatant over time (Fig. 2i). These results indicate that most of the GCNCs were coated or hybridized with CS. In this study, the nanoparticles were intratumorally injected into mouse tumors for in vivo therapy. The size of the particles was appropriate for tumor therapy as it does not affect the injection management and also 11
prevents the release of particles from the tumor tissue.
Fig. 2. Characterization of GCNC and CS-GCNC nanoparticles. (a) HRTEM image, (b) EDS spectrum, (c) XRD pattern, and (d) FT-IR spectrum of GCNCs. TEM images of (e) CS and (f) CS-GCNC nanoparticles. (g) Hydrodynamic size of CS-GCNCs. (h) Suspension stability of GCNCs and CS-GCNCs in HEPES buffer solution. (i) Relative optical absorption at 808 nm of the supernatant of CS-GCNCs in HEPES buffer solution with time. (j) Absorption spectra of GCNCs, CS nanoparticles, and CS-GCNCs. (k) The relationship between absorbance at 808 nm and the concentrations of GCNCs and CS-GCNCs. Photostability of (l) GCNCs and (m) CS-GCNCs (CNC concentration: 0.1 mg/mL) in water over six 808-nm laser irradiation on/off cycles. For the measurement of absorption and photothermal conversion in aqueous solution, GCNCs were dispersed in water containing 2% PVP.
3.2. Absorption coefficient, photothermal conversion, and photostability of GCNCs and CS-GCNCs at 808 nm The absorption coefficient of the GCNCs at 808 nm was 0.83±0.06 L/g·cm. After the 12
GCNCs were incorporated into a CS polymer shell, the resulting CS-GCNCs nanoparticles exhibited similar absorbance to that of the GCNCs between ~300 and 1100 nm (Fig. 2j). The absorption intensities of different concentration CS-GCNC suspensions at 808 nm were consistently higher than those of GCNCs under the same conditions (Fig. 2k). This is because the absorption of the CS-GCNCs had contributions from both the GCNCs and CS. The absorption coefficient of CS-GCNCs at 808 nm was 0.88±0.08 L/g·cm, which was slightly higher than that of the GCNCs (P>0.05), which indicates that CS-GCNCs may have potential for use as photothermal agents. Aqueous suspensions of both GCNC and CS-GCNC nanoparticles exhibited rapid photothermal conversion upon laser irradiation (Fig. 2l, 2m). The photothermal conversion efficiency of CS-GCNCs (23.7%) was similar to that of the GCNCs (23.9%), and other nanoparticles such as prophyrin immobilized nanographene oxide (19.9%) [25] and gold nanorods (22.3%) [26]. The GCNCs exhibited good photostability under 808-nm laser irradiation both before and after coating with CS (Fig. 2l, 2m), which is advantageous for long-term cancer PTT.
3.3. Thermal conversion of GCNCs and CS-GCNCs treated with microwaves or microwave and 808-nm laser co-irradiation. When an aqueous dispersion of CS-GCNCs (200 μL) was exposed to microwave irradiation (the microwave probe was placed 1 cm away from the sample), the temperature of the aqueous suspension increased rapidly with prolonged exposure time and when the microwave power was increased from 2 to 10 W (2450 MHz) (Fig. 3a and b). The temperature increase profiles of distilled water alone (200 μL) under the same microwave irradiation conditions were similar to those of water-dispersed CS-GCNCs. In addition, the 13
temperature of CS-GCNC aqueous suspensions under microwave irradiation did not increase markedly upon raising the concentration of CS-GCNCs (GCNC concentration was increased from 0.01 to 0.5 mg/mL), as shown in Fig. 3c and d. These results indicate that CS-GCNCs
are
not
microwave-absorbing
materials.
GCNCs
are
also
not
microwave-sensitive materials (Fig. S2). To investigate the contribution of the heat generated by microwave irradiation to the thermal conversion of laser-triggered nanoparticles, CS-GCNC suspensions were irradiated with microwaves until they first reached a particular temperature (e.g. 28, 31, or 34°C). This temperature did not change perceptibly during the subsequent ~20 min of microwave irradiation under the same conditions. The results showed that when a CS-GCNC aqueous suspension (0.1 mg/mL) was exposed to microwave irradiation until the temperature of the suspension increased and remained at a certain level; and then the suspension was co-irradiated with the laser and microwaves (Fig. 3e), the sample was heated more quickly than in the case of a suspension exposed to the laser irradiation alone (Fig. 3f and g). For example, the difference in initial temperature between the groups exposed to the laser only and the laser and microwaves was ~6.0 °C, whereas the difference in final temperature between these two groups was ~10.5 °C. The results described above indicate that although microwave irradiation at the investigated power does not efficiently trigger heat generation by GCNCs or CS-GCNCs, there may be a combined effect of interaction between the photothermal effect and microwave thermal effect. That is, the NIR-absorbing materials may be simultaneously exposed to both microwave and NIR laser irradiation to enhance the final temperature of the materials. This is important for cancer therapy because both microwave and laser irradiation were applied at low power, therefore only the tumor tissue containing the NIR-absorbing 14
material should be damaged by the enhanced thermal effect realized under the co-irradiation of a NIR laser and microwaves.
Fig. 3. Temperature changes of CS-GCNCs in aqueous solution upon laser irradiation, microwave irradiation, and laser and microwave co-irradiation. The temperature increase of CS-GCNC aqueous suspensions and distilled water upon microwave irradiation at (a, b) different microwave powers (GCNCs: 0.2 mg/mL) and (c, d) different GCNC concentrations (microwave power: 10 W). (e) Schematic illustration of the method of laser and microwave co-irradiation. (f, g) Photothermal conversion of distilled water-dispersed CS-GCNCs upon laser, and laser and microwave (3 W) co-irradiation, and controls. (a, c, f) Representative infrared thermal images. (b, d, g) Temperature intensities obtained from the images in a, c, and f, respectively. For the groups exposed to laser and microwave co-irradiation, the CS-GCNC suspensions or mouse tumors containing CS-GCNCs were first exposed to microwave irradiation until the temperature increased to 28 or 31 °C (for aqueous suspensions) or 38 °C (for tumors) and then maintained at these temperatures, followed by 20 min of laser exposure. During the laser exposure, CS-GCNC suspensions and mouse tumors were continuously irradiated with the microwaves. L, MW, and IT are laser, 15
microwave, and initial temperature, respectively. 3.4. Cytotoxicity of GCNCs and CS-GCNCs 3.4.1 Cell cycles of cancer cells treated with nanoparticles. The cell cycles of CNE cells treated with CS-GCNCs (0.1 mg/mL), even for 48 h, did not significantly change compared with those of the untreated cells (control) (P>0.05) (Fig. 4a, 4b). However, when the cells were treated with GCNCs (0.1 mg/mL) without CS coating for 48 h, the G0/G1 and G2/M phases significantly increased and decreased, respectively, compared with the control (P<0.01 for G0/G1 and P<0.001 for G2/M, compared with the control) (Fig. S3). These results imply that the toxicity of GCNCs was lowered by CS coating. 3.4.2 Cell viabilities of cancer cells treated with nanoparticles. As shown in Fig. 4c, the CNE cells after incubation with low-concentration GCNCs (e.g., 0.01 mg/mL) for 48 or 72 h exhibited high viability. However, the viabilities of cells incubated with the GCNCs at higher concentration (e.g., 0.1 mg/mL) decreased to (81.9±7.5)% and (77.1±4.4)% after 48 and 72 h of incubation, respectively. Interestingly, when CS-GCNCs containing the same amount of GCNCs as described above were added to the cells and incubated for 48 and 72 h, the cell viabilities ranged from (96.3±4.3)% to (100.4±4.7)%. These results further indicate that the CS polymer coating reduced the toxicity of the GCNCs. The CS coating is therefore necessary to improve the biocompatibility of GCNCs.
16
Fig. 4. The cell cycle and viability of CNE cells treated with CS-GCNCs. (a, b) The effect of nanoparticles on cell cycle distribution. Cells were incubated with CS-GCNCs (0.1 mg/mL) for 24 or 48 h or left untreated. (c) Viabilities of cells after incubation with GCNCs and CS-GCNCs for 48 or 72 h. Observed significance levels were ***P<0.001, **P<0.01, and *P<0.05.
3.5. Drug release and bioactivity of 5Fu loaded CS-GCNCs 5Fu has been widely used for clinical cancer chemotherapy. In a previous paper [27], we calculated that the loading efficiency of 5Fu was (22.4±2.0)%, i.e., 22.4±2.0 mg of 5Fu was loaded per 100 mg of CS-GCNCs. As shown in Fig. 5a, 5Fu was slowly released from 5Fu-GCNCs at room temperature. After 96 h of dialysis, only 74.13±2.8% of 5Fu was released. 5Fu-CS nanoparticles also exhibited sustained drug release and CS-GCNCs further delayed the drug release. After 96 h of dialysis at room temperature, only (74.1±2.8)% and (31.1 ±0.8)% of 5Fu was released from the 5Fu-GCNCs and CS-GCNCs(5Fu), respectively. At 37 °C, the release of 5Fu from CS-GCNCs(5Fu) increased slightly to ~33.6±0.6% after 96 h of dialysis (Fig. 5b). The release rate of 5Fu from the CS-GCNCs(5Fu) could be accelerated by NIR laser irradiation or laser and microwave co-irradiation. As shown in Fig. 5c, when CS-GCNCs(5Fu) 17
suspended in aqueous solution were exposed to an 808 nm laser for 20 min, the amount of 5Fu released from the nanoparticles [(10.1 ± 0.8)%] was 2.11 times higher than that without irradiation [(4.8 ± 0.6)%]. The microwave irradiation may also have contributed to increasing the 5Fu release rate, that is: under 3 W microwave irradiation, the release rate of 5Fu from the nanoparticles with and without laser irradiation increased slightly to [(10.5 ± 0.6%)] and [(5.1 ± 1.0%)], however neither showed a significant difference (P>0.05) compared with the release rates without microwave irradiation (Fig. 5c). The NIR/microwave-enhanced drug release is related to the increased temperature of the drug-loaded nanoparticles. The aqueous suspension of nanoparticles can be easily heated by NIR laser or microwave irradiation. The higher the temperature, the faster the molecular thermal motion. Therefore, the release rate of 5Fu from CS-GCNCs(5Fu) at an initial temperature of 37 °C with or without laser or microwave irradiation must be faster than that from particles with an initial temperature of ~25 °C. The drug release profile of CS-GCNCs(5Fu) described above is beneficial for long-term dual-modal cancer therapy. That is: when CS-GCNCs(5Fu) in tumor tissue were irradiated with an NIR laser or laser and microwaves, tumors were synchronously attacked by the photothermal effect of the GCNCs and the stimulated release of the 5Fu chemotherapeutic from the nanocarriers; in the absence of irradiation, however, only a small amount of drug was released. As depicted in Fig. 5d–g and h, the populations of free 5Fu (0.17 mg/mL) treated CNE cells in the sub-G0 phase after incubation for 24 or 48 h were significantly enhanced compared with those of the cells without 5Fu treatment in the same phase (P<0.01), which indicates that 5Fu has high toxicity to cells. The cells were arrested in the G0/G1 and S phases after the cells were incubated with CS-GCNCs(5Fu) (containing 0.17 mg/mL of 5Fu) 18
or 5Fu for 24 or 48 h. Cell-cycle defects in free 5Fu-treated cells were more serious than those in CS-GCNCs(5Fu)-treated cells, which may be related to the incomplete release of 5Fu from the CS-GCNCs nanoparticles.
Fig. 5. Release rate of 5Fu from CS-GCNCs and the cytotoxicity of CS-GCNCs(5Fu) and controls. (a) Comparison of the release rates of 5Fu from GCNCs, CS-GCNCs, and CS nanoparticles at room temperature (25 °C). (b) Comparison of the release rates of 5FU from CS-GCNCs at 25 °C and 37°C. (c) Laser, microwave, and both induced 5FU release from the CS-GCNCs. For the cytotoxicity study the cells were treated with CS-GCNCs(5Fu) or free 5Fu (CS-GCNCs: 0.8 mg/mL, 5Fu: 0.17 mg/mL) for 24 or 48 h. (d–g) Representative images and (h) histograms of cell cycle distributions. (i) Activities of caspase-3 extracted from the cells. (j) Cell viabilities. Observed significance levels were ***P and #P<0.001, **P<0.01, and *P<0.05. L and MW are laser and microwave, respectively. 19
The expression level of caspase-3 in CNE cells significantly increased after the cells were treated with 5Fu (Fig. 5i). The level of caspase-3 after 48 h of incubation was significantly higher than that after 24 h of incubation. Caspase-3 is an apoptosis-related cysteine peptidase; the higher the caspase-3 level, the greater the cell apoptosis. The cell viability decreased from 100% to (85.7±3.0)% and (63.4±5.8)% after exposure to 5Fu for 24 and 48 h, respectively (Fig. 5j). CS-GCNCs(5Fu) (GCNCs: 0.2 mg/mL, 5Fu: 0.17 mg/mL) also induced significant CNE cell apoptosis by enhancing the caspase-3 expression level, and significantly lowered cell viability (Fig. 5j). The expression of caspase-3 in the 5Fu-loaded nanoparticle group was less than that in the 5Fu group, which may be a result of the slow release of 5Fu from the nanoparticles. However, 5Fu still exhibited high bioactivity leading to damage of cancer cells after incorporation into CS-GCNCs.
3.6. Ability of nanoparticles to kill cancer cells under laser and microwave co-irradiation Both qualitative and quantitative analyses showed that the cell viabilities were close to 100% when the cells were treated with CS (0.6 mg/mL) or CS-GCNCs (0.8 mg/mL) for 20 min (Fig. 6). However, as shown in Fig. 6a, many apoptotic or dead cells could be observed after the cells incubated with CS-GCNCs were exposed to the 808-nm laser for 20 min. The strong cancer cell killing ability of the CS-GCNCs and laser combination was also confirmed by quantitative analyses [cell viability: (52.4±3.9)%] (Fig. 6b). The main phototoxic mechanism of the CS-GCNCs was their photothermal effect because no toxic reactive oxygen species (ROS) were produced by the laser-irradiated GCNCs (Fig. S4). The low number of red fluorescent cells in Fig. 6a shows that the CS nanoparticles were unable to damage cells with or without laser irradiation. As shown in Fig. 6b, the viabilities of cells 20
(initial temperature: ~25 °C) treated with CS or CS-GCNCs after microwave exposure at 5 W exceeded 98%, which showed no significant difference to cells incubated with HEPES buffer exposed to the same microwave irradiation protocol [cell viability: (100.0±1.8)%]. This is because the materials barely absorb microwave energy and the applied microwave irradiation was only at a power of 5 W. However, after the cells incubated with CS-GCNCs were exposed to microwave irradiation to achieve a final culture medium temperature of ~28 °C, and then co-irradiated with microwaves and laser for 20 minutes, the cell viabilities significantly decreased compared with those of the cases where laser irradiation alone was used (P<0.001). For example, in the group with CS-GCNCs and laser exposure, the cell viability was (52.4±3.9)%, whereas for the group with CS-GCNCs and laser and microwave co-irradiation, the cell viability was only (39.4±3.5)%. The strong cell-killing ability of CS-GCNCs under laser and microwave co-irradiation was also confirmed by qualitative measurements (Fig. 6a). These results imply that laser and microwave co-irradiation is beneficial for cancer PTT, as high efficiency PTT may be achieved using low concentration PTT agents and/or a low power density laser.
Fig. 6. The cell killing efficiency of CS-GCNCs upon laser irradiation, microwave irradiation, or laser and microwave co-irradiation and controls. (a) Qualitative measurements. (b) Quantitative measurements. Observed significance levels were ***P and #P<0.001. In which, #
indicates differences between the corresponding results with and without laser or laser and
microwave co-irradiation. L and MW are laser and microwave, respectively. 21
To demonstrate that thermal stimulation and the toxicity of 5Fu exhibit a synergistic cell-killing effect, CNE cancer cells were incubated with CS-GCNCs(5Fu) for a longer time before irradiation so that the cytotoxicity of 5Fu could be observed. After the cells were treated with CS-GCNCs(5Fu) (GCNCs: 0.2 mg/mL) or free 5Fu (0.17 mg/mL) for 24 h (at this time point 30±2.3% of 5Fu was released from CS-GCNCs(5Fu)), the percentages of cell apoptosis were (15.6±0.9)% and (18.7±1.6)%, respectively (Fig. 7a,7b). The percentage of cell apoptosis increased significantly to (67.6±2.7)% after the cells were treated with CS-GCNCs(5Fu) in the same manner as described above and exposed to the laser for 20 min. A similar result was observed in the CS-GCNCs group. This is because the cytotoxicities of both 5Fu and GCNCs are time dependent. A cell viability assay further confirmed the synergistic cell-killing effect of thermal-chemotherapy in the CS-GCNCs(5Fu) group under laser irradiation (Fig. 7c). Importantly, when CS-GCNCs and CS-GCNCs(5Fu) were pre-incubated with cells for 24 h, NIR laser and microwave co-irradiation further improved the cell-killing efficiency of CS-GCNCs and CS-GCNCs(5Fu) (Fig. 7c). For example, after CS-GCNCs(5Fu) were incubated with cells for 24 h and then the cells were exposed to microwave and laser irradiation for 20 min, the cell viability decreased to just (19.2±1.0)%. In contrast, 5Fu and nanomaterials exposed to the microwaves alone did not significantly accelerate cell death (Fig. 7c).
22
Fig. 7. Effects of pre-incubation, laser irradiation, microwave irradiation, and laser and microwave co-irradiation of CS-GCNCs(5Fu), CS-GCNCs, or free 5Fu on CNE cell viability. CNE cells were treated with the samples for 24 h before laser and/or microwave irradiation (5 W, 2450 MHz). (a) Dot plot representation of dual staining of CNE cells with annexin FITC-A and propidium iodide and (b) Cell apoptosis percentages obtained by flow cytometry. (c) Cell viabilities. L and MW are laser and microwave, respectively.
3.7. In vivo cancer therapy In a previous paper [27], we showed that the growth of mouse CNE tumors was significantly inhibited by laser light-triggered CS-GCNCs and CS-GCNCs(5Fu). For nanoparticle-mediated PTT, enhancing local temperature can markedly accelerate the inhibition of tumor growth [27]. Because microwave irradiation can substantially increase the final temperature of laser-irradiated mouse tumors containing CS-GCNCs, we explored 808-nm laser and microwave (3 W, 2450 MHz) co-irradiation for CS-GCNCs-mediated cancer therapy. For a mouse tumor injected directly with CS-GCNCs, microwave irradiation 23
alone (the “CS-GCNCs+MW” group, note: MW is microwave) did not efficiently inhibit tumor growth because the average tumor growth rate was similar to those of tumors in the “HEPES+MW” and “HEPES, no irradiation” groups (Fig. 8a, 8b), and the tumor sizes and average weights in the “CS-GCNCs+MW” group 19 days post-injection were also not significantly different to those in the “HEPES+MW” group (Fig. 8d,8e). This is because CS-GCNCs are not microwave-sensitive materials and the microwave irradiation was at a power of just 3 W. This may be beneficial for clinical applications because such microwave irradiation should not harm normal tissue. The growth of tumors injected with 5Fu and exposed to microwave irradiation (the “5Fu+MW” group) was similar to that of 5Fu-injected tumors that were not subjected to further irradiation [27], which implies that 5Fu at the selected dose did not generate heat under microwave irradiation. The tumor growth (Fig. 8a, 8c) and the sizes and average weights of tumors 19 days post-injection (Fig. 8b, 8e) in the “5Fu+MW” and “CS-GCNCs(5Fu)+MW” groups further indicate that low power microwaves alone did not accelerate damage to tumor tissue by 5Fu or CS-GCNCs. However, when mice were intratumorally injected with CS-GCNCs and the tumors were co-irradiated with microwaves and laser (that is, the “CS-GCNCs+MW+L” group, note: L is laser), the tumor growth was significantly slower than that in the “CS-GCNCs+L” group (Fig. 8a, 8d). Tumors (n=4) were no longer detected after 7 days of co-irradiation and only skin tissue was resected after treatment (Fig. 8b), which was further confirmed by the histological images as only skin cells were detected at the tumor site (Fig. 8f). The tumors (n=4) in the “CS-GCNCs+L” group were not detected after 9 days of laser irradiation. The tumor size in the “CS-GCNCs+MW+L” group was significantly reduced compared with that in the “CS-GCNCs+L” group after 3 days of irradiation (P<0.001) (Fig. 8d). We measured the 24
photothermal conversion of CS-GCNCs in mouse tumors upon laser and microwave irradiation. The results showed that the temperature of the tumor injected with HEPES-dispersed CS-GCNCs increased from normal body temperature to 49.5±0.3 °C after 20 min of co-irradiation, which was significantly higher than the temperature of the tumor injected with HEPES buffer alone and exposed to the same irradiation (Fig. S11); and also higher than the temperature of the tumor injected with CS-GCNCs and exposed to laser irradiation alone (47.0±0.1°C) [27]. These observations indicate that the efficiency of CS-GCNCs-mediated cancer PTT can be obviously improved with the assistance of microwave
irradiation.
Tumors
treated
with
“CS-GCNCs(5Fu)+MW+L”
and
“CS-GCNCs(5Fu)+L” were no longer detected after 4 and 6 days of irradiation, respectively. Only skin was resected at the original tumor sites in the “CS-GCNCs(5Fu)+MW+L” group after treatment (Fig. 8b,8e). These results further demonstrated that co-irradiation with microwaves and laser could reduce the tumor treatment time compared with laser irradiation alone, which is meaningful for clinical use. In addition, the tumors treated with the “CS-GCNCs(5Fu)+MW+L” combination also disappeared more quickly than those treated with “CS-GCNCs+MW+L”. As shown in Fig. 8f, some shrunken cancer cells appeared in the tumor of the “5Fu+MW” group 19 days post-injection, which is similar to the results obtained for the “CS-GCNCs(5Fu)+MW” group. At this time point, the tumor tissues in the “HEPES+MW” and “CS-GCNCs+MW” groups were not noticeably damaged. These results further indicate that microwave irradiation alone is safe for cells and tissues, but the assistance of microwave irradiation is important to improve the nanoparticle-mediated cancer PTT effect. The synergistic effect of chemotherapy and the enhanced thermal effect caused by laser and microwave co-irradiation resulted in the strongest tumor ablation activity. 25
Fig. 8. Tumor growth inhibition by CS-GCNCs, 5Fu, and CS-GCNCs(5Fu) under laser irradiation, microwave irradiation, or laser and microwave co-irradiation. (a) Representative photographs of mice (all photographs are shown in Fig. S6–10). The mouse photographs for the “CS-GCNCs+L”, “CS-GCNCs(5Fu)+L”, and HEPEs groups are not shown here because they were reported in our recent work [27]. (b) Morphologies of tumors or skin. (c, d) Growth rates of tumors. (e–f) Tumors and skin (for the tumors that were not detected) at the original tumor site resected from mice 19 days post-injection. (e) Tumor weights and (f) histological images of the tumors or skin. Significance level observed was *P<0.05. L and MW are laser and microwave, respectively. To conveniently observe the differences in tumor growth between groups with laser irradiation and laser and microwave co-irradiation, the growth curves for the “CS-GCNCs+L”, “CS-GCNCs(5Fu)+L”, and HEPES groups, which were reported in our recent work [27], are also shown in this figure.
4. Conclusions We synthesized GCNCs and CS-GCNCs and showed that the changes in cell cycle distribution of nasopharyngeal carcinoma cells influenced by CS-GCNCs were suppressed 26
compared with those induced by the GCNCs without CS coating, and the final toxicity of CS-GCNCs to cells was significantly reduced compared with that of the GCNCs. CS-GCNCs(5Fu) exhibited sustained drug release and NIR laser irradiation or laser and microwave co-irradiation accelerated the drug release. Compared with that of free 5Fu, CS-GCNCs(5Fu) maintained high bioactivity, altering the cell cycle, improving apoptosis proteinase caspase-3 expression, and inducing cell death. The cell viability significantly decreased after photothermal ablation by CS-GCNCs or combined treatment with CS-GCNCs and 5Fu under NIR laser exposure. We combined microwave and photothermal irradiation to significantly enhance the cancer therapy efficiency of CS-GCNCs and CS-GCNCs(5Fu). The combination effect of microwave thermal and photothermal stimuli led to effective CS-GCNCs cell-killing action and inhibited mouse tumor growth. Because the microwave and laser irradiation used in this study were safe for cells when used alone, and owing to the good biocompatibility, high efficacy in cancer therapy, and simple synthesis of CS-GCNCs; the nanosystems (i.e., CS-GCNCs and 5Fu-loaded CS-GCNCs) based on graphitic nanocages possess considerable promise for clinical cancer therapy through enhanced PTT and chemo-thermal synergistic therapy triggered by the combination of NIR laser and microwaves.
Competing Interests The authors declared that no competing interests exist.
Acknowledgements This study was financially supported by the National Natural Science Foundation of China (31370961, 31570960), and the Science and Technology Innovation Foundation of 27
Shanghai (13NM1402000).
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