- Email: [email protected]
Transferrin modified ruthenium nanoparticles with good biocompatibility for photothermal tumor therapy
Journal of Colloid and Interface Science 511 (2018) 325–334
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Regular Article
Transferrin modified ruthenium nanoparticles with good biocompatibility for photothermal tumor therapy Shuang Zhao 1, Xufeng Zhu 1, Chengwen Cao, Jing Sun, Jie Liu ⇑ Department of Chemistry, Jinan University, Guangzhou 510632, China
g r a p h i c a l a b s t r a c t Schematic illustration of the preparation of Tf modified RuNPs. Cell uptake of Tf-RuNPs is mediated by Tf receptor-mediated endocytosis. Upon the NIR laser irradiation, the internalized Tf-RuNPs can expeditious convert the absorbed light energy to local heat for PTT.
a r t i c l e
i n f o
Article history: Received 2 July 2017 Revised 1 October 2017 Accepted 6 October 2017 Available online 7 October 2017 Keywords: Transferrin Ru nanoparticles Tumor target Photothermal therapy
a b s t r a c t In the past two decades, there were various kinds of photothermal agents being synthesised and investigated for their photothermal effect in antitumor applications. However, it is barely reported that the photothermal effect of Ruthenium (Ru) nanoparticles was researched in depth. In this work, we introduced Ru nanoparticles which possess excellent biocompatibility and metabolize easily to the photothermal therapy field. In addition, to improve the cells capacity of absorbing Ru nanoparticles, these Ru nanoparticles were modified by transferrin (Tf-RuNPs). Subsequently, as is expected, the RuNPs exhibit a remarkably integrated and high-quality photothermal property. On the other hand, it is significantly that Tf modification could also strengthen the cells absorptive ability to uptake Ru nanoparticles through endocytosis., Furthermore, both the in vitro cell ablation and in vivo tumor treatment verified that the TfRuNPs became ideal photothermal agents for photothermal tumor ablation therapy owing to their low toxicity and high cell destruction capability. Ó 2017 Published by Elsevier Inc.
1. Introduction ⇑ Corresponding author. 1
E-mail address: [email protected] (J. Liu). Both authors contribute equally to this work.
https://doi.org/10.1016/j.jcis.2017.10.023 0021-9797/Ó 2017 Published by Elsevier Inc.
Nowadays, the growing and aging of world population may be responsible for the increasing cancer-causing behaviours. After
326
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
all, cancer is still a disturbing and global health problem [1–3]. There is one thing that demands our focal attention that cancers were conventionally treated by traditional methods such as chemotherapy, surgery, radiation therapy, etc [4]. Unfortunately, these methods are restricted because of the adverse effects. In recent years, a growing number of scientists focus on the exploitation of nanomaterials for photothermal therapy (PTT), a minimally invasive and selective treatment of cancer. In this PTT protocol, photo-absorbing nanomaterials convert photon energy to heat, producing a local hyperthermia to burn cancer cells and tissues. As for the light source, near-infrared (NIR, 700 to 1100 nm) laser is being widely used. Because it can reach several centimetres in biological tissues due to the relatively low absorption of hemoglobin and water as well as less damage to normal tissues [5]. Hence, strong absorption in the NIR region of nanomaterials will be beneficial for the application of PTT. There are quiet a few, different types of photothermal agents, such as metal nanostructures [6–14], organic compounds [15,16], carbon materials [17–19]. Actually, a multitude of them have been investigated up to now. There are quiet a few different shapes of gold, for typical examples, nanospheres, nanorods, nanoshells, and nanocages, obviously, all of them will bewell-suited to various biomedical applications [20,21]. Even though, almost all the researches pay excessive attention on the narrow range of metal elements like gold, silver, or palladium. On the contrary, studies on the light and heat properties of the precious metal ruthenium (Ru) are rarely reported. According to this, we have studied the photothermal effect of ruthenium nanoparticles with simple spherical morphology. Gold nanomaterials feature the high cost, poor bioassay and thermal stability, while carbon nanomaterials featurepoorly dispersible and may induce oxidative stress as well as immune response, contrarily, the ruthenium nanoparticles have good biocompatibility and exhibit outstanding photothermal effect. Insufficient intracellular particle delivery may be the main obstacle that prevents the using of metal nanoparticles in the fight against cancerous cells [22]. Considering this, nanoscale modification technology was invented timely. More specifically, it is a
promising pathway to enhance the accumulation of nanoparticles. Different ligands have been modified into nanoparticles and significantly enhanced the cellular ability of absorbing via receptormediated pathways, such as folic acid [23], hyaluronic acid [24], peptides [25], etc. Transferrin (Tf), as a serum protein which is non-toxic, non-immunogenic, and biodegradable: it has been widely studied as a cancer-targeting agent [26]. There is an ocean of Transferrin receptor (TfR) existing on the surface of proliferating and malignant cancer cells [27–29]. Tf/TfR-mediated endocytosis has been proved to be an effective strategy to enhance the nanoparticles accumulation in tumor sites [30]. A host of investigations have indicated that different material modified by Tf extends the blood circulation time and achieves a high selectivity for cancer cells, [32,33]. Collectively, the purpose of this study was to investigate the activity of Ruthenium (Ru) nanoparticles in photothermal therapy against cancer cells. In view of this, we envisage to make Tf modified on the surface of Ru nanoparticles. so that the synthesis of Ru nanoparticles would get prone to be absorbed by cells (Scheme 1). Beyond that, the cellular uptake mechanisms of TfRuNPs were detected in cell assays. Also, the photothermal effects of Tf-RuNPs on A549 cells were measured by MTT assay. Furthermore, we observed and assessed the photothermal effects of TfRuNPs on tumor-bearing mice. Afterwards, it is demonstrated that Tf-RuNPs exhibit excellent capability to kill cancer cells, with less side effects owing to the use of Tf-RuNPs. Simply put, these new nanoparticles could be successfully used for highly efficient in vivo PTT under NIR irradiation.
2. Materials and methods 2.1. Materials All reagents and solvents were purchased commercially and used without further purification unless specially noted, and Ultrapure MilliQ water (18.2 MW) was used in all experiments. polyvinyl pyrrolidone (PVP), Cetyltrimethylammonium bromide
Scheme 1. Schematic illustration of the preparation of Tf modified RuNPs. Cell uptake of Tf-RuNPs is mediated by Tf receptor-mediated endocytosis. Upon the NIR laser irradiation, the internalized Tf-RuNPs can expeditious convert the absorbed light energy to local heat for PTT.
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
(CTAB), HAuCl43H2O, 1-(3-Dimethylaminopropyl)-3-ethylcarbodii mide hydrochloride (EDC), RuCl3nH2O, NaBH4, thioglycolate, NHydroxysuccinimide (NHS), thiazolyl blue tetrazolium bromide (MTT), DAPI, Transferrin were purchased Sigma-Aldrich Chemical Co.
327
2 W cm 2 for 10 min. The temperatures of the suspension were recorded by a thermocouple microprobe immersed in the suspension. Photothermal effect of Tf-RuNPs and water were performed at the same parameters. Infrared thermal images for different NPs suspension and water were taken by Fluke T125 Thermal Imaging Camera.
2.2. Preparation of RuNPs, Tf-RuNPs and gold nanorods 2.5. Cell lines and cell culture In a typical synthesis, 0.1434 g RuCl3nH2O dissolved in 10 mL water (0.5 mM), and 0.5828 g of polyvinylpyrrolidone (PVP) was then added, followed an ice cold NaBH4 solution (0.5 mL, 0.1 M) was dropwise added to mixture under stirring. The mixture then continued stirring for another 30 min. To remove the excess reactants, the reaction mixture was then extensively centrifuged (9000 rpm, 5 min), and washed three times with water to obtain the RuNPs (solid). For Tf conjugation, firstly, 8 mg of thioglycolate was added to 10 mL of 10 mM PBS (pH 8.5) containing 5 mg of RuNPs and stirring at room temperature for 24 h, then the reaction mixture centrifuged (9000 rpm, 5 min) and washed with water for three times to remove excess thioglycolate, then thioglycolate decorated RuNPs (RuNPs-COOH) were obtained . Secondly, the RuNPsCOOH redispersed in 10 mL water containing 50 lL of EDC and 125 lL NHS and stirring for 1 h. At last, the mixture solution mixed with of 0.5 mL transferrin (Tf) solution (5 mg mL 1) and stirring for 24 h at 4 °C. The excess Tf, EDC and NHS were removed by extensively centrifugation (9000 rpm, 5 min) and washed with water for three times. The obtained Tf-RuNPs were dispersed in water for further use. The concentration of Ru ions was determined by ICPMS analysis. Gold nanorods were synthesized following a reported method. Briefly, ice-cold NaBH4 (10 mM, 0.6 mL) was add to a solution containing CTAB (100 mM, 7.5 mL) and HAuCl43H2O (10 mM, 0.25 mL) with vigorous stirring for 10 min, the gold seeds were prepared. To prepared gold nanorods, HAuCl43H2O (10 mM, 16 mL), CTAB (100 mM, 80 mL), AgNO3 (10 mM, 0.5 mL), and ascorbic acid (10 mM, 6 mL) were mixed in a 150 mL conical flask. Then seed solution (0.2 mL) was added with gentle stirring, and the entire solution was left still for 10 h. The gold nanorods were collect by centrifugation (9000 rpm, 5 min) and washed by water 3 times. 2.3. Characterization of Tf-RuNPs The as-prepared Tf-RuNPs was characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), UV–vis spectroscopy, dynamic light scattering (DLS) and BCA assays. TEM samples were prepared by placing one drops of nanoparticles suspension onto a holey carbon film on copper grids. The micrographs were obtained on Hitachi (H7650) instrument for TEM operating at an accelerating voltage at 80 kV. The size distribution and zeta potential of the nanoparticles were measured by PCS on a Nano-ZS instrument (Malvern Instruments Limited) at least three times. FTIR (Equinox 55 IR spectrometer) and UV–vis (Carry 5000 spectrophotometer) studies were recorded in the range of 4000–500 cm 1 and 200–900 nm, respectively. Bicinchoninic acid (BCA) kit (Pierce) used to evaluate Tf content was conducted followed by manufacturer’s instructions. The NIR laser for photothermal experiments is an 808 nm high power NIR laser diode (LSR808NL-2010). 2.4. Photothermal performance To study the photothermal effect under 808 nm laser irradiation, 1 mL aqueous suspension containing 10 lg of RuNPs, 10 lg of gold nanorods or 11.4 lg of Tf-RuNPs were placed in square cuvette and irradiated with an 808 nm NIR laser at a power density of
The human lung adenocarcinoma cell lines A549 and Human Embryonic Kidney 293 (HEK-293) cell lines were used in this study were obtained from the Experimental Animal Center at Sun YatSen University. All cell lines were maintained in DMEM media supplemented with penicillin (100 units mL 1), fetal bovine serum (10%), and streptomycin (50 units mL 1) at 37 °C in a humidified incubator with 5% CO2 atmosphere. 2.6. Cellular uptake of Tf-RuNPs in vitro The cellular uptake of nanoparticles was monitored by ICP-MS method. Briefly, A549 and HEK-293 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with or without RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h, respectively. After incubation, the cells were washed with PBS three times and harvested, and digested with 3 mL of concentrated nitric acid and 1 mL of H2O2 in a digestive stove at 150 °C for 2 h. The digested product was diluted to 8 mL with water and used for ICP-MS analysis. To detect the mechanisms of cellular uptake of Tf-RuNPs, A549 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with 5 mM MBCD, 0.45 M hypertonic sucrose and 5 mM cytochalasin D for 1 h before incubated with Tf-RuNPs. The control samples were without the addition of inhibitors. The cells were then incubated with Tf-RuNPs (11.4 lg mL 1) for 24 h. After treatment, the cells were then washed with PBS for three times and analyzed by ICP-MS. 2.7. Intracellular trafficking of Tf-RuNPs A549 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h then fixed using 3% glutaraldehyde and dehydrated using graded ethanol. TEM samples were sectioned by microtome in Araldite resin and observed on a Hitachi (H-7650) instrument for TEM operating at an accelerating voltage at 80 kV. 2.8. Western blot analysis A549 and HEK-293 cells were incubated with lysis buffer (Beyotime) to obtain total cellular proteins. The protein concentration was examined by BCA assay. Equal amount of proteins were electrophoresed in 15% tricine gels and then transferred to nitrocellulose membrane and blocked with 5% non-fat milk in Tris-Buffered Saline (TBS) buffer overnight. Then the membranes were washed with Tris-Buffered Saline Tween-20 (TBST) buffer three times and incubated with primary antibodies s at 1:1000 dilution in TBS at room temperature for 2 h with continuous agitation. Then the membranes were washed with TBST three times and incubated with secondary antibodies conjugated with horseradish peroxidase at 1:2000 dilution for 1 h at room temperature, followed by 5 times washing with TBST. Protein bands were visualized on X-ray film using enhanced chemiluminescence detection regents (Pierce Biotech, Rockford, IL), b-Actin was used to confirm the comparable amount of proteins in each lane.
328
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
2.9. Photothermal performance of RuNPs and Tf-RuNPs in vitro To evaluate biocompatibility of gold nanorods, RuNPs and TfRuNPs, HEK-293 cells were seeded in 96-well tissue culture plates at 2 103 cells per well and cultured for 24 h. Then cells were incubated with gold nanorods, RuNPs and Tf-RuNPs at different concentrations for 24 h. After treatment, 20 lL per well of MTT solution (5 mg mL 1 phosphate buffered saline) was added to the well and incubated for another 2 h. Next the medium was replaced with 150 lL DMSO per well to dissolve the formazan salt formed. The cell viability was reflected by the color intensity of the formazan solution. Absorbance at 570 nm was taken on a 96-well microplate reader. For PTT in vitro, A549 cells were seeded in 96-well tissue culture plates at 2 103 cells per well and cultured for 24 h. Then the cells were incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h. Subsequently, the treated cells were replaced with fresh culture media to remove the excess NPs and exposed to an 808 nm NIR laser (2 W cm 2) for 1 or 2 min. And the cell viability of each sample were measured by MTT assay. In addition, after NIR laser irradiation, the cells were stained by AO/ EB reaction mixture for 20 min and washed with PBS for three times and observed under CLSM to directly observe the live and dead cells.
in control groups were injected with the same volume of saline. The tumors of mice were followed irradiated by NIR laser for 5 min at power density of 1 W cm 2 and simultaneously imaged by a thermal camera. The mice body weights and tumor sizes were recorded every 3 days, and the tumor volume were calculated as Vtumor = length width2/2. All animal procedures were in accord with the guidelines of the Institutional Animal Care and Use Committee. After 15 days of treatments, all the mice were sacrificed, and their tumors and major organs (heart, liver, spleen, lung and kidney) were dissected, washed with saline and fixed in 4% formaldehyde for histological examination. 2.11. Biodistribution assays of Tf-RuNPs For biodistribution study, ten tumor-bearing mice (tumor size 200 mm3) were divided into two groups. Two group was intravenously injected with RuNPs (0.1 mL, 1 mg mL 1) and TfRuNPs (0.1 mL, 11.4 mg mL 1), respectively. After 24 h, the organs (heart, liver, spleen, lung, kidneys) and tumors were removed. The Ru were quantified through ICP-MS method. The tissues were cut into pieces and digested with 3 mL of concentrated nitric acid and 1 mL of H2O2 in a digestive stove at 150 °C for 2 h. The digested product was diluted to 8 mL with water and used for ICP-MS analysis.
2.10. Antitumor activity in vivo 3. Results and discussion The 5–6 week-old severe combined immune deficiency (SCID) male mice (ordered from NIH) weighing 22 g were divided into groups with five mice per group. The animal tumor model was generated by subcutaneous injected with A549 cells (1 107 cells per mouse) into the mice into the right armpit region of nude mice. For PTT in vivo, after the tumors had become established (200 mm3), the mice were intravenous injected with 20 lL of RuNPs (1 mg mL 1) and Tf-RuNPs (11.4 mg mL 1) suspension. The mice
3.1. Preparation, characterization, and photothermal effect of RuNPs The RuNPs were synthesised by a simple preparation procedure mentioned above. Fig. 1A showed TEM image of the RuNPs with a uniform size about 70 nm. To detect the absorption properties of RuNPs, the UV–vis-NIR absorbance of the RuNPs were measured (in water) at the mass concentration of 10 lg mL 1 (Fig. 1B). The
Fig. 1. (A) TEM image and (B) UV–vis-NIR absorbance spectra of of RuNPs. (C) Stability of RuNPs in PBS (pH = 7.4) and cell culture medium. (D) Photothermal effect of RuNPs at a mass concentration of 10 lg mL 1 with an 808 nm NIR laser (2 W cm 2), the inset shows the final temperature of Tf-RuNPs aqueous suspension and distilled water after 10 min irradiation. (E) IR thermal images for RuNPs after irradiation for 0, 1, 5 and 10 min. (F) Photothermal effect of RuNPs at different concentration with an 808 nm NIR laser (2 W cm 2).
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
RuNPs in water showed monotonically rising absorbance from the NIR to the UV–vis region, although the surface plasmon resonance of RuNPs is not located in the vis-NIR region, but the notable absorbance of RuNPs in the NIR region was deemed to be beneficial for the practice of PTT. Since the RuNPs showed broad absorption through the entire NIR regions, various NIR laser could be chosen for PTT. An 808 nm laser was used as the light source in this work. To quantify the absorption property of RuNPs, the molar extinction coefficient was calculated using the method as described in previous reports [14,34]. TEM data showed RuNPs with a spherical-like shape and the size of 70 nm, the molar extinction coefficient was calculated to be 1.16 108 M 1 cm 1 at 808 nm : this value is remarkable compared with many PTT agents such as porous Pd (6.3 107) [14], carbon material (8.2 106) [18], Cu2 xSe (7.7 107) [35], but lower than that of Au nanohexapods (5.5 109) [36], Pd nanosheets (4.1 109) [6] and Au nanocages (3.2 1010) [12,37,38]. The stability of nanoparticles is also very important for its application. As shown in Fig. 1C, the size of RuNPs did not increase in both PBS and cell culture medium, indicating that. aggregation had not occurred and RuNPs s had good stability in PBS and cell culture medium. The photothermal effect of our RuNPs was measured by irradiating 1 mL RuNPs aqueous suspension with an 808 nm laser (2 W cm 2) for 10 min at a mass concentration of 10 lg mL 1. Changes in temperature were detected with a thermocouple microprobe. As depicted in Fig. 1D, pure water did not show a significant temperature change even after irradiation for 10 min. In contrast, the temperature of RuNPs aqueous suspension increased with the extension of irradiation time, reaching at 61.3 °C after10 min. The rising of temperature in RuNPs aqueous suspension was mapped by real-time thermal imaging using a thermal camera
329
which was shown in Fig. 1E. The color of RuNPs aqueous suspension changed from dark red to bright yellow, indicating the temperature of suspension rapidly increased with the extension of irradiation time. Next, we detected the photothermal effect of RuNPs at different mass concentrations. As shown in Fig. 1F, the rate of temperature rise and the final temperature of RuNPs aqueous suspension were proportional to the mass concentration. These results suggested that the superior photothermal efficiency of RuNPs exhibited provided potential for thermal ablation of tumor tissues. The photothermal conversion efficiency (g) was measured, which used the method developed by Wang [39], at the same time, gold nanorods was used as a photothermal reference. The output of energy was determined by switching off the laser after the RuNPs and gold nanorods aqueous suspension reached a steady state and measuring the temperature decay curve. As shown in Fig. 2A and B, the average size of gold nanorods we synthesized was 50.4 nm in length and 9.7 nm in width, the longitude plasmon resonance wavelength of Au nanorods is centered at 800 nm. As shown in Fig. 2C and D, when 808 nm laser was used as the light source, the temperature curve for RuNPs was similar to that for gold nanorods. The g value of RuNPs was found to be 53.2%, which was lower than that of gold nanorods (87.5%). However, when switch the laser to 660 nm, the rate of temperature rise for RuNPs was slightly higher than that for gold nanorods, and the g value of RuNPs was found to be 60.7%, which was slightly lower than that of gold nanorods (67.4%). As we know, the extinction capability of metal nanoparticles would be significantly enhanced at longitude plasmon resonance wavelength. Thus the extinction capability of gold nanorods was decreased when the light source switch from 808 nm to 660 nm.
Fig. 2. (A) TEM imagine of Au nanorods. (B) UV vis absorption spectra of Gold nanorods. (C) and (D) is temperature plots of the heating and cooling process for RuNPs and Gold nanorods dispersion illuminated by 808 and 660 nm laser (2 W cm 2).
330
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
3.2. Preparation, characterization, and photothermal effect of TfRuNPs Tf conjugated RuNPs with carboxyl of thioglycolate, which has been pre-modified with RuNPs gave the advantage in the method of EDC/NHS mediated acylation such that the by-products, and excess EDC and NHS, can be easily separated from the reaction product by centrifugation and washing in water. The structures of RuNPs, RuNPs-COOH and Tf-RuNPs were evaluated and monitored by transmission electron microscopy (TEM), dynamic light scattering (DLS), FT-IR, UV vis absorption spectra. Fig. 3A shows the TEM images of RuNPs-COOH and Tf-RuNPs: all these nanoparticles presented monodisperse spherical structures. Particle sizes were measured by dynamic light scattering (DLS) as shown in Fig. 3B, the average diameters of the two types of nanoparticles were respectively 74 ± 4.7 and 121 ± 14.5 nm. Compared with the TEM data, the size of Tf-RuNPs measured by DLS was slightly larger than that found by TEM because of different detection condition principles applicable to these two characterizations. TEM images are captured when nanoparticles are in a dried state, whereas DLS returns the hydrodynamic size of nanoparticles. Similar results were found elsewhere [40,41]. In Fig. 3C, the zeta potential of bare RuNPs and
RuNPs-COOH were respectively 1.35 mV and 8.18 mV, the decrease of zeta potential probably was due to the negativecharged of COO groups. The zeta potential kept decreasing to t22.6 mV with the conjugation of Tf onto the RuNPs. These results demonstrated the existence of Tf in our system. Next, Tf-RuNPs were characterized by FTIR to confirm the chemical structure. As shown in Fig. 3D, the FTIR data of RuNPs-COOH showed characteristic peaks 1710 cm 1 and 3452 cm 1 which were assigned to the stretching vibration of CAO and C@O, respectively. The spectrum of Tf-RuNPs exhibited peaks at 1654 cm 1 and 1542 cm 1 corresponded to the first and secondary ACOANHA groups of Tf; furthermore, the Tf conjugated RuNPs were confirmed by UV vis absorbance spectrometry, as shown in Fig. 3E. The Tf-RuNPs had a peak at 280 nm which is the characteristic absorption peak of Tf. Also the BCA protein assay (data not shown), showed that the content of modified Tf on the RuNPs was 12.2 wt%, which further confirmed that Tf has been successfully connected to the RuNPs. The photothermal effect of Tf-RuNPs was also measured by irradiating 1 mL of aqueous suspension with an 808 nm laser (2 W cm 2) for 10 min. To obtain an equal amount of Ru, the mass concentration of RuNPs and Tf-RuNPs were adjust to 10 lg mL 1 and 11.4 lg mL 1, respectively. As shown in Fig. 3F, the
Fig. 3. (A) TEM images of (a) RuNPs-COOH, (b) Tf-RuNPs. (B) Size distributions and (C) zeta potential of RuNPs, RuNPs-COOH and Tf-RuNPs. (D) FT-IR spectra of Tf, RuNPsCOOH and Tf-RuNPs. (E) UV vis absorption spectra of Tf and Tf-RuNPs. (F) Photothermal effect of RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) with an 808 nm NIR laser (2 W cm 2). (G) IR Thermal images of RuNPs, Tf-RuNPs aqueous suspension and distilled water after 10 min of 808 nm laser irradiation (2 W cm 2). Data are shown as mean ± SD (n = 3).
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
temperature increase of Tf-RuNPs was slightly lower than that of RuNPs: this indicated that Tf modification had negligible influence on thermo-activity. Thermal images of RuNPs, Tf-RuNPs and water after 10 min of irradiation were also mapped (Fig. 3G). 3.3. Cellular uptake of Tf-RuNPs Non-selective cellular uptake of drugs still be an obstacle for cancer therapy. Specific Tf/TfR-mediated targeted therapy may provide a promising strategy to solve this problem. Thus, first we examined the expression level of the TfR in cancer and normal cells. The cancer cell A549 and normal cell HEK-293 were used as model cell. As shown in Fig. 4A, the expression levels of TfR in A549 cells significantly higher than that in HEK-293 cells. These results provide a structural basis for Tf-guided selective absorption between cancer and normal cells. Next, we detected the cellular uptake of RuNPs and Tf-RuNPs in A549 cells and HEK-293 cells by using ICP-MS analysis. As shown in Fig. 4B, after 24 h incubation, cellular uptake of RuNPs and Tf-RuNPs showed no obvious difference in HEK-293 cells, this could be due to relatively low expression levels of TfR on the cell membrane which will not induce Tf/TfR-mediated endocytosis. However, the cellular uptake of Tf-RuNPs is almost 3-folds higher than that of RuNPs in A549 cells. We were also able to show the cellular localization of RuNPs and Tf-RuNPs in A549 cells by TEM image (Fig. 4C). After 24 h incubation, both RuNPs and Tf-RuNPs could be found in lysosomal compartments, but the internalized RuNPs of Tf-RuNPs treatment effect is evidently higher than that of RuNPs . These results suggested that Tf modification could significantly enhance the cellular uptake of RuNPs.
331
The aforementioned results indicated that Tf-RuNPs were transported into cells via the pathway of endocytosis, thus we next determined the endocytosis pathway of Tf-RuNPs. As is known, there are three main endocytic pathways for nanoparticles: macropinocytosis, clathrin-mediated endocytosis, and caveolaemediated endocytosis [42]. In this study, we used three different types of endocytosis inhibitor to ascertain which endocytosis pathway was taken by Tf-RuNPs. We used 5 mM MBCD to inhibit caveolae-mediated endocytosis, 0.45 M hypertonic sucrose to inhibit clathrin-mediated endocytosis, and 5 mM cytochalasin D to inhibit macropinocytosis. As shown in Fig. 4D, when treated with hypertonic sucrose, the cellular uptake of Tf-RuNPs notably decreased to 20.8% of control, indicating that the clathrinmediated endocytosis was the main pathway; moreover, MBCD treatment decreased the Tf-RuNPs uptake to 57.8% of control, suggesting caveolae-mediated was involved in the endocytic pathway of Tf-RuNPs. However, the cellular uptake of Tf-RuNPs was only slightly decrease after bening treated with cytochalasin D. Based on different inhibition effects, we can obviously make the conclusion that the internalisation of Tf-RuNPs was mainly dependent on clathrin-mediated endocytosis. As we know, TfR bind to Tf with bound ferric ion through clathrin-coated vesicles [43], since the main pathway of Tf-RuNPs cellular uptake was the clathrinmediated endocytosis, we confirm that the relatively high cellular uptake of Tf-RuNPs was attributed to the binding of Tf to the TfR. 3.4. Photothermal effects of Tf-RuNPs in vitro The excellent biocompatibility of nanoparticles is very important to the cancer therapy. The biocompatibility of gold nanorods,
Fig. 4. ((A) Tf receptor (TfR) expression in A549 and HEK-293 cells. The expression level of TfR was evaluated by western blot analysis. (B) Quantitative analysis of Ru concentrations in A549 and HEK293 cells exposed to RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h by the ICP-MS method. (C) TEM images of A549 cells treated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h, respectively. Internalized RuNPs indicated by the arrows. (D) Ru concentrations in A549 cells under different endocytosis-inhibited conditions. Before the incubation of 11.4 lg mL 1 Tf-RuNPs, cells were incubated with specific endocytosis inhibitors at different periods of time treatment respectively. Control group incubated with 11.4 lg mL 1 Tf-RuNPs only. Data are shown as mean ± SD (n = 3).
332
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
Fig. 5. (A) Viability of HEK293 cells after incubated with different concentrations of gold nanorods (AuNRs), RuNPs and Tf-RuNPs for 24 h. (B) Viability of A549 cells treated with RuNPs and Tf-RuNPs upon NIR laser irradiation for 0, 1 and 2 min. (C) CLSM images of A549 cells incubated with RuNPs and Tf-RuNPs upon 808 nm NIR irradiation for 2 min. Viable cells were stained green with AO, dead cells were stained yellow. Scale bar: 50 lm. Data are shown as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
RuNPs and Tf-RuNPs was determined by MTT assay on Human Embryonic Kidney 293 (HEK293) cells (Fig. 5A). The cell viability of RuNPs and Tf-RuNPs was 84.3% and 82.2%, even at a concentration of 200 lg mL 1, respectively. However, it was observed that gold nanorods exert a higher cytotoxic effect than RuNPs and TfRuNPs, for instance only 38.7% cell viability at a concentration of 200 lg mL 1. These results indicated that both RuNPs and TfRuNPs had excellent biocompatibility compare with gold nanorods. To evaluate the efficacy of the PTT of RuNPs and Tf-RuNPs, A549 cells were incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1). After incubated for 24 h, the cells were irradiated with an 808 nm laser for a certain time. As shown in Fig. 5B, cells without laser irradiation underwent no change in cell viability: those incubated with RuNPs or Tf-RuNPs did. One hand, When cells were incubated with Tf-RuNPs, the cell viability was reduced to 18.3% after 1 min irradiation, and only 3.1% after 2 min. On the other hand, the cell viability when cells incubated with RuNPs was67.7% and 23.4% after 1 and 2 min irradiation, respectively: an approximately three- to fourfold increase over that using TfRuNPs incubation. Furthermore, the cell viability was measured with live (green)/dead (yellow) kit by CLSM, which could detect differences in cell viability though different treatments. As shown in Fig. 5C, no matter whether RuNPs or Tf-RuNPs were used, nanoparticles alone cannot lead to cell death without irradiation. However, after irradiated for 2 min, there were only about five living cells appearing in the picture after incubation with Tf-RuNPs, in contrast, a considerable amount of living cells appeared after incubation with RuNPs. In competition binding experiments, the addi-
tion of Tf significantly decreased the yellow fluorescence of TfRuNPs-treated cells. Combined with the results of earlier cellular uptake experiments, Tf modification increased the intracellular concentration of Ru nanoparticles, resulting in a better photothermal effect. Thus, Tf-RuNPs could be an effective platform for killing cancer cell by photothermal therapy. 3.5. Photothermal effects in vivo To evaluate the targeting ability of Tf-RuNPs, the biodistribution of RuNPs and Tf-RuNPs which were intravenously injected into A549 tumor-bearing nude mice were investigated. After injection with RuNPs and Tf-RuNPs for 24 h, tumor-bearing nude mice were sacrificed . subsequently, the concentration of Ru distributed in the major organs and tumors, which was measured using ICP-MS. As shown in Fig. 6A, a relatively larger fraction of administered TfRuNPs was preferentially accumulated ahead of Ru in the tumors tissue compared to that of RuNPs, which was due to the receptor-ligand interaction between Tf and TfR over-expressing cells. Meantime, certain amount of the Ru present accumulated in liver in both RuNPs and Tf-RuNPs treatments, which was due to the strong phagocytosis in the reticuloendothelial system (RES) organs [44,45]. Finally, we assessed the in vivo antitumor efficacy of Tf-RuNPs, the A549 tumor-bearing nude mice model was used. After the tumor size reached approximately 200 mm3, the photothermal efficacy was studied in three groups of A549 tumor-bearing mice, with tumor size differences minimised among the groups. The average body weight of each mice and average volume of each
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
333
Fig. 6. (A) Biodistribution of RuNPs and Tf-RuNPs in main organs and tumor at 24 h post-injection. Ru concentrations were analyzed by ICP-MS. (B) The average mouse body mass was measured using an electronic balance every three days. (C) In vivo photothermal tumor heating. IR thermal images of tumor-bearing mice treated with saline, RuNPs, and Tf-RuNPs upon 808 nm NIR laser irradiation for 5 min (1 W cm 2). (D) Tumor volumes at different times after tumor inoculation. The tumor volumes were calculated using V = lw2/2. (E) Dislodged tumors removed from mice across five groups after last injection. (F) H&E stained images of heart, liver, spleen, lung, and kidney collected from saline-, RuNPs-, and Tf-RuNPs-treated mice bearing A549 tumors 15 days post injection. Scale bar: 50 lm. Data are shown as mean ± SD (n = 5).
tumor was monitored every 3 days. As shown in Fig. 6B, compared with saline groups, body mass showed no significant difference after RuNPs, and Tf-RuNPs treatments, which indicated that both RuNPs and Tf-RuNPs had good biocompatibility in vivo. The photothermal response of Tf-RuNPs to tumors was assessed by monitoring their relative tumor volumes, a thermal camera was used to monitor the temperature changes in tumor regions. As shown in Fig. 6C and D, after 5 min of NIR laser irradiation (1 W cm 2), the tumors treated with saline exhibited moderately increase to 38.7 °C, and tumors exhibited a rapid increase in size. For treatment with Tf-RuNPs and NIR laser exposure (1 W cm 2, 5 min), the temperature in tumor regions increased rapidly to 57.1 °C, which was high enough to ablate the tumor cells, and tumors were eliminated completely within 12 days. In contrast, for treatment with RuNPs and NIR laser exposure (1 W cm 2, 5 min), the temperature in tumor regions only reached 44.8 °C and the average tumor volume decreased to about 84.5 mm3 after 15 days treatment. The different temperature changes in tumor regions were deemed likely to have been due to the different amounts of accumulated Ru in the tumors regions. After treatment for 15 days, the mice were sacrificed, and the tumors were harvested and photographed (Fig. 6E), no solid tumor tissue were found subcutaneously which indicated the excellent effect of photothermal therapy with Tf-RuNPs. And the major
organs were thus collected for histology analysis. No noticeable sign of organ damage or tumor metastasis was observed from H&E stained organ slices (Fig. 6F), suggesting negligible side effects of RuNPs and Tf-RuNPs for in vivo PTT. Taken together, with low toxicity and high cell elimination capability in vivo, Tf-RuNPs could be used as an efficient PTT agent for tumor therapy.
4. Conclusion In this work, we firstly investigated the photothermal effect of RuNPs. As a new photothermal agent, RuNPs showed the high absorption under NIR irradiation and the efficient heat transformation for photothermal therapy. We successfully synthesised Ru nanoparticles modified by Tf, and then we investigated their photothermal cancer treatment effect in vitro and in vivo. the modification of Tf remarkably enhanced cellular uptake of RuNPs via caveolae-mediated endocytosis and clathrin-mediated endocytosis. In cell assay, Tf-RuNPs exhibited less cytotoxicity to HEK-293 and A549 cells. In vitro cancer cell ablation and in vivo xenograft tumor treatment led to significant cell death and 100% tumor elimination, without observing significant toxic side-effects after treatment, which verified the Tf-RuNPs were superior photothermal agents for photothermal tumor ablation therapy. Thus, Tf-RuNPs
334
would be a application.
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
potential
photothermal
agent
for
antitumor
Acknowledgements This work was supported by the National Natural Science Foundation of China (21171070, 21371075), the Natural Science Foundation of Guangdong Province (2014A030311025) and the Planned Item of Science and Technology of Guangdong Province (2016A020217011). References [1] A.M. Alkilany, L.B. Thompson, S.P. Boulos, P.N. Sisco, C.J. Murphy, Gold nanorods: their potential for photothermal therapeutics and drug delivery, tempered by the complexity of their biological interactions, Adv. Drug Deliv. Rev. 64 (2012) 190–199. [2] D. Sun, Y. Liu, Q. Yu, X. Qin, L. Yang, Y. Zhou, et al., Inhibition of tumor growth and vasculature and fluorescence imaging using functionalized rutheniumthiol protected selenium nanoparticles, Biomaterials 35 (2014). 1572-1538. [3] Q. Cheng, H. Shi, H. Huang, Z. Cao, J. Wang, Y.Z. Liu, Oral delivery of a platinum anticancer drug using lipid assisted polymeric nanoparticles, Chem. Commun. 51 (2015) 17536–17539. [4] P.K. Jain, IH. ES, MA. ES, Au nanoparticles target cancer, Nano Today 2 (2007) 18–29. [5] J. Nam, N. Won, H. Jin, H. Chung, S. Kim, pH-induced aggregation of gold nanoparticles for photothermal cancer therapy, J. Am. Chem. Soc. 131 (2009) 13639–13645. [6] K. Dong, Z. Liu, Z.H. Li, J.S. Ren, X.G. Qu, Hydrophobic anticancer drug delivery by a 980 nm laser-driven photothermal vehicle for efficient synergistic therapy of cancer cells in vivo, Adv. Mater. 25 (2013) 4452–4458. [7] X. Yang, X. Liu, Z. Liu, F. Pu, J.S. Ren, X.G. Qu, Near-infrared light-triggered, targeted drug delivery to cancer cells by aptamer gated nanovehicles, Adv. Mater. 24 (2012) 2890–2895. [8] H. Cui, P. Zhang, W. Wang, et al., Near-infrared (NIR) controlled reversible cell adhesion on a responsive nano-biointerface, Nano Res. 10 (4) (2017) 1345– 1355. [9] Y. Yang, F. Liu, X. Liu, B. Xing, NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles, Nanoscale 5 (2013) 231–238. [10] H. Zhang, H. Wu, J. Wang, Y. Yang, D. Wu, Y. Zhang, Y. Zhang, Z. Zhou, S.P. Yang, Graphene oxide-BaGdF 5 nanocomposites for multi-modal imaging and photothermal therapy, Biomaterials 42 (2015) 66–77. [11] X. Huang, S. Tang, X. Mu, Y. Dai, G. Chen, Z. Zhou, et al., Freestanding palladium nanosheets with plasmonic and catalytic properties, Nat. Nano 6 (2011) 28– 32. [12] J. Chen, C. Glaus, R. Laforest, Q. Zhang, M. Yang, M. Gidding, et al., Gold nanocages as photothermal transducers for cancer treatment, Small 6 (2010) 811–817. [13] W. Wang, H. Cui, P. Zhang, J. Meng, F. Zhang, S. Wang, Efficient capture of cancer cells by their replicated surfaces reveals multiscale topographic interactions coupled with molecular recognition, ACS Appl. Mater. Interfaces 9 (12) (2017) 10537–10543. [14] J.W. Xiao, S.X. Fan, F. Wang, L.D. Sun, X.Y. Zheng, C.H. Yan, Porous Pd nanoparticles with high photothermal conversion efficiency for efficient ablation of cancer cells, Nanoscale 6 (2014) 4345–4351. [15] Z. Sheng, D. Hu, M. Zheng, P. Zhao, H. Liu, D. Gao, et al., Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy, ACS Nano 8 (2014) 12310–12322. [16] X. Du, Z. Li, Y. Liu, S.P. Yang, Y. Cui, Chiral porous metal–organic frameworks containing l-oxo-bis [Ti (salan)] units for asymmetric cyanation of aldehydes, Dalton Trans. 44 (2015) 12999–13002. [17] H.K. Moon, S.H. Lee, H.C. Choi, ACS Nano 3 (2009) 3707–3713. [18] Y. Chen, J.L. Shi, Mesoporous carbon biomaterials, Sci. China Mater. 58 (2015) 241–257. [19] Q. Zhang, F. Liu, K.T. Nguyen, X. Ma, X. Wang, B. Xing, Y. Zhao, Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery, Adv. Func. Mater. 22 (2012) 5144–5156. [20] G. Li, G. Yang, P. Zhang, et al., Rapid cell patterning induced by differential topography on silica nanofractal substrates, Small 11 (2015) 5642–5646.
[21] M. Hu, J. Chen, Z.Y. Li, L. Au, G.V. Hartland, X. Li, et al., Gold nanostructures: engineering their plasmonic properties for biomedical applications, Chem. Soc. Rev. 35 (2006) 1084–1094. [22] H. Yuan, A.M. Fales, T. Vo-Dinh, TAT peptide-functionalized gold nanostars: enhanced intracellular delivery and efficient NIR photothermal therapy using ultra-low irradiance, J. Am. Chem. Soc. 134 (2012) 11358–11361. [23] Y. Lee, H. Lee, Y.B. Kim, J. Kim, T. Hyeon, H. Park, et al., Bioinspired surface immobilization of hyaluronic acid on monodisperse magnetite nanocrystals for targeted cancer imaging, Adv. Mater. 20 (2008) 4154–4157. [24] J.H. Lee, K. Lee, S.H. Moon, Y. Lee, T.G. Park, J. Cheon, . All-in-One target-cellspecific magnetic nanoparticles for simultaneous molecular imaging and siRNA delivery, J Angew. Chem. 121 (2009) 4238–4243. [25] Z.M. Qian, H. Li, H. Sun, K. Ho, Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway, Pharmacol. Rev. 54 (2002) 561–587. [26] Y. Wu, D.S. Wang, Y.D. Li, Understanding of the major reactions in solution synthesis of functional nanomaterials, Sci. China Mater. 59 (2016) 938–996. [27] R. Prior, G. Reifenberger, W. Wechsler, Transferrin receptor expression in tumours of the human nervous system: relation to tumour type, grading and tumour growth fraction, Virchows Arch. A 416 (1990) 491–496. [28] J. Wu, Y. Lin, H. Li, Q. Jin, J. Ji, Zwitterionic stealth peptide-capped 5aminolevulinic acid prodrug nanoparticles for targeted photodynamic therapy, J. Colloid Interface Sci. 485 (2017) 251–259. [29] M. Zhen, C. Shu, J. Li, et al., A highly efficient and tumor vascular-targeting therapeutic technique with size-expansible gadofullerene nanocrystals, Sci. China Mater. 58 (2015) 799. [30] J. Wang, S. Tian, R.A. Petros, M.E. Napier, J.M. DeSimone, The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies, J. Am. Chem. Soc. 132 (2010) 11306–11313. [32] M. Zenke, P. Steinlein, E. Wagner, M. Cotten, H. Beug, M.L. Birnstiel, Receptormediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells, Proc. Natl. Acad. Sci. 87 (1990) 3655– 3659. [33] J.Y. Yhee, S.J. Lee, S. Lee, S. Song, H.S. Min, S.W. Kang, et al., Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumorbearing mice, Bioconjugate Chem. 24 (2013) 1850–1860. [34] S. Zhao, L.T. Ma, C.W. Cao, Q.Q. Yu, L.M. Chen, J. Liu, Curcumin-loaded redox response of self-assembled micelles for enhanced antitumor and antiinflammation efficacy, Int. Nanomed. 12 (2017) 2489–2504. [35] J. Mou, Y. Chen, M. Ma, K. Zhang, C.Y. Wei, H. Chen, J.L. Shi, Facile synthesis of liposome/Cu2 xS-based nanocomposite for multimodal imaging and photothermal therapy, Sci. China Mater. 58 (2015) 294–301. [36] J. Chen, B. Wiley, Z.Y. Li, D. Campbell, F. Saeki, H. Cang, L. Au, J. Lee, X. Li, Y. Xia, Gold nanocages: engineering their structure for biomedical applications, Adv. Mater. 17 (18) (2005) 2255–2261. [37] F. Zhang, Y. Jiang, X. Liu, et al., Hierarchical nanowire arrays as threedimensional fractal nano-biointerfaces for high efficient capture of cancer cells, Nano Lett. 16 (1) (2016) 766. [38] Q. Shao, X. Li, P. Hua, G. Zhang, Y. Dong, J. Jiang, Enhancing the upconversion luminescence and photothermal conversion properties of ～800 nm excitable core/shell nanoparticles by dye molecule sensitization, J. Colloid Interface Sci. 486 (2017) 121–127. [39] J.L. Shen, H.-C. Kim, C.F. Mu, E. Gentile, J.H. Mai, J. Wolfram, L.-N. Ji, M. Ferrari, Z.-W. Mao, H.F. Shen, Multifunctional gold nanorods for siRNA gene silencing and photothermal therapy, Adv. Healthcare Mater. 3 (2014) 1629–1637. [40] Z.H. Li, L. Zhu, Q. Liu, Y. Du, F. Wang, A facile approach to synthesize SiO2 Re2O3 (Re = Y, Eu, La, Sm, Tb, Pr) hollow sphere and its application in drug release, Nanoscale Res. Lett. 8 (2013) 435–445. [41] Y. Cui, H. Dong, X. Cai, D. Wang, Y. Li, Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release, ACS App. Mater. Interfaces 4 (2012) 3177–3183. [42] O.P. Perumal, R. Inapagolla, S. Kannan, R.M. Kannan, The effect of surface functionality on cellular trafficking of dendrimers, Biomaterials 29 (2008) 3469–3476. [43] B.D. Grant, J.G. Donaldson, Pathways and mechanisms of endocytic recycling, Nat. Rev. Mol. Cell Biol. 10 (597–608) (2009) 597–608. [44] L. Guo, D.D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, et al., Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles, ACS Nano 8 (2014) 5670–5681. [45] H.S.S. Qhattal, T. Hye, A. Alali, X. Liu, Hyaluronan polymer length, grafting density, and surface poly (ethylene glycol) coating influence in vivo circulation and tumor targeting of hyaluronan-grafted liposomes, ACS Nano 8 (2014) 5423–5440.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis
Regular Article
Transferrin modified ruthenium nanoparticles with good biocompatibility for photothermal tumor therapy Shuang Zhao 1, Xufeng Zhu 1, Chengwen Cao, Jing Sun, Jie Liu ⇑ Department of Chemistry, Jinan University, Guangzhou 510632, China
g r a p h i c a l a b s t r a c t Schematic illustration of the preparation of Tf modified RuNPs. Cell uptake of Tf-RuNPs is mediated by Tf receptor-mediated endocytosis. Upon the NIR laser irradiation, the internalized Tf-RuNPs can expeditious convert the absorbed light energy to local heat for PTT.
a r t i c l e
i n f o
Article history: Received 2 July 2017 Revised 1 October 2017 Accepted 6 October 2017 Available online 7 October 2017 Keywords: Transferrin Ru nanoparticles Tumor target Photothermal therapy
a b s t r a c t In the past two decades, there were various kinds of photothermal agents being synthesised and investigated for their photothermal effect in antitumor applications. However, it is barely reported that the photothermal effect of Ruthenium (Ru) nanoparticles was researched in depth. In this work, we introduced Ru nanoparticles which possess excellent biocompatibility and metabolize easily to the photothermal therapy field. In addition, to improve the cells capacity of absorbing Ru nanoparticles, these Ru nanoparticles were modified by transferrin (Tf-RuNPs). Subsequently, as is expected, the RuNPs exhibit a remarkably integrated and high-quality photothermal property. On the other hand, it is significantly that Tf modification could also strengthen the cells absorptive ability to uptake Ru nanoparticles through endocytosis., Furthermore, both the in vitro cell ablation and in vivo tumor treatment verified that the TfRuNPs became ideal photothermal agents for photothermal tumor ablation therapy owing to their low toxicity and high cell destruction capability. Ó 2017 Published by Elsevier Inc.
1. Introduction ⇑ Corresponding author. 1
E-mail address: [email protected] (J. Liu). Both authors contribute equally to this work.
https://doi.org/10.1016/j.jcis.2017.10.023 0021-9797/Ó 2017 Published by Elsevier Inc.
Nowadays, the growing and aging of world population may be responsible for the increasing cancer-causing behaviours. After
326
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
all, cancer is still a disturbing and global health problem [1–3]. There is one thing that demands our focal attention that cancers were conventionally treated by traditional methods such as chemotherapy, surgery, radiation therapy, etc [4]. Unfortunately, these methods are restricted because of the adverse effects. In recent years, a growing number of scientists focus on the exploitation of nanomaterials for photothermal therapy (PTT), a minimally invasive and selective treatment of cancer. In this PTT protocol, photo-absorbing nanomaterials convert photon energy to heat, producing a local hyperthermia to burn cancer cells and tissues. As for the light source, near-infrared (NIR, 700 to 1100 nm) laser is being widely used. Because it can reach several centimetres in biological tissues due to the relatively low absorption of hemoglobin and water as well as less damage to normal tissues [5]. Hence, strong absorption in the NIR region of nanomaterials will be beneficial for the application of PTT. There are quiet a few, different types of photothermal agents, such as metal nanostructures [6–14], organic compounds [15,16], carbon materials [17–19]. Actually, a multitude of them have been investigated up to now. There are quiet a few different shapes of gold, for typical examples, nanospheres, nanorods, nanoshells, and nanocages, obviously, all of them will bewell-suited to various biomedical applications [20,21]. Even though, almost all the researches pay excessive attention on the narrow range of metal elements like gold, silver, or palladium. On the contrary, studies on the light and heat properties of the precious metal ruthenium (Ru) are rarely reported. According to this, we have studied the photothermal effect of ruthenium nanoparticles with simple spherical morphology. Gold nanomaterials feature the high cost, poor bioassay and thermal stability, while carbon nanomaterials featurepoorly dispersible and may induce oxidative stress as well as immune response, contrarily, the ruthenium nanoparticles have good biocompatibility and exhibit outstanding photothermal effect. Insufficient intracellular particle delivery may be the main obstacle that prevents the using of metal nanoparticles in the fight against cancerous cells [22]. Considering this, nanoscale modification technology was invented timely. More specifically, it is a
promising pathway to enhance the accumulation of nanoparticles. Different ligands have been modified into nanoparticles and significantly enhanced the cellular ability of absorbing via receptormediated pathways, such as folic acid [23], hyaluronic acid [24], peptides [25], etc. Transferrin (Tf), as a serum protein which is non-toxic, non-immunogenic, and biodegradable: it has been widely studied as a cancer-targeting agent [26]. There is an ocean of Transferrin receptor (TfR) existing on the surface of proliferating and malignant cancer cells [27–29]. Tf/TfR-mediated endocytosis has been proved to be an effective strategy to enhance the nanoparticles accumulation in tumor sites [30]. A host of investigations have indicated that different material modified by Tf extends the blood circulation time and achieves a high selectivity for cancer cells, [32,33]. Collectively, the purpose of this study was to investigate the activity of Ruthenium (Ru) nanoparticles in photothermal therapy against cancer cells. In view of this, we envisage to make Tf modified on the surface of Ru nanoparticles. so that the synthesis of Ru nanoparticles would get prone to be absorbed by cells (Scheme 1). Beyond that, the cellular uptake mechanisms of TfRuNPs were detected in cell assays. Also, the photothermal effects of Tf-RuNPs on A549 cells were measured by MTT assay. Furthermore, we observed and assessed the photothermal effects of TfRuNPs on tumor-bearing mice. Afterwards, it is demonstrated that Tf-RuNPs exhibit excellent capability to kill cancer cells, with less side effects owing to the use of Tf-RuNPs. Simply put, these new nanoparticles could be successfully used for highly efficient in vivo PTT under NIR irradiation.
2. Materials and methods 2.1. Materials All reagents and solvents were purchased commercially and used without further purification unless specially noted, and Ultrapure MilliQ water (18.2 MW) was used in all experiments. polyvinyl pyrrolidone (PVP), Cetyltrimethylammonium bromide
Scheme 1. Schematic illustration of the preparation of Tf modified RuNPs. Cell uptake of Tf-RuNPs is mediated by Tf receptor-mediated endocytosis. Upon the NIR laser irradiation, the internalized Tf-RuNPs can expeditious convert the absorbed light energy to local heat for PTT.
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
(CTAB), HAuCl43H2O, 1-(3-Dimethylaminopropyl)-3-ethylcarbodii mide hydrochloride (EDC), RuCl3nH2O, NaBH4, thioglycolate, NHydroxysuccinimide (NHS), thiazolyl blue tetrazolium bromide (MTT), DAPI, Transferrin were purchased Sigma-Aldrich Chemical Co.
327
2 W cm 2 for 10 min. The temperatures of the suspension were recorded by a thermocouple microprobe immersed in the suspension. Photothermal effect of Tf-RuNPs and water were performed at the same parameters. Infrared thermal images for different NPs suspension and water were taken by Fluke T125 Thermal Imaging Camera.
2.2. Preparation of RuNPs, Tf-RuNPs and gold nanorods 2.5. Cell lines and cell culture In a typical synthesis, 0.1434 g RuCl3nH2O dissolved in 10 mL water (0.5 mM), and 0.5828 g of polyvinylpyrrolidone (PVP) was then added, followed an ice cold NaBH4 solution (0.5 mL, 0.1 M) was dropwise added to mixture under stirring. The mixture then continued stirring for another 30 min. To remove the excess reactants, the reaction mixture was then extensively centrifuged (9000 rpm, 5 min), and washed three times with water to obtain the RuNPs (solid). For Tf conjugation, firstly, 8 mg of thioglycolate was added to 10 mL of 10 mM PBS (pH 8.5) containing 5 mg of RuNPs and stirring at room temperature for 24 h, then the reaction mixture centrifuged (9000 rpm, 5 min) and washed with water for three times to remove excess thioglycolate, then thioglycolate decorated RuNPs (RuNPs-COOH) were obtained . Secondly, the RuNPsCOOH redispersed in 10 mL water containing 50 lL of EDC and 125 lL NHS and stirring for 1 h. At last, the mixture solution mixed with of 0.5 mL transferrin (Tf) solution (5 mg mL 1) and stirring for 24 h at 4 °C. The excess Tf, EDC and NHS were removed by extensively centrifugation (9000 rpm, 5 min) and washed with water for three times. The obtained Tf-RuNPs were dispersed in water for further use. The concentration of Ru ions was determined by ICPMS analysis. Gold nanorods were synthesized following a reported method. Briefly, ice-cold NaBH4 (10 mM, 0.6 mL) was add to a solution containing CTAB (100 mM, 7.5 mL) and HAuCl43H2O (10 mM, 0.25 mL) with vigorous stirring for 10 min, the gold seeds were prepared. To prepared gold nanorods, HAuCl43H2O (10 mM, 16 mL), CTAB (100 mM, 80 mL), AgNO3 (10 mM, 0.5 mL), and ascorbic acid (10 mM, 6 mL) were mixed in a 150 mL conical flask. Then seed solution (0.2 mL) was added with gentle stirring, and the entire solution was left still for 10 h. The gold nanorods were collect by centrifugation (9000 rpm, 5 min) and washed by water 3 times. 2.3. Characterization of Tf-RuNPs The as-prepared Tf-RuNPs was characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), UV–vis spectroscopy, dynamic light scattering (DLS) and BCA assays. TEM samples were prepared by placing one drops of nanoparticles suspension onto a holey carbon film on copper grids. The micrographs were obtained on Hitachi (H7650) instrument for TEM operating at an accelerating voltage at 80 kV. The size distribution and zeta potential of the nanoparticles were measured by PCS on a Nano-ZS instrument (Malvern Instruments Limited) at least three times. FTIR (Equinox 55 IR spectrometer) and UV–vis (Carry 5000 spectrophotometer) studies were recorded in the range of 4000–500 cm 1 and 200–900 nm, respectively. Bicinchoninic acid (BCA) kit (Pierce) used to evaluate Tf content was conducted followed by manufacturer’s instructions. The NIR laser for photothermal experiments is an 808 nm high power NIR laser diode (LSR808NL-2010). 2.4. Photothermal performance To study the photothermal effect under 808 nm laser irradiation, 1 mL aqueous suspension containing 10 lg of RuNPs, 10 lg of gold nanorods or 11.4 lg of Tf-RuNPs were placed in square cuvette and irradiated with an 808 nm NIR laser at a power density of
The human lung adenocarcinoma cell lines A549 and Human Embryonic Kidney 293 (HEK-293) cell lines were used in this study were obtained from the Experimental Animal Center at Sun YatSen University. All cell lines were maintained in DMEM media supplemented with penicillin (100 units mL 1), fetal bovine serum (10%), and streptomycin (50 units mL 1) at 37 °C in a humidified incubator with 5% CO2 atmosphere. 2.6. Cellular uptake of Tf-RuNPs in vitro The cellular uptake of nanoparticles was monitored by ICP-MS method. Briefly, A549 and HEK-293 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with or without RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h, respectively. After incubation, the cells were washed with PBS three times and harvested, and digested with 3 mL of concentrated nitric acid and 1 mL of H2O2 in a digestive stove at 150 °C for 2 h. The digested product was diluted to 8 mL with water and used for ICP-MS analysis. To detect the mechanisms of cellular uptake of Tf-RuNPs, A549 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with 5 mM MBCD, 0.45 M hypertonic sucrose and 5 mM cytochalasin D for 1 h before incubated with Tf-RuNPs. The control samples were without the addition of inhibitors. The cells were then incubated with Tf-RuNPs (11.4 lg mL 1) for 24 h. After treatment, the cells were then washed with PBS for three times and analyzed by ICP-MS. 2.7. Intracellular trafficking of Tf-RuNPs A549 cells were seeded in 6-well tissue culture plates at a density of 1 105 cells per well and cultured for 24 h, then incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h then fixed using 3% glutaraldehyde and dehydrated using graded ethanol. TEM samples were sectioned by microtome in Araldite resin and observed on a Hitachi (H-7650) instrument for TEM operating at an accelerating voltage at 80 kV. 2.8. Western blot analysis A549 and HEK-293 cells were incubated with lysis buffer (Beyotime) to obtain total cellular proteins. The protein concentration was examined by BCA assay. Equal amount of proteins were electrophoresed in 15% tricine gels and then transferred to nitrocellulose membrane and blocked with 5% non-fat milk in Tris-Buffered Saline (TBS) buffer overnight. Then the membranes were washed with Tris-Buffered Saline Tween-20 (TBST) buffer three times and incubated with primary antibodies s at 1:1000 dilution in TBS at room temperature for 2 h with continuous agitation. Then the membranes were washed with TBST three times and incubated with secondary antibodies conjugated with horseradish peroxidase at 1:2000 dilution for 1 h at room temperature, followed by 5 times washing with TBST. Protein bands were visualized on X-ray film using enhanced chemiluminescence detection regents (Pierce Biotech, Rockford, IL), b-Actin was used to confirm the comparable amount of proteins in each lane.
328
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
2.9. Photothermal performance of RuNPs and Tf-RuNPs in vitro To evaluate biocompatibility of gold nanorods, RuNPs and TfRuNPs, HEK-293 cells were seeded in 96-well tissue culture plates at 2 103 cells per well and cultured for 24 h. Then cells were incubated with gold nanorods, RuNPs and Tf-RuNPs at different concentrations for 24 h. After treatment, 20 lL per well of MTT solution (5 mg mL 1 phosphate buffered saline) was added to the well and incubated for another 2 h. Next the medium was replaced with 150 lL DMSO per well to dissolve the formazan salt formed. The cell viability was reflected by the color intensity of the formazan solution. Absorbance at 570 nm was taken on a 96-well microplate reader. For PTT in vitro, A549 cells were seeded in 96-well tissue culture plates at 2 103 cells per well and cultured for 24 h. Then the cells were incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h. Subsequently, the treated cells were replaced with fresh culture media to remove the excess NPs and exposed to an 808 nm NIR laser (2 W cm 2) for 1 or 2 min. And the cell viability of each sample were measured by MTT assay. In addition, after NIR laser irradiation, the cells were stained by AO/ EB reaction mixture for 20 min and washed with PBS for three times and observed under CLSM to directly observe the live and dead cells.
in control groups were injected with the same volume of saline. The tumors of mice were followed irradiated by NIR laser for 5 min at power density of 1 W cm 2 and simultaneously imaged by a thermal camera. The mice body weights and tumor sizes were recorded every 3 days, and the tumor volume were calculated as Vtumor = length width2/2. All animal procedures were in accord with the guidelines of the Institutional Animal Care and Use Committee. After 15 days of treatments, all the mice were sacrificed, and their tumors and major organs (heart, liver, spleen, lung and kidney) were dissected, washed with saline and fixed in 4% formaldehyde for histological examination. 2.11. Biodistribution assays of Tf-RuNPs For biodistribution study, ten tumor-bearing mice (tumor size 200 mm3) were divided into two groups. Two group was intravenously injected with RuNPs (0.1 mL, 1 mg mL 1) and TfRuNPs (0.1 mL, 11.4 mg mL 1), respectively. After 24 h, the organs (heart, liver, spleen, lung, kidneys) and tumors were removed. The Ru were quantified through ICP-MS method. The tissues were cut into pieces and digested with 3 mL of concentrated nitric acid and 1 mL of H2O2 in a digestive stove at 150 °C for 2 h. The digested product was diluted to 8 mL with water and used for ICP-MS analysis.
2.10. Antitumor activity in vivo 3. Results and discussion The 5–6 week-old severe combined immune deficiency (SCID) male mice (ordered from NIH) weighing 22 g were divided into groups with five mice per group. The animal tumor model was generated by subcutaneous injected with A549 cells (1 107 cells per mouse) into the mice into the right armpit region of nude mice. For PTT in vivo, after the tumors had become established (200 mm3), the mice were intravenous injected with 20 lL of RuNPs (1 mg mL 1) and Tf-RuNPs (11.4 mg mL 1) suspension. The mice
3.1. Preparation, characterization, and photothermal effect of RuNPs The RuNPs were synthesised by a simple preparation procedure mentioned above. Fig. 1A showed TEM image of the RuNPs with a uniform size about 70 nm. To detect the absorption properties of RuNPs, the UV–vis-NIR absorbance of the RuNPs were measured (in water) at the mass concentration of 10 lg mL 1 (Fig. 1B). The
Fig. 1. (A) TEM image and (B) UV–vis-NIR absorbance spectra of of RuNPs. (C) Stability of RuNPs in PBS (pH = 7.4) and cell culture medium. (D) Photothermal effect of RuNPs at a mass concentration of 10 lg mL 1 with an 808 nm NIR laser (2 W cm 2), the inset shows the final temperature of Tf-RuNPs aqueous suspension and distilled water after 10 min irradiation. (E) IR thermal images for RuNPs after irradiation for 0, 1, 5 and 10 min. (F) Photothermal effect of RuNPs at different concentration with an 808 nm NIR laser (2 W cm 2).
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
RuNPs in water showed monotonically rising absorbance from the NIR to the UV–vis region, although the surface plasmon resonance of RuNPs is not located in the vis-NIR region, but the notable absorbance of RuNPs in the NIR region was deemed to be beneficial for the practice of PTT. Since the RuNPs showed broad absorption through the entire NIR regions, various NIR laser could be chosen for PTT. An 808 nm laser was used as the light source in this work. To quantify the absorption property of RuNPs, the molar extinction coefficient was calculated using the method as described in previous reports [14,34]. TEM data showed RuNPs with a spherical-like shape and the size of 70 nm, the molar extinction coefficient was calculated to be 1.16 108 M 1 cm 1 at 808 nm : this value is remarkable compared with many PTT agents such as porous Pd (6.3 107) [14], carbon material (8.2 106) [18], Cu2 xSe (7.7 107) [35], but lower than that of Au nanohexapods (5.5 109) [36], Pd nanosheets (4.1 109) [6] and Au nanocages (3.2 1010) [12,37,38]. The stability of nanoparticles is also very important for its application. As shown in Fig. 1C, the size of RuNPs did not increase in both PBS and cell culture medium, indicating that. aggregation had not occurred and RuNPs s had good stability in PBS and cell culture medium. The photothermal effect of our RuNPs was measured by irradiating 1 mL RuNPs aqueous suspension with an 808 nm laser (2 W cm 2) for 10 min at a mass concentration of 10 lg mL 1. Changes in temperature were detected with a thermocouple microprobe. As depicted in Fig. 1D, pure water did not show a significant temperature change even after irradiation for 10 min. In contrast, the temperature of RuNPs aqueous suspension increased with the extension of irradiation time, reaching at 61.3 °C after10 min. The rising of temperature in RuNPs aqueous suspension was mapped by real-time thermal imaging using a thermal camera
329
which was shown in Fig. 1E. The color of RuNPs aqueous suspension changed from dark red to bright yellow, indicating the temperature of suspension rapidly increased with the extension of irradiation time. Next, we detected the photothermal effect of RuNPs at different mass concentrations. As shown in Fig. 1F, the rate of temperature rise and the final temperature of RuNPs aqueous suspension were proportional to the mass concentration. These results suggested that the superior photothermal efficiency of RuNPs exhibited provided potential for thermal ablation of tumor tissues. The photothermal conversion efficiency (g) was measured, which used the method developed by Wang [39], at the same time, gold nanorods was used as a photothermal reference. The output of energy was determined by switching off the laser after the RuNPs and gold nanorods aqueous suspension reached a steady state and measuring the temperature decay curve. As shown in Fig. 2A and B, the average size of gold nanorods we synthesized was 50.4 nm in length and 9.7 nm in width, the longitude plasmon resonance wavelength of Au nanorods is centered at 800 nm. As shown in Fig. 2C and D, when 808 nm laser was used as the light source, the temperature curve for RuNPs was similar to that for gold nanorods. The g value of RuNPs was found to be 53.2%, which was lower than that of gold nanorods (87.5%). However, when switch the laser to 660 nm, the rate of temperature rise for RuNPs was slightly higher than that for gold nanorods, and the g value of RuNPs was found to be 60.7%, which was slightly lower than that of gold nanorods (67.4%). As we know, the extinction capability of metal nanoparticles would be significantly enhanced at longitude plasmon resonance wavelength. Thus the extinction capability of gold nanorods was decreased when the light source switch from 808 nm to 660 nm.
Fig. 2. (A) TEM imagine of Au nanorods. (B) UV vis absorption spectra of Gold nanorods. (C) and (D) is temperature plots of the heating and cooling process for RuNPs and Gold nanorods dispersion illuminated by 808 and 660 nm laser (2 W cm 2).
330
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
3.2. Preparation, characterization, and photothermal effect of TfRuNPs Tf conjugated RuNPs with carboxyl of thioglycolate, which has been pre-modified with RuNPs gave the advantage in the method of EDC/NHS mediated acylation such that the by-products, and excess EDC and NHS, can be easily separated from the reaction product by centrifugation and washing in water. The structures of RuNPs, RuNPs-COOH and Tf-RuNPs were evaluated and monitored by transmission electron microscopy (TEM), dynamic light scattering (DLS), FT-IR, UV vis absorption spectra. Fig. 3A shows the TEM images of RuNPs-COOH and Tf-RuNPs: all these nanoparticles presented monodisperse spherical structures. Particle sizes were measured by dynamic light scattering (DLS) as shown in Fig. 3B, the average diameters of the two types of nanoparticles were respectively 74 ± 4.7 and 121 ± 14.5 nm. Compared with the TEM data, the size of Tf-RuNPs measured by DLS was slightly larger than that found by TEM because of different detection condition principles applicable to these two characterizations. TEM images are captured when nanoparticles are in a dried state, whereas DLS returns the hydrodynamic size of nanoparticles. Similar results were found elsewhere [40,41]. In Fig. 3C, the zeta potential of bare RuNPs and
RuNPs-COOH were respectively 1.35 mV and 8.18 mV, the decrease of zeta potential probably was due to the negativecharged of COO groups. The zeta potential kept decreasing to t22.6 mV with the conjugation of Tf onto the RuNPs. These results demonstrated the existence of Tf in our system. Next, Tf-RuNPs were characterized by FTIR to confirm the chemical structure. As shown in Fig. 3D, the FTIR data of RuNPs-COOH showed characteristic peaks 1710 cm 1 and 3452 cm 1 which were assigned to the stretching vibration of CAO and C@O, respectively. The spectrum of Tf-RuNPs exhibited peaks at 1654 cm 1 and 1542 cm 1 corresponded to the first and secondary ACOANHA groups of Tf; furthermore, the Tf conjugated RuNPs were confirmed by UV vis absorbance spectrometry, as shown in Fig. 3E. The Tf-RuNPs had a peak at 280 nm which is the characteristic absorption peak of Tf. Also the BCA protein assay (data not shown), showed that the content of modified Tf on the RuNPs was 12.2 wt%, which further confirmed that Tf has been successfully connected to the RuNPs. The photothermal effect of Tf-RuNPs was also measured by irradiating 1 mL of aqueous suspension with an 808 nm laser (2 W cm 2) for 10 min. To obtain an equal amount of Ru, the mass concentration of RuNPs and Tf-RuNPs were adjust to 10 lg mL 1 and 11.4 lg mL 1, respectively. As shown in Fig. 3F, the
Fig. 3. (A) TEM images of (a) RuNPs-COOH, (b) Tf-RuNPs. (B) Size distributions and (C) zeta potential of RuNPs, RuNPs-COOH and Tf-RuNPs. (D) FT-IR spectra of Tf, RuNPsCOOH and Tf-RuNPs. (E) UV vis absorption spectra of Tf and Tf-RuNPs. (F) Photothermal effect of RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) with an 808 nm NIR laser (2 W cm 2). (G) IR Thermal images of RuNPs, Tf-RuNPs aqueous suspension and distilled water after 10 min of 808 nm laser irradiation (2 W cm 2). Data are shown as mean ± SD (n = 3).
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
temperature increase of Tf-RuNPs was slightly lower than that of RuNPs: this indicated that Tf modification had negligible influence on thermo-activity. Thermal images of RuNPs, Tf-RuNPs and water after 10 min of irradiation were also mapped (Fig. 3G). 3.3. Cellular uptake of Tf-RuNPs Non-selective cellular uptake of drugs still be an obstacle for cancer therapy. Specific Tf/TfR-mediated targeted therapy may provide a promising strategy to solve this problem. Thus, first we examined the expression level of the TfR in cancer and normal cells. The cancer cell A549 and normal cell HEK-293 were used as model cell. As shown in Fig. 4A, the expression levels of TfR in A549 cells significantly higher than that in HEK-293 cells. These results provide a structural basis for Tf-guided selective absorption between cancer and normal cells. Next, we detected the cellular uptake of RuNPs and Tf-RuNPs in A549 cells and HEK-293 cells by using ICP-MS analysis. As shown in Fig. 4B, after 24 h incubation, cellular uptake of RuNPs and Tf-RuNPs showed no obvious difference in HEK-293 cells, this could be due to relatively low expression levels of TfR on the cell membrane which will not induce Tf/TfR-mediated endocytosis. However, the cellular uptake of Tf-RuNPs is almost 3-folds higher than that of RuNPs in A549 cells. We were also able to show the cellular localization of RuNPs and Tf-RuNPs in A549 cells by TEM image (Fig. 4C). After 24 h incubation, both RuNPs and Tf-RuNPs could be found in lysosomal compartments, but the internalized RuNPs of Tf-RuNPs treatment effect is evidently higher than that of RuNPs . These results suggested that Tf modification could significantly enhance the cellular uptake of RuNPs.
331
The aforementioned results indicated that Tf-RuNPs were transported into cells via the pathway of endocytosis, thus we next determined the endocytosis pathway of Tf-RuNPs. As is known, there are three main endocytic pathways for nanoparticles: macropinocytosis, clathrin-mediated endocytosis, and caveolaemediated endocytosis [42]. In this study, we used three different types of endocytosis inhibitor to ascertain which endocytosis pathway was taken by Tf-RuNPs. We used 5 mM MBCD to inhibit caveolae-mediated endocytosis, 0.45 M hypertonic sucrose to inhibit clathrin-mediated endocytosis, and 5 mM cytochalasin D to inhibit macropinocytosis. As shown in Fig. 4D, when treated with hypertonic sucrose, the cellular uptake of Tf-RuNPs notably decreased to 20.8% of control, indicating that the clathrinmediated endocytosis was the main pathway; moreover, MBCD treatment decreased the Tf-RuNPs uptake to 57.8% of control, suggesting caveolae-mediated was involved in the endocytic pathway of Tf-RuNPs. However, the cellular uptake of Tf-RuNPs was only slightly decrease after bening treated with cytochalasin D. Based on different inhibition effects, we can obviously make the conclusion that the internalisation of Tf-RuNPs was mainly dependent on clathrin-mediated endocytosis. As we know, TfR bind to Tf with bound ferric ion through clathrin-coated vesicles [43], since the main pathway of Tf-RuNPs cellular uptake was the clathrinmediated endocytosis, we confirm that the relatively high cellular uptake of Tf-RuNPs was attributed to the binding of Tf to the TfR. 3.4. Photothermal effects of Tf-RuNPs in vitro The excellent biocompatibility of nanoparticles is very important to the cancer therapy. The biocompatibility of gold nanorods,
Fig. 4. ((A) Tf receptor (TfR) expression in A549 and HEK-293 cells. The expression level of TfR was evaluated by western blot analysis. (B) Quantitative analysis of Ru concentrations in A549 and HEK293 cells exposed to RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h by the ICP-MS method. (C) TEM images of A549 cells treated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1) for 24 h, respectively. Internalized RuNPs indicated by the arrows. (D) Ru concentrations in A549 cells under different endocytosis-inhibited conditions. Before the incubation of 11.4 lg mL 1 Tf-RuNPs, cells were incubated with specific endocytosis inhibitors at different periods of time treatment respectively. Control group incubated with 11.4 lg mL 1 Tf-RuNPs only. Data are shown as mean ± SD (n = 3).
332
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
Fig. 5. (A) Viability of HEK293 cells after incubated with different concentrations of gold nanorods (AuNRs), RuNPs and Tf-RuNPs for 24 h. (B) Viability of A549 cells treated with RuNPs and Tf-RuNPs upon NIR laser irradiation for 0, 1 and 2 min. (C) CLSM images of A549 cells incubated with RuNPs and Tf-RuNPs upon 808 nm NIR irradiation for 2 min. Viable cells were stained green with AO, dead cells were stained yellow. Scale bar: 50 lm. Data are shown as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
RuNPs and Tf-RuNPs was determined by MTT assay on Human Embryonic Kidney 293 (HEK293) cells (Fig. 5A). The cell viability of RuNPs and Tf-RuNPs was 84.3% and 82.2%, even at a concentration of 200 lg mL 1, respectively. However, it was observed that gold nanorods exert a higher cytotoxic effect than RuNPs and TfRuNPs, for instance only 38.7% cell viability at a concentration of 200 lg mL 1. These results indicated that both RuNPs and TfRuNPs had excellent biocompatibility compare with gold nanorods. To evaluate the efficacy of the PTT of RuNPs and Tf-RuNPs, A549 cells were incubated with RuNPs (10 lg mL 1) and Tf-RuNPs (11.4 lg mL 1). After incubated for 24 h, the cells were irradiated with an 808 nm laser for a certain time. As shown in Fig. 5B, cells without laser irradiation underwent no change in cell viability: those incubated with RuNPs or Tf-RuNPs did. One hand, When cells were incubated with Tf-RuNPs, the cell viability was reduced to 18.3% after 1 min irradiation, and only 3.1% after 2 min. On the other hand, the cell viability when cells incubated with RuNPs was67.7% and 23.4% after 1 and 2 min irradiation, respectively: an approximately three- to fourfold increase over that using TfRuNPs incubation. Furthermore, the cell viability was measured with live (green)/dead (yellow) kit by CLSM, which could detect differences in cell viability though different treatments. As shown in Fig. 5C, no matter whether RuNPs or Tf-RuNPs were used, nanoparticles alone cannot lead to cell death without irradiation. However, after irradiated for 2 min, there were only about five living cells appearing in the picture after incubation with Tf-RuNPs, in contrast, a considerable amount of living cells appeared after incubation with RuNPs. In competition binding experiments, the addi-
tion of Tf significantly decreased the yellow fluorescence of TfRuNPs-treated cells. Combined with the results of earlier cellular uptake experiments, Tf modification increased the intracellular concentration of Ru nanoparticles, resulting in a better photothermal effect. Thus, Tf-RuNPs could be an effective platform for killing cancer cell by photothermal therapy. 3.5. Photothermal effects in vivo To evaluate the targeting ability of Tf-RuNPs, the biodistribution of RuNPs and Tf-RuNPs which were intravenously injected into A549 tumor-bearing nude mice were investigated. After injection with RuNPs and Tf-RuNPs for 24 h, tumor-bearing nude mice were sacrificed . subsequently, the concentration of Ru distributed in the major organs and tumors, which was measured using ICP-MS. As shown in Fig. 6A, a relatively larger fraction of administered TfRuNPs was preferentially accumulated ahead of Ru in the tumors tissue compared to that of RuNPs, which was due to the receptor-ligand interaction between Tf and TfR over-expressing cells. Meantime, certain amount of the Ru present accumulated in liver in both RuNPs and Tf-RuNPs treatments, which was due to the strong phagocytosis in the reticuloendothelial system (RES) organs [44,45]. Finally, we assessed the in vivo antitumor efficacy of Tf-RuNPs, the A549 tumor-bearing nude mice model was used. After the tumor size reached approximately 200 mm3, the photothermal efficacy was studied in three groups of A549 tumor-bearing mice, with tumor size differences minimised among the groups. The average body weight of each mice and average volume of each
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
333
Fig. 6. (A) Biodistribution of RuNPs and Tf-RuNPs in main organs and tumor at 24 h post-injection. Ru concentrations were analyzed by ICP-MS. (B) The average mouse body mass was measured using an electronic balance every three days. (C) In vivo photothermal tumor heating. IR thermal images of tumor-bearing mice treated with saline, RuNPs, and Tf-RuNPs upon 808 nm NIR laser irradiation for 5 min (1 W cm 2). (D) Tumor volumes at different times after tumor inoculation. The tumor volumes were calculated using V = lw2/2. (E) Dislodged tumors removed from mice across five groups after last injection. (F) H&E stained images of heart, liver, spleen, lung, and kidney collected from saline-, RuNPs-, and Tf-RuNPs-treated mice bearing A549 tumors 15 days post injection. Scale bar: 50 lm. Data are shown as mean ± SD (n = 5).
tumor was monitored every 3 days. As shown in Fig. 6B, compared with saline groups, body mass showed no significant difference after RuNPs, and Tf-RuNPs treatments, which indicated that both RuNPs and Tf-RuNPs had good biocompatibility in vivo. The photothermal response of Tf-RuNPs to tumors was assessed by monitoring their relative tumor volumes, a thermal camera was used to monitor the temperature changes in tumor regions. As shown in Fig. 6C and D, after 5 min of NIR laser irradiation (1 W cm 2), the tumors treated with saline exhibited moderately increase to 38.7 °C, and tumors exhibited a rapid increase in size. For treatment with Tf-RuNPs and NIR laser exposure (1 W cm 2, 5 min), the temperature in tumor regions increased rapidly to 57.1 °C, which was high enough to ablate the tumor cells, and tumors were eliminated completely within 12 days. In contrast, for treatment with RuNPs and NIR laser exposure (1 W cm 2, 5 min), the temperature in tumor regions only reached 44.8 °C and the average tumor volume decreased to about 84.5 mm3 after 15 days treatment. The different temperature changes in tumor regions were deemed likely to have been due to the different amounts of accumulated Ru in the tumors regions. After treatment for 15 days, the mice were sacrificed, and the tumors were harvested and photographed (Fig. 6E), no solid tumor tissue were found subcutaneously which indicated the excellent effect of photothermal therapy with Tf-RuNPs. And the major
organs were thus collected for histology analysis. No noticeable sign of organ damage or tumor metastasis was observed from H&E stained organ slices (Fig. 6F), suggesting negligible side effects of RuNPs and Tf-RuNPs for in vivo PTT. Taken together, with low toxicity and high cell elimination capability in vivo, Tf-RuNPs could be used as an efficient PTT agent for tumor therapy.
4. Conclusion In this work, we firstly investigated the photothermal effect of RuNPs. As a new photothermal agent, RuNPs showed the high absorption under NIR irradiation and the efficient heat transformation for photothermal therapy. We successfully synthesised Ru nanoparticles modified by Tf, and then we investigated their photothermal cancer treatment effect in vitro and in vivo. the modification of Tf remarkably enhanced cellular uptake of RuNPs via caveolae-mediated endocytosis and clathrin-mediated endocytosis. In cell assay, Tf-RuNPs exhibited less cytotoxicity to HEK-293 and A549 cells. In vitro cancer cell ablation and in vivo xenograft tumor treatment led to significant cell death and 100% tumor elimination, without observing significant toxic side-effects after treatment, which verified the Tf-RuNPs were superior photothermal agents for photothermal tumor ablation therapy. Thus, Tf-RuNPs
334
would be a application.
S. Zhao et al. / Journal of Colloid and Interface Science 511 (2018) 325–334
potential
photothermal
agent
for
antitumor
Acknowledgements This work was supported by the National Natural Science Foundation of China (21171070, 21371075), the Natural Science Foundation of Guangdong Province (2014A030311025) and the Planned Item of Science and Technology of Guangdong Province (2016A020217011). References [1] A.M. Alkilany, L.B. Thompson, S.P. Boulos, P.N. Sisco, C.J. Murphy, Gold nanorods: their potential for photothermal therapeutics and drug delivery, tempered by the complexity of their biological interactions, Adv. Drug Deliv. Rev. 64 (2012) 190–199. [2] D. Sun, Y. Liu, Q. Yu, X. Qin, L. Yang, Y. Zhou, et al., Inhibition of tumor growth and vasculature and fluorescence imaging using functionalized rutheniumthiol protected selenium nanoparticles, Biomaterials 35 (2014). 1572-1538. [3] Q. Cheng, H. Shi, H. Huang, Z. Cao, J. Wang, Y.Z. Liu, Oral delivery of a platinum anticancer drug using lipid assisted polymeric nanoparticles, Chem. Commun. 51 (2015) 17536–17539. [4] P.K. Jain, IH. ES, MA. ES, Au nanoparticles target cancer, Nano Today 2 (2007) 18–29. [5] J. Nam, N. Won, H. Jin, H. Chung, S. Kim, pH-induced aggregation of gold nanoparticles for photothermal cancer therapy, J. Am. Chem. Soc. 131 (2009) 13639–13645. [6] K. Dong, Z. Liu, Z.H. Li, J.S. Ren, X.G. Qu, Hydrophobic anticancer drug delivery by a 980 nm laser-driven photothermal vehicle for efficient synergistic therapy of cancer cells in vivo, Adv. Mater. 25 (2013) 4452–4458. [7] X. Yang, X. Liu, Z. Liu, F. Pu, J.S. Ren, X.G. Qu, Near-infrared light-triggered, targeted drug delivery to cancer cells by aptamer gated nanovehicles, Adv. Mater. 24 (2012) 2890–2895. [8] H. Cui, P. Zhang, W. Wang, et al., Near-infrared (NIR) controlled reversible cell adhesion on a responsive nano-biointerface, Nano Res. 10 (4) (2017) 1345– 1355. [9] Y. Yang, F. Liu, X. Liu, B. Xing, NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles, Nanoscale 5 (2013) 231–238. [10] H. Zhang, H. Wu, J. Wang, Y. Yang, D. Wu, Y. Zhang, Y. Zhang, Z. Zhou, S.P. Yang, Graphene oxide-BaGdF 5 nanocomposites for multi-modal imaging and photothermal therapy, Biomaterials 42 (2015) 66–77. [11] X. Huang, S. Tang, X. Mu, Y. Dai, G. Chen, Z. Zhou, et al., Freestanding palladium nanosheets with plasmonic and catalytic properties, Nat. Nano 6 (2011) 28– 32. [12] J. Chen, C. Glaus, R. Laforest, Q. Zhang, M. Yang, M. Gidding, et al., Gold nanocages as photothermal transducers for cancer treatment, Small 6 (2010) 811–817. [13] W. Wang, H. Cui, P. Zhang, J. Meng, F. Zhang, S. Wang, Efficient capture of cancer cells by their replicated surfaces reveals multiscale topographic interactions coupled with molecular recognition, ACS Appl. Mater. Interfaces 9 (12) (2017) 10537–10543. [14] J.W. Xiao, S.X. Fan, F. Wang, L.D. Sun, X.Y. Zheng, C.H. Yan, Porous Pd nanoparticles with high photothermal conversion efficiency for efficient ablation of cancer cells, Nanoscale 6 (2014) 4345–4351. [15] Z. Sheng, D. Hu, M. Zheng, P. Zhao, H. Liu, D. Gao, et al., Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy, ACS Nano 8 (2014) 12310–12322. [16] X. Du, Z. Li, Y. Liu, S.P. Yang, Y. Cui, Chiral porous metal–organic frameworks containing l-oxo-bis [Ti (salan)] units for asymmetric cyanation of aldehydes, Dalton Trans. 44 (2015) 12999–13002. [17] H.K. Moon, S.H. Lee, H.C. Choi, ACS Nano 3 (2009) 3707–3713. [18] Y. Chen, J.L. Shi, Mesoporous carbon biomaterials, Sci. China Mater. 58 (2015) 241–257. [19] Q. Zhang, F. Liu, K.T. Nguyen, X. Ma, X. Wang, B. Xing, Y. Zhao, Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery, Adv. Func. Mater. 22 (2012) 5144–5156. [20] G. Li, G. Yang, P. Zhang, et al., Rapid cell patterning induced by differential topography on silica nanofractal substrates, Small 11 (2015) 5642–5646.
[21] M. Hu, J. Chen, Z.Y. Li, L. Au, G.V. Hartland, X. Li, et al., Gold nanostructures: engineering their plasmonic properties for biomedical applications, Chem. Soc. Rev. 35 (2006) 1084–1094. [22] H. Yuan, A.M. Fales, T. Vo-Dinh, TAT peptide-functionalized gold nanostars: enhanced intracellular delivery and efficient NIR photothermal therapy using ultra-low irradiance, J. Am. Chem. Soc. 134 (2012) 11358–11361. [23] Y. Lee, H. Lee, Y.B. Kim, J. Kim, T. Hyeon, H. Park, et al., Bioinspired surface immobilization of hyaluronic acid on monodisperse magnetite nanocrystals for targeted cancer imaging, Adv. Mater. 20 (2008) 4154–4157. [24] J.H. Lee, K. Lee, S.H. Moon, Y. Lee, T.G. Park, J. Cheon, . All-in-One target-cellspecific magnetic nanoparticles for simultaneous molecular imaging and siRNA delivery, J Angew. Chem. 121 (2009) 4238–4243. [25] Z.M. Qian, H. Li, H. Sun, K. Ho, Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway, Pharmacol. Rev. 54 (2002) 561–587. [26] Y. Wu, D.S. Wang, Y.D. Li, Understanding of the major reactions in solution synthesis of functional nanomaterials, Sci. China Mater. 59 (2016) 938–996. [27] R. Prior, G. Reifenberger, W. Wechsler, Transferrin receptor expression in tumours of the human nervous system: relation to tumour type, grading and tumour growth fraction, Virchows Arch. A 416 (1990) 491–496. [28] J. Wu, Y. Lin, H. Li, Q. Jin, J. Ji, Zwitterionic stealth peptide-capped 5aminolevulinic acid prodrug nanoparticles for targeted photodynamic therapy, J. Colloid Interface Sci. 485 (2017) 251–259. [29] M. Zhen, C. Shu, J. Li, et al., A highly efficient and tumor vascular-targeting therapeutic technique with size-expansible gadofullerene nanocrystals, Sci. China Mater. 58 (2015) 799. [30] J. Wang, S. Tian, R.A. Petros, M.E. Napier, J.M. DeSimone, The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies, J. Am. Chem. Soc. 132 (2010) 11306–11313. [32] M. Zenke, P. Steinlein, E. Wagner, M. Cotten, H. Beug, M.L. Birnstiel, Receptormediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells, Proc. Natl. Acad. Sci. 87 (1990) 3655– 3659. [33] J.Y. Yhee, S.J. Lee, S. Lee, S. Song, H.S. Min, S.W. Kang, et al., Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumorbearing mice, Bioconjugate Chem. 24 (2013) 1850–1860. [34] S. Zhao, L.T. Ma, C.W. Cao, Q.Q. Yu, L.M. Chen, J. Liu, Curcumin-loaded redox response of self-assembled micelles for enhanced antitumor and antiinflammation efficacy, Int. Nanomed. 12 (2017) 2489–2504. [35] J. Mou, Y. Chen, M. Ma, K. Zhang, C.Y. Wei, H. Chen, J.L. Shi, Facile synthesis of liposome/Cu2 xS-based nanocomposite for multimodal imaging and photothermal therapy, Sci. China Mater. 58 (2015) 294–301. [36] J. Chen, B. Wiley, Z.Y. Li, D. Campbell, F. Saeki, H. Cang, L. Au, J. Lee, X. Li, Y. Xia, Gold nanocages: engineering their structure for biomedical applications, Adv. Mater. 17 (18) (2005) 2255–2261. [37] F. Zhang, Y. Jiang, X. Liu, et al., Hierarchical nanowire arrays as threedimensional fractal nano-biointerfaces for high efficient capture of cancer cells, Nano Lett. 16 (1) (2016) 766. [38] Q. Shao, X. Li, P. Hua, G. Zhang, Y. Dong, J. Jiang, Enhancing the upconversion luminescence and photothermal conversion properties of ～800 nm excitable core/shell nanoparticles by dye molecule sensitization, J. Colloid Interface Sci. 486 (2017) 121–127. [39] J.L. Shen, H.-C. Kim, C.F. Mu, E. Gentile, J.H. Mai, J. Wolfram, L.-N. Ji, M. Ferrari, Z.-W. Mao, H.F. Shen, Multifunctional gold nanorods for siRNA gene silencing and photothermal therapy, Adv. Healthcare Mater. 3 (2014) 1629–1637. [40] Z.H. Li, L. Zhu, Q. Liu, Y. Du, F. Wang, A facile approach to synthesize SiO2 Re2O3 (Re = Y, Eu, La, Sm, Tb, Pr) hollow sphere and its application in drug release, Nanoscale Res. Lett. 8 (2013) 435–445. [41] Y. Cui, H. Dong, X. Cai, D. Wang, Y. Li, Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release, ACS App. Mater. Interfaces 4 (2012) 3177–3183. [42] O.P. Perumal, R. Inapagolla, S. Kannan, R.M. Kannan, The effect of surface functionality on cellular trafficking of dendrimers, Biomaterials 29 (2008) 3469–3476. [43] B.D. Grant, J.G. Donaldson, Pathways and mechanisms of endocytic recycling, Nat. Rev. Mol. Cell Biol. 10 (597–608) (2009) 597–608. [44] L. Guo, D.D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, et al., Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles, ACS Nano 8 (2014) 5670–5681. [45] H.S.S. Qhattal, T. Hye, A. Alali, X. Liu, Hyaluronan polymer length, grafting density, and surface poly (ethylene glycol) coating influence in vivo circulation and tumor targeting of hyaluronan-grafted liposomes, ACS Nano 8 (2014) 5423–5440.