The Effect of Photothermal Therapy on Osteosarcoma With Polyacrylic Acid–Coated Gold Nanorods

Materials

HAuCl₄•3H₂O (99.99%), AgNO₃ (99.8%), polyacrylic acid (PAA, Molecular weight-15 000 g/mol and MW-45 0000 g/mol), NaBH₄ (98%), cetyltrimethylammonium bromide (99%), and ascorbic acid (99%) which were analytical reagents were purchased from Alfa Aesar (USA). Propidium iodide (PI) and Tris (99.0%) was bought from Sigma (USA). Analytical grade reagents such as NaOH, HCl, hydroquinol (98%), and sodium citrate (99.0%) were used immediately. Annexin V-FITC (Fluorescein isothiocyanate) Apoptosis Detection Kit (Vazyme Biotech Co, Ltd. China). Cell Counting Kit-8 (CCK-8; Dojindo, China), fetal bovine serum (FBS), trypsin, phosphate buffered saline (PBS), and dulbecco-modified eagle medium with high glucose (H-DMEM 12430-054) were purchased from Gibco (USA). Ultrapure water was used in all reagent and material dispensing. Human osteosarcoma cell line (MG63) was purchased from Shanghai Institute for Biological sciences, Chinese Academy of Science.

Preparation of GNRs@PAA

Gold nanorods were prepared according to the seed-mediated growth method by El-Sayed and coworkers. Briefly, a seed solution was prepared by mixing 7.5 mL of CTAB (hexadecyl trimethyl ammonium bromide) (0.1 mol/L) and 2.5 mL of HAuCl4 (1 mmol/L) with 0.6 mL freshly prepared 10 mmol/L NaHB4 solution under vigorous stirring at 28°C. After 2 hours, this seed solution was used for the synthesis of the GNRs. In a flask, 13.2 mL of 0.1 mol/L CTAB was mixed with 12 mL of 1 mmo/L HAuCl₄, then 240μL of 10 mmol/L silver nitrate aqueous solution, and 220 μL of 2 mol/L hydrochloric acid were added to the flask. After gently mixing the solution, 192 μL of 0.1 mol/L ascorbic acid was added. On continuously stirring this mixture, various concentrations of seed solution were added finally to initiate the growth of the GNRs. These nanorods were aged for 5 hours to ensure full growth at 28°C. After preparation, excess CTAB was removed by centrifuging twice at 12 000 rpm for 10 minutes and then redispersed in deionization water. ¹⁵

Polyacrylic acid–coated GNRs were prepared by the versatile layer-by-layer approach. In brief, 2 mg/mL PAA-450 000g/moL was prepared in a 10 mmol/L NaCl solution (which was sonicated previously for 30 minutes). Then, 2 mL GNRs (2×) were added drop wise to the PAA solution (v/v = 1:1), and the mixture was stirred vigorously for 6 hours. The PAA-coated GNRs were separated by centrifugation 3 times to remove excess polyelectrolyte and then redispersed in 2 mL of PBS (pH 7.4). The PAA-15000g/moL–coated GNRs were prepared according to the method by Michael D. Wyatt. ²²

Nanoparticles Characterization

A Ultraviolet (UV)-1800 UV-vis-NIR spectrophotometer (Shimadzu, Japan) was used to detect the spectra of UV visible absorption of all samples for GNRs@PAA preparation ranging from 400 nm to 1100 nm wavelength in common experimental environment with room temperature. Transmission electron microscopy (TEM) images of PAA-coated GNRs were implemented by a Hitachi H-800 electron microscope (Japan) under 200 kV accelerating voltage with a charge coupled device camera. Fourier transform infrared (FTIR) spectra were measured using an IR Affinity-1 spectrophotometer (Avatar, Nicolet, USA). Bright-field and fluorescent mappings of MG63 cells were obtained using an Olympus IX51 inverted fluorescence microscope (Olympus, Japan).

Cell Culture

MG63 human osteosarcoma cells were cultured in H-DMEM and supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were cultivated with humidified atmosphere of 5% CO₂ at 37°C which were split using trypsin medium when cell had grown to approximate 80% confluent in 6-well culture plate.

Cytotoxicity Assay

Colorimetric assay is one of the popular methods for quantifying the percentage of survival cells. The cytotoxicity of GNRs@PAA on MG63 cells were analyzed using the CCK-8 assay in this experiment. Briefly, MG63 cells (5 × 10⁵ cells/mL density) were planted into 96-well plates with 100 μL H-DMEM medium and cultured for 24 hours with humidified atmosphere of 5% CO₂ at 37°C. Furthermore, the MG63 cells were incubated for 24 hours with GNRs@PAA conjugates with different concentrations (20 μg/mL, 40 μg/mL, 80 μg/mL, 120 μg/mL, and 160 μg/mL), and the MG63 cells in the control group were incubated in H-DMEM medium. Then, MG63 cells in all groups were treated with H-DMEM medium including 10% CCK-8 reagent for 1 hour to 4 hours. Finally, the absorbance of CCK-8 was determined at absorption wavelength of 450 nm by a microplate reader. The detected cell viability was presented as a relative percentage to control.

Photothermal Therapy

The efficiency of photothermal therapy with GNRs was investigated by detecting mortality rate of MG63 cells after being irradiated with the continuous wave laser at 808 nm. MG63 cells (5 × 10⁵ cells/mL density) were planted into 96-well plates with 100 μL H-DMEM medium and cultured for 24 hours with humidified atmosphere of 5% CO₂ at 37°C. Then, the MG63 cells were incubated for 24 hours with GNRs@PAA conjugates at 80 μg/mL density, and the cell viabilities were detected by CCK-8 assay after laser irradiation at 808 nm with the power density of 1.5 W/cm² for various times (0 minute, 2 minutes, 5 minutes, 10 minutes, and 15 minutes).

Apoptosis Staining

The images of apoptosis or dead cells after photothermal therapy with GNRs@PAA were recorded using a fluorescence microscope of Olympus (Japan). MG63 cells were cultured in monolayer into 6-well plates with the density of 1 × 10⁵ cell in each well for 24 hours in H-DMEM. Then, GNRs@PAA with 60 μg/mL density were added to each well and coincubated in H-DMEM medium for 24 hours. Furthermore, the unbound GNRs in H-DMEM medium were removed followed by washing 3 times with PBS. And thenMG63 cells were exposed to 808 nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately. The Apoptosis Detection kit with Annexin V-FITC and PI were used to obtain the image of cell. All experiments were done in triplicate.

Apoptosis Detection by Flow Cytometry

MG63 cells were cultured into 6-well plates for 24 hours, and then GNRs@PAA were added to each well in different concentrations (0, 10, 20, 40, 60, and 80 μg/mL) in triplicate and coincubated in H-DMEM medium for 24 hours. Furthermore, the unbound GNRs in H-DMEM medium were removed followed by washing 3 times with PBS. Then, MG63 cells were exposed to 808-nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately. The apoptosis rate of MG63 cells were determined by flow cytometry (FCM) using the Annexin V-FITC apoptosis detection kit flowing the protocol.

Statistical Analysis

Group data were presented as mean ± standard error of the mean. Unpaired t test was used to compare between the groups. The means of multiple groups were compared by analysis of variance followed by Least Significant Difference post hoc test. Differences with a 2-tailed P < .05 were considered statistically significant.

Preparation of GNRs Coated With PAA

Gold nanorods predominantly absorb NIR light because of SPR, and the NIR light can penetrate deeply into human tissues, so GNRs can be used in photothermal therapy of carcinoma in human body. To reduce the toxicity and increase the biocompatibility during photothermal therapy, GNRs were modified with PAA for photothermal ablation in human osteosarcoma cells. Figure 1A primitively illustrated the prepared procedure of GNRs@PAA and photothermal therapy for carcinoma.

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Figure 1.

Scheme of GNRs@PAA photothermal therapy on cells and the characters of GNRs@PAA. A., Scheme indicating the process involved in the preparation of GNRs coated with PAA and photothermal therapy on cells. B., Transmission electron microscopy TEM image of GNRs@PAA. C., Absorption spectroscopy of GNRs@ PAA. D., Fourier transform infraredFTIR spectra of GNRs@PAA. E., The temperature increment of GNRs@PAA.

Transmission Electron Microscopy Image and UV–vis Spectroscopy Test of GNRs@ PAA

In order to study the dispersity of GNRs@PAA in solution such as PBS, the TEM micrograph of the GNRs@PAA is shown in Figure 1B. The results showed that the GNRs@PAA was homogeneous and monodispersed, and the average aspect ratio of the GNRs were around 4.2 (length 44.3 nm and width 10.8 nm). Because the PAA polymer was a thin layer on GNRs, there were no obvious changes between the unmodified nanorods and GNRs@PAA.

The UV–vis spectroscopy test of GNRs@PAA is shown in Figure 1C. The results showed that there was a 3-nm red-shift in the plasmon band maximum caused by the coating of polymer layer. The 810 nm was for CTAB-GNRs, while the 813 nm was for the GNRs@PAA. There was no broadening of plasmon band in the UV–vis spectroscopy of GNRs@PAA.

Fourier Transform Infrared Spectra and Temperature Increment in GNRs@PAA

The FTIR measurements were used to determine the formation of PAA-modified GNRs which is shown in Figure 1D. The results showed that there was an apparent carboxyl band at −1710 cm⁻¹ that proved the successful modification of PAA to GNRs by electrostatic interaction and demonstrated that there was apparently no influence on the position and intensity of NIR absorptions for GNRs.

The temperature increment in GNRs@PAA NPs solution was tested through different power density of NIR laser at 808 nm, and the results are shown in Figure 1E. The results showed that during the test process, by an 808 nm laser with a power density of 0.5, 1.0, 1.5, and 2.0 W/cm², respectively, to irradiate the NPs solution, the temperature increased with the power density and time of irradiation, and the highest temperature could reach 63°C. The result demonstrated that the prepared GNRs@PAA was suitable for further photothermal therapy through hyperthermia.

Cytotoxicity Assay of GNRs@PAA

The biosafety is a fundamental factor to estimate NPs before in vivo application, so the cytotoxicity of GNRs@PAA on MG63 cell lines was detected by CCK-8 assay in this experiment (Figure 2A). Cells incubated with GNRs@PAA with different concentrations (20, 40, 80, 120, and 160 μg/mL) for 24 hours, and the results showed that the cell viability rates were all >80% in 20, 40, 80, and 120 μg/mL groups. It demonstrated that GNRs@PAA had superior biocompatibility.

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Figure 2.

The cytotoxicity and photothermal therapies of GNRs@PAA in MG63 cells A, The cytotoxicity of GNRs@PAA in MG63 cells. B, Photothermal therapies of GNRs@PAA in MG63 Cells. MG63 cells are incubated with 80 μg/mL GNRs@PAA for 24 hours, and the cell viabilities were detected by CCK-8 assay after 808-nm laser irradiation with the power density of 1.5 W/cm² by different times. Data are shown as means ± standard error of the means, *p < 0.05 and ** p < 0.01.

Photothermal Ablation

To study the hyperthermia therapy efficiency of GNRs@PAA in vitro, MG63 cells were cultured with 80 μg/mL GNRs@PAA for 24 hours, and then, the culture medium including redundant GNRs@PAA was removed. Furthermore, cells were irradiated with laser power density of 1.5W/cm² for different periods of 0, 10, 15, and 20 minutes, respectively, followed by CCK-8 assay (Figure 2B). The results demonstrated that the cell viabilities decreased with irradiation time, 55% of the MG63 cells were dead at 5 minutes, while 95% MG63 cells were dead at 15 minutes. It demonstrated that GNRs@PAA had superior efficiency of hyperthermia therapy for human osteosarcoma cell.

Apoptosis Staining

To study the hyperthermia therapy efficiency of GNRs@PAA in vitro, MG63 cells were cultured with GNRs@PAA for 24 hours, and then the unbound GNRs in the medium were removed. Furthermore, MG63 cells were exposed to 808-nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately followed by assay of apoptosis detection kit with Annexin V-FITC and PI as shown in Figure 3. The results demonstrated that there were only several apoptosis cells (representation as green as shown in Figure 3) and modicus necrotic cells (representation as red as shown in Figure 3) in the irradiation area with laser of 808 nm for 2 minutes, and it demonstrated that the GNRs@PAA only caused little injuries to the MG63 cells in this group. Furthermore, compared to the abovementioned group after irradiation with laser at 808 nm for 5 minutes, the “green” apoptosis cells and the “red” necrotic cells increased significantly, and the results demonstrated that the efficiency of hyperthermia therapy by GNRs@PAA on MG63 cell increased with the elongation of irradiation time. Although it was proposed by some researchers that the death of carcinoma cell was caused by DNA damage when hyperthermia conditions got to 42°C to 43°C, the mechanism is still unclear up to now. The cell images in bright field of this experiment indicated that cells irradiated with laser of 808 nm showed plasmalemma disruption including bubble formation and thermic fragmentation. Furthermore, the cell image in dark imaging field showed DNA condensation which represented as high green or high red, and the results indicated that the cell death caused by GNRs@PAA was because of the damaged membrane of MG63 cell and the integrated DNA.

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Figure 3.

Apoptosis staining of MG63 cell. Fluorescent and bright field images of MG63 cells after photothermal therapies by an 808-nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately. The scale bar is 100 μm. Dose –response page 20 of 21.

Quantification of Apoptosis Rate

MG63 cells incubated with GNRs@PAA with different concentration (0, 10, 20, 40, 60, and 80 μg/mL) for 24 hours and were exposed to 808-nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately, followed by apoptosis detection of FCM. The apoptotic rate was significantly increased by GNRs@PAA (0.11% ± 0.12%; 10 μg/mL; 0.23% ± 0.09%; 20 μg/mL; 0.46% ± 0.19%; 40 μg/mL; 8.12% ± 0.32%; 60 μg/mL; 12.02% ± 0.33%; 80 μg/mL; P < .05) in comparison to 0 μg/mL GNRs@PAA for 2 minutes. And the apoptotic rate was significantly increased by GNRs@PAA (0.21% ± 0.18%, 10 μg/mL; 0.18% ± 0.16%, 20 μg/mL; 0.51% ± 0.27%, 40 μg/mL; 10.62% ± 0.41%, 60 μg/mL; 15.34% ± 0.49%, 80 μg/mL; P < .05) compared to 0 μg/mL GNRs@PAA for 5 minutes. The results indicated that the apoptosis rate of MG63 cells had been promoted by in vitro photothermal therapy with GNRs@PAA at 60 and 80 μg/mL concentrations, and it exhibited time–dose dependence (Figure 4).

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Figure 4.

Flow cytometry of apoptosis MG63 cell Aafter Pphotothermal Ttherapies by an 808-nm laser with the power density of 1.5 W/cm² for 2 minutes and 5 minutes separately with GNRs@PAA at different concentration.