Absorption-dependent generation of singlet oxygen from gold bipyramids excited under low power density

Abstract

Metal nanoparticles (MNPs) can be used as a kind of new photodynamic therapy (PDT) agent because singlet oxygen (¹O₂) can be generated through directly sensitizing MNPs. Gold nanorods, gold nanoshells and gold nanoechinus were confirmed to be efficient PDT agents in vivo. However, the major excitation spectra of ¹O₂ from all of them are not in the optical biological window (650–900 nm). Herein, gold bipyramids (GBPs) with tunable absorption wavelength were prepared and used to explore ¹O₂ generation capability. ¹O₂ can be generated when GBPs were excited by continuous-wave light within a wide range of wavelengths (660–975 nm). The highest ¹O₂ generation capabilities were obtained when they were excited at the wavelength of the absorption peak, which was quite different from those of other gold nanomaterials. It was also found that ¹O₂ can be generated efficiently by a laser of very low power density (200 mW cm⁻²). The capability of GBPs for PDT has been demonstrated through the destruction of cancer cells. The synergistic effect of PDT and photothermal therapy for the destruction of cancer cells was also demonstrated.

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Introduction

Photodynamic therapy (PDT) has attracted much attention as an efficient non-invasive treatment for the destruction of various cancers cells and tumors.¹ PDT is a photo-therapeutic modality with three main components (the light, the photosensitizers and the oxygen). When photosensitizers are exposed to light of a specific wavelength, they will generate reactive oxygen species (ROS), especially singlet oxygen (¹O₂).² The ¹O₂ generated by photosensitizers is regarded as the main reason for treatment following light absorption and transfer of the excited state energy to oxygen molecules.^3–5 However, the conventional PDT technology (using the organic photosensitizers) suffers from some drawbacks such as poor instability (including photo-induced degradation and enzymatic degradation) and poor water-solubility.^6,7 In addition, the excitation of organic photosensitizers is always outside the range of the biological transparency window (650–900 nm). This results in significant reduction in light penetration depth and thus reduces the capabilities of organic photosensitizers for PDT.⁸

The use of metal (such as gold and silver) nanoparticles for PDT was reported since the observation that ¹O₂ can be generated directly by metal nanoparticles (MNPs).^9–12 The mechanism may involve plasmon mediated electron emission from the MNPs into the surrounding media that lead to ROS generation.^10–12 Several studies have shown that gold nanorods (GNRs),^11,13,14 gold nanoshells,¹⁵ gold nanoechinus¹⁶ and gold nanocages¹² could generate the ¹O₂ and thus be suited for PDT application. Compared to the organic photosensitizers, the advantages of using MNPs for PDT are as follows: (1) they are far more stable than organic dyes under irradiation and in biological environments; (2) the extinction coefficients of MNPs were much higher than those of the commonly used organic photosensitizers. The high extinction coefficient of MNPs can compensate for the slightly low yield of ¹O₂ and reduce the excitation power to promote the reperfusion of tissue oxygen and compensate for oxygen depletion.^17,18 (3) MNPs are solvable and stable in aqueous media. (4) Thanks to the improvement of synthesis techniques, MNPs can be synthesized easily.¹⁹ The excitation wavelengths of ¹O₂ can be easily tuned to near infrared (NIR). This can highly enhance the penetration depth of excitation light into bio-tissue and improve the in vivo photo-therapy capability. Furthermore, MNPs can be functionalized easily. Therefore, MNPs are expected to be an efficient PDT reagent and will promote the PDT application. However, the reported gold nanomaterials suffer from some disadvantages as PDT agents. Because the region of the biological window reaches a minimum scattering and absorbance for all biomolecules, the use of a laser with an output wavelength in this region such as 800 nm will significantly improve the penetration depth for deep-tissue biotherapy.^20–22 The use of the reported gold nanomaterials and their corresponding excitation wavelength will limit the penetration depth of light at the excitation wavelength. In addition, it limits the choices for the light source (such as the low-cost 800 nm continuous-wave (CW) laser) and thus disallows further clinical PDT applications. Currently, rare metal nanomaterials are used as PDT agents in the biological window.

In this work, we report gold bipyramid (GBP) mediated ¹O₂ generation excited by NIR light and the corresponding PDT application. To the best of our knowledge, the study of ¹O₂ generation using GBPs has not been reported. ¹O₂ can be generated when GBPs are excited by a wide range of light (660–975 nm) that amply overlaps with the biological window. The ¹O₂ generation capabilities of GBPs were found to be absorption-dependent, which is quite different from those of other gold nanomaterials.^11,13 We also demonstrated that ¹O₂ can be generated efficiently by a laser of low power density (200 mW cm⁻²). The PDT effects of GBPs on cancer cells were confirmed by the large reduction of cell viability. Considering the ¹O₂ generation capabilities of GBPs were absorption-dependent, the synergistic effect of PDT and photothermal therapy (PTT) for the destruction of cancer cells was also demonstrated.

Experiment

Materials

9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 99.9%), sodium azide (NaN₃, 99.9%), methylene blue (MB, 99.9%), hydrogen tetrachloroaurate trihydrate (HAuCl₄·4H₂O, 99.9%) and polyacrylic acid (PAA, 99.9%) were purchased from Sigma-Aldrich. Hexadecyl trimethyl ammonium bromide (CTAB, 99.0%), ethanol (99.5%) and methanol (99.7%) were purchased from Sino Chemical Reagent Co., Ltd. Sodium borohydride (NaBH₄, 99%), silver nitrate (AgNO₃, 99%), sodium oleate (NaOL, 96.0%), ascorbic acid (VC, 99.7%), hydrochloric acid (HCl, 37 wt% in water) and sodium citrate (99%) were purchased from Aladdin reagent. Regular agarose G-10 (99.7%) was purchased from BIOWEST, Spain. Cell Counting Kit-8 (CCK-8, 99.9%) was purchased from Beyotime Biotechnology. Dulbecco's modified eagle medium (DMEM, 99%) and fetal bovine serum (FBS, 99%) were purchased from Gioco Life technology. All the chemical reagents were used without further purification, and deionized water was used in all of the experiments.

Growth of gold bipyramids

The procedure of synthesizing GBPs is described as follows.²³ 100 μL of 25 mM HAuCl₄ and 100 μL of 25 mM sodium citrate were added into 9.8 mL of deionized water to prepare the gold seeds, and then the ice-cold, freshly prepared NaBH₄ (0.01 M, 300 μL) was added under vigorous shaking. The as-prepared seed solution was stored for at least 4 hours before use. The preparation of the GBP growth solution according to the following procedure: 14 mL of 0.2 M CTAB solution was added into a flask and then the solution was stirred continuously at 700 rpm. 0.5 mL of 25 mM HAuCl₄ was then added. 60 μL of 16 mM AgNO₃ was then added with continuously stirring at 700 rpm. 0.4 mL of 0.1 M ascorbic acid was then added slowly and the color of the solution turned to colorless within 10 s, the citrate-stabilized seed solution with different volume (360 μL, 280 μL, 200 μL, 120 μL) was then added into the GBPs growth solution. The reaction solution was mixed by inversion for 10 s and then left undisturbed for 12 hours in dark. The color of the solution turned from colorless to wine. GBPs with different sizes and shapes were then obtained. The GBPs with absorption wavelengths of 660 nm, 710 nm, 820 nm and 930 nm were prepared here.

Purification of gold bipyramids

The procedure of purifying GBPs is described as follows.²³ 10 mL of the as-prepared GBP solution were centrifuged at 9000 rpm for 10 min, and then the supernatant were removed. The 660 nm, 710 nm, 820 nm and 930 nm GBPs were added into 10 mL of 0.45 M, 0.40 M, 0.35 M, and 0.15 M CTAB solution, respectively. The mixtures were left undisturbed for 12 h to 24 h, and the sediments were redispersed in deionized water.

Preparation of PAA-coated GBPs

The PAA-coated GBPs were prepared according to the following procedure.¹¹ The purified GBPs were washed thoroughly using deionized water and methanol via centrifugation for 3 min (5500 rpm) to remove the CTAB, and then re-dispersed in 10 mL of an aqueous solution containing hydrochloric acid and ethanol (v/v, 15 : 120). The GBP solution was stirred for 3 h at 60 °C three times. 100 uL of PAA (8 μg mL⁻¹) prepared in a 10 mM NaCl solution was added drop by drop into the 10 mL GBP solution. The mixed solution was stirred at 1500 rpm for 30 min, and then isolated by centrifugation at 5500 rpm twice to remove excess polyelectrolytes.

Growth of gold nanorods

Gold nanorods (GNRs) with tunable absorption wavelength were synthesized via the seed-mediated growth process.²⁴ One typical kind of GNRs with a longitudinal LSPR wavelength at 900 nm in a water solution were synthesized as follows. 5 mL of 0.5 mM HAuCl₄ solution and 5 mL of 0.2 M CTAB solution were mixed uniformly. Then 0.6 mL of fresh 0.01 M NaBH₄ solution was diluted to 1 mL and quickly injected into the mixed solution under rapid stirring (1200 rpm) to grow the gold seeds. The seed solution was stored at room temperature for 30 min before use. The growth solution was prepared as follows. 0.2468 g NaOL was dissolved in 50 mL of the 99 mM CTAB solution at 50 °C, and then the solution was cooled down to 30 °C. 4.8 mL of 4 mM AgNO₃ solution was then added into the solution, and it was kept undisturbed for 15 minutes. Afterwards, 50 mL of 1 mM HAuCl₄ solution was added into the solution and then the solution was stirred continuously for 90 minutes at 700 rpm. 0.3 mL of HCl was then added and the solution was slowly and continuously stirred at 400 rpm for 15 min. 0.25 mL of 0.064 M ascorbic acid was added and the solution was vigorously stirred for 30 s. Finally, 0.08 mL of seed solution was injected into the growth solution, and the resultant mixture was stirred for 30 s and left undisturbed at 30 °C for 12 hours for growth. The products were separated by centrifugation at 7000 rpm for 30 minutes twice, followed by being redispersed in deionized water.

Detection of singlet oxygen generation

The laser-excited ¹O₂ generation was characterized by photo-oxidation of ABDA in water. ABDA can react irreversibly with singlet oxygen, inducing a reduction in the ABDA absorption band around 382 nm.¹³ The solutions containing nanoparticles and ABDA were irradiated by a laser point (660 nm) and CW laser (710 nm, 760 nm, 820 nm, 860 nm, 930 nm and 975 nm, respectively). The power density of the laser point is 200 mW cm⁻², and the power density of the CW laser is tunable. The solutions were irradiated for 15 minutes and their absorbance spectra were recorded at 5 min intervals using a spectrometer.

Singlet oxygen quenching

NaN₃ was used as the ¹O₂ quenching reagent in the water solution.¹⁰ In a typical experiment, 2 mL of GBPs solution containing 150 μL ABDA (0.1 mg mL⁻¹) and 150 μL NaN₃ (50 mM) are placed in a quartz cuvette. All the experimental conditions were the same as above. The absorbance spectra were recorded at 5 min intervals.

Tissue phantom preparation

The tissue phantom was prepared as our previous works.²⁵ In brief, the tissue phantom consisted of 0.7 grams per liter of TiO₂ (as scatterers) and 25 × 10⁻⁶ liters of ink per liter (as absorbers). 0.5% agarose was typically used as phantom matrix material. Finally, the optical properties of the phantom are in the range of human epidermis and dermis tissues by controlling the scattering coefficient to be 0.34 mm⁻¹ and the absorption coefficient to be within a range between 0.006 to 0.009 mm⁻¹.^26–28

In vitro cellular PDT experiments

HeLa cells (6 × 10³) were seeded onto 96-well plates and grown in 100 μL of complete medium containing DMEM medium, 10% fetal bovine serum (FBS), 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin at 37 °C for 24 h. Then the cells were incubated with a 10 μL PAA-coated GBP solution with different concentration. After 12 h of incubation, the excess PAA-coated GBPs were washed by PBS twice, and the cells were exposed to a 975 nm laser for 10 min. The 975 nm CW diode laser with a power density of 200 mW cm⁻² was used here. In order to confirm the PDT effects due to the ¹O₂ generation after irradiation, we chose a series of cell groups to be treated by identical amounts of PAA-coated GBPs but no irradiation. After laser irradiating, the medium was changed into a fresh one to be cultured for a further 24 h. After treatment, 10 μL of the CCK-8 solution was added into each well of the plate, followed by incubation for another 1 hour. Then, the cell viability was determined by measuring the absorbance of the wavelength at 450 nm with a microplate reader. All the measurements were conducted in triplicate.

Instrumentations and characterizations

Transmission electron microscopy (TEM) images of the GBPs were obtained by a JEM-2100HR transmission electron microscope (JEOL). Scanning electron microscope (SEM) images were obtained by a ZEISS Ultra 55 scanning electron microscope (Carl Zeiss). The absorption spectra of the GBPs and ABDA were acquired with a UV-vis-NIR spectrophotometer (Lambda 950, PerkinElmer). The CW laser (Mira 900, Coherent) with tunable output wavelength and power was used to excite the GBPs to generate the singlet oxygen. The CW diode laser (MPS8146-10-976, Shanghai B&A Technology Co., Ltd) and microplate Reader (imark, Bio-Rad) were used in cellular PDT experiments.

Results and discussion

Optical properties of GBPs

The PAA coated GBPs were prepared and used in all experiments. Similarly to the gold nanorods, they have two absorption peaks, and their longitudinal localized surface plasmon resonance (LSPR) wavelength could be adjusted from visible to NIR range, as Fig. 1e shows. The representative TEM images of the GBPs are shown in Fig. 1a–d, corresponding to the GBPs with longitudinal LSPR wavelength of 660 nm, 710 nm, 820 nm, and 930 nm, respectively. The sizes of the GBPs are relatively uniform for each sample. The average lengths, widths and aspect ratios of the four types of GBP samples were obtained and recorded in Table 1. For clarification, the GBPs with longitudinal LSPR wavelength at 660 nm, 710 nm, 820 nm and 930 nm are marked GBPs-660, GBPs-710, GBPs-820 and GBPs-930.

Fig. 1 TEM images of (a) GBPs-660, (b) GBPs-710, (c) GBPs-820 and (d) GBPs-930. The scale bar is 100 nm. (e) Absorbance spectra of the purified GBPs.

Table 1 Longitudinal LSPR wavelength, length, width and aspect ratio for a range of GBP samples

Samples	Longitudinal LSPR wavelength (nm)	Length (nm)	Width (nm)	Aspect ratio
GBPs-660	660	68 ± 8	31 ± 3	2.0
GBPs-710	710	65 ± 5	29 ± 2	2.3
GBPs-820	820	95 ± 5	34 ± 3	2.7
GBPs-930	930	170 ± 10	50 ± 3	3.4

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¹O₂ generation under different excitation wavelengths

The application of GBPs in PDT requires effective ¹O₂ generation capability. A common method based on photo-oxidation of ABDA was implemented to assess the capabilities of ¹O₂ generation from GBPs. In this process, the generated ¹O₂ reacts with ABDA, resulting in a decrease in the ABDA absorption band at 382 nm.¹³ This decrease characterizes the ¹O₂ generation capability (not the quantum yield). Here, the optical density (OD) of the GBPs was used to represent their concentration. The OD value of GBPs in each sample solution was 1.0. The excitation power density of 660–975 nm light was 200 mW cm⁻². Fig. 2a–d show the photo-oxidation rate of ABDA in the solution, which contained four types of GBPs and ABDA (see Fig. 3 for more details). For the GBPs-660, GBPs-710, GBPs-820 and GBPs-930, the most efficient excitation wavelength are 660 nm, 710 nm, 820 nm and 930 nm, respectively. The ¹O₂ generation capabilities of GBPs decreased as the excitation wavelengths diverged from their absorption peaks. This indicates that the ¹O₂ generation capability of GBPs is strongly dependent on the excitation and the most efficient excitation wavelength is the absorption peak wavelength. This is quite different from those of other gold nanomaterials like nanorods.¹¹ The results also show that the four types of GBPs have similar ¹O₂ generation capabilities. No decrease in the ABDA absorption occurred neither in the dark condition nor in the condition without GBPs, as shown in Fig. 2 and 4. These experiments indicate that the photo-oxidation of ABDA occured is due to the existence of ¹O₂ rather than other factors such as the reagents and the laser. In order to confirm that the reduction in the absorption of ADBA was indeed caused by the generated ¹O₂, NaN₃ was added into the sample solutions containing the same concentration of GBPs and ABDA. NaN₃ is a very good ¹O₂ quencher.¹⁵ The existence of NaN₃ in the GBP solution will greatly suppress the ¹O₂ level and result in an insignificant absorption decrease of ABDA. As shown in Fig. 5, the absorption peak at 382 nm of ABDA was sharply reduced for all four samples, which were irradiated at their own most efficient excitation wavelengths. This indicates that the GBPs could indeed generate ¹O₂ when excited by laser ranging from 660 to 975 nm. Thus we can easily choose the suitable GBPs and the excitation wavelength (i.e. GBPs-820 and 820 nm light) for PDT application.

Fig. 2 Photo-oxidation of ABDA as a function of irradiation time for (a) the GBPs-660, (b) the GBPs-710, (c) the GBPs-820 and (d) the GBPs-930. The values on the vertical axis are the ratio of the absorption peak of ABDA at 382 nm after and before irradiation. The excitation power density of the 660–975 nm light was 200 mW cm⁻².

Fig. 3 Absorption spectra of ABDA in the presence of GBPs-660 (a), GBPs-710 (b), GBPs-820 (c) and GBPs-930 (d) under irradiation of a laser at 660 nm, 710 nm, 820 nm and 930 nm, respectively. The laser power density is 200 mW cm⁻².

Fig. 4 Photo-oxidation of ABDA as a function of time for the GBPs-660, the GBPs-710, the GBPs-820 and the GBPs-930 in the dark.

Fig. 5 After addition of NaN₃, the photo-oxidation of ABDA as a function of irradiation time for the GBPs-660, GBPs-710, GBPs-820 and GBPs-930 excited at 660 nm, 710 nm, 820 nm and 930 nm, respectively.

The effect of the surrounding temperature on ¹O₂ generation

To determine the effect of the surrounding temperature on the ¹O₂ generation, we measured the ¹O₂ generation capabilities of GBPs-820 excited by an 820 nm laser when the temperature of the sample solutions were arranged from 25 °C to 50 °C. As the temperature rose from 25 °C to 50 °C, the ¹O₂ generation capabilities almost no improved.

The effect of excitation power density on ¹O₂ generation

Using GBPs for PDT also requires a suitable excitation power density. Once the laser power is far higher than the tolerance threshold laser power of the skin, the photo-induced thermal effect will hurt the skin.²⁹ If the threshold laser power of ¹O₂ generation for GBPs is too high, they may not be suitable for PDT application in vivo. Here, we assessed the ¹O₂ generation capabilities for GBPs (GBPs-710, GBPs-820 and GBPs-930) under a series of excitation power densities. As shown in Fig. 6a–c, increasing the excitation power density from 100 mW cm⁻² to 200 mW cm⁻² induced a large improvement in ¹O₂ generation. These power density were still lower than the skin tolerance threshold laser power density of 330 mW cm⁻² at 808 nm.³⁰ No obvious changes in ¹O₂ generation capability were seen from increasing the excitation power density from 200 mW cm⁻² to 1 W cm⁻². The results indicate that a low power density for the laser is sufficient for efficient ¹O₂ generation. Compared to the common photothermal studies in which a high power density of 1–48 W cm⁻² were used,³¹ PDT using GBPs needs a far lower by PDT of GBPs using a laser of low power density.

Fig. 6 Photo-oxidation of ABDA as a function of irradiation time for (a) the GBPs-710 excited at 710 nm, (b) the GBPs-820 excited at 820 nm, and (c) the GBPs-930 excited at 930 nm. The OD values of sample solutions were 1.0 in all cases, and the power density of the laser is ranged from 20 mW cm⁻² to 1 W cm⁻².

The comparison of ¹O₂ generation with GNRs

Here, ¹O₂ generation capabilities of GBPs were assessed by comparing their ¹O₂ generation capabilities with those of three types of GNRs. GNRs with the longitudinal LSPR wavelength at 870 nm, 900 nm and 930 nm were prepared, as shown in Fig. 7 and 8a. It was reported that the excitation spectra of ¹O₂ generated with GNRs with different longitudinal LSPR wavelengths were quite similar and didn't match with their absorption spectra. ¹O₂ can be generated when GNRs were excited by NIR light (875–1100 nm) and 975 nm light is relatively efficient for ¹O₂ generation.¹¹ The three types of GNRs with the same OD as GBPs were irradiated by the 975 nm laser of 200 mW cm⁻², as shown in Fig. 2 and 8b. ¹O₂ generation capability of GBPs was better than GNRs in the same condition.

Fig. 7 Scanning electron microscopy images of GNRs with the longitudinal LSPR wavelength at 870 nm, 900 nm and 930 nm, respectively.

Fig. 8 (a) Absorption spectra of the three types of GNRs. (b) Photo-oxidation of ABDA as a function of irradiation time for the three types of GNRs. The OD of GNR solution were the same as the GBPs-820 solution.

The comparison of ¹O₂ generation with MB

MB is a FDA-approved water-soluble organic photosensitizer with a high quantum yield of singlet oxygen generation (∼0.5).^32,33 In order to assess the ¹O₂ generation capability of GBPs in vivo, the comparison of the ¹O₂ generation capability of MB and GBPs excited at their absorption peaks was implemented. The artificial phantom with the same scattering and absorption coefficients as the human epidermis and dermis tissues was used to simulate the skin, as demonstrated in our previous work.²⁵ The artificial tissue phantom of 500 μm sandwiched between two coverslips was placed before the GBP sample solution and MB solution. The solution of MB with ABDA was irradiated with the 660 nm light passing through the artificial phantom. The solution of GBPs-820 with ABDA was irradiated with the 820 nm light passing through the artificial phantom with the same thickness. The power density of light was 200 mW cm⁻² here. As shown in Fig. 9. The reduction of ABDA after 25 min irradiation is 2.97% in the MB group, while for the GBPs-820 group it is 3.86%. This is because the tissue simulating phantoms will reduced the penetration rate of 660 nm light and thus reduces the capabilities of generation of singlet O₂, while the penetration rate of 820 nm light is much better. This indicates that the GBPs are indeed better than MB for PDT in vivo.

Fig. 9 Photo-oxidation of ABDA as a function of irradiation time for GBPs-820 and MB by tissue phantom. The laser power density is 200 mW cm⁻².

In vitro cellular cytotoxicity

Because of the high ¹O₂ generation capability of GBPs under low power density excitation, we explored their PDT effect on cancer cells. PDT experiments were implemented with HeLa cells treated with different concentration of GBPs (GBPs-820). The measurement of CCK-8 absorbance at 450 nm wavelengths was used to determine the cell viability. Gold nanoparticles have high optical-to-thermal energy conversion efficiency, and thus they were used to kill cells by the PTT effect.^34–37 As shown in Fig. 10a, the temperature for the sample solution with GBPs-820 rose 9 °C to 46 °C when irradiated by the 820 nm laser of 200 mW cm⁻², while no obvious temperature rise was observed for water. This large thermal effect from GBPs-820 will interfere with the confirmation of the PDT effect on cancer cells. However, the temperature of the GBPs-820 sample solution irradiated by the 975 nm laser of 200 mW cm⁻² rose from 37 °C to 40.8 °C after 15 minutes irradiation. The temperature of the water rose from 37 °C to 39.3 °C. The temperature rise was mainly from the water after irradiation. Compared to the 820 nm laser, the use of the 975 nm laser to irradiate the GBPs-820 solution induces far less heat. Considering that the temperature for the destruction of cancer cells throughout PTT effect need to reach the threshold value 42 °C in 15 min,^16,38 a 975 nm laser was used to irradiate the cancer cells treated with GBPs. It can greatly reduce the photothermal effect on cancer cells, and the destruction of cancer cells is mainly due to the PDT effect. As shown by Hwang et al.¹¹ the photothermal effect on cancer cells could be ignored by switching the excitation wavelength to 915 nm for GNRs with a longitudinal absorption peak at 808 nm. In addition, GPBs-820 is also able to generate ¹O₂ at 975 nm excitation although its efficiency is lower than that of at 820 nm excitation. As shown in Fig. 10b, 3.51%, 7.79% and 10.5% of ABDA were reduced after 15 min, 60 min and 120 min irradiation, respectively. Without irradiation, no obvious cancer cells treated with GBPs-820 dead. The cell mortality rates clearly increased when the cancer cells were treated with various concentration of GBPs-820 and irradiated by the 975 nm laser of 200 mW cm⁻² for 10 minutes, as shown in Fig. 10c. The results indicate that the GBPs can indeed kill cancer cells by PDT effect under lower power density excitation. Considering that the excitation wavelengths of the most efficient PDT effect and PTT effect overlap perfectly, the GBPs can be used for bio-therapy by combining PDT and PTT. As shown in Fig. 10c and d, the cell viabilities treated with GBPs-820 (OD = 1) under 808 nm excitation was significantly reduced to 13.2%, which is much lower than the case of 975 nm excitation (29.7%). The viability of HeLa cells has been significantly reduced to 12.8% for the GBPs-660 irradiated by 660 nm laser (Fig. 10f). As 975 nm is not the wavelength for most efficient ¹O₂ generation of GBPs-930, viability of HeLa cells was reduced to 18.7% (Fig. 10e). The reason for the better therapy effect is the combination of PDT and PTT. The best therapy effect was provided when GBPs were excited at their absorbance wavelength due to the best synergistic effect of PDT and PTT. This indicate that GBPs can provide a very good photo-therapy material for the destruction of cancer cells.

Fig. 10 (a) Temperature as a function of irradiation time for a sample solution with GBPs-820 (solid line) and for water (dashed line) irradiated by 820 nm (cyan) and 975 nm (blue) laser, respectively. (b) Photo-oxidation of ABDA as a function of irradiation time for GBPs-820 generated at 975 nm laser. (c) Cell viabilities of HeLa cells incubated with different amounts of GBPs-820 under dark and 975 nm laser irradiation conditions. (d) Cell viabilities of HeLa cells incubated with different amounts of GBPs-820 under dark and 808 nm laser irradiation conditions. (e) Cell viabilities of HeLa cells incubated with different amount of GBPs-930 under dark and 975 nm laser irradiation conditions. (f) Cell viabilities of HeLa cells incubated with different amount of GBPs-660 under dark and 660 nm laser irradiation conditions. The values of abscissa of (c)–(f) are the OD value. The laser power density is 200 mW cm⁻²In vitro cellular cytotoxicity.

Although the selection of GBPs as photosensitizers can overcome some of these drawbacks and thus give a new insight to improve the PDT technology for the cancer destruction, PDT still suffers from a lot of challenges. This study promotes the development of PDT technology and provides a good choice for cancer treatment. More efforts are needed to explore how to improve the PDT technology in the future.

Conclusions

In conclusion, for the first time, ¹O₂ was generated through direct sensitization by GBPs. The excitation wavelength and power were explored and found to have different effects on ¹O₂ generation. ¹O₂ can be generated when GBPs are excited by a laser with a wide range of wavelengths (660–975 nm), which well overlaps with the biological window. The most efficient ¹O₂ generation requires GBPs to be irradiated at the wavelength of the absorption peak. ¹O₂ can be efficiently generated when excited by a laser of low power density (200 mW cm⁻²). The PDT cellular experiment confirmed that GBPs can be a powerful PDT agent. Considering that the excitation wavelengths of the most efficient PDT effect and photothermal effect overlap perfectly, the GBPs thus can be used for bio-therapy by combining PDT and PTT without switching the excitation wavelength.

Acknowledgements

We are grateful to Qiuqiang Zhan, Xianhe Sun and Jing Liu for helpful discussions. The partial support of the Guangdong Innovative Research Team Program (201001D104799318) and the Natural Science Foundation of China (91233208) are gratefully acknowledged.

Notes and references

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Footnote

† These authors contributed equally to this work.

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