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Plasmonic nanoparticles hold great potential in photodynamic therapy (PDT). Herein, a nanoplatform of methylene blue (MB) loaded gold nanobipyramids@SiO2 (GBPs@SiO2-MB) was designed to optimize the generation rate of singlet oxygen (1O2), which was based on the plasmonic effect. The surface plasmon resonance (SPR) of GBPs was finely overlapped with the excitation absorption of MB, a wildly applied organic PDT drug prone to enzymatic degradation. This mesoporous silica coating nanoplatform protects MB molecules against degradation, and this overlap greatly enhances the 1O2 yield of MB by the SPR electron from the GBPs. The GBPs@SiO2-MB nanoparticles exhibit a synergistic effect of PDT and photothermal therapies (PTT) of cancer cells under laser irradiation. This study provides a alternative strategy to improve the classic MB treatment for phototherapy application.
© 2017 Optical Society of America
1. Introduction
Owing to the property of SPR, low-cytotoxic metal-nanomaterials as biomedical materials have attracted much attention for PDT [1,2]. Gold nanoparticles have been one of the ideal materials for PDT owing to their biocompatibility [3]. Among them, Gold nanorods (GNRs) have attracted much more attention than other gold nanostructures. Researchers recently discovered that the edge of GBPs were sharper than GNRs, the local electric-field enhancements of GBPs were larger than GNRs, according to the lightning-rod effect [4]. Thus GBPs should be better than GNRs for PDT. As our previous works reported, 1O2 can be generated while the solution of GBPs were excited by the laser of 660 nm [5].
As a wildly applied organic PDT drug, methylene blue (MB) is a FDA approved water-soluble organic photosensitizer, when excited at the wavelength of 660 nm with a high quantum yield of 1O2 generation. The low price (0.1 dollar/g) is also convenient to clinical applications. However, MB has drawbacks of poor photo-stablity and enzymatic degradation [6].
In this study, we prepare the nanocomposites of MB-embedded GBPs which coating with mesoporous silica (GBPs@SiO2-MB) as a platform. The longitudinal SPR band of GBPs@SiO2 was finely tuned to overlap with the absorbance peak of MB, owing to the photosensitizing response, MB molecules received SPR electron which transfer from the GBPs via laser7. Such overlap can strongly enhance 1O2 yield [7]. This mesoporous silica coating also acted as an effective agent to protect MB molecules against photo degradation and enzymatic degradation [8].
To the best of our knowledge, the GBPs@SiO2-MB nanocomposites have not been reported. This study provides an alternative strategy to enhance the generation rate of 1O2 and improves the classic MB treatment for PDT application.
2. Materials and methods
2.1 Materials
9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 99.9%), methylene blue (MB, 99.9%), sodium azide (NaN3, 99.9%), hydrogen tetrachloroaurate trihydrate (HAuCl4•4H2O, 99.9%) were purchased from Sigma-Aldrich. Ethanol (99.5%) and Hexadecyl trimethyl ammonium bromide (CTAB, 99.0%), were purchased from Sino Chemical Reagent Co., Ltd. Silver nitrate (AgNO3, 99%), sodium borohydride (NaBH4, 99%), sodium citrate (99%) were purchased from Aladdin reagent Co., 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.
2.2 Synthesis and purification of GBPs
The step of synthesizing and purifying GBPs is described as follows: 50 uL of 50 mM sodium citrate and 50 uL of 50 mM HAuCl4 were added into 9.9 mL of deionized (DI) water, and then freshly NaBH4 (0.03 M, 100 mL) were added in the solution under vigorous shaking [9]. This seed solution was stored for 2 hours at room temperature before use. The GBPs growth solution: 14 mL of 0.2 M CTAB was stirred at 1000 rpm, 250 uL of 50 mM HAuCl4 and 48 uL of 20 mM AgNO3 and 200 uL of 0.2 M ascorbic acid were added in sequence. The yellow solution turned to colorless, 360 mL of seed solution was then mixed into the GBPs growth solution and left undisturbed for 12 hours in dark. The color of the solution turned to wine. GBPs with absorption wavelengths of 660 nm were obtained. For purification, 10 mL of the as-prepared GBPs solution were centrifuged at 10000 rpm for 15 min twice, and then added into 10 mL of 0.35 M CTAB solution. The mixtures were deposited for 24 h at 40 °C, the sediments were dispersed in DI water.
2.3 Synthesis of the GBPs@SiO2 solution and GBPs@SiO2-MB nanocomposites
A mesoporous silica coating on GBPs was modified by the Gorelikov protocol [10]. 0.1 mL of 0.1 M NaOH solution was added to 10 mL of the (OD = 2.0) purified GBPs solution. Then, 0.2 mL of 20% tetraethyl orthosilicate (TEOS) in ethanol solution was injected, the solution was shaken 6 times with a vortex in 30 min intervals and then deposited for 24 h. The excess reagents were discarded by centrifugation at 4000 rpm for 15 min twice and washing twice with ethanol. Then, the GBPs@SiO2 solution was mixed with superfluous MB under stirring for 2 days at ambient temperature. To remove the dissociative MB molecules, the GBPs@SiO2-MB solution was centrifuged at 1000 rpm for 10 min and washed with DI water twice, the color of supernatant solution changed from blue to colorless.
2.4 Instrumentations and characterizations
Hela cell were seeded and grown in 200 uL of DMEM medium containing, 10% fetal bovine serum (FBS), 200 U mL−1 penicillin and 200 ug mL−1 streptomycin at 37 °C for 24 h. Then the Hela cell were incubated with a 20 uL of GBPs@SiO2-MB solution. After 12 h the cells were exposed to a 660 nm laser for different time with a power density of 800 mW cm−2. After laser irradiating, the cell medium was changed with new one and cultured for 24 h. And then, 20 uL of the CCK-8 solution was added and measuring the absorbance of 450 nm with a microplate reader. All the measurements were conducted in triplicate.
The morphology of the GBPs and GBPs@SiO2-MB nanoparticles were confirmed by using a JEM 2100HR (JEOL) transmission electron microscope (TEM). The absorption spectra of the GBPs, GBPs@SiO2 and GBPs@SiO2-MB nanoparticle and ABDA solution were acquired by a UV-vis-NIR spectrophotometer (Lamber 950, PerkinElmer). The continuous-wave (CW) diode laser (M-33A660-500-G, Guangzhou MOT-laser Technology Co., Ltd) was used to excite the MB and GBPs@SiO2-MB to generate 1O2.
3. Results and discussion
3.1 Characterization of materials
Figure 1 shows UV-Vis-NIR absorption spectra of the GBPs, GBPs@SiO2, MB and GBPs@SiO2-MB. Figure 1(a) (black line) displays the absorption spectra of the GBPs with a LSPR peak at 640 nm, which match with Fig. 1(b) of GBPs with an aspect ratio of 2.0-2.1. Figure 1(a) (red line) shows absorption spectra of the GBPs@SiO2 with a LSPR peak centered at 650 nm which red–shifted 10 nm. According to the Mie-Gans theory, the increasing dielectric via the silica coating which surrounding GBPs enhances the plasmon wavelength and leads to the red shift [1,9]. Figure 1(a) (blue line) shows the absorption spectra of the MB solution with a peak centered at 664 nm. Figure 1(a) (green line) shows the absorption spectra of GBPs@SiO2-MB nanocomposites match with the TEM image of Fig. 1(c), which red-shifted from 650 nm to 660 nm of GBPs@SiO2. This result was due to the electron transfer from the GBPs to the MB molecules.
Fig. 1 (a) UV-Vis absorption spectra of the purified GBPs (black), GBPs@SiO2 (red), MB (green), GBPs@SiO2-MB (blue). (b) TEM image of the GBPs and (c) TEM image of GBPs@SiO2-MB.
3.2 Detection of 1O2 generation by the GBPs@SiO2-MB
Photo-oxidation of ABDA was used to assess the generation capability of the 1O2. ABDA can react with 1O2, leading to a reduction in the ABDA absorption band around 380 nm [11]. 150 uL (0.1 mg mL−1) of ABDA were added into 2 mL solutions of GBPs@SiO2-MB, then irradiated by laser (660 nm) for 15 seconds at 5 seconds intervals, and their absorbance spectra were recorded. The power density of the laser is 800 mW cm−2. The optical density (OD) value of GBPs@SiO2-MB was 1.0. Figure 2 shows the absorption spectra of ABDA in the presence of GBPs@SiO2-MB under irradiation by laser (660 nm), which showed a gradual spectra peak of 380 nm decline during the 15 seconds, which indicates that 1O2 was produced by the GBPs@SiO2-MB with laser irradiation (660 nm).
Fig. 2 Absorption spectra of ABDA in the presence of GBPs@SiO2-MB under irradiation of a laser at 660 nm. The power density of the laser is 800 mW cm−2.
So as to confirm whether the reduction of ABDA was induced by the GBPs@SiO2-MB. The solutions containing ABDA, GBPs@SiO2-MB and MB were placed in the dark condition. As shown in Fig. 3(a), almost no reduce in the ABDA absorption occurred when in the dark condition with MB or GBPs@SiO2-MB. These results indicate that the reduction of ABDA was due to 1O2 rather than the laser or MB or GBPs@SiO2-MB.
Fig. 3 (a) Photo-oxidation of ABDA as a function of time for GBPs@SiO2-MB and MB in the dark. (b) After addition of NaN3, the photo-oxidation of ABDA as a function of irradiation time for the GBPs@SiO2-MB and MB.
In order to clarify whether the reduction of ABDA was caused by 1O2 which generated by this way, NaN3 as a specific 1O2 quencher was used [12]. 150 uL (50 mM) of NaN3 and 150 uL (0.1 mg mL−1) ABDA were added into 2 mL solutions of GBPs@SiO2-MB. NaN3 quenched the 1O2 level rapidly, which caused the amount of ABDA decreased to about 96%, as shown in Fig. 3(b). This indicates that the GBPs@SiO2-MB could indeed generate 1O2 when irradiated by laser of 660 nm. The power density of the laser is 800 mW cm−2.
3.3 1O2 generation by the MB-GBPs@SiO2 verse MB
As a most classic organic PDT drug, MB with a high quantum yield of 1O2 generation. The highest 1O2 generation rate of MB and GBPs were excited at the wavelength of 660 nm [5,8]. So we can infer that the excitation wavelength of highest 1O2 generation rate of GBPs@SiO2-MB should be 660 nm.
The mesoporous silica layer of GBPs@SiO2 encapsulated with MB. When irradiated by laser, the SPR electron transfer from GBPs to the photosensitizing response MB molecules excite the additional 1O2 [7], which combined with 1O2 produced by MB excited and GBPs excited respectively. This is why more 1O2 can be generated by GBPs@SiO2-MB than that of free MB (with the same MB molecules equivalent), which excited by laser of wavelengths of 660 nm respectively. The power density of the laser is 800 mW cm−2, as shown in Fig. 4. These results indicate that the LSPR band of GBPs overlap with excitation absorption of MB is responsible for the increasing generation of 1O2.
Fig. 4 Photo-oxidation of ABDA as a function of irradiation time for GBPs@SiO2-MB and MB by laser at 660 nm. The laser power density is 800 mW cm−2.
3.4 In vitro cellular cytotoxicity
We next investigated the cancer cell killing facilitated by GBPs@SiO2-MB in vitro. HeLa cell viabilities were assessed by CCK-8 at 450 nm wavelengths. HeLa cell were incubated with GBPs@SiO2-MB (1 nM GBPs@SiO2 containing 50 uM of MB) for 12 h in the dark, and then the excess GBPs@SiO2-MB were washed by PBS twice, and the cells were exposed to a 660 nm laser with a power density of 800 mW cm−2 for different times. As shown in Fig. 5, the cell viability is decreased dramatically with the extension of treatment time, which due to the synergistic effect of PDT and PTT. This indicates that GBPs@SiO2-MB was a good photo-therapy material for the destruction of cancer cells.
Fig. 5 Viability of HeLa cells after GBPs@SiO2-MB treatments with 660 nm laser irradiation, compared to untreated control cells, the power density of the laser is 800 mW cm−2.
4. Conclusion
In this study, we successfully prepared GBPs@SiO2-MB nanocomposites, which acted as not only nanocarrier but also agent for protecting MB molecules against degradation. The LSPR band of the GBPs was finely overlap with the excitation band of MB, due to the SPR performance, the generation rate of 1O2 was remarkably enhanced by the present nanocomposites as compared to the classic MB treatment. Moreover, The GBPs@SiO2-MB nanoparticles exhibit a synergistic effect of PDT and PTT of cancer cells under laser irradiation. Therefore, the present study provides a alternative strategy to improves the classic MB treatment for phototherapy application.
Funding
The National Natural Science Foundation of Guangdong Province, China (2016A030310301); and Medical Scientific Research Foundation of Guangdong Province, China (A2016607).
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