Journal of Materials Chemistry B

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Volume 4 Number 1 7 January 2016 Pages 1–178

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Gold Nanostars for Cancer Cells-Targeted SERS-Imaging and NIR Light-Triggered Plasmonic Photothermal Therapy (PPTT) in the First and Second Biological Windows †
Chunyuan Songa, Fang Lia, Xiangyin Guoa, Wenqiang Chena, Chen Donga, Jingjing Zhanga, Jieyu Zhang*b and Lianhui Wang*a
Cancer cells-targeted imaging and efficient therapy are vital for tumor diagnosis and treatments. However, the development of multifunctional plasmonic nanoparticles with high-performance of SERS-imaging and NIR light-triggered plasmonic photothermal therapy (PPTT) of cancer cells in both the first (NIR-I) and second (NIR-II) biological windows is still a big challenge. In the present work, gold nanostars which possess broad NIR absorption band covering the NIR-I and NIR-II windows with good NIR SERS activity and photothermal effect were synthesized by a seed-mediated growth, using gold chloride (HAuCl4), ascorbic acid (AA) and (1-hexadecyl) trimethylammonium chloride (CTAC) as growth solution. The gold nanostars were further designed to be multifunctional nanoagents by labeling Raman molecules and then conjugating arginine-glycine-aspartic acid (RGD), which can be served as cancer cells-targeted SERS-imaging tags and photothermal nanoagents in both the NIR-I and NIR-II windows. The investigation of in vitro SERS-mapping and PPTT of the A549 human lung adenocarcinoma cells indicates that the proposed multifunctional gold nanostars have a great potential for a wide spectrum of light-mediated applications, such as optical imaging and image-guided phototherapy in both the NIR-I and NIR-II biological windows.

1. Introduction
Nanotheranostics, integrating diagnostic and therapeutic functions into a single nanoagent, have received considerable attentions for diagnosis and therapy of different diseases, since they can provide attractive means to improve the curative effect of cancer by allowing identification, real-time tracking of biodistribution, image-guided specific therapy, and continuous monitoring of therapeutic response.[1-7] Up to now, many inorganic or polymer nanoparticles (NPs) have been used for nanotheranostics applications.[8, 9] Among them, noble-metal nanomaterials, especially gold NPs have been widely investigated as nanotheranostic agents due to their unique properties including strong and tunable near-infrared (NIR) localized surface plasmon resonance (LSPR), good stability, well-established biocompatibility, and facile surface functionalization.[10-13]
Photothermal therapy (PTT) employing PTT agents to kill cancer cells by reaching sufficient hyperthermia (>42°C) under laser irradiation has been considered as a highly specific and

a Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, China.
b School of Science, China Pharmaceutical University, Nanjing, 211198, China.
* Corresponding author: [email protected], [email protected].
†Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x

minimally invasive cancer therapy relative to the traditionally chemotherapy and radiotherapy.[14-16] Recent advances in the field of plasmonics present new opportunities for PTT using noble metal NPs as plasmonic photothermal therapy (PPTT) agents.[17, 18] By photo-exciting conduction electrons which oscillate at the surfaces of noble metal nanostructures (surface plasmons), highly efficient local heating can be achieved by non-radiative relaxation through electron-phonon and subsequent phonon-phonon coupling processes.[19] Surface- enhanced Raman scattering (SERS) as a sensitive spectroscopic technique associating with localized surface plasmon resonance of noble-metal nanomaterials is known as a non- invasive tool with highly valuable for molecular and living cell imaging.[20-24] Moreover, SERS offers excellent resolution for monitoring of intracellular microenvironments and tracking of the cellular distribution of extrinsic molecules, which is promising as a new theranostic platform for multimode imaging and visualizing therapy of cancers.[25, 26] Thus, the design and fabrication of efficient PPTT nanoagents for cancer cells-targeted NIR SERS-mapping and photothermal therapy are extremely useful and important.[27]
To avoid the fluorescence interference of biological tissue, and achieve photothermal treatment of deep-tissue-buried tumors, as well as balance efficacy with safety concerns, it is necessary to develop PPTT agents that not only are able to strongly absorb NIR light in the biological windows I (650-950 nm) and II (1000-1700 nm), ensuring minimal light absorption by surrounding tissues, but also possess high NIR SERS activity

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and tumor-specific accumulation with minimal retention in other normal organs.[11, 28, 29]
In recent years, efforts to improve SERS-activity and light-to- heat conversion efficiency have focused on the size, shape, or surface coating of plasmonic NPs, as well as tunable NIR- LSPR.[30-32] Gold NPs exhibiting NIR-triggered plasmon- based high photothermal conversion efficiency are obtained great attentions as good agents for PPTT applications.[33] The potential uses of gold NPs in NIR PPTT have been reported using a variety of nanostructures, including gold nanoshells,[12, 34, 35] gold nanorods,[15, 36] and recently gold nanocages, et al.[37, 38] Among them, gold nanostars (Au NSs) with branched tips which are known as excellent SERS “hot spots” have been prepared and integrated multiple functionalities in a single nanosystem for simultaneously achieving cancer cell-targeted SERS-imaging and PPTT.[1, 39, 40] However, up to now, few star- like gold NPs have been reported to be suitable for SERS- and PPTT-application in both the NIR-I and NIR-II (NIR-I/II) windows.[41-43] It should be noted that the equipment required for the NIR-II window, such as the laser and the detector, is more expensive than that for the NIR-I window, which limits the practical utilization of the NIR-II window. Therefore, the development of NIR-I/II light-responded PPTT nanoagents is very important to requirements of a wider range of applications in both the NIR-I and NIR-II windows.
In the present work, a new type of Au NSs was synthesized to prepare multifunctional Au NSs for cancer cells-targeted SERS-imaging and NIR-I/II light-triggered PPTT. The Au NSs were synthesized by a seed-mediated growth, using gold chloride (HAuCl4), ascorbic acid (AA) and (1-hexadecyl) trimethylammonium chloride (CTAC) as growth solution. Comparing with the most reported Au NSs with relatively narrow NIR absorption band,[1, 39, 40, 44-46] the as-synthesized star-shaped Au NPs have broad LSPR band that covers both the NIR-I and II windows, and their multiple sharp branches can act as ‘hot spots’ to greatly enhance the local electromagnetic field under NIR illumination.[36] Besides, their large surface area also facilitates surface loading with more cargoes including targeting moieties, Raman reporters and other functional molecules. Basing on these unique properties, the Au NSs were further utilized to design cancer cells-targeted optically active nanoagents for simultaneous molecular SERS- imaging and PPTT. The multifunctional Au NSs with broad NIR working wavelengths can be used as bright SERS tags and efficient NIR-I/II PPTT nanoagents for selective cancer cell imaging and destruction.

2. Experimental
2.1 Materials
Gold chloride trihydrate (HAuCl4·3H2O) and (1-hexadecyl) trimethylammonium chloride (CTAC) were purchased from Alfa Aesar. Ascorbic acid (AA) was obtained from Sinopharm Chemical Reagent Co., Ltd. 4-Mercaptobenzoic acid (4-MBA), N-hydroxysuccinimide (NHS) and 1-ethyl-3-[3- dimethylamino) propyl] carbodiimide (EDC) were obtained

from Sigma-Aldrich. Bovine serum albumin Vie(wBASrAtic)le Ownlianes purchased from Bio Sharp. Arginine-glycinDeO-Ia: s10p.a10rt3i9c/Ca9cTidB0(0R0G6D1E) was bought from Top-peptide Co., Ltd. LDH-cytotoxicity assay kit was purchased from Biovision. A549 lung adenocarcinoma cell line was obtained from Cobioer Biosciences Co., Ltd. Ultrapure Millipore water (18.2 MΩ·cm) was used as the solvent throughout.

2.2 Preparation of multifunctional Au NSs
Synthesis of Au NSs: The Au NSs were synthesized by a seed- mediated growth method. In the first step, Au seeds were synthesized. Briefly, 0.5 mL of 5 mM HAuCl4 and 10 mL of 100 mM CTAC were mixed together and then 0.6 mL of 10 mM NaBH4 were added into the mixture followed by stirring for 5 min. The Au seeds were then left statically at room temperature to age for one day. The synthesized Au seeds were diluted one hundred times for the further usage. In the second step, 1 mL of 10 mM HAuCl4 was mixed with 10 mL of 200 mM CTAC under vigorous stirring. Then 0.05 mL Au seeds were added into the mixture. After 2 min, 0.5 mL of 300 mM AA was added into the mixture and stirred vigorously for 2 min. The mixture was then left undisturbedly for 3 h at 15°C to grow Au NSs. The product was centrifuged at 3000 rpm for 5 min to remove the excess reactants. The precipitates were washed three times and finally re-dispersed to 2 mL with water.
Labelling 4-MBA and RGD onto Au NSs: Herein, 4-MBA
molecules were selected as Raman reporters, and RGD molecules acted as ligands selectively targeted to αvβ3 integrin (a membranous receptor that was overexpressed in A549 human lung adenocarcinoma cells) on the cell membrane and thus succeeded in achieving active targeting of cancer cells and ligand-mediated endocytosis.[47] The multifunctional Au NSs-agents were prepared by labelling 4-MBA and RGD onto Au NSs in sequence. Specifically, 400 μL of 1 mM 4-MBA were added slowly into 2 mL Au NSs under vigorous stirring and the resultant mixture was allowed to react overnight. After that, the product was washed three times by centrifugation (3000 rpm for 6 min) and then 2 mL 4-MBA-labeled Au NSs (Au- 4MBA) were obtained by re-dispersing the precipitates with water. Subsequently, 59.4 μL of 100 mM NHS and 24 μL of 100 mM EDC were added into the Au-4MBA colloid to activate the carboxylate-terminations for conjugating RGD via amidation.

Fig. 1 Schematic illustration of the structure and application of multifunctional Au NSs for cancer cells-targeted NIR SERS-imaging and PPTT.

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To link RGD to Au-4MBA, 200 μL of 1 mM RGD were added into the NHS-activated Au-4MBA and incubated at 4°C for 6 h.

2.9 Instruments

The product was then blocked by 500 μL of 3% BSA for 1 h, followed by centrifugal purification for three times. Finally, 2 mL Au-4MBA-RGD were obtained after re-dispersion with water. The schematic structure of the multifunctional Au NS is shown in Fig. 1.

2.3 Characterization of photothermal effect
For characterizing the photothermal effect of the Au NSs, 200 μL of 1 mg/mL Au NSs (characterized by ICP-MS) were pipetted into each well of 96-well plate. The Au NSs were irradiated by 785 nm NIR-I laser (390 mW/cm2) and 1064 nm NIR-II laser (1160 mW/cm2), respectively. The temperature of the Au NSs solution was monitored by an infrared radiation thermometer with 1 min interval. The control experiment of H2O was also measured under the same conditions.

2.4 Cell culture
A549 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin and penicillin) at 37°C in 5% CO2. The cell- culture medium was renewed after every 48 h.

2.5 Incubation of cells with multifunctional Au NSs
Generally, 1×105 cells were seeded into 96-well plates and incubated with 100 μL of 1, 0.5, 0.2 and 0.1 mg/mL multifunctional Au NSs at 37°C in 5% CO2 for 24 h, respectively.

2.6 Cellular SERS mapping
Before SERS mapping, the cells incubated with multifunctional Au NSs for 4 h were rinsed by PBS for three times to remove medium and dissociative particles. After that, fresh medium was added and SERS mapping were then carried out. The characteristic Raman band of 4-MBA from 1062-1088 cm-1 was selected to create SERS images.

2.7 NIR PPTT
The cells incubated with multifunctional Au NSs were rinsed three times with PBS and then received irradiations from a continuous wave 785 nm laser and a 1064 nm laser for NIR-I and NIR-II PPTT, respectively.

2.8 Cytotoxicity assay
The cytotoxicity assays were tested by a LDH-cytotoxicity assay kit. Specifically, several aliquots of 100 µL supernatant fluid taken from each well were transferred carefully into corresponding wells of a new 96-well plate. Then 100 µL of the reaction mixture of the LDH-cytotoxicity assay kit was added into each well, followed by incubation at 37°C in 5% CO2 for 30 min. The absorbance of the mixture in each well at 490 nm were recorded by a microtiter plate reader. The results of cytotoxicity assay were expressed as the percentage of cell viability.

The UV-vis-NIR spectrophotometer (UV-3600, Shimadzu,
Japan) was used to monitor the absorption properties of the nanoparticles. Transmission electron microscopy (TEM) (HT7700, Hitachi, Japan) and field emission scanning electron microscopy (FESEM) (S-4800, Hitachi, Japan) were utilized to characterize the morphologies of nanoparticles. X-ray diffraction (XRD) analysis was carried out using an X-ray diffractometer (D8 Advance, Bruker, Germany). A 785 nm laser (DS2-11312-103, BWT, China) and a 1064 nm laser (MIL-N- 1064-5W, CNI, China) were used to generate NIR-I and NIR-II light for heating the nanoparticles. Zeta potential measurements and dynamic light scattering (DLS) were conducted by Zeta potential analyzer (ZetaPALS, Brookhaven, America). The temperature was measured by an infrared radiation thermometer (VT02, Fluke, USA). SERS spectra were detected by a confocal Raman microscope (InVia, Renishaw, England) equipping with 785 nm and 1064 nm lasers. The laser beams with 1% laser power were focused by a 50× long-focus lens, and the SERS signals were collected with 1 s exposure time. The SERS mapping were performed by an inverted microscope (ECLIPSE Ti, Nikon, Japan) equipped on the confocal Raman microscope. The 785 nm laser beam (1% power) was focused on the samples by a 50× objective lens, and the Raman signals collected by the objective lens were projected to CCD with 1 s exposure time. Fluorescence images were taken by an inverted fluorescence microscope (IX73, Olympus, Japan) with a 4× objective lens. A microplate reader (ELx808, BioTek, America) was employed for the LDH assay.

3. Results and discussion
3.1 Synthesis and Characterization of Au NSs
Fig. 2a shows a SEM image of the as-synthesized Au NSs. The nanoparticles looking like nanostars have good monodispersity and each of them possesses more than 10 tips with broad LSPR band covering the NIR-I and II windows. The absorbance at 785 nm and 1064 nm were 95.7% and 80.6% of the primary absorption peak at 725 nm (Fig. 2b). XRD characterization shown in Fig. 2c indicates that these Au NSs have a polycrystalline structure, from which four peaks for the (111), (200), (220), and (311) diffraction planes of face-centered- cubic (fcc) Au were observed.[48, 49] The photothermal effect of the Au NSs induced by a 785 nm irradiation with 390 mW/cm2 power is shown in Fig. 2d (square). The temperature of Au NSs colloid increased by 7.1°C after exposure for 1 min, and a temperature increase of 16.4°C was obtained after 10 min of irradiation. As a control, only a negligible temperature increase (1.5°C) was observed for the negative sample (H2O) after exposure for 10 min (triangle). The square in Fig. 2e plots the photothermal effects of the Au NSs under the illumination of 1064 nm NIR laser with 1160 mW/cm2 power. After 10 min of irradiation, the temperature increased by 14.7°C. In contrast, the temperature increase of water was very limited under the same irradiation (triangle). The photothermal conversion efficacy of Au NSs under 785 nm and 1064 nm were calculated

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to be 34.5% and 13.0% respectively, following the reported method.[37] The NIR-triggered plasmon-based photothermal conversion was ascribed to the strong surface plasmon resonance under the illumination of 785 nm and 1064 nm laser beams, which resulted in enhanced photon absorption.[33] The Au NSs also have good photothermal and physical stabilities under the irradiation of 785 nm and 1064 nm laser beams as shown in Fig. S1 and Fig. S2 (ESI†). These results indicate that the as-synthesized Au NSs can be used as effective PPTT agents both in the NIR-I and NIR-II biological windows. Fig. 2f plots the SERS spectra of 4-MBA absorbed on the Au NSs under the illuminations of 785 nm and 1064 nm lasers, respectively. Each SERS spectrum was averaged from eight measurements at different positions. The distinct SERS peaks at 1078 cm-1 and 1589 cm-1 are designated to the two characteristic Raman peaks of 4-MBA. SERS characterization demonstrates that the Au NSs possess strong surface enhancement effect which can be utilized to prepare bright SERS tags for NIR-I and NIR-II applications.

Fig. 3 (a) Absorption spectra of Au NSs, 4-MBA labeled Au NSs (Au-4MBA), EDC/NHS
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activated Au-4MBA (Au-4MBA EDC/NHS), and the multiDfuOncI:ti1o0n.a1l0A3u9/NCS9sT(BAu0-040M6B1AE-
RGD); (b) Cytotoxicity assays of A549 cells incubated with various concentrations of Au NSs and Au-4MBA-RGD.

3.2 Characterization of the multifunctional Au NSs
The multifunctional Au NSs were prepared by two steps, i.e. labelling Raman reporters onto the Au NSs and then conjugating RGD. Herein, 4-MBA molecules were mixed with Au NSs to link molecules onto gold surface by thiol groups, which made the primary LSPR peak redshift from 725 to 734 nm (Fig. 3a). Subsequently, the 4-MBA-labeled Au NSs (Au- 4MBA) with carboxyl groups outside were activated by EDC and NHS. These activated carboxyl groups can covalently bond with the amino groups of polypeptides, yielding stable conjugation of the two molecules, i.e. multifunctional Au NSs (Au-4MBA-RGD).[50, 51] The activation process and the following linkage of RGD resulted in a significant redshift of the primary LSPR to 760 nm, and the multifunctional Au NSs showed a broad NIR-LSPR covering both the NIR-I and II windows. Zeta potential and DLS characterizations also confirmed that 4-MBA molecules and RGD were successfully immobilized onto the Au NSs (Table S1, ESI†). Since partial original CTA+ ions were exchanged by 4-MBA molecules, leaving carboxyl groups outside the particles, the zeta potential of colloid decreased from 48.68 mV to 40.14 mV, and then reduced to 27.57 mV after activation of EDC/NHS, finally changed to -29.82 mV after conjugation with RGD. Besides, with the increase of molecule layers modified on the surface of particles, the hydrodynamic diameter of particles increases gradually (Fig. S3, ESI†). As shown in Fig. S4 (ESI†), the multifunctional Au NSs shows good stabilities in water, PBS and serum for more than five days, by monitoring their absorption spectra.

3.3 In vitro experiments
Cell viability assay. To evaluate the cytotoxicity of the multifunctional Au NSs (Au-4MBA-RGD), A549 cells were incubated with the Au-4MBA-RGD for 24 h to allow a sufficient cellular uptake of Au NSs. Fig. 3b shows the results of cell

viability assay of 1×105 cells incubated with 100 μL of 1, 0.5,
0.2 and 0.1 mg/mL multifunctional Au NSs, respectively. By

contrast, the cytotoxicity assays of equivalent amount of naked (without 4-MBA and RGD) Au NSs are also shown in Fig. 3b. The cytotoxicities of the naked Au NSs and Au-4MBA-RGD

Fig. 2 Characterizations of the as-synthesized Au NSs. (a) SEM image, (b) optical
property, (c) XRD pattern, (d) and (e) photothermal curves of equivalent Au NSs and H2O under the irradiation of NIR-I (785 nm) and NIR-II (1064 nm) light respectively, (f) SERS characterizations.

were significantly dependent on the concentration of nanoparticles. The viability of Au-4MBA-RGD treated cells increased from 67.2% to 77.1%, 88.3% and 97.5% respectively, corresponding to the decreasing concentration. Besides, the Au-4MBA-RGD showed significantly improved biocompatibility relative to the naked Au NSs, which means that the surface modifications of 4-MBA and RGD can reduce the toxicity distinctly, mainly since the previous toxic CTA+ ions on the surface were replaced by 4-MBA and biological molecules RGD. It should be noted that considering the severe toxicity of

1 mg/mL multifunctional Au NSs, in subsequent in vitro studies

Concentration (mg/mL)

the cells were treated by 0.5, 0.2 and 0.1 mg/mL Au-4MBA- RGD.

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SERS mapping. As shown in Fig.2f, the Au NSs exhibited good surface enhancement effect under 785 nm and 1064 nm laser irradiation. As an example, cancer cells-targeted SERS mapping was conducted by 785 nm irradiation. Fig. 4 (left) shows the bright-field (BF) images and the corresponding SERS images taken from the A549 cells after incubation with 0.5, 0.2 and 0.1 mg/mL Au-4MBA-RGD, respectively. The SERS signal and the distribution of Au NSs in A549 cells were enhanced continuously with the increase of particle concentration. Since signal intensity is directly proportional with the amount of SERS tags, these SERS images clearly indicate that more nanoparticles were internalized into the A549 cells when the cells were incubated with higher concentration of multifunctional Au NSs. Some representative SERS spectra collected at the different spots corresponding to the SERS images are shown in Fig. S5 (ESI†). In contrast, Fig. 4 (right) shows the similar images of A549 cells incubated with equivalent amount of Au-4MBA without conjugation with RGD. Very weak SERS signal was observed from the cells even incubated with the highest concentration of RGD-free Au- 4MBA (0.5 mg/mL), which indicates that very small amount of Au-4MBA were captured by the cells. It is known that RGD is a good guider for the identification of cancer cell by targeting towards the over expressing high-affinity αvβ3 integrin.[52] Therefore, the RGD conjugated-particles had better performance on specifically binding to A549 cells than the RGD-free Au NSs. Besides, the Au-4MBA-RGD were effectively internalized by A549 cells through a RGD-mediated endocytosis, which is in agreement with the previously reported results.[47, 52] To further verify the specific targeting of the multifunctional Au NSs to cancer cells, the immortalized normal human oral keratinocyte (HOK) cells were selected as negative control and incubated with the multifunctional Au NSs under the same conditions. The SERS mappings of the HOK cells were performed to track the distribution of Au NSs in the living HOK cells (Fig. S6, ESI†). Obviously, very weak SERS signals were observed from 0.5 mg/mL Au-4MBA-RGD

Fig. 4 Bright-field (BF) images and the corresponding SERS images of A549 cells
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incubated with 0.5, 0.2 and 0.1 mg/mL multifunctional AuDNOSIs: (1A0u.-140M3B9A/C-R9GTDB,0le0ft0)6a1nEd
RGD-free Au NSs (Au-4MBA, right).

NIR PPTT of cancer cells. The multifunctional Au NSs not only can be used as bright SERS tags for cancer cells-targeted imaging but also as NIR PPTT nanoagents for therapy. The NIR-I PPTT efficacy (785 nm, 390 mW/cm2, 6 min irradiation) of the multifunctional Au NSs was qualitatively evaluated via fluorescence imaging by using calcein-AM (a non-fluorescent dye which can permeate the cell membrane and be hydrolyzed by intracellular esterases to a green fluorescent calcein dye in live cells) to stain living cells and propidium iodide (PI) with red fluorescence to stain dead cells for 30 min, respectively. After washing, fluorescent images of the cells were acquired. Fig. 5a shows the fluorescent images of the control (A549 cells without incubation with multifunctional Au NSs) and the A549 cells incubated with 0.5, 0.2 and 0.1 mg/mL multifunctional Au NSs, respectively. According to the fluorescence images, much more dead cells can be observed from the PPTT agents treated cells. In contrast, the control shows bright green fluorescence, implying that most cells were alive. Fluorescence imaging result is consistent with the cell viability tests shown in Fig. 5b. A monotonic decrease of cell viability from 86.8% (control) to 32.5% (0.1 mg/mL), 20.7% (0.2 mg/mL), and 4.3% (0.5 mg/mL) was obtained. All these results indicate that the proposed multifunctional Au NSs hold great potential for the NIR-I PPTT of cancer cells.

incubated cells, which indicates that few multifunctional Au NSs were captured by HOK cells since HOK cells do not ‘over’ express αvβ3 integrin. As a conclusion, the proposed multifunctional Au NSs show a good selectivity of the A549 cells, and can be used as bright SERS tags for A549 cells- targeted imaging.

Exposure time (min) Concentration (mg/mL)

Fig. 5 (a) Fluorescent images of calcein-AM and PI stained A549 cells which were treated with 0 (control), 0.1, 0.2 and 0.5 mg/mL multifunctional Au NSs, followed by NIR-I 785 nm laser irradiation. (b) Cytotoxicity assays of 0, 0.1, 0.2 and 0.5 mg/mL multifunctional Au NSs-treated A549 cells after irradiating by 785 nm laser with 390 mW/cm2 power for 6 min. (c) Cytotoxicity assays of 0.5 mg/mL multifunctional Au NSs-

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treated A549 cells after irradiating by 785 nm laser with 0, 160, 260 and 390 mW/cm2 power for 6 min. (d) Cytotoxicity assays of 0.5 mg/mL multifunctional Au NSs-treated A549 cells after irradiating by 785 nm laser with 390 mW/cm2 power for 0, 1, 3 and 6 min. (e) Cytotoxicity assays of the 0, 0.1, 0.2 and 0.5 mg/mL multifunctional Au NSs- treated A549 cells after irradiating by 1064 nm laser with 1 W/cm2 power for 5 min.

The influences of laser power and exposure time on NIR-I PPTT were also investigated. The results shown in Fig. S7 (ESI†) indicates that the higher concentration of Au NSs were illuminated by the laser, the better heating efficiency can be obtained. However, the higher concentration of multifunctional Au NSs was incubated with cancer cells, the more serious cytotoxicity and non-specificity (Fig. 4 and 5) were resulted. Given these factors, A549 cells were incubated with 0.5 mg/mL multifunctional Au NSs to investigate the optimal laser power and exposure time of NIR-I PPTT. The cell viability assays were performed after PPTT and the results are shown in Fig. 5c and 5d, respectively. When PPTT was conducted at a low power (160 mW/cm2), the irradiated cells remained relatively high viability of 88.3% relative to 90.6% of the control cells which were incubated with multifunctional Au NSs but without laser exposure. Once the power increased to 390 mW/cm2, the cell viability decreased distinctly from 63.4% of 260 mW/cm2 irradiation to 4.3%, as shown in Fig. 5c. The exposure time also showed significant effect on the therapeutic effect of PPTT. Under the irradiation of 390 mW/cm2 the cell viability decreased fast to 35.6% and 29.6% after exposure for 1 min and 3 min respectively, and finally decayed to 4.3% when illumination for 6 min. Generally, the appropriate 785 nm (NIR-I) PPTT can be obtained by heating the cancer cells with 390 mW/cm2 power for 6 min.
The application of the Au NSs in the NIR-II PPTT was verified by conducting the cytotoxicity assays of the A549 cells treated with 0, 0.1, 0.2 and 0.5 mg/mL multifunctional Au NSs after irradiating for 5 min under 1064 nm light with 1 W/cm2 power which was a relatively low and more favourable working power without hurt the skin.[53] The results shown in Fig. 5e indicate that the cell viability decreased by 19% after exposure to 1064 nm light. When the cells were incubated with 0.1, 0.2 and 0.5 mg/mL multifunctional Au NSs, the NIR-II PPTT shows an enhanced cytotoxicity with the increase of the amount of PPTT agents, and the PPTT by using 0.5 mg/mL multifunctional Au NSs killed ~88% A549 cells. Therefore, the proposed multifunctional Au NSs also can be used as good NIR-II PPTT agents for cancer therapy. In comparison with the previous reports, the proposed PPTT agents exhibited obvious advantage of working in both the NIR-I and NIR-II windows and the therapies conducted in the two windows have good effective in killing tumor cells with the working power below the skin-tolerance threshold.

Conclusions
In the present work, a new type of Au NSs were synthesized in high yield by a seed-mediated growth with the growth solution containing gold chloride, ascorbic acid and CTAC surfactant. The products possess star-shaped morphologies and broad NIR

LSPR band covering the NIR-I and NIR-II windowVsiewwAirttihcle gOonloinde NIR-light photothermal effect and SERS DacOtIi:v1i0ti.1e0s3.9T/Ch9eTBA0u00N6S1Es were further designed to be multifunctional Au NSs as SERS tags and PPTT nanoagents by labelling Raman reporter and conjugating RGD for cancer cell-targeted SERS-imaging and NIR-I/II PPTT. Absorption spectra and zeta potentials were used to monitor the successful surface modifications of Au NSs. The Au NSs show good SERS-activities under the NIR-I (785 nm) and NIR-II (1064 nm) lasers excitation, and the SERS mapping results indicate that the proposed multifunctional Au NSs have bright SERS and good uptake specificity of A549 cells. Besides, the multifunctional Au NSs shows good therapeutic effect of both NIR-I and NIR-II PPTT. As a summary, the proposed multifunctional Au NSs has a great potential for cancer cell-targeted SERS-imaging and NIR-I/II photothermal therapy.

Acknowledgements
This work was financially supported by the National Key Research and Development Program of China (2017YFA0205300), National Natural Science Foundation of China (61871236, 61501522), Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R37), Natural Science Foundation of Jiangsu Province of China (BK20181395), Key Research and Development Program of Jiangsu (BE2018732), and Priority Academic Program Development of Jiangsu Higher Education Institutions (YX03001).

Notes and references

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