Rational Design of Ultrabright SERS Probes with Embedded

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Rational Design of Ultrabright SERS Probes with Embedded Reporters for Bioimaging and Photothermal Therapy Xiulong Jin,†,# Boris N. Khlebtsov,∥,# Vitaly A. Khanadeev,∥ Nikolai G. Khlebtsov,*,∥,⊥ and Jian Ye*,†,‡,§ †

State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, ‡Shanghai Med-X Engineering Research Center, School of Biomedical Engineering, and §Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, P. R. China ∥ Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia ⊥ Saratov National Research State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia S Supporting Information *

ABSTRACT: Plasmonic nanoparticles can be utilized as surface-enhanced Raman scattering (SERS) probes for bioimaging and as photothermal (PT) agents for cancer therapy. Typically, their SERS and PT efficiencies reach maximal values under the on-resonant condition, when the excitation wavelength overlaps the localized surface plasmon resonance (LSPR) wavelength preferably in the near-infrared (NIR) biological window. However, the photogenerated heat may inevitably disturb or even destroy biological samples during the imaging process. Herein, we develop ultrabright SERS probes composed of metallic Au@Ag core−shell rodlike nanomatryoshkas (RNMs) with embedded Raman reporters. By rationally controlling the Ag shell thickness, the LSPR of RNMs can be tuned from UV to NIR range, resulting in highly tunable SERS and PT properties. As bright NIR SERS imaging nanoprobes, RNMs with a thick Ag shell are designed for minimal PT damage to the biological targets under the off-resonance condition, as illustrated through monitoring the changes in mitochondrial membrane potential of cancer cells during SERS imaging procedure. By contrast, RNMs with a thin Ag shell are designed as multifunctional NIR theranostic probes that combine enhanced photothermal therapy capability, as exemplified by efficient PT killing of cancer cells, with reduced yet still efficient imaging properties at the on-resonance excitation. KEYWORDS: plasmonics, SERS, bioimaging, photothermal therapy, core−shell nanoparticles classical EM models,13−16 which can be further modified to account for nonlocal and quantum tunneling effects.17,18 Such SERS probes with more hot spots strongly enhance the efficiency of the spontaneous Raman scattering process and therefore allow reducing the acquisition time and performing fast SERS imaging, which is of great importance in practice. For example, gap-enhanced Raman tags (GERTs)19 have been recently applied successfully for high-speed and high-resolution (50 × 50 pixels) single-cell confocal Raman imaging within 30 s with an acquisition time of 10 ms/pixel under near-infrared (NIR) irradiation due to built-in hot spots in the nanomatryoshka (NM) geometry. The acquisition time can be possibly further reduced to 1 ms/pixel when different Raman reporters are encoded or a multishell geometry is employed.20,21

1. INTRODUCTION Surface-enhanced Raman scattering (SERS) spectroscopy exhibits important features, including high sensitivity and specificity, which are especially useful for biosensing and bioimaging.1,2 SERS spectroscopy has additional advantages for multiplexed biomedical applications due to the much narrower spectral line width compared to that of the fluorescent spectrum.3,4 Together with extensive explorations of SERS for biosensing,5−7 more recently, SERS has shown great potential in in vivo intraoperative bioimaging as a guiding tool for tumor surgery due to unique optical codes from the fingerprint Raman spectra of SERS probes.8−11 These promising perspectives drive us to consider the strategy to design practically optimal SERS probes for bioimaging. Typically, SERS probes are designed with high local electromagnetic (EM)-field regions, namely, EM hot spots, because it is the dominant mechanism of the Raman scattering enhancement.12 A rational design of SERS probes with efficient EM hot spots can be guided by simulation tools based on the © 2017 American Chemical Society

Received: June 17, 2017 Accepted: August 21, 2017 Published: August 21, 2017 30387

DOI: 10.1021/acsami.7b08733 ACS Appl. Mater. Interfaces 2017, 9, 30387−30397

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ACS Applied Materials & Interfaces

Figure 1. Design strategy to fabricate SERS probes with embedded Raman reporters for bioimaging and photothermal therapy (PTT). On-resonant probes with a thin Ag shell show a moderate SERS performance and maximized photothermal (PT) effect, whereas off-resonant probes with a thick Ag shell show superhigh SERS performance and minimized PT effect.

that, through rationally choosing a thick Ag shell, RNMs may exhibit superstrong NIR SERS enhancement and minimized PT damage to the biological cells under the off-resonance condition or act as multifunctional NIR theranostic agents that combine the imaging and photothermal therapy (PTT) capability under the on-resonance condition, when a thin Ag shell is chosen.

However, the SERS probes only with strong EM local-field hot spots are not optimal for bioimaging because they may cause a decreased photostability of probes or induce side effects, such as photodamage to biological samples during the imaging process.22,23 Typically, the maximum SERS enhancement occurs at excitation wavelengths quite close to the localized surface plasmon resonance (LSPR) wavelength of individual nanoparticles (NPs).24−27 Consequently, very high photothermal (PT) conversion by NPs is produced at the same resonance wavelengths because the optical absorption coefficient is maximal at the LSPR wavelength. The unwanted heat generated by laser irradiation may induce the degradation and photoreaction of Raman reporters as well as inevitably destroy biological cells or tissues being imaged. Surprisingly enough, this important issue has been underappreciated in previous studies; although, some aspects have been discussed recently. For instance, it has been noted19,28 that the LSPR spectrum of complex nanostructures is no longer a good indicator for the maximal SERS enhancement due to the plasmon coupling effect. Therefore, it would be desirable to decouple the LSPR spectrum with the SERS performance, combining maximal SERS performance and minimal photothermal conversion efficiency under the off-resonance condition. For example, the mesoporous silica-coated spherical GERTs have been designed with built-in Raman reporters for off-resonance NIR laser excitation and reduced PT effects, leading to the ultrahigh photostability during laser irradiation.20 Another example is a bioenabled synthesis of complex nanostructures with built-in EM hot spots formed by densely packed satellite NPs grown on a plasmonic core.29 At the off-resonance excitation, such SERS probes produce minimal heating and perturbation of cells being imaged. On the other hand, the on-resonant SERS probes would demonstrate acceptably high SERS performance combined with a strong photoheating effect, thus making a new platform for multifunctional theranostic applications.30−33 Thus, there is a great demand in rational design of SERS probes with great SERS enhancement and tailorable PT effect. In this work, we propose a new strategy to design ultrabright SERS probes composed of Au@Ag core−shell rodlike nanomatryoshkas (RNMs) with embedded reporters (Figure 1). Such nanostructures with controllable Ag shell thickness exhibit strong SERS intensity, high tunable optical plasmon resonances from UV to NIR range, and enhanced spectrally tunable photothermal properties. Furthermore, we demonstrate here

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. All chemicals were obtained commercially and used without further purification. Cetyltrimethylammonium bromide (CTAB, >98.0%), cetyltrimethylammonium chloride (CTAC, 25% water solution), sodium oleate (NaOL, technical grade >82% fatty acid), L-ascorbic acid (AA, >99.9%), benzenedithiol (BDT), 4-nitrobenzenethiol (NBT), naphtalenethiol (NT), hydrochloric acid (HCl, 37 wt % in water), tetraethylorthosilicate (TEOS, 98%), and sodium borohydride (NaBH4, 99%) were purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O) and silver nitrate (AgNO3, >99%) were purchased from Alfa Aesar. Ultrapure water obtained from a Milli-Q Integral 5 system was used in all experiments. 2.2. Synthesis of Au@Ag Rod Nanomatryoshkas (RNMs). Nanorods (NRs) were first prepared by a seed-mediated growth in binary surfactant mixture as described elsewhere34,35 and redispersed in 10 mM CTAB solution with a [Au] of 1 mM. Next, 300 μL of a 2 mM Raman reporter (NT, BDT, or NBT) solution in ethanol was added to 10 mL of NRs under vigorous sonication. The mixtures were incubated for 20 min at 50 °C and resuspended in 0.1 M CTAC after washing three times with 0.1 M CTAC solution. Then, various amounts of 100 mM AgNO3 solution were added into the mixture of 4 mL of functionalized NRs and 12 mL of H2O, followed by the addition of four times molar excess of AA as reductant. The mixture was incubated unstirred at 70 °C for 3 h for NT and NBT Raman reporters and 12 h for BDT. Mesoporous silica coating of NMs was performed using the method reported by Gorelikov and Matsuura.36 NaOH (0.1 M) was added to 16 mL of NMs to adjust pH to 11. Three 30 μL injections of 20% TEOS in methanol were carried under gentle stirring at 30 min intervals, and the reaction was kept for 12 h. After that, the obtained NMs were washed three times and finally redispersed in 4 mL of water or serum-free Dulbecco’s modified Eagle’s medium (DMEM). AuNR@Ag@NBT particles with different Ag shell thicknesses were fabricated following the RNM synthesis protocol, in which NBT molecules were attached to the outer Ag surface in the last step. 2.3. NP Characterization. Extinction spectra were measured with a SPECORD 250 spectrophotometer (Analytik Jena, Germany). Transmission electron microscopy (TEM) images were recorded on 30388


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5 μL of 200× stock solution (Beyotime Biotechnology, China) with 0.8 mL of ultrapure water and 0.2 mL of JC-1 staining buffer solution. After incubation at 37 °C for 20 min, the cells were washed with PBS twice. The mitochondrial fluorescence was detected immediately on a confocal laser scanning microscope (40× oil immersion objective, numerical aperture = 1.2, Leica TCS SP5 confocal microscope; Germany). JC-1 was excited at 488 and 561 nm, and the emissions were collected at 515−535 nm (green, J-mono) and 570−600 nm (red, J-aggr). All image acquisitions and further data analysis were performed with the software of Leica LAS AF. Normal HeLa cells without SERS mapping were set as the control group. 2.9. PTT Experiments. HeLa cells were plated on flat-bottom sixwell plates. After the cells reached monolayer, they were washed with PBS and incubated with silica-coated NMs dispersed in serum-free DMEM at a concentration of 8.3 × 1010 mL−1 for 4 h. Next, the cells were resuspended in full cultural medium and irradiated with an 808 nm continuous wave (CW) laser with a power density of 4 W/cm2 during 30−300 s. Fluorescence-based live−dead assay was used to evaluate the viability of cells after PTT in the region of irradiation and outside. For this end, a staining solution containing 5 mL of serumfree DMEM, 8 μL of fluorescein diacetate (5 mg/mL), and 50 μL of propidium iodide (2 mg/mL) was prepared. The cells were incubated with the staining solution for 5 min, washed with serum-free DMEM, and analyzed by fluorescence microscopy at 460 nm excitation. The PTT concentration effects of silica-coated NMs were also taken under same parameters.

the instruments of Libra-120 (Carl Zeiss, Jena, Germany) and JEM2100F (JEOL, Japan). The average nanorod (NR) size (width and length) and Ag shell thickness were determined from high-resolution TEM (HRTEM) imaging by counting more than 50 NPs. High-angle annular dark-field scanning transmission electron microscopy (STEM), energy-dispersive X-ray spectrum (EDS), and element mapping images were recorded on a JSM 100CX (n) transmission electron microscope at an accelerating voltage of 200 kV. Normal Raman and SERS spectra were acquired with a PeakSeeker Pro 785 Raman spectrometer (Ocean Optics) in 1 cm quartz cuvettes using 785 nm irradiation (30 mW). The acquisition interval was 30 s, and all spectra were averaged over 10 independent runs. Spectral and TEM measurements were done at the Simbioz Center for the Collective Use of Research Equipment in the Field of Physical-Chemical Biology and Nanobiotechnology at the IBPPM RAS. 2.4. Photothermal Measurements of Colloids. To measure the laser heating kinetics for NM suspension, an 808 nm diode laser with an optic fiber output and a maximal continuous output power of 2 W (Opto Power Corp.) was used. NIR thermographic measurements were made using the IRISYS 4010 system (U.K.), which records the temperature in the range of −10 to 250 °C with a sensitivity of 0.15 °C. The duration of the heating was usually 5 min. The temperature space distribution was registered by a thermovision camera on the side (37 cm away) of the tube in the direction, which was perpendicular to the laser beam. The thermograms were registered at an interval of 1 min. The dynamic image from the thermovision camera was recorded with a photocamera at an interval of 10 s. 2.5. Cell Culture. HeLa human cancer cells were purchased from Biolot (Russia) and cultured in a DMEM with 10% fetal bovine serum (FBS) and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin) (Sigma, St. Louis, MO). The cells were grown in a water-jacketed incubator at 37 °C with 5% CO2-humidified atmosphere in 25 cm2 tissue culture flasks. 2.6. Cell Viability Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to check NM toxicity. HeLa cells were grown in 96-well immunological plates. After the cells reached the monolayer, silica-coated NM suspension with various concentrations was added. After incubation with NPs, the cells were washed with phosphate-buffered saline (PBS) and 0.2 mL of MTT solution was added (Sigma) followed by incubation for 3 h in the dark at 37 °C. Then, the cells were washed with PBS and 0.2 mL of dimethyl sulfoxide was added to dissolve formazan crystals. Absorbance spectra were collected by Tecan spectrophotometer with microplate reader. All experiments were repeated six times. 2.7. SERS Imaging. HeLa cells were plated at a density of 4 × 104 cells on 0.5 cm × 0.5 cm silicon substrates in flat-bottom 24-well plates. For single-label imaging, 200 μL of silica-coated NMs with different embedded reporters was resuspended in 1 mL of cultural medium and added to each well. For multiplex imaging, a mixture of NMs with different Raman reporters (70 μL of each) was applied. Then, the cells were incubated with NMs for 4 h at 37 °C, washed with PBS, and fixed with 1% glutaraldehyde. The SERS scanning was performed with a confocal InVia Renishaw Raman microscope (785 nm, 63× water immersion objective, 7 μW, and 10 ms integration time per pixel). 2.8. Photodamage Test of On- and Off-Resonance SERS Probes. For detecting photodamage effects of on- and off-resonance RNMs to HeLa cells during SERS mapping test, a mitochondrial fluorescent probe JC-1 was introduced to monitor the changes of mitochondrial membrane potential. HeLa cells were plated at a density of 3 × 105 in a 3.5 cm confocal coordinate culture disk and cultured for 20 h at 37 °C. After removing the culture medium (DMEM), 100 μL of on- or off-resonance silica-coated RNMs was mixed with 900 μL of the culture medium and then added to culture disk. The final RNM concentration was about 0.05 nM. The cells were incubated for 4 h at 37 °C, and their culture medium was replaced. The as-prepared samples were directly utilized for SERS mapping tests. For the SERS mapping, the scanning region was located by the coordinate labeled at the bottom of the culture disk with a mapping area of 80 μm × 80 μm. After SERS mapping tests, the cells were stained with JC-1 by diluting

3. RESULTS AND DISCUSSION The initial Au nanorod (NR) suspension was prepared in a binary surfactant mixture as described elsewhere.34,35 According to TEM images, the fabricated Au NRs have a width of 16 ± 3 nm and a length of 92 ± 17 nm (Figure 2a-i). The average axial ratio of Au NRs is about 5.7, in good agreement with the theoretical estimation37 based on the experimental dipolar longitudinal (937 nm) and transversal (507 nm) resonance wavelengths (Figure 2b-i). The high peak ratio (∼6) between the longitudinal and the transverse modes indicates a minimal percentage (17 nm) Ag shell because such RNMs become too large and polydispersed, although their SERS performance can be further improved (data not shown). Herein, we try to understand the enhancement mechanism of SERS in the RNMs combined with the particle morphologies gained from the TEM images discussed above. Although thicker Ag shell may improve the LSPR quality factor of entire Au−Ag hybrid core−shell NPs and also possibly enhance the chemical effect (CHEM mechanism), we are not able to evaluate these contributions quantitatively. More importantly, the EM effect, as the dominant SERS mechanism, also cannot explain the SERS properties of RNMs, that is, the linear dependence of the SERS intensity on the Ag shell thickness when the particles are offresonance. Indeed, the classical electromagnetic theory turns to be useless for this purpose as there are no gaps between the Au and Ag layers and we cannot calculate the enhanced 30391


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Figure 4. SERS bioimaging with off-resonant RNMs. (a) Schematics and TEM images of RNMs with different embedded (i) NBT, (ii) NT, and (iii) BDT Raman reporters. Insets show HRTEM images of corresponding RNMs coated with a mesoporous silica layer. All scale bars are 50 nm. (b) Evaluation of cell viability after incubation with the off-resonant SERS probe of silica-coated NBT-RNMs. (c) Bright-field (BF) images and their overlays with SERS imaging of HeLa cells incubated with (i) NBT-, (ii) NT-, and (iii) BDT-RNMs. (d) BF image and its overlay with the multiplexed SERS imaging of HeLa cells incubated with the mixture of three types of RNMs.

3a. The distance between the end of the fiber and the liquid surface in the tube was about 30 mm, and the thermograms were registered by an infrared (IR) thermovision camera (Figure 3b). The photothermal kinetic curves (Figure 3c) show that the NBT-RNM particles with an Ag shell thickness of 3.3 nm has the best PT efficiency with a maximal temperature up to 65 °C, which corresponds to about 40 °C increment after the irradiation during 300 s (Figure 3b,c). In contrast, the sample with the thickest (16.7 nm) Ag shell has the worst PT efficiency with a slight temperature increment of about ∼8 °C (Figure 3b,c). The significant difference in photothermal efficiency among RNMs can be rationalized from the relationship between the temperature increase and the LSPR wavelength (Figure 3d), which demonstrates significantly lower absorption efficiency of the RNMs due to the LSPR mismatching to the laser wavelength of 808 nm (Figure 3b,c). Combining the tunable extinction spectra and SERS performance of RNMs (Figure 2) with their highly tunable PT efficiency (Figure 3), we conclude that the off-resonant RNMs at the NIR excitation show a remarkably high SERS performance but a low absorption coefficient, thus ensuring minimal perturbation to the biological species during the imaging process. On the other hand, the on-resonant RNMs can be designed for multifunctional NIR theranostic probes that can image and kill the target cells due to photothermal effects. We further employed these highly SERS-active off-resonant RNMs for the demonstration of multiplexed cell-imaging ability. RNMs were embedded with three types of Raman

NBT NPs with different Ag shell thicknesses obtained by adding 10−80 μL of 100 mM AgNO3 to reaction mixture. In general, the extinction spectra for both particle types are close, although some differences were observed for maximal amount of AgNO3 added. In contrast, the SERS responses of AuNR@ Ag@NBT particles (Figure S4b) differ dramatically from those shown in Figure 2b. Roughly speaking, the intensities of main peaks at 1343 cm−1 for AuNR@Ag@NBT NPs are about of 1 order of magnitude less compared to peak intensities for NBTRNMs. Specifically, for particles of both types fabricated with 10, 20, 40, 60, and 80 μL of 100 mM AgNO3 added to reaction mixture, the ratios of the main SERS peaks (Iinside/Isurface) are 4.1, 1.5, 14.9, 14.9, and 35.3, respectively. We also notice different evolution of SERS peaks with an increase in Ag shell thickness (Figures 2h and S4c). For NBT-RNMs, the peak magnitude increases monotonously, whereas for AuNR@Ag@ NBT NPs, the maximal SERS response is observed for intermediate shell thickness corresponding to 20 μL of 100 mM AgNO3. This can be explained by plasmon resonance excitation of SERS at 785 nm laser light as the extinction resonance is located at 796 nm for particles obtained after addition of 20 μL of 100 mM AgNO3. Out of plasmon resonance, the SERS peak intensity varies from 755 (10 μL) to 1205 counts (80 μL). Next, we studied the PT properties of aqueous RNMs with different Ag shell thicknesses. The RNMs were irradiated in a glass cuvette with a sample volume of 4 mL, which was placed vertically, and the NIR radiation (808 nm, 2 W/cm2) was delivered with a light diode from the top as illustrated in Figure 30392


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ACS Applied Materials & Interfaces reporters, NBT, BDT, and naphtalenethiol (NT), with a suitable off-resonance Ag shell thickness at 785 nm excitation laser (Figure 4a). BDT and NT molecules were selected due to their high Raman cross sections and the distinguishable Raman fingerprints of NBT (Figure 4d).59,66 For example, the Raman bands at 1340 cm−1 (for NBT, blue), 1385 cm−1 (for NT, green), and 1589 cm−1 (for BDT, red) were utilized herein for SERS mapping. To improve the probe biocompatibility, all RNMs were coated with a mesoporous silica shell (insets in Figure 4a). The silica shell shows a clear mesoporous morphology with an averaged thickness of about 10 nm for all types of RNMs (Figure S5 in the Supporting Information). The RNMs retain the off-resonant property after the silica coating because such a coating usually causes only a small red shift of about 8 nm.67 The biocompatibility of silica-coated RNMs was tested by MTT assay with different NP concentrations and incubation time. As shown in Figure 4b, the percentage of living cells cocultured with RNMs at a concentration up to 6.5 × 1010 mL−1 for 96 h remains more than 80% compared to that without RNMs. As the concentration of RNMs was increased to 8.5 × 1010 mL−1, the cell viability dropped to about 40% after 96 h of incubation and was considered to be cytotoxic. Therefore, according to the above cell viability study, RNMs with a concentration from 0 to 6.5 × 1010 mL−1 can be considered to be noncytotoxic. The bright-field (BF) images of HeLa cells incubated with silicacoated (i) NBT-, (ii) NT-, and (iii) BDT-RNMs indicate that all SERS probes are nonspecifically swallowed and accumulated around the cell nucleus, which is similar to the previous observation.68 The overlaid images show that the SERS images fit well with the corresponding BF images for all three types of off-resonant RNMs. The possibility of multiplexed SERS imaging using the mixture of three RNMs is demonstrated in Figure 4d. It should be noted that such a single-cell SERS image was acquired within 50 s (2500 spectra acquired from an area of 27 μm × 28 μm) with a 10 ms exposure time per pixel. During the above NIR SERS imaging process, NBT-RNMs may generate PT effects under optical excitation and thus inevitably cause photodamage to biological cells. To the best of our knowledge, such unwanted photodamage to cells during the SERS imaging was not evaluated in detail before. Here, we tried to assess the photodamage effects on cells from on- and off-resonance NBT-RNMs by monitoring the change in mitochondrial membrane potential with JC-1 staining. Normal cells have a higher mitochondrial membrane potential, which decreases when cells suffer from damage.69,70 JC-1 dye can form aggregates (J-aggr) in the mitochondrial matrix and emits red fluorescence when mitochondria have a high membrane potential. By contrast, in the cell cytoplasm, JC-1 is present in the form of monomers (J-mono) and exhibits green fluorescence when the membrane potential of cells is low.70,71 The ratio of the red/green fluorescence intensities can characterize quantitatively the change in mitochondrial membrane potential and the photodamage effects of NBTRNMs to cells during the SERS imaging. For on- and offresonance NBT-RNMs, the HeLa cells were performed for SERS imaging procedure before the JC-1 staining. Normal HeLa cells without nanoparticle and SERS treatments were set as control. From confocal images (Figure 5a), the intensities of red and green fluorescence of HeLa cells imaged with offresonance SERS probes and control HeLa cells are nearly the same, indicating that off-resonance NBT-RNMs have very little photodamage to cells after SERS imaging procedure. However,

Figure 5. Assessment of cell photodamage effects of on- and offresonance NBT-RNMs by monitoring the change in mitochondrial membrane potential with JC-1 staining. (a) Confocal microscopic images of HeLa cells after the SERS imaging with on- and offresonance NBT-RNMs and control groups. (b) Data for the changes of mitochondrial membrane potential by comparing J-aggr/mono ratio statistics. “J-aggr” and “J-mono” are the abbreviations for the aggregate and monomer states of JC-1 dye. All scale bars are 20 μm.

for on-resonance NBT-RNMs, cells exhibit stronger green fluorescence and much weaker red fluorescence in contrast to off-resonance NBT-RNMs and the control cells. This suggests that on-resonance NBT-RNMs cause evidently larger photodamage to HeLa cells. Quantitative evaluations of the red/ green fluorescence intensity ratio normalized to that for control cells (Figure 5b) further confirm a stronger photodamage to HeLa cells caused by on-resonance NBT-RNMs as compared to off-resonance particles. Specifically, the red/green ratios for on- and off-resonance NBT-RNMs are 0.62 and 0.83, respectively. The last value is very close to that of control cells, indicating much less photodamage produced during SERS imaging procedure. Therefore, off-resonant NBT-RNMs are greatly favorable SERS imaging probes due to much less photodamage effect and improved SERS signals. The in vitro photothermal therapy of on-resonant NBTRNM probes was further evaluated in HeLa cells with an 808 nm laser excitation. The cells were incubated with NBT-RNMs at a concentration of 8.3 × 1010 mL−1 in the serum-free culture medium. After 4 h, the cells were washed and irradiated with an 30393


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Figure 6. (a) Schematics and (b−f) results of in vitro PTT experiments using on-resonant RNM SERS probes. Viabilities of HeLa cells after NIR irradiation without SERS probes (b) and with on-resonant silica-coated RNM probes (8.3 × 1010 mL−1) for 0 (c), 30 (d), 120 (e), and 300 s (f) irradiation times. Dotted lines indicate the boundaries between illuminated and nonilluminated areas.

808 nm CW laser (4 W/cm2) during different time intervals from 30 to 300 s. Then, they were stained with calcein acetoxymethyl and propidium iodide. The control cells without probes retain their viability after 300 s irradiation by the same laser light (Figure 6a,b). In contrast, for cells incubated with NBT-RNMs, we observed apoptotic features within the illuminated region already after 30 s irradiation (Figure 6d). Longer irradiation time further leads to complete destruction of cells in the illuminated region while nonirradiated cells remain alive (Figure 6e,f). For a plasmonic Au@Ag RNM, the maximal light absorption and photothermal properties are determined by the global particle structure and the core/shell morphology, whereas the dielectric properties of the embedded molecules play a minor role. A direct evidence for similarity of extinction spectra of three NBT-, NT-, and BDT-RNMs comes from their experimental extinction spectra with increased Ag shell thickness (Figure S6). In particular, their general profile of spectra and blue shift of the major plasmonic peak are quite similar. This means that the change of embedded Raman reporters between Au core and Ag shell will not influence the PTT effects of RNMs. To evaluate the PTT effect for different RNM concentrations, we repeated PTT in vitro experiments for three NBT-RNM concentrations. All experimental conditions were the same as in Figure 6 (4 W/cm2; irradiation times: 0, 30, 120, and 300 s), except for a smaller cell concentration to make available single-cell observation. In general, we did not find any notable effects of the RNM concentration, at least for concentrations used here (Figure S7 in the Supporting Information). The SERS stability of NPs under storage conditions and in different physiological environments is important for biomedical applications. To address this point, we performed several stability tests (Figure S8 in the Supporting Information). In particular, the silica-coated off-resonant NBT-RNMs (Figure 2a-vi) showed adequate stability in aqueous solution at different pH values ranging from 4 to 11, ultrapure water, and ethanol (see Figure S8). In 10% fetal bovine serum (FBS), the Raman intensity decreased quickly to about 50% in the first 24 h, but then RNMs still exhibited decent SERS signals and remained stable during the next 72 h incubation. These data are promising for future biomedical applications.

4. CONCLUSIONS In conclusion, we have developed a new type of plasmonic ultrabright SERS probes composed of Au@Ag core−shell rodlike nanomatryoshkas (RNMs) with embedded Raman reporters for bioimaging and cancer photothermal therapy. By controlling the Ag shell thickness, we have fabricated RNMs with highly tunable LSPR from UV to NIR range and highly tunable photothermal properties with the NIR excitation. Furthermore, the unique core−shell junction geometry with embedded reporters allows decoupling the LSPR spectrum with the SERS performance. By monitoring the changes in mitochondrial membrane potential with JC-1 staining, we have confirmed quantitatively a low photothermal damage to HeLa cells from off-resonance RNMs during SERS imaging procedure. Thus, the developed off- and on-resonance RNMs can be used as supercontrast NIR SERS agents for bioimaging with minimized photothermal influence to the biological samples when choosing a thick (e.g., ∼17 nm) Ag shell, or as multifunctional NIR theranostic probes that can image and photothermally kill the cancer cells when choosing a thin (e.g., ∼2 nm) Ag shell.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08733. Extinction spectra of Au NRs and Au NRs@NBT; TEM images of Au NRs, Au@BDT@Au RNMs, and NBTRNMs with different Ag shell thicknesses and mesoporous silica-coated BDT-, NT-, and NBT-RNMs; demonstration of AuNR(core)@gap@Au(shell) structure for nanomatryoshkas with BDT molecules embedded between gold core and shell; extinction and SERS spectra of Au@Ag@NBT particles with different Ag shell thicknesses; detailed calculations of SERS enhancement factors of RNMs; extinction spectra of AuNR@NBT@ Ag, AuNR@NT@Ag, and AuNR@BDT@Ag nanoparticles with different Ag shell thicknesses; dependence of the PTT efficiency on the concentration of onresonant NBT-RNMs; SERS stability data for silicacoated NBT-RNMs (PDF) 30394


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (N.G.K.). *E-mail: [email protected] (J.Y.). ORCID

Jian Ye: 0000-0002-8101-8362 Author Contributions #

X.J. and B.N.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS X.J. and J.Y. gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21375087, 81571763, and 81622026) and Shanghai Jiao Tong University (No. YG2016MS51). This work was also supported by the Grant from the Shanghai Key Laboratory of Gynecologic Oncology. The work by B.N.K., V.A.K., and N.G.K. was supported by the Russian Foundation for Basic Research (project nos. 16-02-00054 and 17-02-00075). The work by V.A.K. was also supported by a grant from the President of RF (project no. MK-2617.2017.2).

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Rational Design of Ultrabright SERS Probes with Embedded

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