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Aptamer-Conjugated Au Nanocage/SiO2 Core-Shell Bifunctional Nanoprobes with High Stability and Biocompatibility for Cellular SERS Imaging and Near-Infrared Photothermal Therapy Shengping Wen, Xuran Miao, Gao-Chao Fan, Tingting Xu, Li-Ping Jiang, Ping Wu, Chenxin Cai, and Jun-Jie Zhu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00682 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019
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Aptamer-Conjugated Au Nanocage/SiO 2 Core-Shell Bifunctional Nanoprobes with High Stability and Biocompatibility for Cellular SERS Imaging and Near-Infrared Photothermal Therapy Shengping Wen, † Xuran Miao, † Gao-Chao Fan,‡ Tingting Xu,† Li-Ping Jiang,† Ping Wu,*,§ Chenxin Cai, § and Jun-Jie Zhu*,† †
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing 210023, PR China ‡
Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE;
Shandong Key Laboratory of Biochemical Analysis; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China §
Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of
Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Mate rials Science, Nanjing Normal University, Nanjing 210023, PR China
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ABSTRACT: The combination of surface-enhanced Raman scattering (SERS) imaging technology with near- infrared (NIR) light-triggered photothermal therapy is of outmost importance to develop novel theranostic platforms. Herein, an aptamer-conjugated Au nanocage/SiO 2 (AuNC/SiO 2 /Apt) core-shell Raman nanoprobe has been rationally designed as the bifunctional theranostic platform to fulfill this task. In this theranostic system, the Raman- labelled Au nanocage (AuNC) was encapsulated into a bio- inert shell of SiO 2 , followed by conjugating aptamer AS1411 as the target-recognition moiety. AuNC served as the SERS-active and photothermal substrate due to its large free volume, built- in plasmon effect and NIR photothermal capacity, while the SiO 2 coating endowed the nanoprobes with good stability and biocompatibility, as well as abundant anchoring sites for surface functionalization. Considering their prominent SERS and photothermal properties, the application potential of the AuNC/SiO 2 /Apt nanoprobes was investigated. The proposed nanoprobes could be applied to targeted detection and SERS imaging of nucleolin-overexpressing cancer cells (MCF-7 cells as the model) from normal cells, and also exhibited acceptable photothermal efficacy without systematic toxicity. This theranostic nanoplatform provided a possible opportunity for in situ diagnosis and noninvasive treatment of cancer cells by SERS imaging- guided photothermal therapy. KEYWORDS: bifunctional theranostic nanoplatform, AuNC/SiO2 /Apt nanoprobes, SERS imaging, photothermal therapy, cancer cells
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Cancer is one of the momentous causes of mortality that seriously threaten human health and life.1 Although cancer therapy is frequently frustrated for multiple challenges, the survival rate of most cancer patients will be significantly improved through the early diagnosis and effective therapy. At present, conventional strategies like radiography and biopsy are less sensitive to diagnose cancer in the nascent stage, and are arduous to implement in-situ oncotherapy. To surmount these bottlenecks, ongoing endeavors have been exerted to design various theranostic modalities, which integrate diagnostic and therapeutic functio ns for precise cancer treatment.25 Recently, surface-enhanced Raman scattering spectroscopy (SERS) has catalyzed an upsurge of interest as a pursued tool in biosensing and bioimaging fields, due to its incomparable virtues of ultrahigh sensitivity, photostability, and narrow fingerprint signature (~1 nm) over the traditional fluorescence method.6 Furthermore, this technique holds high spatial resolution without background interference from water. Hence, these merits render SERS great promise for cancer diagnosis.7,8 Photothermal therapy is well acknowledged as a remote-controlled and noninvasive therapeutic paradigm that converts near- infrared (NIR) light energy into locoregional hyperthermia, inducing the irreparable cellular damage and tumor eradication. 9 Unlike chemotherapy and radiotherapy, photothermal therapy shows unparalleled advantages in terms of site-specific controllability, low cost, high safety, and remarkable curative effect. 911 Given above exciting characteristics, the integration of SERS imaging and photothermal therapy into a single nanoplatform is expected to be a feasible and attractive alternative in cancer dia gnostics and annihilation. The SERS imaging- guided photothermal therapy is a useful protocol to implement efficient and personalized medicine. Ultrasensitive SERS imaging can directly visualize the fine margin, size, and evolution of cancer cells or tumor tissues via Raman-based theranostic probes,12-15 providing the guidance for choosing the optimal photothermal therapy and real-time monitoring of the therapeutic process. The rational design of SERS-based photothermal probes is of grand significance to realize SERS imaging and photothermal ablation of cancer cells. Ideally, an effective SERS-integrated 3 ACS Paragon Plus Environment
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photothermal nanoplatform needs to possess both the high NIR photothermal effect and a strong electromagnetic enhancement. So far, several types of NIR-active materials such as metal nanoparticles,1618 organic compounds or polymers,19,20 and carbon nanomaterials15,21,22 have been extensively exploited to achieve the aforementioned goals. Besides unstable optical properties after prolonged illumination, some organics still suffer from the fundamental problems of complicated synthesis and weak Raman responses. In contrast, metal nanostructures with tunable plasmon resonance in the NIR regime have commanded considerable attention in bioimaging and therapeutic applications, as their unique nanoscale natures are strongly dependent on size, shape, and structure. 23,24 This is important because the NIR laser excitation (700-900
nm) offers a deeper tissue penetration with
minimal trauma and
low
autofluorescence.25 Meanwhile, the inherent “hot spots” created by anisotropic metal nanoparticles are conducive to stimulating the electromagnetic amplification of Raman signals. In these regards, Au nanocages (AuNCs) represent a potent class of plasmonic nanostructures that are peculiarly appealing to theranostic applications.26 An individual AuNC possesses the superior coupling electromagnetic field between interior and exterior surface walls, as well as a huge free volume to accommodate more Raman molecules on account of its hollow interior and porous wall, giving rise to intensive signal enhancement and in turn improving the SERS sensitivity. On the other hand, because of their larger absorption cross sections, AuNCs exhibit higher optical absorption and photothermal conversion efficiency in comparison to traditional organic dyes and spherical or rod counterparts.27,28 Consequently, AuNCs are wonderful candidates for the construction of SERS imaging- integrated photothermal theranostic nanoprobes. The direct use of naked Raman-encoded AuNCs nanoprobes for theranostic purposes may suffer from random self-aggregation in complex physiological environments, which decreases the colloidal stability of nanoprobes and the reproducibility of detection signals,29,30 limiting their bioimaging effectiveness. Moreover, the competitive adsorption of external species on the 4 ACS Paragon Plus Environment
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surface of AuNCs inevitably detaches Raman reporters and brings unwanted interferences.31,32 To ensure the stability and signal fidelity of Raman-encoded AuNCs nanoprobes, one straightforward strategy is to wrap AuNCs with a protective shell. Due to its optical transparency and bio- inertness, silica (SiO 2 ) is one of the hottest coating materials that not only endows AuNCs-based Raman nanoprobes with remarkable chemical and mechanical stability but also facilitates versatile surface functionalization. Furthermore, the outer SiO 2 shell preserves Raman reporters from harsh surroundings or contamination,33 hence evading a possible loss and signal fluctuation of reporter molecules. Such a protective shell also guarantees that the detectable signal solely arises from Raman nanoprobes. 34 The tumor-targeting ability of Raman probes is another critical issue that should be taken into account during the theranostic process. As biorecognition elements, aptamers are immensely adopted for the actively targeted delivery of nanoprobes to the desired tumor site and enhancing treatment outco mes, since they are more easily available compared to costly and instable antibodies. 3537 Herein, we engineered an aptamer-conjugated AuNC/SiO 2 core-shell Raman nanoprobe (AuNC/SiO 2 /Apt) as the bifunctional theranostic nanoplatfom for cellular SERS imaging and NIR-mediated photothermal therapy (Scheme 1). In this study, 4- mercaptobenzoic acid (pMBA) worked as a bright Raman reporter due to its large cross-section, simple Raman signal, and excellent affinity for metallic surfaces via thiol bind ing. AuNCs accounted for amplifying the Raman signal of the absorbed pMBA and inducing local heat to kill cancer cells upon NIR laser exposure. The outer SiO 2 shell enabled pMBA-encoded AuNCs with high stability and provided bioconjugation sites for tagging a nucleolin-specific aptamer AS1411. The SERS activity and photothermal transformation ability of the rationally designed AuNC/SiO 2 /Apt nanoprobes were theoretically and experimentally studied. And the practicability of AuNC/SiO 2 /Apt nanoprobes was investigated for SERS imaging and photothermal therapy towards nucleolin-positive breast cancer cells (MCF-7 cells as the model). The presented bifunctional nanoprobes showed robust and high SERS/photothermal effect, as well as low cytotoxicity. 5 ACS Paragon Plus Environment
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Scheme 1. Schematic diagram of cellular SERS imaging and photothermal therapy.
EXPERIMENTAL SECTION
Preparation of AuNC/SiO2 /Apt Nanoprobes The preparation of AuNC/SiO 2 NPs and their modifications with amino
groups
(AuNC/SiO 2 -NH2 ) were described in the Supporting Information. AuNC/SiO 2 /Apt nanoprobes were fabricated as follows. A 0.5 mL of AS1411 (6.0 µM in PBS) was activated at room temperature for 30 min with 50 µL EDC (30 mg mL1 ) and 50 µL of NHS (30 mg mL1 ) solutions. After introducing 2.0 mL of AuNC/SiO 2 -NH2 (1.8×1013 particles mL1 ), the mixture was stirred overnight at 4 °C. The obtained AuNC/SiO 2 /Apt nanoprobes were then incubated with 0.5 mL of mPEG-SPA (1.0 µM in PBS) to block non-specific binding sites for 6 h. The mPEG-SPA was engrafted
onto
the
nanoprobes
after being
hydrolyzed
to
yield
N-hydroxysuccinimide in water, which further reacted with the residual amino groups on the SiO 2 shell. The final product was purified by three centrifugation/washing cycles, redispersed in 0.5 mL of PBS, and stored at 4 °C until use. The particle concentration was 7.2×1013 particles mL1 , corresponding to 147.2 mg mL−1 . For comparison, the PEGylated SiO 2 /Apt NPs (~ 69 nm 6 ACS Paragon Plus Environment
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in size) were prepared using the similar procedure. The synthesis of SiO 2 NPs was detailed in the Supporting Information. Both AuNC/SiO 2 /mApt NPs and AuNC/SiO 2 /rDNA NPs were fabricated using the same strategy excepted for replacing Apt with three-base mismatched AS1411 (mApt) and random single chain DNA (rDNA), respectively.
Cellular SERS Imaging For cellular SERS mapping, MCF-7 (or NIH 3T3) cells were plated at a density of 2×10 3 cells on a silicon wafer (5 mm × 5 mm) in a confocal dish (diameter, 35 mm) and cultured at 37 °C for 24 h. Then, these cells were incubated in 200 µL of DMEM containing the AuNC/SiO 2 /Apt nanoprobes at a designated concentration (35 µg mL−1 ). After incubation for 4 h, the cells were washed with PBS to remove unbound nanoprobes, and were fixed with 4% of formaldehyde before Raman imaging.
Photothermal Therapy
To explore the photothermal therapeutic efficacy of Raman nanoprobes, MCF-7 cells were seeded into a 96-well plate at a density of 1×104 cells per well and cultured for 24 h at 37 °C in a 5% CO2 incubator. After being rinsed with PBS, the cells were treated with the specific concentration of AuNC/SiO 2 /Apt nanoprobes in 200 μL DMEM for 4 h. Following this step, the supernatant was replaced by 200 μL of fresh medium, and the cells were subsequently exposed to an 808-nm laser (1.5 W cm−2 , 6 mm of beam diameter) for 5 min. After incubation for another 20 h, the cell viability was quantified by MTT assays.
RESULTS AND DISCUSSION Characterization of AuNC/SiO2 /Apt Nanoprobes In our design, plasmonic AuNC/SiO 2 /Apt nanoprobes were intentionally selected as photothermal transducers and SERS generators owning to their extraordinary features, such as 7 ACS Paragon Plus Environment
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high photothermal transformation efficiency, hot spot density, and stability. 27 Typically, AuNCs were prepared by taking advantages of the galvanic replacement between HAuCl4 and Ag nanocubes (AgNCs). Primitively, AgNCs were synthesized using a NaHS- mediated polyol protocol with ethylene glycol as the reductant and polyvinylpyrrolidone as the capping agent. Underwent the galvanic replacement, AgNCs would be nicely transformed into AuNCs with the porous and void nanostructure composed of an extremely thin wall. Then, pMBA molecules were assembled on AuNCs via Au-S bonds. Subsequently, pMBA- labelled AuNCs were encapsulated into a rigid shell of SiO 2 , which can effectively prevent the leakage of the labelled pMBA, leading to an improvement of detection specificity and Raman signal stability. Finally, AS1411 and a PEG monolayer were covalently conjugated at the surface of SiO 2 shell. The resulted AuNC/SiO 2 /Apt Raman probes were endowed with the target-recognition ability, good stability, and adequate biocompatibility.
Figure 1. Typical TEM images of AgNCs (A), AuNCs (B), AuNC/SiO 2 NPs (C), and AuNC/SiO 2 /Apt (D). Insets: HRTEM images of AgNCs (A) and AuNCs (B). Scale bars: 20 (Insets) and 50 nm.
Transmission electron microscopy (TEM) and scanning electron microcopy (SEM) images gave the first confirmation of AuNC/SiO 2 /Apt nanoprobes. Figure 1A and S1 are TEM and SEM images of AgNCs, respectively, and the as-prepared AgNCs showed well-defined cubic 8 ACS Paragon Plus Environment
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morphology with an average edge length of ~29.1 nm (Figure S2). Upon galvanic replacement, the obtained AuNCs displayed a regular cubic geometry with an outer edge length of approximately 29 nm and a wall thickness of about 3.5 nm, as well as a slight corner truncation and tiny pores on the side face (Figure 1B). In addition, the yield of the cubic cage was found to be about 89% by calculating the number ratio (NCubic
naocage
/NTotal
nanoparticles)
from different
batches of colloidal AuNCs based on their TEM images. As revealed from Figure 1C, we observed AuNCs to be fully insulated by a dense shell of SiO 2 (~17 nm in thickness). After conjugation with aptamer AS1411, the size of AuNC/SiO 2 /Apt nanoprobes increased to 66.5 nm as compared to 63 nm of AuNC/SiO 2 NPs (Figure S3). These morphology changes demonstrated the formation of AuNC/SiO 2 /Apt nanoprobes. The formation of AuNC/SiO 2 /Apt nanoprobes was also verified by monitoring the surface charge alterations during the synthesis process. The corresponding zeta potentials of bare AuNCs, AuNC/SiO 2 , and AuNC/SiO 2 /Apt were detected to be −17.6, −41.7, and −22.5 mV, respectively (Figure S4), demonstrating the formation of AuNC/SiO 2 /Apt nanoprobes. To gain deeper insights in the formation of AuNC/SiO 2 /Apt nanoprobes, their optical properties were exhaustively investigated. UV-visible spectra were measured to confirm the fabrication of the AuNC/SiO 2 /Apt nanoprobes. As listed in Figure S5A, the localized surface plasmon resonance (LSPR) of AuNCs red-shifted to 745 nm compared with 415 nm of AgNCs. Then, a 25 nm bathochromic shift of the absorption peak was observed after SiO 2 coating, which resulted from a higher refractive index of SiO 2 (n=1.57) relative to water (n=1.33).34,38 Upon conjugation of aptamer AS1411, a new peak appeared at 260 nm, which belongs to the characteristic absorption peak of AS1411, indicating the successful conjugation of aptamer at the surface of AuNC/SiO 2 NPs. Noteworthily, the extinction spectrum of AuNC/SiO 2 /Apt nanoprobes was found to have a minor red-shift (3.4 nm) compared to that of AuNC/SiO 2 NPs. A broadened adsorption peak of 774 nm presented, which matches well with the central wavelength (785 and 808 nm) of the NIR laser, setting the solid ground for SERS detection and photothermal therapy. Similarly, the FTIR results supported 9 ACS Paragon Plus Environment
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the successful preparation of the AuNC/SiO 2 /Apt nanoprobes (Figure S5B). All above observations indicated that the AuNC/SiO 2 /Apt nanoprobes were reasonably constructed.
Raman Activity and Photothermal Performance of SERS Probes
The AuNC/SiO 2 NPs were rationally engineered as bifunctional theranostic agents for cellular SERS imaging and NIR- mediated photothermal therapy. Therefore, we studied Raman activity and photothermal transition behavior of the AuNC/SiO 2 NPs after their synthesis. Firstly, we used FDTD simulation to evaluate the SERS enhancement of the engineered AuNC/SiO 2 NPs. It is generally considered that the electromagnetic field contributes greatly to the SERS enhancement. As a result, we simulated the electric field intensity and distribution of the AuNC/SiO 2 NPs (Figure 2A, inset). Regions of enhanced field strength were not only observed at the frame of the AuNC, but also at edge of the holes distributed at the surface of the AuNC, suggesting more hot spots provided by our nanocage structure compared to the nanocube due to its porous structure. A resonant mode locating at 782 nm was observed (Figure 2A), which matches well with the Raman excitation wavelength (Note, 785 nm laser was chose in case of cells detection.), implying a predicable SERS enhancement effect of the AuNC/SiO 2 NPs. Hence, we further evaluated the enhancement of the Raman signal by quantifying the maximum electric field intensity (Emax /E0 )2 . The (Emax /E0 )2 at the Au/SiO 2 interface of the AuNC/SiO 2 NPs was estimated to be ~2250, inferring that AuNC/SiO 2 NPs can give an enhancement of ~5 ×106 according to the relation of 𝐺𝑆𝐸𝑅𝑆 = 𝐸𝑚𝑎𝑥 𝐸0 4 , where Emax is the maximum electric field intensity at the laser excitation wavelength (λL, 785 nm), and E0 is the amplitude of the incident field. Therefore, the engineered AuNC/SiO 2 NPs can be expected to function as a highly active SERS platform for cells detection. We also computed the absorption and scattering cross section because the LSPR peak position in the NIR as well as the ratio of absorption cross section (Cabs ) relative to scattering cross section (Csca) is significant to photothermal therapy agents. We can find that AuNC/SiO 2 NPs 10 ACS Paragon Plus Environment
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show a LSPR peak at 770 nm (green curve, Figure 2B), agreeing well with the experimental result (black curve, Figure 2B). Please note that there is a profile difference between the simulated extinction spectrum and the experimental one. The experimentally recorded spectra have a wider LSPR peak than simulated ones. This is because that the FDTD simulation approximates an ideal and monodisperse particle shape that does not interact with other nanoparticles as occurred in experimental measurements. In addition, the experimental extinction intensity of colloidal nanoparticles is high sensitive to the nanoparticle concentration. Therefore, the experimentally measured extinction intensity is lower compared to that simulated result. Also, the dimers and higher aggregates of AuNCs (Figure 1C) inside the silica may broaden the LSPR peak in the experimental extinction spectrum. To a certain extent, these aggregates might facilitate the SERS response and strengthen photothermal performance, probably due to their high absorption cross sections and plasmonic coupling between adjacent inner cores. Moreover, the calculated ratio of Cabs/Csca was 18, indicating that a larger contribution of the light absorption to the extinction cross section of AuNC/SiO 2 NPs. The results implied the predictable high photothermal conversion efficiency of AuNC/SiO 2 NPs. Along with the high SERS activity, AuNC/SiO 2 NPs showed great potential as bifunctional nanoprobes in SERS imaging-guided cancer photothermal therapy.
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Figure 2. (A) Maximum electric field intensity (Emax /E0 )2 at the Au/H2 O interface of the AuNC/SiO 2 NP as a function of the wavelength. Inset: the electric field distribution of the AuNC/SiO 2 NPs obtained at the wavelength of 782 nm. (B) The experimental results for the extinction spectrum (green curve) and the calculated results for the extinction (black curve), absorption (red curve), and scattering cross sections (blue curve) of AuNC/SiO 2 NP with an outer edge length of 29 nm, a wall thickness of 3.5 nm, and a SiO 2 shell thickness of 17 nm. (C) Raman spectra of pMBA- labelled AuNCs, AuNC/SiO 2 , and AuNC/SiO 2 /Apt under identical conditions. (D) The SERS stability of AuNC/SiO 2 /Apt nanoprobes. (E) Temperature change of SiO 2 /Apt and AuNC/SiO 2 /Apt nanoprobes at required concentrations under laser irradiation (808 nm, 1.5 W cm−2 ). (F) Cyclic temperature profiles of AuNC/SiO 2 /Apt nanoprobes and indocyanine green (ICG) at the same concentration (35 µg mL−1 ) over 10 laser-on/off cycles of 808-nm laser illumination. Note: 20, 35, 40, 80, and 120 µg mL−1 of the AuNC/SiO 2 /Apt suspension contained the particle concentration of 9.78×10 9 , 1.71×1010 , 1.96×1010 , 3.91×1010 , and 5.87×1010 particles mL1 , respectively.
After theoretical predictions of high SERS activity and photothermal conversion efficiency, proof-of-concept studies were carried out firstly by probing the SERS spectra of pMBA- labelled 12 ACS Paragon Plus Environment
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AuNC/SiO 2 NPs under 785- nm excitation. Figure 2C clarifies that the strong SERS profile could be well resolved on AuNC/SiO 2 NPs (red curve). As expected, two fingerprint peaks of 1078 and 1588 cm−1 are attributed to the aromatic ring breathing vibration and C=C stretching vibration modes of pMBA, respectively.39 For comparison, Raman measurements were conducted to collect the SERS signal of pMBA- labelled AuNC/SiO 2 /Apt nanoprobes, which was consistent with that of pMBA- labelled AuNC/SiO 2 NPs (Figure 2C), elucidating that the conjugation with aptamer would not affect the Raman activity. We also compared the Raman enhancement of the as-synthesized AuNCs with Au nanorods (AuNRs), since AuNRs were typically regarded as highly SERS-active substrates. We synthesized AuNRs (I) whose length was almost the same as the edge size of AuNCs (Figure S6A and S6B) and used pMBA as the Raman reporter to probe their SERS activities. In Figure S7, the Raman intensities at 1078 and 1588 cm−1 for pMBA- labelled AuNCs were 2.5 and 3.1- fold higher than those for pMBA- labelled AuNRs (I), respectively. In parallel, AuNRs (II) with a longitudinal LSPR band at 745 nm were fabricated (Figure S6A and S6C), which overlapped well with the absorption peak of AuNCs. AuNRs and AuNCs were used as SERS substrates with same concentrations (4.5×1012 particles mL‒1 ). It was found that the Raman signal of pMBA on AuNRs (II) was much lower than that on AuNCs (Figure S7). These results suggested that the as-synthesized AuNCs were preferential for Raman enhancement. Furthermore, the Raman stability of the AuNC/SiO 2 /Apt nanoprobes was deliberately studied for different durations. Figure 2D explicates that these nanoprobes exhibited highly reproducible and reliable Raman signals during five days of storage. The relative standard deviations of the signal intensities at 1078 and 1588 cm−1 were 5.0% and 4.2%, respectively. Likewise, the SERS response of the AuNC/SiO 2 /Apt nanoprobes after NIR irritation (5 min) did not show any appreciable variations compared with those of untreated one, underlining the substantial antibleaching ability and structural stability of the nanoprobes (Figure S8). This is very favorable to the repeated SERS imaging of cancer cells. These phenomena could be rationalized by the fact 13 ACS Paragon Plus Environment
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that pMBA would not be obliterated from AuNCs after coated with a protective shield o f SiO 2 . Besides pMBA, other Raman molecules can be embedded between the AuNC core and the SiO 2 shell for multiplexed SERS imaging thanks to the facile synthetic protocol for AuNC/SiO 2 /Apt nanoprobes. Based on the exceptional NIR absorption feature of AuNC/SiO 2 /Apt nonoprobes, we embarked on evaluating their photothermal effects under the continuous exposure with an 808-nm laser (1.5 W cm−2 ). As plotted in Figure 2E, the solution temperature escalated quickly from ambient temperature to 50 °C within the first 4 min and eventually tended to reach a plateau in the presence of AuNC/SiO 2 /Apt (20 µg mL−1 ), whereas both the PEGylated SiO 2 /Apt NPs suspension (120 µg mL−1 , Figure S9) and the water control gave a trifling rise (~5 °C) in temperature. As well known, the bare SiO 2 NPs are not the NIR-active material, so that the PEGylated SiO 2 /Apt NPs could not convert the NIR light energy into heat. These results indicated that the AuNC/SiO 2 /Apt nanoprobes could capture the NIR light and efficiently convert it into thermal energy. Such a distinguishing photothermal activity was mainly attributed to their plasmon resonance absorption at the tested wavelength. More interestingly, the photothermal curves of AuNC/SiO 2 /Apt nanoprobes exhibited both time- and concentration-dependent photothermal behaviors. The optimal irradiation time was set at 5 min. The photothermal stability of AuNC/SiO 2 /Apt nanoprobes was studied (Figure 2F). The AuNC/SiO 2 /Apt nanoprobes presented a repeatable temperature elevation over 10 successive laser-on/off cycles, indicative of their good photothermal reversibility and stability. The photothermal stability was also validated by no changes in spectroscopic characteristic and morphology upon NIR irritation (Figure S10). In Figure S11, no evident spectral shift reflected that the AuNC/SiO 2 /Apt nanoprobes were well dispersive in different media, such as purified water, PBS, DMEM medium, fetal bovine serum (10%), and NaCl (0.9%). It was primarily ascribed to a hydrophilic PEG coating on the surface of nanoprobes. Altogether, the AuNC/SiO 2 /Apt nanoprobes showed great promise for simultaneous SERS imaging and 14 ACS Paragon Plus Environment
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photothermal therapy of tumor cells.
SERS Imaging of Cancer Cells
The high Raman activity of the synthesized AuNC/SiO 2 /Apt nanoprobes is useful for the specific detection of cancer cells because one can realize the Raman detection of target cancer cells based on the Raman signal of pMBA. Before performing Raman detection, the MTT assay was applied to assess the cytotoxicity of AuNC/SiO 2 /Apt nanoprobes by using MCF-7 and NIH3T3 cells as models, and ICG as the control. It was found that more than 89% of MCF-7 or NIH3T3 cells stayed alive even with a dose as high as 300 µg mL−1 (Figure S12), highlighting the nontoxicity of AuNC/SiO 2 /Apt. This benefited from the SiO 2 shell which is chemically stable and biocompatible in organisms. The feasibility of AuNC/SiO 2 /Apt nanoprobes for SERS imaging of MCF-7 cells (as the model) was then evaluated. As depicted in Figure 3A (upper panel), Raman images mapped at 1588 cm−1 are outlined. An intensified SERS signal in the Raman mapping image was achieved from MCF-7 cells after incubation with AuNC/SiO 2 /Apt NPs. However, MCF-7 cells cultured in pure culture medium did not exhibit any Raman signals (Figure 3A, middle panel), signifying that the as-synthesized AuNC/SiO 2 /Apt nanoprobes can be used to detect MCF-7 cells. To monitor the distribution of nucleolin in the cells, we acquired Raman spectra at different spots (Figure 3B). The characteristic bands of pMBA at 1078 and 1588 cm−1 were detected at spots 1 and 2 on the surface of the MCF-7 cell after incubation with AuNC/SiO 2 /Apt, while no measurable signal was obtained at the spot 4 outside the cell. This is due to the high binding affinity of AS1411 to nucleolin on the MCF-7 cell membrane.40 Thus, the distribution of the nucleolin could be tracked by using the AuNC/SiO 2 /Apt NPs as SERS imaging probes. Furthermore, the Raman signal of pMBA was clearly observed at the randomly selected bright spot 3 (Figure 3A). But similar bright spots were not appeared in the controls. Accordingly, the bright spots originated from Raman signals rather than the autofluorescence. 15 ACS Paragon Plus Environment
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The selectivity of this detection was demonstrated by use of the NIH3T3 cells (nucleolin- negative normal cells) as controls. After being incubated with the AuNC/SiO 2 /Apt nanoprobes, NIH3T3 cells showed a Raman image with the low background, which was almost the same as that of MCF-7 cells alone (Figure 3A, under panel), because the probes cannot bind to these nucleolin- negative expressed cells. In other controls, we studied the SERS mapping images of MCF-7 cells incubated with pMBA-labelled AuNC/SiO 2 NPs, AuNC/SiO 2 /mApt NPs, and AuNC/SiO 2 /rDNA NPs, respectively. Nevertheless, SERS signals were negligible in those controls due to the absence of the specific interaction between aptamer and nucleolin (Figure S13). These findings indicated that the AuNC/SiO 2 /Apt nanoprobe was endowed with the specific selectivity for targeting cancer cells.
Figure 3. (A) SERS images of single MCF-7 cell (or NIH3T3) with and without AuNC/SiO 2 /Apt nanoprobes (Left: bright field; Right: SERS mapping images based on the intensity of 1588 cm−1 ). (B) Representative Raman spectra at different spots indicated in Figure 3A. Dotted rectangles represent SERS imaging areas. Scale bars: 20 µm.
NIR Photothermal Therapy of Tumor Cells
Motivated by their excellent photothermal effects, the practicability of AuNC/SiO 2 /Apt nanoprobes for photothermal therapy (PTT) of MCF-7 cells was estimated. The MCF-7 cells 16 ACS Paragon Plus Environment
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incubated with the AuNC/SiO 2 /Apt nanoprobes (100 μg mL‒1 ) were irradiated under an 808 nm laser (1.5 W cm−2 ) for 5 min. The therapeutic effect of PTT for MCF-7 cells was verified by calcein-AM/PI double staining, which is a universal approach to differentiate live/dead cells. As well known, PI cannot penetrate live cell membranes, but can insert into nuclear DNA of dead or late apoptotic cells and give a red fluorescence. Figure 4 illustrates that the negative control and the cells treated only with a NIR light (1.5 W cm−2 for 5 min) or the AuNC/SiO 2 /Apt nanoprobes (100 µg mL−1 ) emitted bright green fluorescence, which suggested that the cell viability was virtually unaffected in these groups. On the contrary, the strong red emission was observed in AuNC/SiO 2 /Apt nanoprobe- incubated cells after NIR light radiation, owing to the cellular destruction induced by the photothermal effect. These results confirmed that the AuNC/SiO 2 /Apt nanoprobes possessed striking photothermal potency for killing cancer. The dramatic cell death was likely caused by following reasons: first, the local heat from AuNC/SiO 2 /Apt nanoprobes exceeded the damage threshold (42 °C), resulting in the irreversible cell necrosis. Second, the cellular uptake of nanoprobes could be improved via nucleolin- mediated endocytosis,41 thus promoting the incident light absorption. Figure S14 summarizes different photothermal agents used in SERS and PTT. It is noteworthy that the irradiation power density (1.5 W cm−2 ) used in this study was lower than previously reported values for popcorn-shaped AuNPs (12.5 W cm−2 ),42 magnetic Au nanoshells (6.67 W cm−2 ),43 and AuNRs (8.5 W cm−2 ).44 Also, the as-prepared nanoprobes offered a higher Raman enhancement factor than most of mentioned Au-based nanostructures. Because the AuNC core in the nanoprobes could create a vast number of “hot spots” together with a larger free volume and also give a higher photothermal conversion efficiency, which played vital roles in simultaneously achieving the excellent electromagnetic enhancement and high phothothermal effect. The PTT effect was also evidenced by the MTT analysis. As described in Figure S15, the relative cell morality increased with the elevated concentration of AuNC/SiO 2 /Apt, and 93% of MCF-7 cells were killed by 100 µg mL−1 of the nanoprobes upon laser illumination (1.5 W cm−2 ) 17 ACS Paragon Plus Environment
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for 5 min. However, the therapeutic efficacy of AuNC/SiO 2 NPs or ICG on MCF-7 cells was much less than that of AuNC/SiO 2 /Apt. There was no obvious reduction in cell viability observed with NIR light only. These results re-confirmed the effective photothermal performance of the as-synthesized AuNC/SiO 2 /Apt nanoprobes in PTT, and it is even higher than traditional fluorescent dye ICG. Therefore, it undoubtedly showed that the AuNC/SiO 2 /Apt nanoprobes could effectively kill malignant cells in PTT.
Figure 4. Fluorescence images of MCF-7 cells treated with PBS (negative control), only laser, AuNC/SiO 2 /Apt nanoprobes, and laser + AuNC/SiO 2 /Apt nanoprobes. Scale bars: 50 µm. CONCLUSIONS
In summary, we have successfully fabricated the AuNC/SiO 2 /Apt NPs as bifunctional theranostic platforms for cellular SERS imaging and NIR-triggered photothermal therapy. Due to their built- in plasmon effects and hollow core nanostructures, the AuNC/SiO 2 /Apt NPs were capable of providing a higher SERS enhancement and sufficient hyperther mia. Moreover, they demonstrated great photostability and biocompatibility during the photothermal treatment 18 ACS Paragon Plus Environment
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because of an inert SiO 2 shell. These unique traits made the current AuNC/SiO 2 /Apt nanoprobes as promising theranostic agent for both cellular SERS mapping and photothermal therapy without noticeable adverse effects. The proposed theranostic nanoprobes could selectively recognize nucleolin-overexpressing MCF-7 cells through SERS imaging, and also showed the acceptable therapy efficacy upon NIR laser irradiation at a relatively low power density. Collectively, the AuNC/SiO 2 /Apt nanoprobes held the enormous potential toward the SERS imaging- guided diagnosis and remote-controlled photothermal annihilation of cancer cell. In addition, this nanoprobe could be applied to trace the tumor sites and biodistribution in vivo.
ASSOCIATED CONTENT
Supporting Information Supporting Information Available: The following files are available free of charge. Supplementary experimental; The SEM image of AgNCs; Particle size distribution, zeta potential, UV-Vis-NIR, and FTIR characterizations of the AuNC/SiO 2 /Apt nanoprobes; Characterization of AuNRs, SiO 2 NPs, and SiO 2 /Apt NPs; Raman spectra of pMBA on AuNCs and AuNRs; Optical and TEM data of AuNC/SiO 2 /Apt nanoprobes before/after NIR irradiation; Colloidal stability of AuNC/SiO 2 /Apt nanoprobes in different media; Cytotoxicity assays; Cellular SERS images; Comparison of different photothermal agents used in SERS and PTT; The PTT evaluation of ICG, AuNC/SiO 2 NPs, and AuNC/SiO 2 /Apt nanoprobes.
AUTHOR INFORMATION Corresponding Authors * E- mail:
[email protected]. * E- mail:
[email protected]. ORCID Shengping Wen: 0000-0002-7907-4445 19 ACS Paragon Plus Environment
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Gao-Chao Fan: 0000-0002-4645-3115 Jun-Jie Zhu: 0000-0002-8201-1285 Notes The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are gratefully for the financial support from the National Natural Science Foundation of China (21775070, 21424807), the International Cooperation Foundation from Ministry of Science and Technology (2016YFE0130100), the Jiangsu Province Postdoctoral Science Foundation (1501033B), the China Postdoctoral Science Foundation (2015M581767), the State Key Laboratory of Analytical Chemistry for Life Science Foundation (5431ZZXM1705), and the Fundamental Research Funds for the Central Universities (020514380082, 020514380127, 020514380115, and 020514380102). Thank for the support from Guangdong Provincial Key Platform
and
Major
Scientific
Research
Projects
for
Colleges
and
Universities
(2015KCXTD029).
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