Bioconjugation of Gold Nanobipyramids for SERS Detection and

Feb 1, 2017 - (4-6) Hence these advantages endow SERS imaging with great potential for the detection of cancer cells even at the early stage. ... (29-...
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Bioconjugation of Gold Nanobipyramids for SERS Detection and Targeted Photothermal Therapy in Breast Cancer Jie Feng,†,‡,§,⊥ Limin Chen,†,‡,§ Yuanzhi Xia,⊥ Jie Xing,⊥ Zihou Li,⊥ Qiuping Qian,§ Yan Wang,§ Aiguo Wu,⊥ Leyong Zeng,*,⊥,∥ and Yunlong Zhou*,‡,§ ‡

School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou 325035, P. R. China § Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Wenzhou 325000, P. R. China ⊥ Division of Functional Materials and Nano Devices, Ningbo Institute of Materials Technology & Engineering, Chinese Academy of Sciences, Ningbo, Zhejiang 315201, P. R. China ∥ Key Laboratory of Medical Chemistry and Molecular Diagnosis of Ministry of Education, Chemical Biology Key Laboratory of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, P. R. China S Supporting Information *

ABSTRACT: Gold nanobipyramids (Au NBPs) with two sharp tips present an extremely strong electric field enhancement, which endows them with more advantages in biomedical photonics than other gold nanostructures. The application of Au NBPs for diagnosis and therapy is now under intensive investigation. Here, we report Au NBPs for surface-enhanced Raman scattering (SERS) detection and photothermal therapy (PTT) of MCF-7 cancer cells both in vitro and in vivo via bioconjugation with Raman reporter 2-naphthalenethiol (2-NAP) and folic acid (FA). The results showed that bioconjugated Au NBPs not only could be used for the quantitative detection of the MCF-7 cells in the range of 5−500 cells/mL, but also lead to the enhanced Raman signal in MCF-7 tumor-bearing nude mice with high specificity. Moreover the bioconjugated Au NBPs exhibited excellent photothermal performance in both in vitro and in vivo therapies, in which the cell viability decreased to 6.44% and the relative volume of MCF-7 tumors treated with bioconjugated Au NBPs reduced to 0.037 under the irradiation of an 808 nm laser. Our results indicate that bioconjugated Au NBPs offer an excellent nanoplatform for PTT and SERS detection in the future. KEYWORDS: Au nanobipyramids, bioconjugation, SERS detection, photothermal therapy

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INTRODUCTION Multidisciplinary diagnoses and treatments have been employed to improve the efficacy of cancer therapy.1,2 Among various tumor imaging methods, SERS imaging, as a newly emerged technique, has become a fascinating and powerful tool for cell detection and imaging without rapid photobleaching and autofluorescence of biological samples.3 It can noninvasively detect the biological samples using near-infrared (NIR) excitation to ensure laser powers low enough for living cells to withstand. In addition, this technique can be used to detect a very low concentration analyte with extraordinary specificity.4−6 Hence these advantages endow SERS imaging with great potential for the detection of cancer cells even at the early stage.7,8 Meanwhile, among the existing cancer treatment methods, photothermal therapy has recently emerged as a promising method to treat cancer with minimal invasion and high selectivity.9−11 This technique involves employing the plasmonic nanocrystals (NCs) into the tumor as a transducer that can convert NIR light to heat and generate a localized temperature increase,12 which causes the irreversible cellular damage and subsequent tumor destruction.13−15 To improve © 2017 American Chemical Society

therapeutic efficacy, integrating SERS imaging function during PTT, has been accepted as a promising strategy in cancer therapy because of its precise localization, subsequent accurate therapy, and monitoring of therapeutic response.16−18 Up to now, various shapes of gold nanostructures such as nanospheres, nanorods, nanocages, and nanostars have all been employed as SERS active substrates and hyperthermia agents because of their excellent properties including strong localized surface plasmon resonance (LSPR), in vivo stability, and wellestablished biocompatibility.18−24 Au NBPs, a kind of elongated plasmonic gold nanostructures with two sharp tips, have recently gained increasing attention. Compared with the gold nanostructures above, Au NBPs with better shape and size homogeneity, excellent chemical stability, and tunable absorption wavelengths, can be easily tuned to NIR region by controlling the aspect ratio during synthesis.25−27 The strong optical absorption within the NIR region where living tissue has a minor absorption coefficient Received: January 10, 2017 Accepted: February 1, 2017 Published: February 1, 2017 608

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

Article

ACS Biomaterials Science & Engineering

were added into the above solution orderly. Finally, 2.74 mL of the resulting seed solution was added into the above growth solution. These solutions were kept at 28 °C for several hours and their color gradually changed from almost clear to dark pink and remained. Prior to encoding Au NBPs with the Raman reporter 2-NAP, Au NBPs were further purified according to a previous report with only slight modification.25 Basically, 420 mL of the as-synthesized Au NBPs were first centrifuged at 8000 rpm for 10 min, then the precipitates were redispersed in 315 mL of 0.08 M CTAC solution, which were followed by the subsequent addition and mixing 52 mL of 0.01 M AgNO3 and 26 mL of 0.1 M ascorbic acid. The resultant solution was kept in an oil bath at 65 °C for 4 h to produce Au@Ag nanorods. The bimetallic Au@Ag nanorods were centrifuged at 5000 rpm for 10 min, the precipitates were then dispersed in 315 mL of 0.05 M CTAB and left undisturbed for about another 4 h under room temperature, during which the Au@Ag nanorods aggregated together and precipitated to the bottom of the container, whereas the spherical-like NCs remained in the supernatant. The supernatant was then discarded and the remaining precipitate was dispersed in 200 mL of 0.05 M CTAB solution. The resulting solution was subsequently mixed gently with 4.2 mL of NH3·H2O (25 wt %) and 3.2 mL of 1.6 M H2O2 and kept undisturbed for another 6 h. Then the clear supernatant was taken out and centrifuged at 5000 rpm for 10 min and finally the purified Au NBPs were dispersed in 100 mL of 5 mM CTAB solution for further use. Before folic acid conjugation, the purified Au NBPs were first labeled with Raman reporter 2-NAP by incubating 100 mL of Au NBPs solution with 2 mL of ethanol solution of 5 mM 2-NAP. The resulting 2-NAP labeled Au NBPs were denoted as NAP-Au NBPs later. After 2 h, 0.05 g of SH-PEG-NH2 was added to the above solution and kept stirring under room temperature for 24 h until the system reached equilibrium, after which the resulting NCs were further dialyzed for another 24 h in a 5 kDa cutoff cellulose ester membrane to remove the excess 2-NAP and SH-PEG-NH2. The resulting products were denoted as PEG-NAP-Au NBPs hereafter. The PEG-NAP-Au NBPs were then dispersed in 100 mL of distilled water. For folic acid conjugation, 0.25 mg of folic acid dispersed in 0.5 mL of MES buffer (pH 6.0, 10 mM) was first activated with the help of 15 μL of EDC (10 mg/mL) and 15 μL of NHS (10 mg/ mL), After activation for 30 min, 100 mL of the as-synthesized PEGNAP-Au NBPs were added to the above solution and kept stirring under room temperature for 24 h and the final products (denoted as FA-NAPAu NBPs hereafter) were collected and washed with distilled water by centrifugation at 8000 rpm for 10 min. And then they were dispersed in 100 mL of distilled water for further use. Characterization. The morphology and size of Au NBPs and FANAP-Au NBPs were acquired by transmission electron microscopy (TEM). Before the measurement, samples were prepared by drying a drop of dispersion of suspension on a carbon-coated copper grid at room temperature and were analyzed using a JEM-2100F electron microscope equipped with operating voltage of 200 kV. UV−vis−NIR spectra of both Au NBPs and FA-NAP-Au NBPs were recorded on a PerkinElmer Lambda 35 UV−vis spectrophotometer between 400 and 900 nm wavelength. And the samples were measured in a 1 cm quartz cuvette using the corresponding pure solvent as a reference. Zeta potential measurement was conducted in Zetasizer-NanoS from Malvern Instruments. The photothermal performance of FA-NAP-Au NBPs was investigated using an 808 nm laser and an infrared (IR) thermal imaging system. An IR thermal imaging system (MAG-V30, Vst Light & Technology Ltd., Wuhan of China) was employed for the recording temperature change. Before the measurement, the particles were directly dispersed in deionized water. SERS spectra were obtained on a Renishaw Raman Microscope configured with a 785 nm excitation laser line. Laser power at the sample was measured to be 28 mW. Cell Culture and in Vitro Cell Cytotoxicity Study. Human breast cancer cells (MCF-7) and human hepatoma cells (HepG2) were cultured in DMEM culture medium supplemented with 10% of FBS, 100 units/mL of penicillin, and 100 mg/mL of streptomycin at 37 °C in 5% CO2 environment. MCF-10A cells were cultured in DMEM/F-12 medium containing 5% horse serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 100 ng/mL cholera toxin, 10 ng/mL epidermal growth

makes Au NBPs potentially to act as a hyperthermia agent for photothermal therapy. Previous studies have already proven that SERS enhancement mainly originates from chemical and predominant electromagnetic (EM) contributions.28 More recent theoretical calculations have further revealed that Au NBPs possess even stronger local electric-field enhancements than nanorods or other shapes owing to the sharper tips.29−31 Therefore, it can be expected that Au NBPs can induce large increase in SERS signal by the strong local EM field enhancement near their sharp tips after excitation of the localized SPR. Despite these fascinating properties of Au NBPs, only a few studies have reported the application of Au NBPs for photothermal treatment of cancer, and even fewer made use of all their integrated functionalities simultaneously due to the limitation of the preparation and modification of Au NBPs.32,33 Therefore, it is of great interest to integrate these functionalities and further explore its potential as a SERS/PTT based bifunctional theranostic probe for cancer therapy. Breast cancer is one of the leading causes of cancer-related death among women.34,35 The development of the effective theranostic probe for breast cancer for the early diagnosis, treatment is of great importance and will greatly improve survivorship. Here, as a proof of concept, we reported an Au NBPs based bifunctional probe to specifically detect and kill MCF-7 breast cancer cells based on the SERS and photothermal properties of Au NBPs. The sharp tips of Au NBPs endow itself with ultrasensitivity in in vitro detection of MCF-7 cells and in vivo SERS imaging of MCF-7 tumor. Meanwhile, the bioconjugation of Au NBPs enabled Au NBPs specifically to target and kill breast cancer cells via photothermal therapy. Our work serves as an experimental verification that the bioconjugated Au NBPs are a promising candidate for SERS detection and PTT for breast cancers.

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MATERIALS AND METHODS

Reagents and Materials. Gold chloride trihydrate (HAuCl4·3H2O, 48%), sodium borohydride (NaBH4, 98%), silver nitrate (AgNO3, 99%), L-ascorbic acid (99%), hexadecyltrimethylammonium bromide (CTAB, 98%), cetyltrimethylammonium (CTAC solution 25% in water), trisodium citrate (99%), 2-naphthalenethiol (2-NAP, 99%), Nhydroxysuccinimide (NHS), and ammonia solution (28−30 wt %) were purchased from Sigma-Aldrich. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC·HCl), folic acid (FA), and hydrogen peroxide solution (30 wt %) were purchased from Aladdin Reagent Co. Ltd. and used as received. Amino-group-terminated PEGylated thiol (SH-PEG-NH2, MW = 5000) was purchased from Shanghai Yanyi Biotechnology Corporation. Trypsin-EDTA (0.25%), fetal bovine serum (FBS, Gibco), Dulbecco’s modified Eagle medium (DMEM) and 5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from obtained from Gibco Life Technologies. All glassware and Teflon-coated magnetic stirring bars were thoroughly cleaned with aqua regia, followed by copious rinsing with purified water. Water was purified with a Millipore system. Preparation of FA-NAP-Au NBPs. The Au NBPs were synthesized by a seed-mediated method according to a previous report with only slight modification.30 Briefly, the citrate-stabilized seed solution was prepared by adding 0.15 mL of 0.01 M freshly prepared ice-cold NaBH4 solution into an aqueous solution composed of 0.125 mL of 0.01 M HAuCl4, 0.25 mL of 0.01 M trisodium citrate, and 9.625 mL of water under vigorous stirring. The resulting seed solution was then heated in a water bath regulated at 30 °C for at least 2 h. During this step, the seeds became more reddish, indicating a slight increase in size. They were then removed from the water bath and stored at 4 °C. Second, 20 mL of 10 mM HAuCl4 and 400 mL of 100 mM CTAB solution were mixed with 4.0 mL of 10 mM silver nitrate to prepare the growth solution. Then, 8.0 mL of 1.0 M hydrochloric acid and 3.2 mL of 100 mM L-ascorbic acid 609

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

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Scheme 1. Schematic Illustration of the Preparation of Bioconjugated Au NBPs and Its Application in SERS Detection and NIRTriggered PTT of Breast Cancers

which the photodensity was 0, 0.4, 0.8, 1.2, 1.6, and 2.0 W/cm2. (2) MCF-7 cells incubated with FA-NAP-Au NBPs (150 μg/mL) were irradiated by an 808 nm laser (2.0 W/cm2), in which the irradiation time was 0, 1, 2, 3, 4, and 5 min. (3) MCF-7 cells incubated with FA-NAP-Au NBPs were irradiated by an 808 nm laser (2.0 W/cm2) for 5 min, in which the concentration of FA-NAP-Au NBPs were 9.375, 18.75, 37.5, 75, and 150 μg/mL. Alternatively, MCF-7 cells in the control were also irradiated by an 808 nm laser with different power densities and different times. Finally, the viabilities of MCF-7 cells were measured by an MTT assay. Furthermore, the viabilities of MCF-7 cells in the control and incubated with FA-NAP-Au NBPs (150 μg/mL) under the irradiation of an 808 nm laser (2.0 W/cm2) for 0, 1, 3, and 5 min were also assessed by calcein-AM/ethidium homodimer-1 double staining as reported in the literature.28 Animal Models and In Vivo Systematic Biocompatibility. Female Balb/c (nu/nu) nude mice (18−21 g, 4−6 weeks old) were used in this work. All animal experiments were performed in compliance with the guidelines for the care and use of research animals established by Wenzhou medical university. For biocompatibility measurements, 18− 21 g healthy nude mice (4−6 weeks old) were injected with 100 μL of PBS and FA-NAP-Au NBPs (750 μg/mL in PBS) via the tail vein. After 14 days, they were sacrificed, and the main organs (the heart, liver, spleen, lung, and kidney) were removed for histological analysis. Tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The histological sections were then imaged under an inverted optical microscope. In this work, the tumor model of MCF-7 was established and used. Briefly, MCF-7 cells were diluted with PBS and then injected subcutaneously into each mouse at the right back with about 1 × 107 cells. When the tumors grew to be about 5 mm in diameter, the in vivo PTT therapy was conducted. In Vivo SERS Imaging of MCF-7 Tumor-Bearing Nude Mice. MCF-7 tumor-bearing nude mice were injected with 50 μL of PBS, 2NAP (2.5 mM in PBS/ethanol), FA-NAP-Au NBPs (1.5 mg/mL in PBS). After 30 min, the Raman spectra of mice were measured with a

factor, 0.5 mg/mL hydrocortisone, 10 mg/mL insulin, and 1% Lglutamine at 37 °C under an atmosphere of 5% CO2. Prior to study the in vitro PTT performance, we first investigated its cytotoxicity against the MCF-7 cells. Basically, MCF-7 cells in logarithmic growth were cultured in 96-well plates for 24 h and then incubated with FA-NAP-Au NBPs at 37 °C for another 24 h, in which the concentration of FA-NAP-Au NBPs were 9.375, 18.75, 37.5, 75, 150 μg/mL. Another set (without any treatment) was used as control. After incubation, 10 μL of 5 mg/mL MTT solution was added to each well and fully mixed. About 4 h later, the fromazan’s absorbance at 490 nm was measured. We assigned the viability of control cells as 100%, base on which we could estimate the relative viability of cells treated with FANAP-Au NBPs. In Vitro SERS Detection of MCF-7 Cancer Cells Using FA-NAPAu NBPs SERS Scattering Assay. For in vitro SERS detection of MCF-7 cancer cells, we evaluated the specificity and sensitivity of our designed FA-NAP-Au NBPs SERS scattering assay for the detection of MCF-7 cells, respectively. In a typical sensitivity experiment, 100 μL of 0.9 mg/mL FA-NAP-Au NBPs were added into 5 mL of preseeded cells in DMEM (80000, 40000, 20000, 10000, 5000, 2500, 500, 250, 50, 25, and 5 MCF-7 cells/mL) and then incubated for 30 min at 37 °C. Afterward, the samples were centrifuged (400 g, 5.0 min), washed with PBS for three times, mixed with paraformaldehyde, and then concentrated to 200 μL for SERS detection. The same method was also used to study the specificity. The only difference is that 100 μL of 0.9 mg/mL FA-NAP-Au NBPs was incubated with 5.0 mL of cells in DMEM containing 10 000 HepG2 cells/mL (or MCF-10A cells). In Vitro PTT Studies of FA-NAP-Au NBPs. In vitro PTT performance of FA-NAP-Au NBPs was then evaluated on MCF-7 cells. Basically MCF-7 cells in logarithmic growth were cultured in 96well plates for 24 h and then added FA-NAP-Au NBPs. After incubation for 24 h, cells were washed with PBS to remove excess unassociated FANAP-Au NBPs and MCF-7 cells were then irradiated by an 808 nm laser under different conditions. (1) MCF-7 cells incubated with FA-NAP-Au NBPs (150 μg/mL) were irradiated by an 808 nm laser for 5 min, in 610

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

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ACS Biomaterials Science & Engineering

Figure 1. Characterization of bioconjugated Au NBPs. (A, B) Typical TEM images of Au NBPs and FA-NAP-Au NBPs (scale bar: 100 nm). (C) UV− vis−NIR spectra of Au NBPs and FA-NAP-Au NBPs. (D) IR spectra of (a) Au NBPs, (b) PEG-NAP-Au NBPs, and (c) FA-NAP-Au NBPs. (E) Zeta potential of Au NBPs, PEG-NAP-Au NBPs and FA-NAP-Au NBPs. (F) SERS spectra of pure 2-NAP, Au NBPs, and FA-NAP-Au NBPs. wavelength of 785 nm with an integration time of 1 s and laser power of 28 mW. As a comparison, the Raman spectrum of a mouse without the injection was set as control and measured at the same conditions. In Vivo Photothermal Therapy on MCF-7 Tumor-Bearing Nude Mice. MCF-7 tumor-bearing nude mice were randomly divided into four groups (n = 5, per group): injected with 50 μL of PBS, PBS +NIR, FA-NAP-Au NBPs (1.5 mg/mL in PBS), and FA-NAP-Au NBPs +NIR (1.5 mg/mL in PBS) for groups of 1, 2, 3 and 4, respectively. Especially, the mice in groups of 2 and 4 were irradiated by an 808 nm laser (1.6 W/cm2) for 5 min after 24 h injection. Thermal images were taken every minute using IR thermal imaging system. The tumor volume and bodyweight were measured every other day, and the volume was calculated to be V = (length of tumor) × (width of tumor)2/2. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). On the 14th day, the mice were sacrificed, and the tumors and the main organs (heart, liver, spleen, lung, and kidney) were dissected for H&E staining.

SERS detection and NIR-triggered PTT in breast cancer is illustrated in Scheme 1. In general, AuNBPs were first encoded with Raman reporter 2-NAP via the Au−S bond. To enhance its specificity for the recognition of MCF-7 cancer cells, we functionalized NAP-Au NBPs with heterofunctional thiol-PEGamine (SH-PEG-NH2), subsequently FA was covalently grafted to the PEG-NAP-Au NBPs through the reaction between carboxyl group of FA and free amine group of SH-PEG-NH2. Finally, the resulting bioconjugated Au NBPs were used for SERS detection and NIR-triggered PTT in breast cancers. The morphology of Au NBPs before and after bioconjugation was first examined by transmission electron microscopy (TEM). Au NBPs are about 117.05 ± 4.45 nm and 36.08 ± 2.55 nm in length and width (Figure 1A), respectively. After FA conjugation, the FA-NAP-Au NBPs still kept almost identical size and shape (Figure 1B), which could be further confirmed by the corresponding DLS data of Au NBPs and FA-NAP-Au NBPs. As shown in Figure S1, two peaks of the size distribution (intensity) appeared because of the rodlike shape of Au NBPs. And it also could be seen that FA-NAP-Au NBPs had exhibited a

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RESULTS AND DISCUSSION Strategy for the Preparation of Bioconjugated Au NBPs for SERS Detection and Photothermal Therapy of Breast Cancers. The preparation of bioconjugated Au NBPs for 611

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

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ACS Biomaterials Science & Engineering

Figure 2. (A) SERS spectra of (a) MCF-7 cells, (b) FRα negative HepG2 cells incubated with FA-NAP-Au NBPs, (c) FRα negative MCF-10A cells incubated with FA-NAP-Au NBPs, (d) FRα positive MCF-7 cells incubated with PEG-NAP-Au NBPs, and (e) FRα positive MCF-7 cells incubated with FA-NAP-Au NBPs. (B, C) SERS signal of in the range of 5−10 000 cell/mL after incubated with FA-NAP-Au NBPs. (D) Plot of the SERS intensity at 1065 cm−1 as a function of the concentration of MCF-7 cells stock solution.

to the phenyl ring of folic acid and 2-NAP, and the band at 1180 cm−1 can be ascribed to stretching vibration of −C-O groups in free carboxyl group from conjugated FA. Overall, these confirmed the successful linkage between FA and PEG-NAPAu NBPs via amidation reaction. Zeta potential was further conducted to track the evolvement of linked groups on the surface of Au NBPs. Figure 1E shows the zeta potentials of Au NBPs, PEG-NAP-Au NBPs and FA-NAPAu NBPs, respectively. The zeta potential of Au NBPs is +37 mV due to the existence of remaining CTAB molecule. After grafting with 2-NAP and SH-PEG-NH2, the zeta potential decreases to +17.7 mV, indicating that some CTAB molecules have been replaced by both 2-NAP and SH-PEG-NH2 due to stronger affinity of thiol group to Au surface than that between amine group to Au surface. As a piece of evidence for the successful covalent conjugation of FA, FA-NAP-PEG-Au NBPs have a zeta potential of 2.9 mV, indicating that amine group on the surface of PEG-NAP-Au NBPs had been reacted with FA, which is in consistent with previous IR analysis. The representative Raman spectra of pure 2-NAP, Au NBPs and FA-NAP-Au NBPs (Figure 1F)showed sharp and narrow peaks at a wavenumber of 516, 598, 636, 766, 843, 1065, 1378, and 1451 cm−1, and so on, which could be clearly distinguished from pure 2-NAP and Au NBPs. This fact indicates that FANAP-Au NBPs might have potential application in SERS detection. To study the photothermal effect of FA-NAP-Au NBPs induced by NIR irradiation, different concentrations of FA-NAP-Au NBPs were irradiated by an NIR laser (808 nm, 2 W/cm2) for 5 min. The temperature of FA-NAP-Au NBPs solution increases with the irradiation time, and the temperature

slight red shift in size, indicating that surface modification does not cause the aggregation of NCs. The bioconjugation of Au NBPs were then characterized via UV−vis−NIR spectroscopy, infrared spectroscopy (IR), Raman spectroscopy, and Zeta potential. UV−vis−NIR spectra indicate that Au NBPs have a longitudinal surface plasmon resonance peak at 786 nm (Figure 1C). After FA conjugation, an 18 nm red shift was observed due to the increased local refractive index of the surrounding medium for FA-NAP-Au NBPs after replacing CTAB with SH-PEG-NH2 and FA.36,37 The FA-NAP-Au NBPs exhibit a strong extinction band at 804 nm, making it highly promising in photothermal therapy under the irradiation of an 808 nm laser. Curve a in Figure 1D shows the IR spectra of Au NBPs before modification. Two bands at ∼2917 and ∼2849 cm−1 are ascribed to the asymmetric and symmetric vibrations of CH2 units from the remaining CTAB molecules binding to the surface of Au NBPs. The adsorption at 1105 and 1350 cm−1 in curve (b) (Figure 1D) can be assigned to the stretching and antisymmetric stretching of C−O−C from SH-PEG-NH2, whereas the bands at 1650 and 1459 cm−1 come from the aromatic ring C−C stretching mode of the 2-NAP, together with the band at 847 cm−1 belongs to out-of-plane bending modes of the aromatic C−H group. These results clearly indicate that both 2-NAP and SH-PEG-NH2 have been chemically conjugated to the NAP-Au NBPs. The IR spectra of FA-NAP-Au NBPs (curve c, Figure 1D) are characterized by a number of characteristic bands located at 1655, 1618, 1450, and 1180 cm−1. The band at 1655 cm−1 can be assigned to the amide carbonyl groups, and the band at 1618 cm−1 can be assigned to the bending mode of NHvibration. The characteristic band at 1450 cm−1 can be assigned 612

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

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ACS Biomaterials Science & Engineering

Figure 3. In vitro viabilities of MCF-7 cells under different conditions characterized by the MTT assay and live/dead cell staining. (A) Cytotoxicity of MCF-7 cells incubated with FA-NAP-Au NBPs. (B) Viabilities of MCF-7 cells in the control and incubated with FA-NAP-Au NBPs under the irradiation of an 808 nm laser with different power densities, in which the concentration of FA-NAP-Au NBPs were 150 μg/mL, and the irradiation time was 5 min. (C) Viabilities of MCF-7 cells in the control and incubated with FA-NAP-Au NBPs under the irradiation of an 808 nm laser with different irradiation times, in which the concentration of FA-NAP-Au NBPs were 150 μg/mL, and the photodensity was 2 W/cm2. (D) Viabilities of MCF-7 cells incubated with FA-NAP-Au NBPs with different concentrations under the irradiation of an 808 nm laser, in which MCF-7 vrlld without irradiation were set as control, the photodensity was 2 W/cm2 and the irradiation time was 5 min. (E) Fluorescence images of MCF-7 cells in the control (a) and incubated with FA-NAP-Au NBPs (b) under the irradiation of an 808 nm laser with different irradiation time for 0, 1, 3, and 5 min by calcein-AM/ethidium homodimer-1 live/dead cell staining, in which green fluorescence from calcein and red fluorescence from ethidium homodimer-1 indicate live and dead cells, respectively. Scale bar = 50 μm.

increases more rapidly as the concentration of FA-NAP-Au NBPs increase (Figure S2B). Moreover, the temperature of FANAP-Au NBPs solution at a concentration of 75 μg/mL raises from 27.7 to 61 °C after irradiation for 5 min. In comparison, the temperature of pure water increases by only 0.3 °C under the same laser exposure condition. As difference indicates FA-NAPAu NBPs might have potential application as an effective hyperthermia agent for cancer therapy. In Vitro SERS Detection of MCF-7 Cancer Cells by FANAP-Au NBPs. In order to investigate whether FA-NAP-Au NBPs could be specifically used for the detection of MCF-7 cancer cells, we then studied how SERS intensity changes after

the addition of folate receptor alpha (FRα) negative human hepatocellular carcinoma cell line (HepG2), human normal breast epithelial cells (MCF-10A) and FRα-positive MCF-7 cells. As shown in curve a in Figure 2A, MCF-7 cells did not generate any SERS characteristic peaks. After binding with the FA-NAPAu NBPs, the SERS spectra of the MCF-7 cells display characteristic peaks of 2-NAP (curve e). In contrast, SERS signal of 2-NAP is negligible in the presence of both HepG2 cells (curve b and MCF-10A cells (curve c)). Such kind of drastic differences could be explained as follow: MCF-7 cancer cells overexpressed FRα,38,39 so in the presence of MCF-7 cells, several FA-NAP-Au NBPs could specifically bind to the surface of MCF-7 cancer 613

DOI: 10.1021/acsbiomaterials.7b00021 ACS Biomater. Sci. Eng. 2017, 3, 608−618

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ACS Biomaterials Science & Engineering

Figure 4. H&E staining images of main organs (heart, liver, spleen, lung, and kidney) of healthy nude mice injected with (a) PBS and (b) FA-NAP-Au NBPs. Images were obtained under Leica microscope using a 40× objective.

= 0.9914), the good linear relationship indicated that FA-NAPAu NBPs SERS scattering assay could be used to quantitatively analyze the MCF-7 cancer cells, and the limit of detection (LOD) in this report could be as low as 5 cells/mL. In Vitro Biocompatibility and in Vitro PTT of MCF-7 Cancer Cells. Encouraged by both excellent photothermal performance and in vitro targeted detection ability of FA-NAPAu NBPs, the in vitro PTT of FA-NAP-Au NBPs against MCF-7 cells were then investigated under different conditions. A standard cell toxicity test was carried out to determine the cytotoxicity of FA-NAP-Au NBPs before they were used for further biological experiments. Figure 3A shows the cell viability of MCF-7 cancer cells after 24 h incubation with different concentration of FA-NAP-Au NBPs; the results show that even the concentration of FA-NAP-Au NBPs increased to 150 μg/mL, the cell viability of MCF-7 cells was still higher than 96%, indicating that FA-NAP-Au NBPs exhibit negligible toxicity to MCF-7 cells at the studied doses. Figure 3B shows the viabilities of MCF-7 cells in the control and incubated with FA-NAP-Au NBPs (150 μg/mL) under irradiation of an 808 nm laser with different power densities for 5 min. A single 808 nm laser irradiation is not harmful for MCF-7 cells in the control, but the MCF-7 cells incubated with FA-NAP-Au NBPs could be obviously killed under the irradiation of an 808 nm laser, in which the cell viability of MCF-7 cells could drastically decrease to 20.56, 11.28, and 6.44% with the photodensity of 0.8, 1.6, and 2.0 W/cm2, respectively. By changing the irradiation time, the viabilities of MCF-7 cells in the control and the one incubated with FA-NAP-Au NBPs (150 μg/mL) were also measured (Figure 3C). Similar to the results described in Figure 3B, the viability of MCF-7 cells in the control hardly decreased, but the viability of MCF-7 cells incubated with FA-NAP-Au NBPs decrease to 41.14, 15.85, 15.2, 10.28, and 8.39%, with the irradiation time extending from 1 to 5 min, respectively. Furthermore, under the irradiation of an 808 nm laser with the photodensity of 2.0 W/cm2 for 5 min, the viabilities of MCF-7 cells were measured to be 93.37, 66.66, 6.79, 6.55, and 1.5%, when the concentration of FA-NAP-Au NBPs increased from 9.375 to 150 μg/mL (Figure 3D). These above in vitro MTT results clearly indicate that FA-NAP-Au NBPs could act as an excellent hyperthermia agent for in vitro PTT under the irradiation of an 808 nm laser. In addition, the in vitro PTT efficiency of MCF-7 cells incubated with FA-NAP-Au NBPs (150 μg/mL) under different irradiation time was further evaluated by Calcein-AM/Ethidium Homodimer-1 double staining assay. As shown in Figure 3E, the MCF-7 cells in the control all remained healthy with green calcein fluorescence even we extend the irradiation time to 5 min, suggesting that an 808 nm laser irradiation is not harmful for

Figure 5. In vivo SERS spectra of MCF-7 tumor-bearing nude mice in the control and injected with PBS, pure 2-NAP, and FA-NAP-Au NBPs under the excitation of a 785 nm laser (28 mW, 1 s).

cells, which lead to the aggregation of NCs.40,41 As a result, several hot pots could be formed, which in turn resulted in a significant enhancement of Raman signal intensity thorough electromagnetic enhancement. However, in contrast, both HepG2 cells and MCF-10A cells did not overexpress FRα, therefore there could be a weak interaction between FA-NAP-Au NBPs and HepG2 cells or MCF-10A cells. Due to the lack of strong interaction, FA-NAP-Au NBPs did not produce enough hot spots on the surface of HepG2 cells or MCF-10A cells, thereby Raman enhancement could not be observed. To further demonstrate that FA-NAP-Au NBPs could bind on the surface of the MCF-7 cells via specific interaction of FA and FRα of MCF-7 cells, we performed another control experiment, in which nontargeting PEG-NAP-Au NBPs were incubated with MCF-7 cells, and no clear SERS signal of 2-NAP could be observed due to the lack of specific interaction (curve d). All these above contrast differences clearly demonstrate that FA-NAP-Au NBPs SERS scattering assay is highly specific for the detection of MCF7 cells. To further evaluate the sensitivity of FA-NAP-Au NBPs SERS scattering assay, we then evaluated different concentrations of MCF-7 cells stock solution. As shown in Figure 2B, C, the SERS intensity increases with an increase in the concentration of MCF7 cells in the range of 5−10000 cells/mL. It was further demonstrated that the concentration of MCF-7 cells increases to above 20000 cells/mL, the SERS intensity remained almost static (Figure 2D), which was probably due to the fact that small size aggregates were better for the formation of hot spots.40 The inset plot of Figure 2D further gives the SERS intensity versus different concentration of MCF-7 cells in the range of 5−500 cells/mL (R2 614

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ACS Biomaterials Science & Engineering

Figure 6. In vivo PTT characterization of MCF-7 tumor-bearing nude mice injected with PBS, PBS+NIR irradiation, FA-NAP-Au NBPs, and FA-NAPAu NBPs+NIR irradiation (n = 5, per group). (A) Change in relative tumor volume in different groups of PBS, PBS+NIR, FA-NAP-Au NBPs, and FANAP-Au NBPs+NIR. (B) Change of body weight in different groups of PBS, PBS+NIR, FA-NAP-Au NBPs, and FA-NAP-Au NBPs+NIR. (C) IR thermal imaging of tumor-bearing nude mice injected with (a) PBS and (b) FA-NAP-Au NBPs under the irradiation of an 808 nm laser for 0, 1, 3, and 5 min. (D) Representative digital photographs of MCF-7 tumor-bearing nude mice in different groups ((a) PBS, (b) PBS+NIR, (c) FA-NAP-Au NBPs, and (d) FA-NAP-Au NBPs+NIR) at the beginning and at the end of 14 days.

Figure 7. H&E staining images of the tumor and main organs (heart, liver, spleen, lung, and kidney) of MCF-7 tumor bearing nude mice injected with PBS (a), PBS+NIR (b), FA-NAP-Au NBPs (c) and FA-NAP-Au NBPs+NIR (d). Images were obtained under a Leica microscope using a 40× objective.

MCF-7 cells. Remarkably enhanced cancer cell killing efficiency could be observed for the MCF-7 cells incubated with FA-NAPAu NBPs when the irradiation time was only 3 min. Further

extension of the irradiation time to 5 min resulted in the complete killing of MCF-7 cells. These staining results are also consistent with that of the MTT assay. 615

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ACS Biomaterials Science & Engineering In Vivo Biocompatibility and in Vivo SERS Imaging of MCF-7 Tumor Bearing Nude Mice. The toxicity is always a great concern for nanoparticulate system used in biomedicine.42 To clarify whether FA-NAP-Au NBPs could cause any detrimental effect, the in vivo biocompatibility was then investigated by injecting PBS and FA-NAP-Au NBPs in healthy nude mice via the tail vein, and the tissue sections of main organs (heart, liver, spleen, lung, and kidney) were then evaluated by H&E staining. As shown in Figure 4, no noticeable pathological tissue damage or abnormality of major organs could be observed in both the groups of FA-NAP-Au NBPs and PBS, indicating that FA-NAP-Au NBPs possess excellent in vivo compatibility at the studied doses, which could be further applied in vivo SERS imaging and in vivo PTT of breast cancers with low risk. On the basis of the performance of FA-NAP-Au NBPs for the targeted in vitro SERS detection of MCF-7 cells, we further investigated its potential for the in vivo SERS imaging of MCF-7 tumor. The MCF-7 tumor-bearing mice injected with PBS, 2NAP or FA-NAP-Au NBPs were subjected to 30 min scanning after injection. As shown in Figure 5, The Raman spectra of tumors without injection or with injection of PBS and pure 2NAP are similar, in which no obvious peaks could be observed. However, many sharp and narrow peaks located at 516, 598, 636, 766, 843, 1065, 1378, and 1451 cm−1 could be seen in the Raman spectra of MCF-7 tumors injected with FA-NAP-Au NBPs. These above results clearly demonstrate that FA-NAP-Au NBPs could be promising as a SERS imaging probe for the in vivo diagnosis of breast cancer. In Vivo PTT of MCF-7 Tumor-Bearing Nude Mice. The in vitro PTT performance together with preferable tumor accumulation of FA-NAP-Au NBPs as detected by the in vivo Raman imaging inspired us to test it for further in vivo cancer photothermal therapy. The in vivo PTT of FA-NAP-Au NBPs against MCF-7 tumor bearing mice were then assessed in 14 days. We randomly divided the MCF-7 tumor-bearing nude mice into four groups and then injected and treated with PBS, PBS +NIR, FA-NAP-Au NBPs and FA-NAP-Au NBPs+NIR, respectively. Figure 6A shows the change of relative tumor volume of MCF-7 tumor-bearing nude mice in 14-day treatments. At the 14th day, the relative volume of MCF-7 tumors is 8.28, 8.38, and 8.86 in groups of PBS, PBS+NIR, and FA-NAP-Au NBPs, respectively, but is 0.037 in the group of FANAP-Au NBPs+NIR. Moreover, the body weight of tumorbearing nude mice in all four groups also does not decrease significantly (Figure 6B). To compare the temperature change of nude mice injected with PBS and FA-NAP-Au NBPs under the irradiation of an 808 nm laser, we also measured the in vivo IR thermal images of MCF-7 tumors (Figure 6C). In 5 min of irradiation, the temperature of the MCF-7 tumor in the group of FA-NAP-Au NBPs rapidly increases to 70 °C above, which is high enough to kill all kinds of cancer cells, whereas negligible increment in temperature could be observed in the group of PBS. Figure 6D further shows the representative digital photographs of MCF-7 tumor-bearing nude mice in four groups at the beginning and at the end of 14 days period, the tumors in the group of FA-NAP-Au NBPs+NIR were completely ablated, leaving a small scar at the original tumor site, whereas the other group showed rapid tumor growth. These results clearly reveal that FA-NAP-Au NBPs could also effectively kill the tumor cells in vivo by the photothermal effect under the laser irradiation of an 808 nm laser. Finally, to evaluate the therapeutic efficacy and the potential risk of FA-NAP-Au NBPs and NIR lights for the tumor

treatment, the tissue sections of the tumor, heart, liver, spleen, lung, and kidney of nude mice in four groups were analyzed by H&E staining. As shown in Figure 7, a large area of dead cells without nuclei in the tumor could be clearly seen in the group of FA-NAP-Au NBPs+NIR, whereas many nuclei existed in other groups, indicating that the destruction of MCF-7 cells is indeed caused by the PTT effect of FA-NAP-Au NBPs under the irradiation of an 808 nm laser. In addition, histological analysis of major organ shows that no noticeable signal of organ damage could be observed from H&E stained organ slices including heart, liver, spleen, lung, and kidney. These imply that FA-NAPAu NBPs is a safe and effective hyperthermia agent for in vivo photothermal therapy of breast cancers.

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CONCLUSION In summary, we have developed a facile and feasible method for SERS detection and photothermal therapy of breast cancer. In this method, we conjugated Au NBPs with Raman reporter 2NAP and FA. The resulting bioconjugated Au NBPs could be used for both in vitro SERS detection of MCF-7 cells and in vivo SERS imaging of MCF-7 tumor with high specificity. Moreover, we demonstrated that the bioconjugated Au NBPs were valuable as a new theranostic system for both SERS detection and PTT in breast cancers with low risk.

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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/acsbiomaterials.7b00021. DLS data of Au NBPs and FA-NAP-Au NBPs, photothermal performance, and IR thermal imaging of FA-NAPAu NBPs under different conditions (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Aiguo Wu: 0000-0001-7200-8923 Yunlong Zhou: 0000-0001-5654-1170 Author Contributions †

J.F. and L.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is financially supported by Natural Science Foundation of China (NSFC-21573162 and NSFC-21603166) and WIBEZD 2014001-02. J.F. thanks W.X. for her assistance with Raman imaging experiment.

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