Accepted Manuscript Surface-enhanced Raman Scattering (SERS)-active Gold Nanochains for Multiplex Detection and Photodynamic Therapy of Cancer Linlin Zhao, Tae-Hyun Kim, Hae-Won Kim, Jin-Chul Ahn, So Yeon Kim PII: DOI: Reference:

S1742-7061(15)00157-9 http://dx.doi.org/10.1016/j.actbio.2015.03.036 ACTBIO 3649

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

30 September 2014 13 March 2015 31 March 2015

Please cite this article as: Zhao, L., Kim, T-H., Kim, H-W., Ahn, J-C., Kim, S.Y., Surface-enhanced Raman Scattering (SERS)-active Gold Nanochains for Multiplex Detection and Photodynamic Therapy of Cancer, Acta Biomaterialia (2015), doi: http://dx.doi.org/10.1016/j.actbio.2015.03.036

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Surface-enhanced Raman Scattering (SERS)-active Gold Nanochains for Multiplex Detection and Photodynamic Therapy of Cancer Linlin Zhaoa, Tae-Hyun Kimb, c, Hae-Won Kimb, c, Jin-Chul Ahnd, So Yeon Kima, e, *

a

Graduate School of Energy Science and Technology, Chungnam National University, Daejeon 305-764, South Korea

b

Department of Nanobiomedical Science and WCU Research Center, Dankook University, Cheonan 330-714, South Korea

c

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330714, South Korea

d

Beckman Laser Institute Korea, Dankook University, Cheonan 330-714, South Korea

e

Department of Chemical Engineering Education, College of Education, Chungnam National University, Daejeon 305-764, South Korea

* Corresponding author. Tel.: +82 42 821 5892 E-mail address: [email protected] (S. Y. Kim)

ABSTRACT Multifunctional nanomedicine holds considerable promise as the next generation of medicine that will enable early detection of diseases, as well as simultaneous monitoring and therapy with minimal toxicity. In particular, surface-enhanced Raman scattering (SERS) technology with high sensitivity and multiplexing capabilities is emerging as a powerful alternative for identifying specific biological targets in live cells. In this paper, we present the synthesis of SERS-active gold nanochains (AuNCs) as a potential theranostic system for multiplex detection and photodynamic therapy (PDT) of cancer. AuNCs were prepared by a simple physical mixing method to assemble citrate-stabilized gold nanoparticles into nanochains using hyaluronic acid and hydrocaffeic acid (HA-HCA) conjugates as templates. In addition, Raman reporters and photosensitizers (PSs) were conjugated onto the surface of the AuNCs for multiplex detection and PDT action. After mixing with HA-HCA conjugates, citrate-stabilized gold nanoparticles formed the AuNC structure, and AuNC length was controlled by the HCA conjugation ratio in the HA-HCA conjugates. AuNCs exhibited maximal absorption in the near-infrared (NIR) spectral region and effective SERS property. Confocal microscopy, flow cytometry, Raman spectroscopy and Bio-TEM measurements were used to determine cellular uptake of the Raman reporter, PS and AuNCs in HeLa cells. AuNCs conjugated with Raman reporter and PS (HA-HCAn-Au-Pheo-NPT) showed more than 99% cellular uptake and exhibited excellent phototoxicity even at low PS concentrations compared with free PS after laser irradiation. This SERS-active AuNC (HA-HCAn-Au-PheoNPT) shows promise for applications in theranostics, integrating SERS imaging and PDT.

KEYWORDS: gold nanochains, SERS, photodynamic therapy, theranostics, multiplex detection, self-assembly

1. Introduction The development of nano-structured materials with unique optical, electronic, and magnetic properties has opened up new possibilities for molecular imaging and spectroscopic detection of specific targets in biomedical applications. In particular, surface-enhanced Raman scattering (SERS) spectroscopy is one of the most powerful analytical techniques for identification of molecular species, with the potential of reaching single-molecule detection under ambient conditions. Conventional Raman spectroscopy produces intrinsically weak scattering signals [1,2]. However, on roughened surfaces, especially on plasmonic metal nanostructures, surface plasmon resonance (SPR) leads to a strongly enhanced electric field, resulting in significantly greater efficiency of a range of surface optical processes such as Raman, fluorescence, and second-harmonic effects [3-6]. SERS is one of the most important spectroscopies inherently originated from the SPR of metal nanostructures. SERS has received intense interest since its discovery in 1974 due to various salient attributes such as ultrasensitivity (a detection sensitivity that is 10-14 orders of magnitude higher than conventional Raman spectroscopy), excellent selectivity, rapid detection capability, high signal-to-noise ratio, non-photobleaching features and its use of single photoexcitation [7-10]. Several SERS nanoprobes have been developed for biological analysis, and are capable of providing fingerprint information for probe molecules at the single-molecule level, such as in vivo tumor targeting and spectroscopic detection [11], and bacterial capture and culture-free analysis in human blood [12]. In addition, the wavelength used for SPR is strongly related to their shape and size. As compared to spherical gold nanoparticles, which exhibit SPR at wavelengths in the visible light range, anisotropically grown gold nanorods have a strong longitudinal SPR mode along the elongated direction under near-infrared (NIR) light, and their absorption wavelength can be finely tuned by controlling the aspect ratio of the gold nanorod [13,14]. Since NIR light has much longer

penetration depth into biological tissue than visible or infrared light, the fabrication of various gold nanostructures has drawn more attention for biomedical applications [11,12,15-19]. With these new tools, there is growing interest in theranostic nanomedicine with integrated imaging and therapeutic modalities for simultaneous diagnosis, disease treatment, and monitoring of therapeutic efficacy. In this study we designed SERS-active AuNCs as a novel theranostic system for cancer multiplex detection and photodynamic therapy (PDT). To form AuNCs composed of gold nanoparticles, we used the biocompatible polymer conjugate hyaluronic acid-hydrocaffeic acid (HA-HCA). HA, which consists of repeating disaccharide units of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine, is a linear, nonsulfated glycosaminoglycan found ubiquitously in the extracellular matrix (ECM) of virtually all mammalian connective tissues. The use of HCA, 3,4-dihydroxyhydrocinnamic acid was inspired by mussel adhesion phenomena in nature. Mussels secrete specialized adhesive proteins containing a high content of the catecholic amino acid, and it and its derivatives have demonstrated strong interfacial adhesion strength [20,21]. We utilized HCA as a high-affinity anchor for gold nanoparticle stabilization and AuNC formation. Our SERSactive AuNCs are not only useful for cancer detection, but also for delivery of a PS for PDT action. PDT, a noninvasive treatment modality for a range of diseases including cancers [22], combines three components (light, PS, and oxygen) to treat malignant diseases. Upon irradiation at the appropriate wavelength, the PS can be excited and transfers energy to surrounding tissue oxygen, generating highly reactive oxygen species (ROS) such as singlet oxygen (1O2). The ROS can react with biomolecules such as unsaturated lipids, amino acid residues in proteins, and nucleic acid bases in DNA, initiating an apoptotic or necrotic response and eventually leading to oxidative damage and cell death [23-27]. Despite its advantage over traditional treatments, PDT has not been accepted for general

treatment in clinics because of technical difficulties in application. Conventional PSs often randomly distribute in vivo and lack selectivity to tumors [28]. A significant challenge that needs to be overcome for most treatments is the hydrophobic nature of the PS, which severely hampers intravenous administration through the bloodstream. Because of their low water solubility, hydrophobic PSs cannot be directly injected intravenously. Here, to improve solubility and enhance cellular uptake and PDT efficiency, we conjugated a hydrophobic PS, pheophorbide a (Pheo), onto our AuNCs. Although various conventional SERS nanoprobes have been developed for biomarker assays [5,6,29], a SERS-active AuNC for cancer multiplex detection and therapy has not been reported. The main objective of this study was to describe a novel multifunctional SERSactive AuNC prepared by self-assembling of gold nanoparticles into AuNCs for simultaneous cancer detection and PDT. Raman reporter (2-naphthalenethiol (NPT)) and thiol groupfunctionalized PS (Pheo-SH) were conjugated onto AuNCs through thiol groups. The chemical composition of intermediates and the final product were determined by 1H NMR. The photophysical properties of AuNCs in aqueous solution and morphology were determined by UV-visible spectroscopy and TEM. In vitro cellular localization in HeLa cells was investigated by confocal microscopy, flow cytometry, SERS and Bio-TEM. The phototoxicity of free Pheo and SERS-active AuNCs was also investigated.

2. Materials and methods 2.1. Materials and characterization HA (molecular weight: about 1.65×106 Da), sodium citrate tribasic dihydrate, cysteamine hydrochloride, triethylamine (TEA), and N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma Chemical Co. (St. Louis,

MO, USA). Chloroauric acid, HCA, 2-naphthalenethiol (NPT), and 4-(dimethylamino) pyridine (DMAP) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Nhydroxysuccinimide (NHS) was purchased from Fluka (Buchs, Switzerland). Dimethyl sulfoxide (DMSO) and acetone were obtained from Samchun Pure Chemical Co., Ltd. (Gyeonggi-do, Korea). Pheo was purchased from Frontier Scientific, Inc. (Logan, UT, USA). Spectra/Por membranes were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco BRL (Invitrogen Corp., CA, USA). All chemicals were analytical grade and used as received without further purification. The chemical structure was determined by 600 MHz 1H NMR (AVANCE Ⅲ 600, Bruker, Rheinstetten, Germany) using D2O, DMSO-d6 as the solvent. FT-IR spectra were also recorded on a FT/IR-460 PLUS spectrometer (JASCO) ranging between 4000 and 400 cm-1, with a resolution of 2 cm-1 and 64 scans. The photophysical properties of AuNCs in aqueous solution were confirmed by UV-visible spectrophotometry (UVmini-1240, Shimadzu, Japan). Raman measurements were performed with a high resolution Raman system (LabRAM HR800, Horiba Jobin Yvon, France). A He-Ne laser emitting at 785nm served as the excitation source. The morphologies, sizes, and size distributions of the chain-like nanostructures were determined by field emission transmission electron microscopes (FE-TEM) (Tecnai G² F30, FEI, Amsterdam, the Netherlands) and dynamic light scattering (DLS) (ELS-Z, OTSUKA, Japan) at 25ºC using a He-Ne laser (633 nm) as a light source. The morphology of AuNCs in HeLa cells was determined by Bio-transmission electron microscope (Bio-TEM) (Tecnai G² Spirit, FEI, Oregon, USA). For in vitro cellular uptake tests, the cells were visualized using a confocal laser scanning microscope (LSM 510, Carl Zeiss Ltd., Jena, Germany) and the cellular fluorescence was quantified by flow cytometry (FACSCAN, BD Biosciences, CA,

USA).

2.2. Synthesis of citrate-stabilized gold nanoparticles Gold nanoparticles were synthesized using the citrate-reduction method reported by G. Frens [30]. Briefly, 16 ml of 10 mg/ml sodium citrate tribasic dihydrate solution was added to 800 ml of 0.1 mg/ml chloroauric acid solution under stirring and the mixture solution was heated to boiling in an oil bath. After this addition, the color of the boiling mixture solution changed from pale yellow to colorless, indicating the beginning of reduction. In the initial stage of the reaction, the solution color gradually changed from very dark blue to purple, and finally, wine red. When the wine red color appeared, boiling continued for at least 20 min in order to complete the reaction, followed by natural cooling to room temperature and storage in a refrigerator for future use. The concentration of gold nanoparticles in the solution was determined by UV-Vis spectra [31].

2.3. Preparation of HA-HCA conjugates (HA-HCA) After HA (1 g; 2.4 mmol repeat units) was dissolved completely in 150 ml of deionized water, HCA (43.7 mg; 0.24 mmol~873.8 mg; 4.8 mmol), EDC (690.1 mg; 3.6 mmol), and DMAP (439.8 mg; 3.6 mmol) were added. The reaction mixture was stirred at 900 rpm for 48 h at room temperature under nitrogen. The product was harvested by precipitation using cold acetone. The precipitate was removed into a dialysis membrane and purified by dialysis against deionized water for 2 days, and then finally freeze-dried. A series of HA-HCA conjugates were synthesized by controlling the feed ratio of HA and HCA, as shown in Table 1.

2.4. Preparation of thiol group-modified Pheo (Pheo-SH) Pheo (63 mg; 0.11 mmol) was dissolved in DMSO (20 ml). After complete dissolution, NHS (21.9 mg, 0.19 mmol) was added and stirred for 2 h at room temperature under nitrogen. Then, EDC (36.5 mg; 0.19 mmol), cysteamine hydrochloride (18.2 mg; 0.1 mmol), and TEA (223 µl; 1.1 mmol) were added to the Pheo solution. The reaction mixture was stirred at 400 rpm for 24 h at room temperature in the dark. The resulting product was purified by dialysis against deionized water for 2 days and then freeze-dried.

2.5. Fabrication of SERS-active AuNCs composed of gold nanoparticles (HA-HCAnAu-Pheo-NPT) A SERS probe with a chain-like nanostructure was prepared by a simple physical mixing method using HA-HCA conjugates as templates to assemble citrate-stabilized gold nanoparticles into nanochains. After 50 ml citrate-stabilized gold nanoparticle solution (1.98×10-9 mmol/ml) was added into 20 ml HA-HCA aqueous solution (2.5 mg/ml), the mixture was stirred at 600 rpm for 30 min at room temperature. Then 2.5 ml NPT solution in DMSO (1 mg/ml) and 2 ml Pheo-SH solution in DMSO (4 mg/ml) were added in to the mixture, respectively. The resulting mixture was stirred at 600 rpm for 24 h at room temperature in the dark. The SERS-active AuNCs (HA-HCAn-Au-Pheo-NPT) were purified by dialysis against deionized water and then freeze-dried. For systemic comparison, HAHCAn-Au and HA-HCAn-Au-NPT were also synthesized (Table 2). The drug conjugation content (DCC) of Pheo was confirmed by 1H NMR using the relative intensity ratio of the benzene protons of the HA-HCA (7.0 ppm) to the methane protons of Pheo (8.9 ppm, 9.4 ppm, and 9.7 ppm). Raman spectra of an aqueous dispersion of the HA-HCAn-Au-NPT were measured to determine the Raman enhancement factor (EF). EF was calculated using the equation, EF=(Isig/Csig)/(Iref/Cref), where Isig and Iref represent the

intensities of the 1375 cm-1 band for the NPT adsorbed on the quartz substrate with and without AuNCs, respectively, whereas Csig and Cref represent the corresponding concentrations of NPT on these substrates.

2.6. Cellular uptake of HA-HCAn-Au-Pheo-NPT HeLa cells (1×105 cells/well) were seeded onto 6-well plates and cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin at 37oC in a humidified 5% CO2-95% air atmosphere. After 24 h, the medium was replaced with 1.5 ml of fresh medium containing free Pheo (10 µg/ml), HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-Au-Pheo-NPT (110 µg/ml, 10 µg/ml Pheo equiv.), and then incubated for 4 h. The cells were then washed with PBS and harvested using 0.05% trypsin-EDTA. 4’, 6-Diamidine-2-phenylindol (DAPI) was added to stain the cell nucleus at room temperature. All experiments were carried out in a dark room to prevent photodegradation of the probes. The cells were visualized using a confocal laser scanning microscope. For the flow cytometry assay, HeLa cells (1×105 cells/well) were seeded onto 6-well plates in 1.5 ml RPMI 1640 and allowed to attach for 24 h. After cell attachment, the medium was replaced with 1.5 ml of fresh medium containing free Pheo (10 µg/ml), HA-HCA0.3-AuPheo-NPT and HA-HCA2.0-Au-Pheo-NPT (110 µg/ml, 10 µg/ml Pheo equiv.), and then incubated for 4 h. The cells were then washed with PBS and trypsinized. The harvested cells were washed by cold PBS and fixed using 4% paraformaldehyde solution. After fixation, the sample was washed again using PBS, and then the cellular fluorescence was quantified by flow cytometry. The Pheo fluorescence was excited by a laser at 670 nm.

2.7. Monitoring AuNC fate in HeLa cells HeLa cells (1×105 cells/well) were seeded onto 6-well plates in 1.5 ml RPMI 1640 and

allowed to attach for 24 h. After cell attachment, the medium was replaced with 1.5 ml of fresh medium containing HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-Au-Pheo-NPT (1 mg/ml), and then incubated for 4 h. Next, the cells were washed with PBS and fixed in 2.5% glutaraldehyde for 1 h. After several PBS washes, the samples were stained with 2% osmium tetroxide and 0.5% uranyl acetate. The samples were gradually dehydrated in ethanol and embedded in Epon-propylene oxide. Briefly, the samples were dehydrated with a concentration series of ethanol (30, 50, 70, 80, 90, 95, 99, and 100%; 40 seconds per change). Propylene oxide (PO) was added in PO-ethanol solutions at ethanol:PO=50:50, ethanol:PO=25:75, and PO100%. Epon-PO was then added and the samples were held at 40oC overnight. Thin sections were obtained with an ultramicrotome and deposited onto TEM grids. Morphology of AuNCs in HeLa cells was observed by bio-transmission electron microscopy (Bio-TEM; Tecnai G² Spirit, FEI, OR, USA).

2.8. In vitro phototoxicity assay of SERS-active AuNCs HeLa cells (1×104 cells/well) were seeded onto 96-well plates in 200 µl RPMI 1640 and allowed to attach for 24 h. After cell attachment, the medium was replaced with 100 µl of fresh medium containing free Pheo (3 µg/ml), HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0Au-Pheo-NPT (33 µg/ml, 3µg/ml Pheo equiv.), and then incubated for 4 h. The cells were washed with PBS and replace with fresh culture medium. The samples were irradiated at 0.4 mW/cm2 with a He-Ne laser (670 nm) for 0, 10, 15, and 30 min. Then, irradiated cells were incubated at 37ºC for 24 h and cell viability was evaluated by Cell Counting Kit 8 (CCK-8, Dojindo Laboratories, Japan). Untreated cells served as 100% viable cells. Data presented are averaged results of triplicate experiments. In order to determine the influence of Pheo concentration on the cytotoxicity, we also investigated the in vitro dark-toxicity of free Pheo, HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-Au-Pheo-NPT under a series of concentrations (0,

3, 6, and 10µg/ml, Pheo equiv.) without laser treatment using a similar method.

3. Results and Discussion 3.1. Synthesis and characterization of SERS-active AuNCs The procedure to synthesize SERS-active AuNCs is illustrated in Figure 1. The synthesis of intermediates and the final product was confirmed by 1H NMR spectra. First, HCA ligands were introduced to HA backbones by reaction of the carboxyl group of HCA and the hydroxyl of HA (Figure 1A). A series of HA-HCA with various HCA conjugation ratios was prepared by controlling the feed ratio of HCA to HA (Table 1). As shown in Figure 2A (b), peaks at about 1.9 and 3.6 ppm corresponded to the methyl proton and protons in the rings of HA, and peaks at 2.4, 2.8, 7.0, and 7.9 ppm belong to the methylenes, benzene, and hydroxyl protons in HCA, respectively. Compared with the original HA, HA-HCA showed a new peak at ~2924 cm-1 attributed to the aromatic ring of HCA in the FT-IR spectra (Figure 2A (a)). These results confirmed that HCA was conjugated to HA successfully. Second, a thiol group was introduced to Pheo by reaction between the amino group of cysteamine hydrochloride and the carboxyl group of Pheo (Figure 1B). The functionalization of the thiol group to Pheo (Pheo-SH) was confirmed by 1H NMR. As shown in Figure 2B (b), the peaks at 0.97 and 1.08 ppm belong to the thiol group and methylene protons of cysteamine hydrochloride, and peaks at about 6.2, 6.4, 8.2, 8.9, 9.4, and 9.7 ppm belong to Pheo’s characteristic peaks. The reaction was also confirmed from the FT-IR spectrum of Pheo-SH, in which the new peak for S-H stretching appeared at 1988 cm-1, as shown in Figure 2B (a). These results indicate that thiol groups were conjugated successfully to the Pheo molecules. In the final step, SERS-active AuNCs (HA-HCAn-Au-Pheo-NPT; where n is the molar

feed ratio of HCA to HA repeat unit) were prepared by the physical mixing of HA-HCA, citrate-stabilized gold nanoparticles, NPT, and Pheo-SH in aqueous solution (Figure 1C). In addition, HA-HCAn-Au and HA-HCAn-Au-NPT were also synthesized for systemic comparison as described in Table 2. Figure 2C exhibits the 1H NMR spectra of the resulting product HA-HCA2.0-Au-Pheo-NPT, with the characteristic peaks of each part (HA, HCA, NPT and Pheo).

3.2. Photophysical properties of AuNCs Figure 3B shows the UV-visible absorption spectra of citrate-stabilized gold nanoparticles and HA-HCAn-Au in aqueous solution. The absorption peak at 276 nm belongs to HCA. Citrate-stabilized gold nanoparticles (Figure 3B, (a) curve) and HA-HCA0.0-Au (Figure 3B, (b) curve) samples exhibit a characteristic SPR band at 519 nm that indicates the formation and existence of gold nanoparticles. However, as the HCA conjugation ratio of HA-HCA increased (HA-HCA0.1-Au, HA-HCA0.3-Au, HA-HCA0.5-Au, HA-HCA1.0-Au, and HAHCA2.0-Au), the intensity of the transverse band at 519 nm decreased, and a new plasmon band (600-700 nm) appeared on the red side of the transverse band of the citrate-stabilized gold nanoparticles. This new plasmon band can be assigned to the longitudinal band. The appearance of this long-wavelength plasmon band clearly indicates the formation of aggregated nanostructures. The photographs in Figure 3A show different colors of citratestabilized gold nanoparticles and HA-HCAn-Au aqueous solutions due to the different SPR effects of those nanostructures.

3.3. Morphology of AuNCs The morphologies, sizes, and size distributions of citrate-stabilized gold nanoparticles and HA-HCAn-Au were observed by TEM and DLS measurement. As shown in Figure 4,

submicron-sized, nearly spherical nanoparticles were observed. For citrate-stabilized gold nanoparticles, DLS measurement showed a narrow and monodisperse unimodal pattern (Figure 4A, (a)). The average size was 30.1 nm. In the TEM image, the nanoparticle aggregates were found that could be due to surface tension as the water evaporates during sample preparation (Figure 4B, (a)). When gold nanoparticles are prepared by the citratemediated reduction method, citrate ions attach to the surface of gold nanoparticles and form a protective layer with a negative charge. Although citrate-stabilized gold nanoparticles disperse very well in solution and are stable due to electrostatic repulsion for extended periods at room temperature, they aggregate easily under physiological salt conditions, and are therefore not considered suitable for many biological applications [32]. Upon addition of HA-HCA polymer conjugates, dispersibility was improved and linear chain-like nanostructures formed gradually, visualized by TEM imaging. The length of HAHCAn-Au nanochains increased as the HCA conjugation ratio of HA-HCA increased (Figure 4B, (c)-(g)). DLS measurement of HA-HCAn-Au nanochains exhibited a relatively high degree of polydispersion as shown in the (c)-(g) of Figure 4A. These peaks were belonged to the transverse size and longitudinal size of AuNCs. These results corresponded well with the plasmon absorption of the UV-visible spectra (Figure 3B).

3.4. SERS activity of SERS-active AuNCs Figure 5 shows the SERS spectra of free Raman reporter (NPT), HA-HCA2.0-Au, and AuNC samples including NPT (HA-HCAn-Au-NPT, 28 µg/ml NPT equiv.), which were prepared by dropping on the quartz substrate and air drying. AuNC samples without NPT (HA-HCA2.0-Au) exhibited no appreciable signal (Figure 5A) and free NPT also showed a very weak SERS signal (Figure 5B). However, AuNCs samples including NPT (HA-HCAnAu-NPT) showed a much stronger SERS response compared to free NPT and HA-HCA2.0-Au

(Figures 5C, D, E and F). SERS activity increased gradually as the HA-HCAn-Au-NPT nanochains lengthened. HA-HCA2.0-Au-NPT with the longest chains exhibited the strongest SERS signals (Figure 5F), indicating that the formation of nanochain structures led to plasmonic vesicles with strong SERS activity. The Raman signal observed at 1064 cm-1 can be assigned to thiol groups, and indicates that the Raman reporter NPT was directly bonded to the surface of the gold nanoparticles. The bands at 516, 765, 1375, 1447, and 1618 cm-1 are due to the aromatic C-C stretching vibrations of NPT. The surface enhancement factors (EF) for HA-HCAn-Au-NPT were roughly estimated by comparing the peak intensity at 1375 cm-1of HA-HCAn-Au-NPT and free NPT [33-35]. As shown in Table 1, EF increased with values of 2.7×105, 2.2×106, 5.7×107, and 1.8×108 as the length of the HA-HCAn-Au-NPT nanochains increased.

3.5. Multiplex imaging and cellular localization of SERS-active AuNCs in HeLa cells Figure 6 shows the SERS spectra obtained by incubating the SERS-active AuNCs (HAHCAn-Au-Pheo-NPT) with HeLa cells for 4h. HeLa cells treated with HA-HCAn-Au-PheoNPT exhibited the characteristic Raman signals of NPT at 765, 1064, and 1375 cm-1. In addition, the fate of HA-HCAn-Au-Pheo-NPT samples with different chain lengths was monitored in HeLa cells by Bio-TEM. HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-AuPheo-NPT samples were easily found in HeLa cells after 4 h of incubation (Figure 7). Many were trapped in vesicles in the cytoplasm, such as endosomes or lysosomes, allowing visualization at higher magnification (Figure 7A (b), (c), and (d); Figure 7B (b), (c), and (d)). This finding also demonstrates that HA-HCAn-Au-Pheo-NPT samples with chain-like nanostructures were internalized into HeLa cells via the endocytic pathway, and that AuNC length did not significantly influence cellular uptake behavior. The efficiency of PDT treatment is highly dependent on PS cellular uptake and

accumulation in malignant tissues [36]. The intracellular localization of Pheo molecules in HeLa cells were also investigated using confocal laser scanning microscopy and flow cytometry. As shown in Figure 8A, the confocal microscopy assay was based on the red autofluorescence of Pheo, and the blue fluorescence from DAPI bound to the nucleus. HeLa cell samples treated with free Pheo, HA-HCA0.3-Au-Pheo-NPT, and HA-HCA2.0-Au-PheoNPT produced strong fluorescence signals from Pheo around the nucleus and at the inner part of the cells, indicating that Pheo molecules were internalized into the HeLa cells. These strong fluorescence signals from Pheo could be also used to clearly understand the dynamics of intracellular networks and signal transduction in diagnostics. In addition, fluorescenceactivated cell sorting distribution of free Pheo and HA-HCAn-Au-Pheo-NPT fluorescence in HeLa cells was recorded to quantify the cellular uptake efficiency of Pheo. The flow cytometry assay showed relatively high (more than 99%) cellular uptake in not only HAHCAn-Au-Pheo-NPT samples, but also free Pheo (Figure 8B).

3.6. In Vitro phototoxicitytest of SERS-active AuNCs To determine the feasibility of SERS-active AuNCs as PS nanocarriers for PDT, the in vitro cytotoxicity of free Pheo, HA-HCA0.3-Au-Pheo-NPT, and HA-HCA2.0-Au-Pheo-NPT was investigated by CCK-8 assay and the average cell viability was monitored. We investigated the dose-dependent (0, 3, 6, and 10 µg/ml; concentration of Pheo) cytotoxicity of free Pheo and HA-HCAn-Au-Pheo-NPT without laser irradiation (Figures 9A). Here, 0 µg/ml means culture medium for Pheo sample. For HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-AuPheo-NPT samples, 0 µg/ml means free AuNC without Pheo (concentration of free AuNCs: 30 µg/ml). We found that free Pheo and free AuNCs exhibited no significant cytotoxicity under dark conditions without laser exposure. However, for HA-HCAn-Au-Pheo-NPT, cell viability decreased when the concentration of Pheo was higher than 6 µg/ml (Figure 9A) due

to the high concentration of gold nanoparticles [37,38]. Thus, we used 3 µg/ml of Pheo concentration which is did not show adverse effect on cell viability under dark condition for further phototoxicity assay. We also investigated the phototoxicity of free Pheo, HA-HCA0.3-Au-Pheo-NPT, and HAHCA2.0-Au-Pheo-NPT using various laser exposure times (0, 10, 15, and 30 min). With 3 µg/ml Pheo and 0.4 mW/cm2 laser intensity, HA-HCA0.3-Au-Pheo-NPT, and HA-HCA2.0-AuPheo-NPT exhibited significantly enhanced phototoxicity as the laser exposure time increased (Figure 9B). However, free Pheo showed no significant phototoxicity even after 30 min of laser irradiation. Thus, the PDT efficiency of HA-HCAn-Au-Pheo-NPT samples was obviously higher than free Pheo, probably due to improved Pheo solubility in the aqueous environment from the water-soluble HA-HCA polymer conjugates and increasing 1O2 quantum yield [39]. Taken together, these results indicate that our SERS-active AuNCs (HAHCAn-Au-Pheo-NPT) could considerably enhance the efficiency of PDT, even at low Pheo concentrations.

4. Conclusions Recently, various conventional SERS nanoprobes have been developed for biological applications [40-43]. However, a SERS-active AuNC for cancer multiplex detection and therapy has not been reported. In the present study, we demonstrate the applicability of SERS-active AuNCs as a potential theranostic system for multiplex detection and PDT of cancer. A series of HA-HCA polymer conjugates with various HCA conjugation ratios were synthesized successfully. These HA-HCA conjugates were used as templates for the formation of chain-like nanostructures by self-assembly of citrate-stabilized gold nanoparticles. Upon addition of HA-HCA, the dispersibility was improved and linear chain-

like nanostructures were formed. The length of AnNCs increased gradually with the HCA conjugation ratio in HA-HCA conjugates. After chain like nanostructure formation, their photophysical properties were significantly changed. UV-visible spectroscopy demonstrated that a new plasmon band appeared in the near-infrared (NIR) spectral region, associated with the longitudinal mode of the plasmon oscillation along the long axis of the AuNCs. Raman reporter (NPT) and thiol-functionalized PS (Pheo-SH) were also conjugated onto the surface of AuNCs. The AuNCs exhibited significant SERS properties. Here, the HA-HCA polymer conjugates induced the assembly of gold nanoparticles into AuNC structures, which created many hot spots at the junctions of the nanochains. As a result, the plasmon coupling of gold nanoparticles at these hot spots produced a very intense local electromagnetic field and consequently strong SERS signals [44]. The efficiency of PDT treatment is highly dependent on the PS cellular uptake and accumulation in malignant tissues. In the in vitro cellular uptake test, confocal microscopy, flow cytometry and Bio-TEM imaging showed that AuNCs conjugated with NPT and PS (HA-HCAn-Au-Pheo-NPT) could be internalized into HeLa cells. In addition, HA-HCAn-Au-Pheo-NPT exhibited excellent phototoxicity at a low Pheo concentration (3 µg/ml Pheo equiv.). Our findings suggest that HA-HCAn-Au-Pheo-NPT shows promise as a theranostic system for effective multiplex detection and PDT of cancer.

Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A3013658).

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Table 1 Composition of HA-HCA conjugates

a

Samples

Feed ratio HA:HCAa

Conjugation ratio (%)b

Molecular weight (Da)c

HA-HCA0.0

1 : 0.0

0.00

1.65×106

HA-HCA0.1 HA-HCA0.3

1 : 0.1 1 : 0.3

8.96 28.17

1.72×106

HA-HCA0.5

1 : 0.5

38.47

1.95×106

HA-HCA1.0

1 : 1.0

40.70

1.97×106

HA-HCA2.0

1 : 2.0

48.60

2.03×106

1.85×106

Molar feed ratio of HCA to HA repeat unit Percent of HA repeat units were functionalized by HCA, determined by UV-Vis spectra. c Molecular weight of HA-HCAn was calculated based on the average molecular weight of HA (1.65×106 Da) and the conjugation ratio of HCA b

Table 2 Properties of SERS-active AuNCs with different HCA conjugation ratios

Samples

Feed ratioa (HA:HCA)

HA-HCA0.0-Au

1 : 0.0

519,

HA-HCA0.1-Au

1 : 0.1

519, 605

HA-HCA0.3-Au

1 : 0.3

519, 612

HA-HCA0.5-Au

1 : 0.5

519, 627

HA-HCA1.0-Au

1 : 1.0

519, 646

HA-HCA2.0-Au

1 : 2.0

519, 658

HA-HCA0.0-Au-NPT

1 : 0.0

-

HA-HCA0.1-Au-NPT

1 : 0.1

-

HA-HCA0.3-Au-NPT

1 : 0.3

2.7×105

HA-HCA0.5-Au-NPT

1 : 0.5

2.2×106

HA-HCA1.0-Au-NPT

1 : 1.0

5.7×107

HA-HCA2.0-Au-NPT

1 : 2.0

1.8×108

HA-HCA2.0-Au-Pheo-NPT

1 : 0.3

9.0

HA-HCA2.0-Au-Pheo-NPT

1 : 2.0

9.0

Absorption wavelength (nm)b

EFc

DCC (%)d

-

a

Molar feed ratio of HCA to HA repeat unit. UV absorption wavelength of AuNCs (HA-HCAn-Au) in aqueous solution, where n is the molar feed ratio of HCA to HA repeat unit. c Raman Enhancement Factors (EF) (HA-HCAn-Au-NPT), EF=(Isig/Csig)/(Iref/Cref), where Isig and Iref represent the intensities of the 1375 cm-1 band for the NPT adsorbed on the quartz substrate with and without AuNCs, respectively, whereas Csig and Cref represent the corresponding concentrations of NPT on these substrates. d Drug conjugation content (DCC), determined by 1H NMR. b

DCC (%) =

Amount of Pheo in AuNCs Pheo ×100%= ×100% Amount of Pheo-loaded SERS-active AuNCs HA-HCAn-Au-Pheo-NPT

Figure Legends Figure 1.

Synthesis of (A) hyaluronic acid (HA) and hydrocaffeic acid (HCA) conjugates (HA-HCA), (B) thiol group-conjugated Pheo (Pheo-SH), and (C) HA-HCA-Au-Pheo-NPT.

Figure 2.

(A) (a) FT-IR spectra of HA-HCA and HA, (b) 1H NMR spectra of HA-HCA and HA; (B) (a) FT-IR spectra of Pheo-SH and Pheo, (b) 1H NMR spectra of Pheo-SH and Pheo; (C) 1H NMR spectra of HA-HCA-Au-Pheo-NPT.

Figure 3.

(A) Photographs of AuNCs in aqueous solution; (B) UV-visible absorption spectra of AuNCs in aqueous solution, (a) citrate-stabilized gold nanoparticles, (b) HA-HCA0-Au, (c) HA-HCA0.1-Au, (d) HA-HCA0.3-Au,(e) HA-HCA0.5-Au, (f) HA-HCA1.0-Au, and (g) HA-HCA2.0-Au.

Figure 4.

(A) DLS data of gold nanoparticles and gold chain-like nanostructures with different lengths, (a) citrate-stabilized gold nanoparticles, (b) HA-HCA0-Au, (c) HA-HCA0.1-Au, (d) HA-HCA0.3-Au, (e) HA-HCA0.5-Au, (f) HA-HCA1.0Au, and (g) HA-HCA2.0-Au; (B) FE-TEM images of gold nanoparticles and gold chain-like nanostructures with different lengths, (a) citrate-stabilized gold nanoparticles, (b) HA-HCA0-Au, (c) HA-HCA0.1-Au, (d) HA-HCA0.3-Au, (e) HA-HCA0.5-Au, (f) HA-HCA1.0-Au, and (g) HA-HCA2.0-Au

Figure 5.

Raman spectra of SERS-active AuNCs with different chain lengths. (A) HAHCA2.0-Au, (B) free NPT, (C) HA-HCA0.3-Au-NPT, (D) HA-HCA0.5-Au-NPT, (E) HA-HCA1.0-Au-NPT, and (F) HA-HCA2.0-Au-NPT.

Figure 6.

Raman spectra of HeLa cells incubated with SERS-active AuNC samples for 4 h, (A) free Pheo, (B) HA-HCA0.3-Au-Pheo-NPT, and (C) HA-HCA2.0-AuPheo-NPT.

Figure 7.

(A) Bio-TEM images of HeLa cells after incubation with HA-HCA0.3-AuPheo-NPT; (B) Bio-TEM images of HeLa cells after incubation with HAHCA2.0-Au-Pheo-NPT.

Figure 8.

(A) Confocal images and (B) flow cytometry results of cellular uptake of free Pheo, HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-Au-Pheo-NPT against HeLa cells (DAPI [blue color], Pheo [red color)]). Scale bars represent 20 µm.

Figure 9.

In vitro cytotoxicity assay using free Pheo, HA-HCA0.3-Au-Pheo-NPT and HA-HCA2.0-Au-Pheo-NPT against HeLa cells, (A) dark toxicity and (B) phototoxicity depending on laser exposure time.

Au

Au

Au

Au

Au

Au Pheo-SH

HCA

HA

NPT

CH3 OH

(A)

.

O

CH3

O

OH

NH

OH

O

O

n

O

O

ONa

.

+

O HO

HO

OH

Hyaluronic acid (HA)

OH

DMAP, EDC Room temperature, 48h

.

O

NH O

O

n

O

O

HO

O

OH

ONa

Hydrocaffeic acid (HCA)

OH

O

HA-HCA

.

O OH OH

H2C

H3C

H2C

H3C

CH3

H3C

(B)

NH

N

+

N

NH

CH3

H3C H2N

Room temperature, 24h

NH

N CH3

H3C

Cysteamine hydrochloride

O

NH

EDC, NHS, TEA

SH

CH3

H3C

N

O

O

O

O OH

O

O

H3C

O NH

HS

Pheophorbide a (Pheo)

H3C

Pheo-SH CH3

(C)

SH

HA-HCA +

Citrate-stabilized gold nanoparticles

+

Pheo-SH +

OH Room temperature, 24h

.

O

NH .

O

O O

2-Naphthalenethiol (NPT, Raman reporter)

O

OH

n

O ONa

H2C H3C

OH

O

CH3 N HN

H3 C

O

HA-HCA-Au-Pheo-NPT

N

OH NH

N

O

NH

Au

O

O O

H3C

HS

SH

SH

HN

NH H3C

HS

OH

O

CH3

O

CH3

O

O H3C

H3C

CH3

NH

HS O

CH3 NH

N

CH3 CH3 CH2

CH3 CH2

N H

O

H3C O

N O

N H N CH3

H3 C H3C

Figure 1

(A) (a) Transmittance (%)

(b) HA (1) HA-HCA

aromatic ring 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

(b)

b

CH3 OH

.

O

OH

a O O

O NH .

O O

ONa

a

n OH

O

HA-HCA

O

e

c

f

OH OH

d

d

e

a

(1) HA-HCA

d

b

f

c

(2) HA 10

8

6

ppm

Figure 2

4

2

0

(B) (a) Transmittance (%)

(2) Pheo

(1) Pheo-SH

-SH

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

(b) hH C 2

j

g d

jH C 3

NH

jH C

CH3

lNH

N

c

H3C

l

e

N CH3

3

k O

O

f

i

j

k

O

O NH

HS

a b

H3C

Pheo-SH

e d

j

f

c

h

g

b a

l

(1) Pheo-SH

i

(2) Pheo 10

8

6

ppm

4

2

0

(C) CH3 OH O

.

a O O

O

OH

NH .

O O

ONa

a

f

n

H3 C

OH

O

m

b

O

OH NH

N

H3 C

N

CH3

k i

n

l

CH3

CH3

O

h

O

m

O

HS

a

O NH

CH3 CH3 CH2 N H

O

H3 C O

CH2

N

HA-HCA2.0-Au-Pheo-NPT

e

HS

SH CH3

pp N

H3 C

SH

O HN

NH

HN

NH

NH

Au

N

H3 C

HS

OH

O

CH3

c

O

CH3

O

O H3 C

H3 C

g m d

H2 C

m

O

N H N

n

CH3

H3 C H3 C

m e d 10

c g 8

i k

bb

hf

p 6

ppm

l 4

Figure 2

2

0

(B)

(b)

(a)

(A)

(c)

(d)

(e)

(f)

(g)

2.5

Absorption intensity (a.u.)

2.0 (a) Citrate-stabilized gold nanparticles (b) HA-HCA0-Au 1.5

(c) HA-HCA0.1-Au (d) HA-HCA0.3-Au (e) HA-HCA0.5-Au

(a)

(f) HA-HCA1.0-Au

1.0

(b)

(g) (d)

0.5

(c)

(g) HA-HCA2.0-Au

(f) (e)

0.0 300

400

500

600

700

Wavelength (nm)

Figure 3

800

900

1000

(A) (a) Citrate-stabilized gold nanoparticles

(b) HA-HCA0-Au

(c) HA-HCA0.1-Au

(e) HA-HCA0.5-Au

(f) HA-HCA1.0-Au

(g) HA-HCA2.0-Au

(a) Citrate-stabilized gold nanoparticles

(b) HA-HCA0-Au

(c) HA-HCA0.1-Au

(e) HA-HCA0.5-Au

(f) HA-HCA1.0-Au

(g) HA-HCA2.0-Au

(d) HA-HCA0.3-Au

(B)

Figure 4

(d) HA-HCA0.3-Au

SERS intensity (a.u.)

(F) HA-HCA2.0-Au-NPT (E) HA-HCA1.0-Au-NPT (D) HA-HCA0.5-Au-NPT (C) HA-HCA0.3-Au-NPT (B) Free NPT (A) HA-HCA2.0-Au

200

400

600

800

1000

1200

1400 -1

Raman shift (cm )

Figure 5

1600

1800

2000

SERS intensity (a.u.)

(C) HA-HCA2.0-Au-Pheo-NPT

(B) HA-HCA0.3-Au-Pheo-NPT

(A) Free Pheo

200

400

600

800

1000

1200

1400 -1

Raman shift (cm )

Figure 6

1600

1800

2000

(A)

(b)

(a)

(c) (b)

(d)

(B)

(c)

(d)

(a)

(b)

(b) (c)

(d)

(c)

(d)

Figure 7

(A) Confocal images Nucleus

(B) Flow cytometry Pheo

Merged 0.17%

Control

99.44%

Free Pheo

99.90%

HA-HCA0.3-Au -Pheo-NPT

99.96%

HA-HCA2.0-Au -Pheo-NPT

Figure 8

Free Pheo HA-HCA0.3-Au-Pheo-NPT

(A)

HA-HCA2.0-Au-Pheo-NPT

Cell viability (% of Ctrl)

100

80

60

40

20

0 0

3

6

Pheo concentration (ug/ml)

10

Contral Free Pheo HA-HCA0.3-Au-Pheo-NPT

(B) 100

Cell viability (% of Ctrl)

HA-HCA2.0-Au-Pheo-NPT

80

60

40

20

0 0

10

15

Laser exposure time (min)

Figure 9

30

Au

Au

Au

Au

Au

Au HA Citrate-stabilized gold nanoparticles

HA-HCA0.5-Au

HCA

HA-HCA0.1-Au

HA-HCA1.0-Au

Pheo-SH

NPT

HA-HCA0.3-Au

HA-HCA2.0-Au

Surface-enhanced Raman scattering (SERS)-active gold nanochains for multiplex detection and photodynamic therapy of cancer.

Multifunctional nanomedicine holds considerable promise as the next generation of medicine that will enable early detection of diseases, as well as si...
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