Click-Functionalized SERS Nanoprobes with Improved Labeling

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Click-Functionalized SERS Nanoprobes with Improved Labeling Efficiency and Capability for Cancer Cell Imaging Renyong Liu, Jun Zhao, Guangmei Han, Tingting Zhao, Ruilong Zhang, Bianhua Liu, Zhengjie Liu, Cheng Zhang, Linlin Yang, and Zhongping Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10409 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

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ACS Applied Materials & Interfaces

Click-Functionalized SERS Nanoprobes with Improved Labeling Efficiency and Capability for Cancer Cell Imaging Renyong Liu,†,‡ Jun Zhao,† Guangmei Han,† Tingting Zhao,† Ruilong Zhang,§ Bianhua Liu,† Zhengjie Liu,† Cheng Zhang,† Linlin Yang,† Zhongping Zhang*,† ,§ †

CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines, Chinese Academy

of Sciences, Hefei, Anhui 230031, China ‡

Department of Chemistry, University of Science and Technology of China, Hefei, Anhui

230026, China §

School of Chemistry and Chemical Engineering, Anhui University, Hefei, Anhui, 230601,

China KEYWORDS: click functionalization, surface-enhanced Raman scattering, nanoprobes, folate conjugates, labeling, cancer cell imaging

ABSTRACT: Precise identification and detection of cancer cells using nanoparticle probes are critically important for early cancer diagnosis and the subsequent therapy. We herein develop a novel folate receptor (FR)-targeted surface-enhanced Raman scattering (SERS) nanoprobes for cancer cell imaging based on click coupling strategy. A Raman-active derivative (DNBA-N3) is

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designed with a disulfide bond for covalently anchoring to the surface of hollow gold nanoparticles (HAuNPs) and a terminal azide group for facilitating highly efficient conjugation with bioligand. Modification of HAuNPs with DNBA-N3 yields monolayer coverage of Raman labels absorbed on the nanoparticle surface (HAuNP-DNBA-N3) and strong SERS signals. HAuNP-DNBA-N3 can be simply and effectively conjugated with folate bicyclo[6.1.0]nonynes derivatives via copper-free click reaction. The synthesized nanoprobes (HAuNP-DNBA-FA) exhibit excellent targeted capacities to FR-positive cancer cells relative to FR-negative cells through SERS mappings. The receptor-mediated delivery behaviors are confirmed by comparison with the uptake of HAuNP-DNBA-N3 and free FA competition experiments. In addition to its good stability and benign biocompatibility, the developed SERS nanoprobes have a great potential for applications in targeted tumor imaging.

1. INTRODUCTION Target detection and image analysis of cancer cells using spectroscopic tags conjugated with biological ligands are significantly important for improving early cancer detection and treatment.1,2 Folate receptor (FR) is one of the notorious biomarker enriched on the surface of human ovarian carcinoma cell lines which was discovered in 1991.3 As such, folate conjugates and complexes have become a promising class of imaging probes for distinguishing FRoverexpressed cancer cells from the other healthy ones.4-9 Although traditional radioligand binding assays can quantitatively measure FR expression in carcinomas and normal tissues,10 they inevitably require complicated protocols, harmful radioactive materials and specialized instruments. Recently, fluorescence nanoprobes including folic acid (FA)-conjugated gold nanoclusters, quantum dots and carbon nanomaterials have been widely used to target and image FR-overexpressed cancer cells owing to their advantages of high sensitivity and capability of


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visualization.11-14 However, the poor photostability and strong autofluorescence limit their wide applications in both in vitro and in vivo targeted cell imaging. With the integration of high sensitivity and unique fingerprint feature, surface-enhanced Raman scattering (SERS) technique has emerged as a pursued spectroscopic tool for biological detection and imaging.15-21 A typical targeted SERS nanoprobe is built on metal substrates (e.g. gold nanoparticles, AuNPs) conjugated with Raman-active molecules for signal response and targeted species for cell recognition.22 Taking FR-targeted SERS nanoprobes as an example, there are mainly three advances in labeling strategies for targeted imaging of FR-overexpressed cell lines. (1) Direct conjugation metal/nanomaterial hybrids (e.g. Au/graphene oxide, Au/carbon nanotubes) with FA for SERS imaging utilizing the characteristic signals of nanomaterials, such as the Raman G-band peak of graphene oxide and single-walled carbon nanotube composites.23,24 (2) SERS nanoparticles are firstly modified with Raman-active molecules and encapsulated in the silica or polymer shells for further coupling to FA molecules.25-30 (3) Folate-targeted SERS nanoprobes are engineered by coating with a thiolate-based monolayer that has a terminal group for allowing reaction with FA molecules.31,32 Obviously, the third strategy has a significant improvement in imaging capabilities for amplifying the responses and binding affinities due to the increase of the concentrations of Raman labels and available recognition moieties. However, to date only para-aminothiophenol molecules are used as linkers for the formation of amide bonds with FA molecules.31,32 Meanwhile, the carbodiimide conjugation protocols need complicated activation processes and may affect the biological activity of special ligands.33 As a result, a general and mild approach for constructing folate-targeted SERS probe is urgently needed.


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In this work, for the first time, we report a novel FR-targeted SERS nanoprobe based on click functionalization of substrates with FA derivatives. It is known that click chemistry is initially named for the copperI-catalyzed 1,3-dipolar cycloaddition reaction between azides and terminal alkynes, which has been widely used for bioconjugation purposes with high yields and synthetic facility.34,35 However, the presence of copperI is detrimental to living cells.34 As an activated variant, a strain-promoted azide-alkyne cycloaddition reaction (called copper-free click chemistry) has been exploited to conjugate biomolecules without observable cytotoxicitiy.36-40 The SERS nanoprobe is constructed by modifying hollow gold nanoparticles (HAuNPs) with a monolayer of Raman-active labels, which composed of disulfides for covalently anchoring to the surface of HAuNPs and azide groups for clicking with folate bicyclo[6.1.0]nonynes derivatives (BCN-Folate). By using cyclooctynes activated by ring strain, the click coupling reaction can occur rapidly in mild condition and does not require a cytotoxic copper catalyst. The strengths of the strategy are demonstrated in the selective targeting and imaging of FR-positive cancer cells. We anticipate that combining the ultrasensitive properties of SERS nanoparticles with the universal surface functionality of click chemistry will allow for improving targeting efficiency for specific cancer cell lines.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. Folic acid (FA), 11-azido-3,6,9-trioxaundecan-1-amine (NH2PEG-N3), 5,5’-dithiobis(2-nitrobenzoic acid) (DNBA), N-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane

(BCN-amine)

and

fluoresceine

5-

isothiocyanate (FITC) were obtained from Sigma-Aldrich. N-hydroxy succinimide (NHS), dicyclohexyl carbodiimide (DCC), gold(III) chloride trihydrate (HAuCl4·3H2O), cobalt(II) chloride hexahydrate (CoCl2·6H2O), trisodium citrate (C6H5O7Na3·2H2O), sodium borohydride


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(NaBH4), N,N-dimethylformamide (DMF), trimethylamine and dimethyl sulfoxide (DMSO) were purchased from Shanghai Chemicals Ltd. Human nasopharyngeal epidermal carcinoma cell line (KB), human cervical carcinoma cell line (HeLa), and human lung carcinoma cell line (A549) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HRP-conjugated monoclonal mouse anti-beta actin was purchased from KangChen Bio-tech Inc. (Shanghai, China). Folate receptor alpha antibody (F-15) was purchased from Santa Cruz Biotechnology, Inc. (USA), and anti-goat IgG-peroxidase antibody (SigmaAldrich) was used as a secondary antibody in Western blotting. Ultrapure water with a resistivity of 18.2 MΩ·cm was used. 2.2. Click Synthesis of FR-Targeted SERS Nanoprobes (HAuNP-DNBA-FA). HAuNPs were firstly prepared by the sacrificial galvanic replacement of cobalt nanoparticles.41 Briefly, 100 mL of water, 100 µL of 0.4 M aqueous CoCl2·6H2O, and 500 µL of 0.1 M aqueous C6H5O7Na3·2H2O were added into a three-neck flask. The mixture was degassed with nitrogen for 1 h. Then, 300 µL of a freshly prepared 1 M aqueous NaBH4 solution was added quickly. After NaBH4 was completely reacted, a 0.1 M aqueous HAuCl4·3H2O solution was added in 50µL aliquots to a final volume of 500 µL. Upon the completion of gold addition, nitrogen flow was stopped and the color of the solution changed to deep blue. The resulting HAuNPs were centrifuged at 4000 rpm for 40 min, and redispersed in water (～5.0×10-10 M). Secondly, the Raman-active azide derivatives (DNBA-N3) were synthesized as follows. The carboxyl groups of DNBA were activated with DCC and NHS to couple to the primary amines of azide linker. Briefly, 0.059 g of the activated DNBA (0.1 mmol) was then dissolved in 2 mL of DMSO, and 80 µL of NH2-PEG-N3 (5.0 M) was dropwise added. The reaction mixture was stirred overnight. The resulting mixture was poured into water to form a precipitate. The orange-yellow precipitate


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was centrifuged, washed with water, and dried under vacuum (see MS spectrum in Figure S1). Thirdly, to 1 mL of HAuNPs solution was added 40 µL of DNBA-N3 (0.03 M in DMSO), and the reaction mixture was stirred for 3 h. The azide-functionalized HAuNPs (HAuNP-DNBA-N3) was centrifuged, washed with DMSO three times and resuspended into 1 mL of PBS for the following click bioconjugation. Then folate bicyclo[6.1.0]nonynes derivatives (BCN-Folate) were synthesized by the reaction of the carboxyl groups of FA and amino groups of BCN-amine. Typically, 0.05 g of FA (0.12 mmol) was first dissolved in 2 mL of DMSO. Then 0.025 g of DCC (0.11 mmol) and 0.013 g of NHS (0.11 mmol) were added and the resulting mixture was stirred for 3 h. A solution of BCNamine (0.036 g, 0.11 mmol) was added dropwise with vigorous stirring overnight. Then the reaction mixture was filtered to remove the white precipitate (the byproduct dicyclohexylurea), and the filtrate was poured into water (30 mL) and stirred for 30 min to form a precipitate. The orange-yellow precipitate (BCN-Folate) was filtered, washed with acetone, and freeze-dried under vacuum (see MS spectrum in Figure S2). Finally, HAuNP-DNBA-FA was prepared by copper-free click reaction between HAuNPDNBA-N3 and BCN-Folate. To a 1 mL aliquot of HAuNP-DNBA-N3 was added 60 µL of BCNFolate (0.02 M in DMSO) and the mixture was stirred for 1 h. The resulted nanoprobes were centrifuged, washed with DMSO three times, resuspended into 1 mL of PBS and stored at 4 ºC before use. As a control, the carbodiimide coupling approach was used to directly conjugate FA onto DNBA modified HAuNPs (named as HAuNP-DNBA-FA*). Briefly, 40 µL of a 0.03 M activated DNBA solution in DMSO was added to 1 mL of HAuNPs solution and the mixture was stirred for 3 h. The functionalized HAuNPs were then centrifuged, washed with DMSO three times and


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resuspended into 1 mL of PBS. Finally, 60 µL of a 0.02 M FA solution in DMSO was added to 1 mL of DNBA modified HAuNPs and the mixture reacted for 6 h. After centrifugation and washing procedures, the obtained HAuNP-DNBA-FA* was resuspended into 1 mL of PBS and stored at 4 ºC before use. 2.3. Click Synthesis of FR-Targeted Fluorescent Probes (FITC-FA). Firstly, FITC-azide derivatives (FITC-N3) were synthesized as follows. 100 mg of FITC (0.26 mmol) was first dissolved in 1.5 mL of DMF, and 30 µL of trimethylamine (0.21 mmol) was added at 0 °C. Then, a solution of NH2-PEG-N3 (43 µL, 0.21 mmol) in DMF (1.5 mL) was added dropwise with vigorous stirring under argon. The resulting mixture was stirred for 12 h at room temperature in the dark, concentrated in vacuo and purified by silica gel column chromatography (CH2Cl2/CH3OH = 9/1). The orange-yellow product (FITC-N3) was concentrated and freezedried under vacuum (see MS spectrum in Figure S3). Finally, FITC-FA was prepared by copperfree click reaction between FITC-N3 and BCN-Folate. To a 0.5 mL of FITC-N3 (0.01 M in the mixture of DMSO/PBS = 2:8) was added 0.5 mL of BCN-Folate (0.02 M in DMSO) and the mixture was stirred at room temperature in the dark for 1 h, before it was diluted in 10 mL of DMSO/PBS (2:8) to give a stock solution of FITC-FA. 2.4. Characterization and Instruments. The size and the morphology of the HAuNP-DNBAFA were examined by JEOL 2010 transmission electron microscopy (TEM) operated at 200 kV accelerating voltage. UV-vis absorption spectra were recorded with a Shimadzu UV-2550 spectrometer. The infrared spectra were recorded with Nicolet Nexus-670 FT-IR spectrometer using KBr method. High-resolution mass spectra (HR-MS) were obtained using an Agilent QTOF

6540

mass

spectrometer.

Immunoblotting

was

performed

with

automatic

chemiluminescence gel imaging analysis system (Tanon, Fine-do X6). Fluorescent images were


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acquired on confocal microscope (Zeiss LSM 710), and the excitation wavelength was 488 nm. SERS measurements were performed with a DXR confocal microscopy Raman system (Thermo Fisher Scientific Inc.). SERS spectra and imaging were collected with a 633-nm excitation laser, 50-µm slit and 50× objective len for confocality. The laser power and accumulation time were 10 mW and 10 s, respectively. 2.5. Cell Culture and Imaging. All cells were respectively cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. To study the uptake and imaging of the FR-targeted SERS nanoprobes, the cells were cleaved by trypsin and transferred to each glass culture dish. After 24 h of incubation, the cells were washed with PBS twice and resuspended into 2 mL of the fresh medium. Subsequently, 200 µL of HAuNP-DNBA-FA or HAuNP-DNBA-FA* solutions were separately added into each dish and mixed properly. Dishes were returned to the incubator (37 °C, 5% CO2) and incubated for different times, washed with PBS and kept in 2 mL of PBS for dark-field and Raman imaging experiments. To confirm the receptor-mediated uptake of HAuNP-DNBA-FA, two control experiments were performed in cells. (1) Equal volumes of HAuNP-DNBA-N3 (200 µL, 5.0×10-10 M) solution were respectively added into the cells instead of HAuNP-DNBA-FA solution, and the results were analyzed by DXR confocal microscopy Raman system. (2) The cell cultures were pretreated with 50 µL of 1 mM FA solution for 1 h prior to HAuNP-DNBA-FA treatment. In addition, similar fluorescent imaging experiments were also performed by using FITC-FA. 2.6. Western Blot Analysis of Cells. For whole-cell extract preparation, cells were washed twice with PBS, scraped and centrifuged (1500 rpm, 5 min), and the pellet was resuspended in 500 µL of denaturation buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 7.4, 0.2%


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SDS) containing protease inhibitor cocktail. Loading buffer was added to each sample, and samples were loaded onto 12% SDS-PAGE gel after heating at 95 °C for 5 min. Subsequently, proteins were transferred to nitrocellulose membranes (Millipore, Immobilon Transfer Membrane, 0.45 µm) and analyzed by immunoblotting. The relative FR-α content was calculated with respect to the amount of β-actin. 2.7. MTT Assay. To measure cell viability, the cells were firstly seeded on 96-well plates (5.0×103 cells/well) and incubated for 24 h. Then, they were treated with various concentrations of HAuNP-DNBA-FA. After incubation for another 24 h, the cells were washed three times with PBS, and freshly prepared MTT (0.5 mg/mL) solution in culture medium was added to each well. After incubation for 4 h at 37 °C, the media was removed and cells were dissolved in 200 µL of DMSO. Then the absorbance of each well was measured at 570 nm.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Click-Functionalized SERS Nanoprobes. Figure 1 illustrates the click synthesis route of HAuNP-DNBA-FA. HAuNPs employed here were firstly synthesized in aqueous solution by the reported sacrificial galvanic replacement of cobalt nanoparticles and used as substrates.41 The nanoparticles with the hollow structures exhibit strong Raman enhancement effects.42-44 The Raman-active azide derivatives (DNBA-N3) obtained from the reaction between DNBA and NH2-PEG-N3 were then covalently modified onto the surface of HAuNPs via the dithiol group, thereby forming a monolayer coverage of Raman reporter molecules and providing clickable azide groups for further bioconjugation. Finally, HAuNP-DNBA-FA was obtained by attaching BCN-Folate on the terminal azide groups of HAuNPs via copper-free click reaction. The constructed SERS nanoprobes with increased labeling concentration and available targeting species are expected to improve their capabilities


9


in cell imaging. It is noted that the presence of the PEG linker can help to minimize nonspecific adsorption and potentially provide more accessibility to the targeted cancer cells. In principle, the universal surface functionality based on click chemistry will allow for controlled synthesis of any other bioconjugated SERS imaging agents with desired ligands. N3

N3 3

O 3

NO2

NH

O

NH

O

N N

NO2

DNBA-N3 (1)

HAuNP HGN

O

S

S

S

S

N (2) BCN-Folate

N

O2N O

O2 N

N

N

N

HN

N3

N3

O

O

=

HAuNP-DNBA-N HGN-DNBA-N33

3

N3

N

N

O

HN

N

N

N

Copper-Free Click Reaction

3

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N3 N3

O

DNBA-N3

N3

= H BCN-Folate

O

N H O2 N

3

O S S

N H NO2

O 3

N3

H N

O N

H O O

HAuNP-DNBA-FA HGN-DNBA-FA

N3

O N H

O

2N H

HO

N

H N O

NH2

N N

O

Figure 1. Schematic illustration for the click synthesis of HAuNP-DNBA-FA. (1) HAuNPs were firstly modified with DNBA-N3, and (2) the resultant azide groups were further reacted with alkyne moieties of BCN-Folate via copper-free click reaction. The as-prepared HAuNPs have an average diameter of ~35 nm, and the wall thickness is ~10 nm as shown in Figure 2A, the size of which is suitable for intracellular imaging.45 Furthermore, the high-magnification TEM image inserted in Figure 2A is taken to clearly demonstrate the hollow architecture of the nanoparticles. It can be seen that there is strong contrast difference in the nanosphere with a bright center surrounded by a much darker edge. Conjugation HAuNPs


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with DNBA-N3 and BCN-Folate red-shifts the absorbance peak of HAuNPs from 628 nm to 635 nm due to the change in the dielectric surrounding (Figure 2B). Compared to HAuNP-DNBA-N3, the clicked HAuNP-DNBA-FA is still well-dispersed and stable in aqueous solution (as shown in

A 20 nm

100 nm

Normalized Absorbance (a.u.)

the inset of Figure 2B), which is essential for the following cellular imaging.

B (2) HAuNP-DNBA-FA

(1) HAuNPs

C

2105 ( νN3 )

(4) HAuNP-DNBA-FA (3) HAuNP-DNBA-N3 (2) DNBA-N3 (1) DNBA

4000

Raman Intensity (a.u.)

500 550 600 650 700 750 800 Wavelength (nm)

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


D

1332

851

1069

1556

(2) HAuNP-DNBA-FA

(1) HAuNP-DNBA-N3

3200 2400 1600 800 -1 Wavenumber (cm )

600

900 1200 1500 1800 -1 Raman Shift (cm )

Figure 2. (A) TEM image of HAuNPs (the inset is the high-magnification TEM image of individual HAuNP). (B) UV-vis spectra of (1) HAuNPs and (2) HAuNP-DNBA-FA (the inset is the optical photograph of HAuNP-DNBA-FA solution). (C) FT-IR spectra of (1) DNBA, (2) DNBA-N3, (3) HAuNP-DNBA-N3 and (4) HAuNP-DNBA-FA. (D) SERS spectra of (1) HAuNP-DNBA-N3 and (2) HAuNP-DNBA-FA. Meanwhile, FT-IR spectra were used to study the evolution of surface functionalization of HAuNPs (Figure 2C). Compared to DNBA, both DNBA-N3 and HAuNP-DNBA-N3 displayed the asymmetric stretching peak of the azide group (νN3) at 2105 cm-1. While the further treatment


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with BCN-Folate resulted in the disappearance of the absorption band, indicating that the azide groups on the surface of HAuNP have been completely consumed with the alkyne groups of BCN-Folate to form triazoles via copper-free click reaction. Figure 2D shows SERS spectra of the solutions of HAuNP-DNBA-N3 and HAuNP-DNBA-FA. The dominant peaks (851 cm-1, 1069 cm-1, 1332 cm-1 and 1556 cm-1) of DNBA from HAuNPs before and after click reaction could be clearly recorded. Notably, the higher SERS intensity of HAuNP-DNBA-N3 relative to HAuNP-DNBA-FA might be partly from the aggregates due to their poor dispersity in aqueous solution. Furthermore, the stability of the SERS nanoprobes was examined. As shown in Figure S4, HAuNP-DNBA-FA with PEG protection had good stability in PBS and cell culture medium. In contrast, the absorbance of HAuNP-DNBA-FA* without PEG linker immediately decreased to a low value, indicating rapid aggregation and precipitation of nanoprobes. We also assessed the effects of a variety of external conditions on SERS signatures and intensities, and the results showed that there was no significant change when HAuNP-DNBA-FA was transferred from pure water to 0.2 M NaCl, pH 4.5 and pH 9.0 aqueous solutions (Figure S5). Even after long-term storage (three months) in aqueous solution, the SERS signals of HAuNP-DNBA-FA kept almost identical (Figure 3). This click-functionalized nanoparticle with stable Raman readout and specific biological ligands offers a robust platform for SERS-based bioassays. For the following cellular imaging experiments, the strongest peak of the symmetric nitro stretch at 1332 cm-1 was used to map SERS images of different cancer cells.


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1000 Raman Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


800 600 400 200 0 5

10

20

30

60

90

Time (days)

Figure 3. The SERS stability of HAuNP-DNBA-FA. The Raman intensity of the strongest peak at 1332 cm-1 kept almost identical after the HAuNP-DNBA-FA solution was stored at 4 ºC for three months. 3.2. Fluorescent Imaging of Cancer Cells Using FITC-FA. The levels of FRs expressed on the surface membranes of typical cancer cells (KB, HeLa and A549) were firstly determined by Western blot analysis (Figure S6). Of the three cancer types tested, KB cells have the highest FR expression level followed by HeLa cells, and A549 cells express very low to nondetectable levels of FRs, which is consistent with the reported results.8 Then fluorescent imaging agent FITC-FA synthesized by the click reaction between FITC-N3 and BCN-Folate was used to target and image the above cells. The specific binding can take place between FITC-FA and FRpositive cancer cells since the folate conjugates possess similar affinity to FRs as free FA molecules.5 As shown in Figure 4, a bright green fluorescence was observed on the whole KB cells and the membranes of HeLa cells after the incubation with FITC-FA (50 nM) for 30 min, respectively. While FR-negative A549 cells showed very weak or no fluorescence at the same experimental conditions. The preliminary results seemingly indicate that the fluorescent brightness is directly related to the levels of FRs expressed on the cell membranes, and giving the distinguishable fluorescent images. However, FITC-N3 without the attachment of FA


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conjugates could enter KB cells (Figure S7). Meanwhile, the green fluorescence could also be observed for FA pretreated KB cells (Figure S8). These negative results indicated that fluorescent probes could also freely diffuse into KB cells, in addition to the receptor-mediated delivery. The nonspecific results might be due to the round morphology characteristic of KB cells themselves, which induces the rapid uptake and efflux of the organic agents.36,46,47 Therefore, the free diffusion behavior of the organic agents through specific cell membranes makes the molecular probes still face a challenge in cell-targeting images.

Figure 4. (A) Chemical structures of FITC-FA and FITC-N3. (B) Confocal images of KB, HeLa and A549 cells after incubated with FITC-FA (50 nM) for 30 min, respectively. 3.3. SERS Imaging of Cancer Cells Using HAuNP-DNBA-FA. The prepared HAuNPDNBA-FA was further used as imaging agents for targeting and imaging of cancer cells. As expected, the HAuNP-DNBA-FA can be internalized into the cells via receptor-mediated delivery, and the molecular Raman imaging with high spatial resolution can directly identify cancer cells that express the membrane protein FRs. Here, the 633 nm line of a HeNe laser was used for SERS imaging experiments due to the enhanced surface plasmon resonance with HAuNPs and the degradation of the signal-to-background ratio. The viabilities of different


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cancer cells treated with HAuNP-DNBA-FA showed no obvious cytotoxic effects (Figure S9). Figure 5 shows time-dependent dark-field images of the three cancer cells after incubated with 50 pM of HAuNP-DNBA-FA for different times. It was noted that the bright dots observed in A549 cells after 0.5 h incubation with HAuNP-DNBA-FA were not from the scattering light of SERS nanoprobes, since they also appeared without the addition of any nanoprobes (Figure S10). When the incubation time was prolonged from 0.1 to 3 h, the amounts of uptake and the intracellular distribution of the nanoprobes increased in KB and HeLa cells. While only few nanoprobes were attached on the surface of A549 cells even after 3 h incubation with the nanoprobes. As shown in Figure 5, the brightness of the light scattering was in the order of KB > HeLa > A549. Although unknown bright scattering dots presented in A549 cells, control experiments with the unclicked HAuNP-DNBA-N3 and free FA competition experiments demonstrated that the internalization was FR-dependent and FA could significantly enhance the cellular uptake after being click-functionalized onto the surface of HAuNPs (the left images of Figure S11 and Figure S12). Meanwhile, the same experiments were performed by using directly *

conjugated HAuNP-DNBA-FA . The results showed that the nanoprobes without the PEG linker were unstable in culture medium, and the agglomerates were inconvenient to access to the targeted cancer cells and might led to uncertain results (Figure S13). Therefore, HAuNP-DNBAFA with available bioligands and good stability can improve the targeted capacities to FRpositive cancer cells.


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Figure 5. Dark-field images of KB, HeLa and A549 cells after incubated with HAuNP-DNBAFA (50 pM) for 0.5, 1, 3 h, respectively. In order to clearly reveal the capability of HAuNP-DNBA-FA in targeting and imaging of FRpositive cancer cells, the corresponding colorful SERS images mapped with the Raman bands at 1332 cm-1 were measured and analyzed. The laser beam was focused onto a single cell and the SERS mappings were obtained with 10 s integration time for each pixel. As shown in the middle images of Figure 6, even though the distribution of the nanoprobes was uneven, the bright Raman images for FR-positive cancer cells could be observed after the incubation with the nanoprobes for 1 h. At the same time, SERS images of the cells incubated with the unclicked HAuNP-DNBA-N3 or pretreated with FA were mapped to confirm the receptor-mediated endocytosis of the nanoprobes (the middle images of Figure S11 and Figure S12). Compared with the results of the above fluorescent experiments, the visibly reduced colorful SERS images were observed for KB cells in the control experiments, indicating that the delivery of nanoparticle probes was FR-dependent and the developed nanoprobes could effectively avoid the negative diffusion behavior.


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Figure 6. Left: dark-field images of KB, HeLa and A549 cells after incubated with HAuNPDNBA-FA nanoprobes (50 pM) for 1 h, respectively. Middle: SERS images of the selected areas in the left images (mapped with the Raman bands at 1332 cm-1 and the color bar indicates the Raman signal intensity). Right: the corresponding Raman spectra at the indicated sites in the middle images. Here, the typical sites 1, 2 and 3 in the middle images were chosen according to the Raman intensity from the weakest to the strongest. The corresponding SERS spectra collected from the sites 1, 2 and 3 were shown in the right of Figure 6, Figure S11 and Figure S12. The Raman intensities of 1332 cm-1 from KB, HeLa and A549 cells in the indicated three sites were analyzed and summarized in Figure 7. It can be estimated that the strongest Raman intensity of HAuNPDNBA-FA in KB cells is ∼1.60- and 3.56-fold those in HeLa and A549 cells. As for the unclicked HAuNPs and FR saturated cells, only the weak or negligible Raman intensities were detected. It is foreseeable that the folate-targeted SERS nanoprobes with improved labeling


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efficiency and imaging capability have a great application prospect in the field of tumortargeting and therapeutics.

Figure 7. Comparison of the Raman intensities at 1332 cm-1 in the indicated sites of cells (without FR saturation) after the incubation with (A) HAuNP-DNBA-FA, (B) unclicked HAuNPs for 1 h, and (C) FR saturated cells after the incubation with HAuNP-DNBA-FA for 1 h. The concentrations of HAuNP-DNBA-FA and HAuNP-DNBA-N3 are the same (50 pM).

4. CONCLUSIONS In summary, we have fabricated a novel folate-targeted SERS nanoprobe for selective imaging and diagnosis of FR-overexpressed cancer cells. The monolayer coverage of Raman-active azide derivatives anchored at the surface of HAuNPs increases the number of label molecules and further undergoes highly efficient bioconjugation with folate cyclooctynes derivatives through copper-free click reaction. It has been demonstrated that the folate-conjugated nanoprobes have a significant improvement in imaging capabilities for amplifying the response and binding affinities. The SERS nanoprobes can selectively bind to the FR-positive cancer cells, and the dark-field and SERS images can visualize the distribution of the nanoprobes in typical cancer cells with different levels of FRs and give the distinguishable images between the FR-positive cells and the FR-negative cells. The engineering SERS nanoparticles with good cellular targeting


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ability to specific cancerous cells have great potential to serve as ideal imaging agents for tumortargeting and therapeutics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge. MS spectra of the synthesized compounds, Western blot analysis, metabolic viability of cells, control experiments (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (61205152, 21335006, 21475135 and 21605145), and the Natural Science Foundation of Anhui Province (1608085QB32 and 1508085SQB200).

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Click-Functionalized SERS Nanoprobes with Improved Labeling

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