Preparation of Novel Fluorescent Nanocomposites Based on Au

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Preparation of Novel Fluorescent Nanocomposites Based on Au Nanoclusters and Their Application in Targeted Detection of Cancer Cells Xiaoyu Wang, Junhan Xia, Chun Wang, Lu Liu, Shuxian Zhu, Wei Feng, and Lidong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16457 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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ACS Applied Materials & Interfaces

Preparation of Novel Fluorescent Nanocomposites Based on Au Nanoclusters and Their Application in Targeted Detection of Cancer Cells Xiaoyu Wang,#,† Junhan Xia,#,† Chun Wang,† Lu Liu,† Shuxian Zhu,† Wei Feng*,‡,§ and Lidong Li*,† †

State Key Laboratory for Advanced Metals and Materials, School of Materials Science and

Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China. ‡

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, P. R. China.

§

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, P. R. China.

ABSTRACT

Fluorescent gold nanoclusters (AuNCs) have drawn considerable research interests owing to their unique emission properties. However, the environment surrounding the nanocluster greatly influences its luminous behavior. In this work, a novel nanocomposite based on AuNCs with bright fluorescence and high biocompatibility was prepared. In the nanocomposite, mesoporous silica (mSiO2) nanospheres provided a mesoporous framework, which helped to template the 1 ACS Paragon Plus Environment

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formation of ultra-small AuNCs and also prevented their aggregation in different solutions. These nanocomposites emitted stable fluorescence even in complex biological environments. After self-assembly of folic acid-conjugated poly(L-lysine), the presence of folic acid on the nanocomposites guaranteed a good recognition in folate receptor (FR)-positive cells, improving detection selectivity. Cellular experiments demonstrated that the nanocomposites had good dispersity in physiological environment and could be internalized by FR-positive cancer cells, resulting in bright fluorescence. We believe that this research provides a simple approach to the fabrication of stable fluorescent AuNCs nanocomposite, which show good compatibility with complex biological systems and great potential for applications in biological imaging and cell detection. KEYWORDS: fluorescence, nanocomposites, gold nanoclusters, self-assembly, cell detection INTRODUCTION Fluorescence imaging is an important optical analytical technique that is widely applied in biological and medical research.1-6 This imaging technology offers not only high sensitivity but also high spatial resolution. Nanoparticles, with sizes smaller than 200 nm, can be internalized into cells,7 and fluorescent nanoparticles have become the most rapidly developed class of fluorescent probes for fluorescence detection. To date, various nanoparticle systems including organic small molecule nanoparticles,8,9 conjugated polymer nanoparticles,10,11 quantum dots,12,13 and metal nanostructures14,15 have been reported and used as fluorescent probes. Among these nanomaterials, metal nanoclusters (NCs), such as Au, Ag, and Cu nanoclusters, with large Stokes shifts, strong luminescence, and good biocompatibility are now being extensive studied.16-18 Metal NCs contain several to a hundred metal atoms and their sizes are typically smaller than 2 2 ACS Paragon Plus Environment

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nm.19 Because the size of metal NCs is comparable to the Fermi wavelength of electrons, metal NCs exhibit discrete energy levels and molecular-like properties such as strong luminescence.20 Therefore, tuning the size of metal NCs provides a means of tailoring their light-emission properties for applications in fluorescence imaging. Moreover, metal NCs do not contain toxic elements and exhibit good biocompatibility, which makes them particularly attractive for biological imaging. The properties of metal NCs are highly sensitive to their size and the preparation of ultra-small metal NCs is highly desirable. A typical method used to prepare metal NCs is the reduction of M(I)-thiolate complexes, where M is a metal.21 This method involves two stages: fast reductive growth of the metal NC precursors followed by etching of the metal NC precursors by thiolate to form metal NCs.22 Metal NCs are protected by thiolate ligands, which maintain their stability. However, changes to the local environment can influence the interaction between the thiolate and metal core, leading to aggregation of the metal NCs and corresponding changes to their lightemission properties.23 This problem greatly limits the use of metal NCs as a fluorescence imaging material in complex biological environments. Recently, biomolecules, including proteins, peptides, and DNA have been used to replace thiolate as protective ligands for metal NC synthesis.24-26 However, further surface modification of metal NCs with functional groups may affect the structure of the biomolecules and the light-emission properties of the metal NCs.27 Moreover, owing to the ultra-small size of metal NCs, it is difficult to remove residual precursors and toxic reductants by centrifugation, which can limit their further applications in cellular imaging. Therefore, there is an urgent need to prepare novel nanocomposites based on metal NCs to expand their applications in biomedical fields.

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The combination of matrix materials with metal nanostructures to construct nanocomposites has been shown to be an effective way of addressing these issues.28-30 Mesoporous silica (mSiO2) has uniform nanopores and high biocompatibility.31,32 The diameter of the nanopores is of the same order as the dimensions of ultra-small metal NCs; hence, mSiO2 is a promising template for preparing metal NCs in situ. The encapsulated reagents and fluorophores in the nanopores are protected from the degradative and fluorescence quenching effects of the biological environment.33 Meanwhile, mSiO2 is an optically transparent material and has a large band gap (~9 eV),34 making it an ideal framework for fluorescent probes. Furthermore, silica nanospheres possess good hydrophilicity. It is facile to achieve the surface modification of silica nanoparticles with functional groups. In this paper, we designed and synthesized novel fluorescent nanocomposites for cancer cell detection. As shown in Scheme 1, the AuNCs were in situ reduced within the nanopores of mesoporous silica, which prevented their aggregation and fluorescence attenuation in different solutions. Then, the folic acid-conjugated poly(L-lysine) (PLL-FA) were assembled on the surface of silica nanoparticles by electrostatic interaction. Based on the high affinity of FA for the folate receptor (FR), the polymer PLL-FA afforded an ability to detect FR-positive cancer cells. Thus, the nanocomposites based on AuNCs showed stable fluorescence, high biocompatibility, and good dispersity in simulated physiological solutions. We also showed internalization of the nanocomposites by FR-positive special cancer cells, demonstrating their potential effectiveness in cancer cell detection.


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Scheme 1. Schematic representation of the fabrication of fluorescent nanocomposites based on AuNCs. EXPERIMENTAL SECTION Materials and Measurements. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), 11-mercaptoundecanoic acid (11-MUA), tetra(hydroxymethyl)phosphonium chloride (THPC), ncetyltrimethylammonium bromide (CTAB), glutathione (GSH), tetraethyl orthosilicate (TEOS), PLL (Mw = 30,000–70,000), FA, n-hydroxysuccinimide (NHS) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC) were purchased from J&K Chemical or Sigma-Aldrich. All reagents were used without further purification, unless otherwise stated. The human nasopharyngeal cancer cell line (KB) and normal human liver cells line (L02) was purchased from cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Ultrapure water (18.6 MΩ) was used throughout the experiments. Ultraviolet visible (UV-vis) absorption spectra were recorded at room temperature on a Hitachi U3900 spectrophotometer. Fluorescence spectra were measured on a Hitachi F-7000 fluorescence spectrometer equipped with a Xenon lamp excitation source. A Hitachi H-7650B transmission electron microscope (TEM) operating at an acceleration voltage of 80 kV was used 5 ACS Paragon Plus Environment


to obtain images showing the nanocomposite morphology. High-resolution transmission electron microscope (HRTEM) images were obtained on a JEM 2010 TEM with an accelerating voltage of 200 kV. An energy-dispersive spectrometer (EDS) analyzer attached to the HRTEM was used to analyze the composition of the nanocomposite. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker Hyperion 2000 spectrometer. The average hydrodynamic diameters and zeta-potentials were measured with a Nano ZS90 instrument (Malvern Instrument Ltd., England). Time-domain lifetime measurements were measured with an ultrafast lifetime spectrofluorometer (Delta Flex) based on time-correlated single-photon counting. The absolute fluorescence quantum yields were recorded on a spectrofluorometer (NanologR FluoroLog-3-2iHR320, Horiba Jobin Yvon) equipped with an integrating sphere. Fluorescence images of the cells were recorded on Olympus 1X73 fluorescence microscopy and using a 420/40 nm excitation filter with 50 ms exposure time. Synthesis of mSiO2 Nanospheres. The mSiO2 nanospheres were synthesized following a published procedure.35 CTAB (0.25 g) was first dissolved in ultrapure water (120 mL) under constant stirring and NaOH (1.75 mL, 1 M) was then added. TEOS (1.25 mL) was injected dropwise into the solution, and the reaction was maintained at 80 °C with stirring for 2 h. The resulting solution was filtered and washed with methanol and water to obtain a white precipitate. The solid product was dried in air to a weight of 0.3 g. The synthesized nanospheres were then refluxed for 24 h in a solution of 32 mL methanol and 1.79 mL HCl (37.4%). To remove the remaining surfactant template, the solution was centrifuged at 9300 × g for 15 min. Three more cycles of centrifugation/washing were conducted with ethanol. The nanospheres were redispersed in ultrapure water (25 mL) and the final concentration of the mSiO2 solution was 12 mg/mL. 6 ACS Paragon Plus Environment

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Synthesis of mSiO2-AuNCs nanocomposites. The above mSiO2 solution (1.76 mL) was mixed with ultrapure water (7.84 mL) under stirring for 5 min and HAuCl4 (20 mM, 0.3 mL) was added. After stirring for 30 min at room temperature, NaOH (1 M, 0.1 mL) was added to the mixture. The appearance of the solution changed from light yellow to colorless. THPC solution (80%, 2.45 µL) was then injected into the mixture to reduce the Au(III) and form Au nanoparticles. The reaction lasted for 60 min and resulted in the formation of a light yellow opaque solution. Ultrapure water (7.8 mL), sodium tetraborate (50 mM, pH 9.2, 2 mL), and 11MUA (1 M, 0.2 mL) were then added to the mixture.36 The reaction mixture was stirred for 48 h in the dark. The product was purified by two centrifugation cycles for 15 min at 3300 × g and redispersed in ultrapure water to remove excess ligands. The purified nanospheres were stored at 4 °C in the dark for further use. A free AuNC solution was also prepared for control experiments. The preparation of the free AuNCs was the same as that described for the mSiO2-AuNCs nanocomposites except that the mSiO2 solution was replaced with an equivalent volume of ultrapure water. We used GSH as both a reducing and capping agent to synthesize AuNCs in mSiO2 nanospheres. The mSiO2 solution (12 mg/mL,1 mL), HAuCl4 (20 mM,0.50 mL) and GSH (100 mM, 0.15 mL) were mixed with 3.35 mL of ultrapure water at 25 °C. The reaction mixture was heated to 70 °C under stirring for 24 h. The resulting solution was centrifuged two times at 3300 × g for 15 min. Preparation of mSiO2-AuNCs/PLL-FA nanocomposites. Firstly, FA was conjugated onto PLL through EDC/NHS reaction. The EDC solution (200 µL, 10 mM) and NHS solution (200 µl, 10 mM) were injected into 2.2 mL FA solution (0.5 mM). After 30min, PLL (2 mg/mL, 400 µl) 7 ACS Paragon Plus Environment


were added to the mixture and stirred at room temperature for 10 h under darkened conditions. The product was dialyzed against ultrapure water for 2 days to remove free FA and obtain PLLFA solution. Then, 100 µL FA-PLL solution was quickly injected into 2 mL SiO2-AuNCs nanoparticles solution under stirring. After 0.5 h, the resulting solution centrifuged at 3300 × g for 5 min to obtain mSiO2-AuNCs/PLL-FA nanocomposites. Stability Assay. To study the influence of the solution on the mSiO2-AuNCs/PLL-FA nanocomposites, we dispersed mSiO2-AuNCs/PLL-FA, mSiO2-AuNCs and free AuNCs in aqueous solution and phosphate buffered solution (PBS), respectively, and measured their quantum yields. To explore the influence of pH on the mSiO2-AuNCs/PLL-FA nanocomposites, we prepared four mixtures of the nanocomposites adjusted to pH 4.0, 5.0, 6.0, and 7.4 via addition of different volumes of HCl (1 M) or NaOH (1 M). The emission intensity at 535 nm was detected under 370 nm excitation. We performed parallel control experiments with mSiO2AuNCs and free AuNC solution under the same conditions. Cellular Imaging Assay. The KB was seeded in 35 mm × 35 mm culture plates and grown in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum (FBS). The plates were then incubated at 37 °C in a 5% CO2 humidified atmosphere for 24 h. A portion of the Au NCs, mSiO2-AuNCs and mSiO2-AuNCs/PLL-FA nanocomposites (100 µL) was added to the medium (900 µL) on the 35 mm × 35 mm plates to a final concentration: (mSiO2) = 0.1 mg/mL. After incubation at 37 °C for 2 h, the medium was removed and the cells were washed twice with PBS (pH = 7.4). The specimens were then observed with the use of an oil immersion lens (100× magnification; NA 1.4) in an Olympus 1X73 fluorescence microscopy. L02 cells


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were incubated with mSiO2-AuNCs/PLL-FA nanocomposites under the same experimental process. Cytotoxicity Assay by MTT Method. The cytotoxicity of the nanocomposite was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. KB and L02 cells were seeded in 96-well tissue culture plates and treated with various concentrations of nanocomposite (0–0.2 mg/mL). The concentration of the NPs was calculated from the initial mSiO2. After incubation for 2 h at 37 °C in a 5% CO2 humidified atmosphere, the medium was poured out, and 100 µL medium was added to each well. After incubation for 22 h, MTT (1 mg/mL in PBS, 100 µL) was injected into each well, and the culture was incubated for 4 h. The MTT medium solution was then removed and the cells were lysed by the addition of DMSO (100 µL). The plate was gently shaken for 5 min, the absorbance values of purple formazan at 570 nm were measured on a Spectra MAX 340PC plate reader. RESULTS AND DISCUSSION Synthesis and Characterization of nanocomposites. Firstly, we prepared mSiO2 nanospheres with uniform nanopores to act as a framework and microreactor for the AuNCs (Scheme 1). TEM images of the mSiO2 nanospheres are depicted in Figure S1. We prepared a type of mSiO2 with spherical nanoparticles with an average diameter of 170.0±5.3 nm, containing mesopores with a diameter of approximately 2.7 nm. The mesopores provided sufficient space to act as a microreactor for formation of AuNCs, with sizes less than 2 nm. We used THPC, which releases formaldehyde, to reduce Au(III) and form the Au nanoparticles. The Au nanoparticles were then etched with 11-MUA to form AuNCs within the mSiO2.36



UV-vis absorption spectra in Figure 1a shows that the absorption peak of the free AuNCs was centered at approximately 370 nm, while mSiO2 had no absorption peak in the wavelength range of 300–800 nm. Thus, the absorption peak between 350 and 400 nm in the mSiO2-AuNCs was attributed to the formation of AuNCs. Next, we measured the emission spectra of the nanocomposites. As shown in Figure 1b, the emission at approximately 535 nm for mSiO2AuNCs was similar to that of the free AuNCs. These results indicate that AuNCs were successfully prepared in the mSiO2 framework and that their optical properties were unaffected by the mSiO2 framework.

Figure 1. (a) UV-vis absorption spectra of mSiO2 nanospheres (black line), mSiO2-AuNCs nanocomposites (red line) and free AuNCs (green dash line). (b) Normalized fluorescence emission of free AuNCs (black line) and mSiO2-AuNCs nanocomposites (red line). [mSiO2AuNCs] = 5 mg/mL, [mSiO2] = 10 mg/mL, [Free AuNCs] = 10 mg/mL. Excitation wavelength was 370 nm. The morphology of mSiO2-AuNCs was characterized by TEM imaging (Figure 2). The TEM images in Figure 2a and 2b show that the nanocomposites formed as mesoporous nanoparticles 10 ACS Paragon Plus Environment

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with a particle diameter of approximately 172 nm. The structure of the mSiO2 in nanocomposite was no different from that of the pure mSiO2 nanospheres, which confirmed that the formation of AuNCs had no effect on the structure of the mSiO2. The box in Figure 2b shows a region where elemental mapping analysis was performed. Signals from S and Au could be attributed to presence of S atoms in the 11-MUA molecules and AuNCs, respectively. The distribution of signals from Si (Figure 2c), O (Figure 2d), S (Figure 2e), and Au elements (Figure 2f) demonstrated that the AuNCs were in situ formed within the mSiO2 nanostructure.

Figure 2. TEM (a) and HRTEM (b) images of the mSiO2-AuNCs nanocomposites. (c–f) Elemental mapping images of Si (c), O (d), S (e), and Au (f) corresponding to the region marked in Figure 2b.



We further examined the crystal structure of the AuNCs in the mSiO2-AuNCs nanocomposites. After etching the mSiO2 framework with NaOH, a TEM image of the remaining internal structure of the AuNCs is shown in Figure 3a. The internal AuNCs showed lattice fringes of 2.4 Å, corresponding to the d-spacing of the (111) crystal plane of face-centered cubic (fcc) Au.37 The crystal structure of the AuNCs was consistent with that of the free AuNCs (Figure 3b). Thus, ultra-small fluorescent AuNCs formed in the nanopores of the mSiO2 nanospheres, indicating the suitability of the mSiO2 nanospheres as a template for the AuNCs.

Figure 3. HRTEM images of AuNCs in nanocomposites after NaOH etching (a) and free AuNCs (b). Scale bars: 5 nm. Inset shows a closeup of the crystalline structure of an individual AuNC. Scale bars: 2 nm. To explore the in situ formation of AuNCs within the mSiO2, we attempted the synthesis of AuNCs in mSiO2 nanospheres using GSH as both a reducing and capping agent, which is a commonly used approach for synthesis of AuNCs.38 However, we detected no obvious fluorescence signal in the mSiO2 nanospheres after centrifugation (Figure S2). One possible reason for this absence is that GSH is a tripeptide, which is more sterically hindered than


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formaldehyde. This effect could limit the diffusion of GSH molecules into the mSiO2 and prevent formation of the Au(I)-GSH complex within. We also examined the emission spectrum of mSiO2 nanospheres after incubation with free AuNCs for 48 h. There is no emission in the mSiO2 nanospheres after centrifugation (Figure S3). Conversely, formaldehyde and 11-MUA can diffuse into nanopores of mSiO2 to facilitate formation of the Au-thiolate complexes and the synthesis of AuNCs in situ. Then, we synthesized PLL-FA and assembled it on the surface of nanocomposites via electrostatic interaction (Scheme 1). The biocompatible PLL was used as the modified layer because of reactive primary amines on its molecular chain39. It not only provides positive charge for self-assemble, but also can bond FA to achieve cell selectivity analysis. Through the typical EDC/NHS reaction, FA was conjugated with amino groups on the PLL. As shown in Figure S4, the PLL-FA showed not only the characteristic peaks of the PLL but also indicative peaks of FA at 1605, 1501 cm-1 (aromatic ring stretching of the pyridine and p-amino benzoic acid moieties), 1340, 850 and 751 cm-1 (aromatic C–H bands)40. The result indicated the successful conjugation of FA to the PLL. Due to dissociation of silanol groups, the mSiO2-AuNCs process the negatively charged surface with a ζ potential about –33.5 mV. So the positively charged PLL-FA can be adsorbed onto the mSiO2-AuNCs surfaces by electrostatic attractions. The ζ potential transferred to +16.4 mV, indicating that the PLL-FA was successfully loaded to form mSiO2AuNCs/PLL-FA. Correspondingly, the average diameter of the nanocomposites increased to 177.4 ± 2.3 nm with good dispersity in water solution. These results demonstrated the successful preparation of mSiO2-AuNCs/PLL-FA nanocomposites.



After modified with PLL-FA, the optical properties of the mSiO2-AuNCs/PLL-FA nanocomposites were investigated. As shown in Figure 4a, the absorption peaks of mSiO2AuNCs/PLL-FA and mSiO2-AuNCs nanocomposites locate at the same position. There is almost no change on the emission of Au NCs after assembly of PLL-FA (Figure 4b). The mSiO2AuNCs/PLL-FA nanocomposites featured a large Stokes shift of approximately 165 nm. We further investigated the fluorescence lifetime of the mSiO2-AuNCs/PLL-FA and mSiO2-AuNCs nanocomposites using time-correlated single-photon counting (Figure 4c). The calculated average lifetimes were 38 and 39 ns for mSiO2-AuNCs/PLL-FA and mSiO2-AuNCs nanocomposites, which are sufficient to prevent autofluorescence (occurs on the 2–3 ns time scale) from the influence of biological species in imaging.41 However, addition of PLL-FA to the Au NCs solution will result in fluorescence decrease (Figure S5). The phenomenon can be explained by the electrostatic interactions between PLL and carboxylic anions from 11-MUA in the AuNCs and formed aggregation.42 Based on the above results, we conclude that assembled PLL-FA layer does not affect the optical properties of AuNCs. The mSiO2 framework promoted the modification of PLL-FA and prevented aggregation of AuNCs. The large Stokes shift and long lifetime will enable the mSiO2-AuNCs/PLL-FA nanocomposites to be used as fluorescent probes for biological imaging.


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Figure 4. (a) UV-vis absorption (b) fluorescence spectra and (c) fluorescence intensity decay of mSiO2-AuNCs (black line) and mSiO2-AuNCs/PLL-FA nanocomposites (red line), respectively. Excitation wavelength was 370 nm. Stability of mSiO2-AuNCs/PLL-FA nanocomposites. The stability of nanoparticles in biological solutions is sensitive to the ionic strength,43 thus, it is essential for nanomaterials used in bioimaging to have good stability in biological solutions for their applications to in vitro or in vivo systems. The performance of mSiO2-AuNCs/PLL-FA nanocomposites in PBS (pH 7.4, 5 mM) solution was tested. As shown in Figure 5a, the mSiO2AuNCs/PLL-FA nanocomposites exhibited relatively high fluorescence intensity in PBS and aqueous solution. It can be seen in the corresponding fluorescent image under 365 nm UV light irradiation. As shown in Figure 5b, there is a slight decrease for mSiO2-AuNCs in PBS. However, we found a considerable decrease in the fluorescence intensity of the free AuNCs in PBS compared with that in aqueous solution (Figure 5c). To determine the emission efficiency of the mSiO2-AuNCs/PLL-FA, mSiO2-AuNCs and free AuNCs, the absolute fluorescence quantum yields (QY) were measured, and the results are summarized in Figure 5d. The mSiO2AuNCs/PLL-FA nanocomposites maintained the same quantum yield about 2% in PBS and aqueous solution. The QY of the mSiO2-AuNCs was determined to be 1.93% in PBS and 2.14% in aqueous solution, indicating only a slight change. However, the free AuNCs showed a decrease in their QY from 2.01% in aqueous solution to 0.45% in PBS. The decreased fluorescence of AuNCs in PBS could be attributed to cation induced AuNCs aggregation based on the electrostatic interactions between cations in PBS and carboxylic anions from 11-MUA.42 However, the AuNCs in the mSiO2-AuNCs/PLL-FA nanocomposites were protected by the 15 ACS Paragon Plus Environment


mesoporous silica framework of the mSiO2. Moreover, the PLL-FA reduced the penetration of cations into the pores of silica based on electrostatic repulsion. These results demonstrated that the mSiO2-AuNCs/PLL-FA nanocomposites improved the stability of the AuNCs in ionic solution.

Figure 5. Fluorescence emission spectra and fluorescent images of mSiO2-AuNCs/PLL-FA nanocomposites (a), mSiO2-AuNCs nanoparticles (b) and free AuNCs (c) in aqueous solution and PBS. (d) QYs of mSiO2-AuNCs/PLL-FA nanocomposites, mSiO2-AuNCs and free AuNCs in different solutions. The excitation wavelength was 370 nm. The high rate of glycolysis under both aerobic and anaerobic conditions leads to the accumulation of lactic acid in rapidly growing cancer cells;44 thus, tumor tissues and intracellular 16 ACS Paragon Plus Environment

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endosomes feature an acidic environment. Hence, we also measured the effects of solution pH on the stability of the mSiO2-AuNCs/PLL-FA nanocomposites. As shown in Figure 6a, the fluorescence intensities of the free AuNCs in acidic solution clearly decreased. The fluorescence intensities of mSiO2-AuNCs nanoparticles decreased to 77% at pH 4.0, while the mSiO2AuNCs/PLL-FA nanocomposites exhibited relatively high fluorescence intensities in the pH range of 4.0 to 7.4. These differences can be attributed to effects of pH on the capping ligands. The carboxyl groups of the alkanethiol ligand were in the form of –COOH in acidic solution resulting in a hydrophobic surface, which promoted aggregation of the free AuNCs and decreased fluorescence.45 Conversely, the AuNCs fixed in the mesoporous mSiO2 structure were protected by PLL-FA and maintained their dispersion at low solution pH. Figure 6b shows images of the solutions in pH 5.0, with and without 365 nm UV light irradiation. The mSiO2AuNCs/PLL-FA nanocomposite maintained its dispersion in acidic solution; however, the free AuNCs tended to aggregate and formed a white precipitate at pH 5.0. The decrease of the emission intensity could be observed by visual inspection. Therefore, the holes and PLL-FA efficiently shielded the internal AuNCs from the aggregation effects in acid solution. The stability of AuNCs in an acidic environment could be improved in mSiO2-AuNCs/PLL-FA nanocomposites.



Figure 6. (a) Effect of pH on relative fluorescence intensity (I/I0) of mSiO2-AuNCs/PLL-FA, mSiO2-AuNCs and free AuNCs in aqueous solution. (b) Fluorescence images of mSiO2AuNCs/PLL-FA, mSiO2-AuNCs and free AuNCs in an acidic solution (pH 5.0) with and without 365-nm UV lamp irradiation. Cellular Imaging and Cytotoxicity Assay The mSiO2-AuNCs/PLL-FA nanocomposites possessed good stability in various solutions, which are beneficial features for biological imaging; hence, cellular imaging experiments were performed. We chose KB cell as the FR-positive cell because of the highly overexpressed FR on their surface.46 As shown in Figure 7a and Figure S6, we observed bright fluorescence in the cytoplasm of KB cells after they were treated with the mSiO2-AuNCs/PLL-FA nanocomposites for 2 h. No aggregates of the nanocomposites in cell culture medium were observed. Conversely, mSiO2-AuNCs cannot be uptaken by the KB cells after the same incubation time. The result demonstrates that the attachment of PLL-FA to the composites can result in more efficient uptake by the KB cells. And the mSiO2-AuNCs/PLL-FA nanocomposites showed good stability under physiological conditions. Then, the mSiO2-AuNCs/PLL-FA nanocomposites incubated with a 18 ACS Paragon Plus Environment

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FR-negative cell L02 was further carried out. Figure 7a shows that there is no fluorescent signal for L02. Comparing the fluorescence images of the nanocomposites incubated with FR-positive and FR-negative cells, it is concluded that the mSiO2-AuNCs/PLL-FA nanocomposites were most likely transported into KB cells via FR-mediated endocytosis process.47 The nanocomposites can be targeted for imaging KB cells based on FA-FR interaction. To investigate the biocompatibility of the nanocomposites, the cell viability of KB and L02 cells after incubation with different amounts of the nanocomposites was measured. As shown in Figure 7b, more than 83% of the KB cells remained alive even at a nanocomposite concentration up to 0.2 mg/mL, which is twice as high as the concentration used for cellular imaging. The normal L02 cells maintained high cell viability over the entire concentration range. Therefore, the nanocomposites show less effect on the non-target cells. These results confirm that the mSiO2AuNCs/PLL-FA nanocomposites have good biocompatibility and good selectivity. They are suitable for applications to cell detection.



Figure 7. (a) Fluorescence, bright field and overlay images of KB and L02 cells after incubation with mSiO2-AuNCs/PLL-FA nanocomposites for 2 h. Scale bars: 5 µm. (b) Cell viability after incubation of KB and L02 cells with various concentrations of mSiO2-AuNCs/PLL-FA nanocomposites. CONCLUSION In this work, stable fluorescent AuNCs were reduced in situ within the nanopores of mSiO2. By self-assembly of PLL-FA on the surface of mSiO2, novel fluorescent nanocomposites were prepared. The resulting nanocomposites were applied to mSiO2 as a framework to protect the AuNCs formed within the nanopores from aggregation and fluorescence attenuation in different 20 ACS Paragon Plus Environment

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solution types. The nanocomposites showed bright fluorescence and good dispersibility in physiological environments. Moreover, the surface modified PLL-FA of nanocomposites improved their interaction with FR-positive cancer cells. Through receptor-mediated endocytosis, the internalization of the nanocomposites in FR-positive cancer cells occurred. A cell viability assay indicated that the nanocomposites possessed low cytotoxicity. These results indicate that our nanocomposites contain highly stable ultra-small AuNCs, which show good compatibility with complex biological systems and great potential for applications in biological imaging and cell detection. ASSOCIATED CONTENT Supporting Information. TEM images of mSiO2 nanospheres and their mesoporous structure characterized by HRTEM. Fluorescence spectra of nanocomposites formed with GSH. Fluorescence spectra of nanocomposites incubation with free AuNCs. FT-IR spectra of PLL, FA and PLL-FA. Fluorescence spectra of Free AuNCs before and after adding PLL-FA. Fluorescence, bright field and overlay images of KB cells after incubation with mSiO2-AuNCs nanocomposites. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions 21 ACS Paragon Plus Environment


#

X.W. and J.X. contributed equally to this study.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51673022), the Fundamental Research Funds for the Central Universities (FRF-TP-16-026A1) and the State Key Laboratory for Advanced Metals and Materials (2017Z-03). REFERENCES 1. Nguyen, Q. T.; Olson, E. S.; Aguilera, T. A.; Jiang, T.; Scadeng, M.; Ellies, L. G.; Tsien, R. Y. Surgery with Molecular Fluorescence Imaging Using Activatable Cell-Penetrating Peptides Decreases Residual Cancer and Improves Survival. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4317–4322. 2.

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Preparation of Novel Fluorescent Nanocomposites Based on Au

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