Facile Synthesis of Biocompatible Fluorescent Nanoparticles for


Nov 2, 2015 - In this work, we report the facile synthesis of functional core–shell structured nanoparticles with fluorescence enhancement, which sh...
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Facile Synthesis of Biocompatible Fluorescent Nanoparticles for Cellular Imaging and Targeted Detection of Cancer Cells Fu Tang, Chun Wang, Xiaoyu Wang, and Lidong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08907 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 3, 2015

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Facile Synthesis of Biocompatible Fluorescent Nanoparticles for Cellular Imaging and Targeted Detection of Cancer Cells Fu Tang, # Chun Wang, # Xiaoyu Wang, and Lidong Li* School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China

ABSTRACT: In this work, we report the facile synthesis of functional core-shell structured nanoparticles with fluorescence enhancement, which show specific targeting of cancer cells. Biopolymer poly-L-lysine was used to coat the silver core with various shell thicknesses. Then the nanoparticles were functionalized with folic acid as a targeting agent for folic acid receptor. The metal-enhanced fluorescence effect

was

observed

when

the

fluorophore

(5-(and-6)-carboxyfluorescein-succinimidyl ester) was conjugated to the modified nanoparticle surface. Cellular imaging assay of the nanoparticles in folic acid receptor-positive cancer cells showed their excellent biocompatibility and selectivity. The as-prepared functional nanoparticles demonstrate the efficiency of the metal-enhanced fluorescence effect and provide an alternative approach for the cellular imaging and targeting of cancer cells.

KEYWORDS: fluorescence, polymer, core-shell nanoparticles, fluorescence enhancement, cellular imaging

INTRODUCTION Fluorescence detection is one of the most commonly used analytical methods in the fields of chemistry and biochemistry, exploiting the fluorescent properties of certain molecules or materials. 1-9 The brightness of the fluorophore is of great importance as a high signal-to-noise ratio yields the desirable characteristics of high sensitivity and detectability. However, conventional fluorescent materials usually have a low 1

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quantum yield and, when used in large quantities, the light emissions from the fluorophores cause self-quenching, restricting their further application.10-12 Functional conjugated polymers and aggregation-induced emission molecule nanoparticles have been fabricated as substitute fluorescent materials because of their improved photostability and brightness. Studies have shown that they are ideal materials for biosensing and imaging13-19, however, their lack of water solubility and biocompatibility limits their application. Some metal nanostructures (such as gold and silver (Ag) nanoparticles) are reported to possess unique spectroscopically favorable properties that can enhance the emissions of fluorophores localized near their surfaces. This phenomenon is defined as metal-enhanced fluorescence (MEF)

20,21

and can be explained by the near-field

interactions of the plasmon. The MEF effect may provide a promising method to address the quenching problems of fluorophores, thus introducing a new way to realize high sensitivity in fluorescence detection. It has been theoretically calculated and experimentally supported that the MEF effect greatly depends on the distance between

the

fluorophore

and

metallic

substrates.

Maximum

fluorescence

enhancement can be achieved when this distance is optimized.22-28 Earlier work about MEF systems have mainly focused on fabricating metal nanostructure films on planar surfaces.29-35 Although obvious fluorescence enhancements have been achieved via the metallic planar substrates, it is imperative to develop MEF colloidal systems so that this unique fluorescence enhancement phenomenon can be applied to biological fields, such as biosensing and bioimaging.36-43 Hybrid nanoparticles with metallic cores have been developed for MEF studies. Silica,38,39 polymers,40,41 proteins,42 as well as DNA43 have been assembled onto metal nanoparticle surfaces to control the distance between the fluorophore and metals; however, few studies have reported efficient MEF in colloidal systems or applied them to biosensing and bioimaging fields. In this work, we reported a facile method to fabricate a core-shell structured fluorescent nanoparticle with efficient fluorescence enhancement effect and specific targeting ability. To prepare this nanoparticle, Ag nanoparticles coated with 2

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biocompatible poly-L-lysine (PLL) shells were synthesized. Folic acid (FA) was then conjugated to the as-prepared [email protected] nanoparticle surfaces through a reaction between the carboxyl groups of FA and the amino groups of PLL chain. Meanwhile, 5-(and-6)-carboxyfluorescein-succinimidyl ester (FAM-SE) was chosen as the dye molecule to further modify the PLL shell surface, resulting in the formation of [email protected]/(FA/FAM-SE) fluorescent nanoparticles. An increase in the fluorescence intensity of FAM-SE was observed because of the MEF effect from the silver core, after optimizing the PLL shell thickness. Folic acid (FA) can bind with the folic acid receptor (FR) with high affinity, meaning that the FA-modified nanoparticles, [email protected]/(FA/FAM-SE), could be useful for the detection of FR-positive cancer cells. A cell imaging assay demonstrated that the as-prepared nanoparticles could target and be taken up by cancerous cells, such as KB cells, the surface of which are rich with FRs. Very few modified nanoparticles were observed in FR-negative MCF 7 cells, which were used as the control. Furthermore, [email protected]/(FA/FAM-SE) possess good

biocompatibility

as

demonstrated

by

a

cell

viability

assay.

[email protected]/(FA/FAM-SE) provides a new approach to realize the efficiency of the MEF effect and for its application in biological fields, such as cellular imaging and targeting of cancer cells.

EXPERIMENTAL SECTION Materials and Measurements. Silver nitrate, di-tert-butyldicarbonate (Boc2O), N-hydroxysuccinimide

(NHS),

1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide

hydrochloride (EDC), folic acid (FA), trifluoroacetic acid (TFA), and Poly(L-lysine) (PLL)

were

purchased

from

Sigma-Aldrich.

3,3’-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) was purchased from 3B Scientific Corporation (USA). 5-(and-6)-Carboxyfluorescein-succinimidyl ester (FAM-SE) was purchased from Life Technologies Co. Other reagents used in the experiments were purchased from Sigma-Aldrich, and were used without further purification, unless otherwise noted. Distilled water was used throughout. The breast carcinoma (MCF 7) and human nasopharyngeal epidermal carcinoma (KB) cells were 3

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purchased from the Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Ultraviolet-visible (UV-Vis) absorption spectra were collected on a Hitachi U-3900H spectrophotometer. Fluorescence spectra were obtained using a Hitachi F-7000 fluorescence spectrometer equipped with a Xenon lamp excitation source. Transmission electron microscope (TEM) images were recorded with Hitachi H-7650B. The fluorescence images of the prepared nanoparticles and the cells were recorded using a confocal laser scanning biological microscope (CLSM; Olympus FV1000-IX81). Cell viability was detected using a Spectra MAX 340PC plate reader. Fluorescence lifetime measurements were performed using an Edinburgh Instruments FLS980 spectrometer with excitation wavelength of 470 nm. The ζ-potential and size distributions of the nanoparticles were measured by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS90 at room temperature. And nanoparticles were dispersed in distilled water during these measurements. Synthesis of [email protected] Nanoparticles with Different Shell Thicknesses. The [email protected] nanoparticles with different shell thicknesses were synthesized in three steps. Briefly, Ag nanoparticles were first synthesized by adding 10 mL of freshly prepared 1% [Ag(NH3)2]NO3 to 10 mL of 2% tannic acid solution, at room temperature, with vigorous stirring. After 30 min, the resulting Ag colloids were collected, purified by centrifugation, and re-dispersed in 20 mL of distilled water. Then, 100 µL of the as-prepared Ag nanoparticles were added into 4 mL of cystamine dihydrochloride solution (0.25 mg/mL) under vigorous stirring. And the reaction was kept under stirring at room temperature for 5 h. Subsequently, the amino-modified Ag nanoparticles were washed and purified three times by centrifugation at 8600 ×g for 20 min, and re-dispersed in 1 mL of distilled water. Finally, the amino-modified Ag nanoparticles (1 mL) were added dropwise into 4 mL of PLL solution under gentle stirring, followed by the quick addition of the DTSSP solution (500 µL, 2.5 mg/mL). The pH value of the whole solution was adjusted to 9–10 with NaOH (1M) to facilitate the reaction, and the mixture was then stirred at room temperature for 12 h. The differing shell thicknesses were obtained by 4

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varying the concentration of PLL solution from 2.5 mg/mL to 10 mg/mL, respectively. The as-prepared nanoparticles were separated from the reaction mixture by centrifugation at 8600 ×g for 20 min, and re-dispersed in 4 mL of distilled water. Synthesis of Folic Acid-Conjugated Nanoparticles ([email protected]/FA). To conjugate FA onto the [email protected] nanoparticle surfaces, Boc-protection of FA was first completed by adding 100 µL of Boc2O (20 mM) into a stirred solution of FA (1 mL, 2 mM). The reaction mixture was stirred at room temperature for 12 h under darkened conditions and nitrogen atmosphere. Then, 100 µL of EDC solution (0.1 M) were added to the mixture, followed by 100 µL of NHS (0.1 M). After stirring in the dark overnight, the resulting solution was dialysis against deionized water for 12 h to remove the excess reactant. Then 100 µL of it was added to 4 mL of [email protected] solution. After reaction for 12 h, the prepared nanoparticles were collected by centrifugation at 8600 ×g for 20 min, and re-dispersed in 4 mL of PBS buffer (pH 7.4). Synthesis of MEF Nanoparticles ([email protected]/(FA/FAM-SE)). To fabricate the [email protected]/(FA/FAM-SE) nanoparticles, 10 µL of 0.5 mM FAM-SE were added to 1 mL of the prepared [email protected]/FA nanoparticles PBS solution under vigorous stirring. [email protected]/(FA/FAM-SE) nanoparticles were obtained after 2 h of incubation. The nanoparticles were washed and centrifuged with PBS buffer (pH 7.4). Then, 6 µL of TFA was added and the mixture was stirred for 2 h. After the reaction, the nanoparticles were washed, centrifuged, and re-dispersed in 100 µL of PBS buffer (pH 7.4). The final concentration of Ag nanoparticles was about 0.8 mg/mL, while that of FA and FAM-SE on [email protected]/(FA/FAM-SE) nanoparticles was about 0.11 mM and 8.3 µM respectively, after monitoring the absorption of the combined supernatant liquid. MEF Measurements. To study the MEF effect, 10 µL of [email protected]/(FA/FAM-SE) PBS solution was added into 1 mL of PBS buffer (pH 7.4), and the fluorescence emissions were measured under excitation of 470 nm. For comparison, FAM-SE molecules in PBS buffer with or without the presence of Ag nanoparticles were also used as the control. The FAM-SE concentration was fixed at 8.3 × 10-8 M Cell Culture and Cellular Imaging Assay. Breast carcinoma (MCF 7) cells that 5

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lack folic acid receptors were incubated in a 10% fetal bovine serum (FBS) containing Dulbecco’s modified eagle medium. KB cells, a variation of cancer cell that originates from nasopharyngeal cancer and is known to over-express folic acid receptors, were incubated in FA-deficient RPMI-1640 culture medium, containing 10% FBS.44-46 For cell imaging observation, 1 mL (100,000 cells/mL) of the two types of cell lines were seeded in separate confocal dishes, and incubated at 37 °C in a 5% CO2 humidified atmosphere for 24 h. Then, 100 µL of [email protected]/(FA/FAM-SE) nanoparticles were added to 900 µL of the medium containing each type of cell line. After incubation at 37 °C for 4 h, the medium was removed and the cells were washed with PBS three times. Each dish was observed with a confocal laser scanning microscopy (FV1200-IX83, Olympus, Japan) upon 488 nm excitation. Cytotoxicity Assay. The cytotoxicity of the [email protected]/(FA/FAM-SE) nanoparticles was

evaluated

using

the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) method. MCF 7 and KB cells were seeded in 96-well tissue culture plates and maintained for 24 h in medium containing FBS. They were then treated with various concentrations of [email protected]/(FA/FAM-SE) nanoparticles and incubated for 12 h. After, 100 µL MTT, 1 mg/mL in PBS, was added to each well, after the medium was removed, and the cells were incubated at 37 °C for another 4 h. Then the MTT solution was removed and 100 µL of dimethyl sulfoxide were added. The plate was gently agitated for 5 min, and the absorbance of purple formazan at 570 nm was then detected using a Spectra MAX 340PC plate reader.

RESULTS AND DISCUSSION Synthesis and Characterization of Fluorescent Hybrid Biocompatible Nanoparticles. Silver nanoparticles were first synthesized via a tannic acid reduction method.47 The maximum surface plasmon absorption of the as-prepared Ag nanoparticles was at 417 nm (shown in Figure 1a, black line). The Ag nanoparticle surfaces were then modified with large amounts of amino groups through the strong affinity between Ag and the disulfide groups of cystamine. The shell was prepared by cross-linking of PLL molecules with a water-soluble cross-linker, DTSSP, which 6

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possesses two activated N-hydroxysulfosuccinimide esters that can react with the primary amine groups of PLL molecular chains. Amino groups on the Ag nanoparticle surfaces facilitated the PLL shell coating via amino group linkage. UV-Vis absorbance spectra (Figure 1a) show a 10-nm red shift of the surface plasmon absorbance of Ag nanoparticles after the PLL shell coating (the red line), which can be attributed to the increase of the refractive index around the Ag nanoparticles after the shell coating. This verified the successful modification with the PLL shell.48 To apply these synthesized nanoparticles to targeted tumor cells, we conjugated FA to the nanoparticle surface. FA is an essential precursor in the synthesis of nucleic acids and some amino acids, but it cannot be produced endogenously by mammalian cells. The internalization of FA by cells is usually achieved via receptor-mediated endocytosis. The FR binds FA with high affinity, and it is known to be over-expressed in many tumors, including malignancies of the breast, ovary, lung, kidney, and brain. The density of FRs also appears to increase as the grade of the cancer increases.45,49 As such, the FR-based mechanism can be exploited to target tumor cells. The conjugation of FA with the synthesized nanoparticles was based on the binding of carboxyl groups on FA molecules and amino groups on the PLL shell surfaces following the usual NHS/EDC reaction. As FA has both carboxyl and amino groups, Boc2O was chosen to protect the amino groups before the carboxyl group activation process so as to avoid self-conjugation of FA molecules. The Boc was later removed by TFA reaction.50 After the FA conjugation, we monitored absorption spectrum of the as-prepared [email protected]/FA nanoparticles (Figure 1, blue line). By comparison with the absorption spectrum of [email protected], we can see that the FA conjugation has little effect on the plasmon absorption band of the nanoparticles. This may be explained by the small size and low molecular weight of FA molecules that are unable to change the refractive index around the Ag nanoparticles more obviously.

7

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Figure 1. UV-Vis absorption spectra of Ag nanoparticles (black line), [email protected] nanoparticles (red line), [email protected]/FA nanoparticles (blue line), [email protected]/(FA/FAM-SE) nanoparticles (orange line), and free FAM-SE molecules (green dash).

As PLL is a relatively big molecule, and there are plenty of reactive primary amines on its molecular chain, we chose an amine-reactive molecule, FAM-SE, as the fluorophore to study the MEF effect of the prepared nanoparticles. The FAM-SE contains N-hydroxysulfosuccinimide ester group at the end of the molecular chain that can covalently bond with amine groups on the PLL shell surface. This bonding can efficiently reduce the leakage of dye molecules. Moreover, it was reported that the MEF effect is highly dependent on the spectral overlap between the surface plasmon peaks of the Ag nanoparticles and the absorption of fluorophores,51 another reason for the selection of FAM-SE. The absorption spectra of Ag nanoparticles (black line) and FAM-SE (the green dash line) in Figure 1 shows that the absorption of FAM-SE overlaps well with the surface plasmon absorption of Ag nanoparticles. The orange line in Figure 1 presents the plasmon absorption spectrum of the as-prepared [email protected]/(FA/FAM-SE) nanoparticles. It can be seen that the absorption spectrum of [email protected]/(FA/FAM-SE) was a superposition of the absorption spectrum of [email protected]/FA nanoparticles and the free FAM-SE solution. The final concentration of FAM-SE on [email protected]/(FA/FAM-SE) nanoparticles was about 8.3 µM after monitoring the absorption of the combined supernatant liquid. 8

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To further prove the successful synthesis of [email protected]/(FA/FAM-SE) nanoparticles, we monitored the synthesis process via DLS measurements. The results (Figure S1a in the Supporting Information) showed an average number mean hydrodynamic diameter of about 35.11 nm for Ag nanoparticles, with a polydispersity index (PDI) of 0.33. After the PLL shell coating procedure, the average diameter of the nanoparticles increased to about 55.27 nm, suggesting a shell with thickness of about 10 nm had been successfully synthesized onto the Ag nanoparticles. Meanwhile, the PDI decrease to about 0.27, indicating the improvement of nanoparticles dispersity after a polymer shell coating. Further conjugation of FA and FAM-SE onto [email protected] nanoparticles showed little influence on the nanoparticle diameter and PDI. This may be due to the small molecular weight and size of FA and FAM-SE molecules. However, the change in ζ-potential of nanoparticles could be clearly observed after monitoring the surface charge during the nanoparticle modification process. As we can see from Figure S1b, the initial ζ-potential of Ag nanoparticles was about -28.5 mV as tannic acid was used as its surfactant. After the PLL shell coating, the ζ-potential showed a great charge reverse to about +54.2 mV, which could be attributed to the protonated amines on the PLL chains that made the nanoparticle surfaces full of positive charge. Conjugation of FA onto [email protected] nanoparticle surfaces decreased the ζ-potential to about +35.9 mV for the consuming of amino groups. And similar phenomenon was observed for further conjugation of FAM-SE, as the final ζ-potential of [email protected]/(FA/FAM-SE) nanoparticles was about +17.1 mV. These results demonstrated the successful preparation of [email protected]/(FA/FAM-SE) nanoparticles. We

have

also

studied

the

morphology

of

the

prepared

Ag

and

[email protected](FA/FAM-SE) nanoparticles with TEM observation. Figure 2a presents the TEM image of the prepared Ag nanoparticles, with an average size of about 30 nm, and nearly spherically shaped. When compared with the TEM image of [email protected](FA/FAM-SE) nanoparticles present in Figure 2b, an obvious core-shell structure can be observed for all the nanoparticles. We can see clearly that the Ag cores (dark contrast) were fully encapsulated by a uniform PLL(FA/FAM-SE) shell 9

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(light contrast), and with a shell thickness of about 8 nm, similar to our DLS observation. To further demonstrate the successful conjugation of FAM-SE molecules with the PLL shell, we observed the [email protected]/(FA/FAM-SE) nanoparticles by confocal laser scanning microscopy (CLSM). [email protected]/(FA/FAM-SE) nanoparticle solution was dropped on a coverslip, and the sample was observed by CLSM under the excitation of 488 nm. Bright green fluorescent faculae could be clearly seen in the CLSM image of [email protected]/(FA/FAM-SE) shown in Figure 2c. The well-dispersed state of green faculae demonstrated that the fluorescence was from the FAM-SE assembled on [email protected]/FA nanoparticles, not from that of free FAM-SE molecules in solution. This result indicated the successful conjugation of FAM-SE molecules with nanoparticles.

Figure 2. TEM images of (a) Ag nanoparticles and (b) [email protected]/(FA/FAM-SE) nanoparticles; (c) confocal laser scanning microscopy images of [email protected]/(FA/FAM-SE) nanoparticles.

MEF Effect Assay. As MEF is a distance-dependent effect, we coated the Ag core with PLL shell to serve as a spacer layer between Ag and the fluorophore FAM-SE. Then, the MEF effect was studied by a fluorescence spectrometer with an excitation wavelength of 470 nm. For comparison, FAM-SE molecules in PBS buffer with or without the presence of Ag nanoparticles were also used for the fluorescence assay. The FAM-SE concentration was fixed at 8.3 × 10-8 M in all experimental solutions. From the fluorescence spectrum shown in Figure 3 we can see that, for the Ag/FAM-SE solution (red line), in which FAM-SE directly interacted with Ag nanoparticles, the fluorescence intensity of FAM-SE was quenched to about 35% of its original value. The fluorescence quenching effect could be attributed to the 10

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resonant energy transfer from the fluorophore to the metal.23,52 However, when a PLL shell spacer was used to separate the fluorophore and metal, that is, the [email protected](FA/FAM-SE) nanoparticle (blue line), an increase of the fluorescence intensity of FAM-SE was observed when compared with the free state of FAM-SE in solution.

Figure 3. Fluorescence intensity spectra of [email protected]/(FA/FAM-SE) dispersion (blue line), free FAM-SE solution (green line), and Ag/FAM-SE dispersion (red line) in PBS buffer (pH 7.4). The concentration of FAM-SE was maintained at 8.3 × 10-8 M.

The distance-dependent fluorescence enhancement of FAM-SE by Ag nanoparticles was further studied by varying the fluorophore-metal distance via controlling the PLL shell thicknesses by changing the PLL concentration from 2.5 mg/mL to 10 mg/mL during the shell formation process. The corresponding PLL shell thicknesses varied from about 3 nm to 13 nm, as calculated by the TEM observations (Figure S2 in the Supporting Information). After monitoring their fluorescence spectra, we found that, with the increase of the shell thicknesses, i.e., the increase of fluorophore-metal distances, the fluorescence intensity of FAM-SE was found to be enhanced, in contrast to fluorescence quenching when the fluorophore and the metal core are in direct contact (Figure S2 in the Supporting Information). An optimal fluorescence enhancement of about 2.1-fold occurred at a fluorophore-metal distance of about 8 nm. Further increase of the fluorophore-metal distance caused a decrease in the enhancement factor. The nanoparticles with PLL shell thickness of 8 nm was then 11

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chosen to perform our next experiment studies. The MEF effect was reported to be distance dependent and achieved mainly by two mechanisms.21 The first is the amplification of electric fields, which provides stronger excitation rates without modifying the fluorescence lifetime; The second mechanism is the increase of radiative decay rate of fluorophores caused by surface plasmon coupling which changes the lifetime of the fluorophores.

38, 39, 53

To demonstrate the

mechanism of the fluorescence enhancement in our nanoparticle system, we studied the fluorescence lifetime of FAM-SE and [email protected]/(FA/FAM-SE) nanoparticles. It can be clearly seen from Figure 4 that the intensity decay of FAM-SE on the [email protected]/(FA/FAM-SE) nanoparticles was much faster than that of FAM-SE molecules in their solution state. The calculated average lifetime of FAM-SE in its solution state was about 3.87 ns, while that of [email protected]/(FA/FAM-SE) nanoparticles reduced to about 1.28 ns, suggesting the fluorescence enhancement in our prepared system derived from the increased radiation decay affected by the Ag nanoparticles.

Figure 4. Fluorescence lifetime measurements of FAM-SE in its solution state and on the [email protected]/(FA/FAM-SE) nanoparticles. The instrument response function (IRF) is also included.

Cell Imaging and Cytotoxicity Assay. The effect of FA modification on the cellular uptake of nanoparticle was studied in the cancer cell lines MCF 7 and KB. KB cells were chosen because of their predisposition to over-expressing FR. In contrast, MCF 7 breast carcinoma cells that lack FR were chosen for the control experiment.44-46 From Figure 5, the CLSM images of MCF 7 and KB cells incubated 12

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with [email protected]/(FA/FAM-SE) nanoparticles for 4 h at 37 °C, we can see that green fluorescence was observed within both cell types, while nearly no fluorescence signal can be detected from the CLSM images for cells incubated without nanoparticles (Figure S3 in the Supporting Information), indicating the internalization of [email protected]/(FA/FAM-SE) nanoparticles. However, the green fluorescence observed from the fluorescence image of MCF 7 cells was extremely weak as cellular uptake was low. In sharp contrast, many more nanoparticles were internalized by FR over-expressed KB cells, evident from the much brighter green fluorescence that could be observed. And when we chose the nanoparticles with unfavorable shell thickness as control experiment (Figure S4 in the Supporting Information), the little weaker fluorescence observed from its fluorescence image demonstrate the efficient MEF effect in cells as shown in Figure 5. This result is consistent with our previous expectations.

Figure 5. CLSM images of MCF 7 cancer cells (the top row) and KB cells (the bottom row) at the presence of [email protected]/(FA/FAM-SE) nanoparticles ([FAM-SE]= 8.3 × 10-7 M). Left: phase contrast bright-field images. Middle: fluorescence images. Right: the over-lapped images of bright-field and fluorescence images. The fluorescence images were obtained using a 488-nm laser. The scale bar represents 20 µm.

For the prepared fluorescent nanoparticles to be applied as targeting vehicles, it is 13

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important that the nanoparticles possess low cytotoxicity. By comparing the bright field images of cells in Figure 5 and Figure S3, we can see that co-incubation with [email protected]/(FA/FAM-SE) nanoparticles affected the morphology of MCF 7 and KB cells little. Then we incubated the prepared [email protected]/(FA/FAM-SE) nanoparticles with both KB cells and MCF 7 cells and investigated the cell viability with various nanoparticle

concentrations

via

the

MTT

method.

The

amount

of

[email protected]/(FA/FAM-SE) nanoparticles used was expressed as the concentration of conjugated FAM-SE molecules. The absorbance of MTT at 570 nm is dependent on the degree of activation of the cells. The cell viability can then be calculated via the ratio of absorbance of the cells incubated with [email protected]/(FA/FAM-SE) nanoparticles to that of the cells incubated with culture medium only. As shown in Figure 6, the cell viability of both cell lines are still as high as about 90% when incubated with [email protected]/(FA/FAM-SE) nanoparticles at the concentration that was used in the cellular imaging assay (the final concentration of FAM-SE was 8.3 × 10-7 M). When the concentration of FAM-SE is as high as 1.66 µM, both cell lines have a viability of more than 80%. Those results indicated the good biocompatibility of the prepared [email protected]/(FA/FAM-SE) nanoparticles.

Figure 6. Cell viability results after incubation of MCF 7 and KB cells with different concentrations of [email protected]/(FA/FAM-SE) nanoparticles using MTT assay. Error bars correspond to standard deviations from three separate measurements. The amounts of nanoparticles were expressed as the concentrations of conjugated FAM-SE molecules. 14

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CONCLUSION In this work, we fabricated core-shell structured fluorescent nanoparticles, with a fluorescence enhancement effect and additional specific cancer cell targeting ability. A biocompatible shell was developed to coat silver nanoparticles and served as a spacer layer to separate fluorophore (FAM-SE) from the metal core. By varying shell thickness, the fluorophore-metal distance could be controlled and optimized. FAM-SE was conjugated onto the surface of [email protected] nanoparticle via the formation of covalent bond with the PLL chain. The fluorescence of FAM-SE was enhanced by about 2.1-fold because of the MEF effect of the silver core after its conjugation. By bonding folic acid on the nanoparticle surfaces, the as-prepared fluorescent nanoparticles show obvious specific internalization of FR-positive KB cells over FR-negative MCF 7 cells. Cell viability assays demonstrate the low cytotoxicity of these nanoparticles. By using the MEF effect, the as-prepared fluorescent nanoparticles show promise for application in cellular imaging and target sensing of cancer cells.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (L.L.) Author Contributions #

F. T. and C.W. contributed equally to this study.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51403018, 51373022), the Fundamental Research Funds for the Central Universities 15

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(FRF-TP-15-002A2). The authors thank Dr. Libin Liu for giving support for the cellular experiments.

ASSOCIATED CONTENT Supporting Information. Size,

polydispersity

and

ζ-potential of

Ag,

[email protected],

[email protected]/FA and

[email protected]/(FA/FAM-SE) nanoparticles in solution. Fluorescence enhancement factor of [email protected]/(FA/FAM-SE) nanoparticles with different shell thickness. CLSM images of MCF 7 cancer cells and KB cells incubated with cell culture medium only and with [email protected]/(FA/FAM-SE) nanoparticles, the shell thickness was about 13 nm.

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