Multifunctional Gold Nanoparticles-Based Fluorescence Resonance

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Biological and Medical Applications of Materials and Interfaces

Multifunctional Gold Nanoparticles-Based Fluorescence Resonance Energy Transfer Probe for Target Drug Delivery and Cell Fluorescence Imaging Qian Zhang, Yan Gong, Xin-jie Guo, Peng Zhang, and Caifeng Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12897 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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ACS Applied Materials & Interfaces

Multifunctional Gold Nanoparticles-Based Fluorescence Resonance Energy Transfer Probe for Target Drug Delivery and Cell Fluorescence Imaging Qian Zhang, Yan Gong, Xin-jie Guo, Peng Zhang* and Cai-feng Ding*

Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China. *E-mail: [email protected]; [email protected].

KEYWORDS: CS-AuNPs-DOX, target drug delivery, fluorescence imaging, glutathione (GSH), cell apoptosis

ABSTRACT: Drug delivery system has profound significance for imaging capabilities and monitoring apoptosis process precisely in cancer therapeutic field. Herein,

we

designed

cysteamine-stabilized

gold

nanoparticles,

CS-AuNPs-doxorubicin, for fluorescence-enhanced cell imaging and target drug delivery. For cancer therapy, doxorubicin (DOX) was incorporated to CS-AuNPs by

1

ACS Paragon Plus Environment

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disulfide linkages which could be cleaved by glutathione (GSH) in cancer cells specifically. In addition, red emissive DOX was quenched effectively by particular quenching effect of Fluorescence Resonance Energy Transfer (FRET) from DOX to AuNPs, rendering monitoring target drug release by visual luminescence. The released DOX-SH acted as an indicator for cancer cells with red fluorescence and was further used for stimuli-responsive drug therapy. After an overall investigation of detection for GSH, pro-apoptosis for cancer cells and inhibition for tumor tissues in vivo, the CS-AuNPs-DOX nanoprobe shows obviously enhanced performance. This proposal provides an intelligent strategy for cell imaging and drug delivery, which serves as a promising candidate for anticancer therapeutic applications.

INTRODUCTION Nowadays, chemotherapy has become the most important treatment for tumors, whereas the drugs are usually nonspecific, targeting all cells in the body and not just cancer cells. Besides the serious side effects on normal tissues, other considerations such as short retention time in human body and poor stability also cause great limitations in the practical use of chemical therapy. Doxorubicin (DOX) has been extensively applied for cancer therapy in clinical, including soft tissue sarcoma,1, 2 breast cancer,3, 4 malignant lymphomas,5 etc., due to its broad anti-tumor spectrum and potent anti-tumor activity.6 However, toxicity toward non-targeted tissues could scarcely be avoided during the doxorubicin treatment, and the drug is rapidly eliminated by enzymolysis and hydrolysis when circulating in the body, thus seriously 2


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limiting the usage of DOX.7, 8 To improve the rapeutic effect of DOX on target tumor chemotherapy and its retention time while reducing normal tissue toxicity, target drug delivery has gained considerable attention.9, 10 Recent years have witnessed the development of drug delivery systems. Various nanomaterials, consisting of liposome,11-13 polymer nanoparticles,14, 15 water soluble polymer,16, 17 vesicles18, 19 and inorganic materials20, have been employed to build up a series of stable carriers for the rapeutic agents. These nano-systems feature enhanced water-solubility and achieve target drug delivery owing to enhanced permeation and retention (EPR)21 effect of tumor tissues. Accumulated drugs and their prolonged retention time improves therapy efficacy and reduces toxicity and side effects on normal tissues.22 It should be pointed out that noble metal (Au, Ag, Pt, etc.) nanomaterials, especially gold nanoparticles (AuNPs), have been adopted in many areas in the light of its versatile and straightforward design strategy, unique optoelectronic

properties,

excellent

biocompatibility

and

potential

non-cytotoxicity.23-28 Nanocarriers can enter cancer cell microenvironment, and the crucial problem is controlled release of drugs from the carriers and inhibition of toxicity for normal cells simultaneously. It is worth mentioning that stimuli responsive release systems could overcome these challenges, which were divided into light, electro, magnetic, thermal and pH-responsive release systems on account of their stimuli conditions.29-34 Controlled release based on tumor makers is an alternative choice with respect to overexpressed biomolecules in cancer cells, among which glutathione (GSH) is 3



considered to be a typical one.35 It is because that GSH concentration in tumor cells is about 4 times higher than that in normal cells, which is attributed to severe anoxia in tumor tissue. Hence, with these considerations in mind, it is challenging and highly desired to prepare novel nanocarriers for target tumor therapy, including precise orientation, target drug delivery and controlled release at the same time. On the basis of our previous research36, simplification of preparation, presice controls over drug release and modification of cell internalization are under considerations. In that regard, the feasible system, CS-AuNPs-DOX, has been successfully fabricated by judicious introduction of disulfide linkages to bridge AuNPs and DOX derivatives decorated with sulfhydryl groups. Break of S-S bonds and release of DOX-SH processes are readily achievable upon the interaction of CS-AuNPs-DOX with GSH. This system is fluorescence-silence, which is ascribed to Fluorescence Resonance Energy Transfer (FRET) process from red emissive DOX to AuNPs, providing a background-free detection for GSH in vitro with a limit of detection as low as 5×10-7 M. Overexpressed intracellular GSH level accounts for selective fluorescence imaging for cancer cells, and the improved apoptosis efficiency results from the enhanced uptake by utilization of AuNPs comparing with free DOX. Further study in vivo offers visual orientation and significant therapy for tumor. This as-prepared therapeutic nano-agent has achieved target drug delivery and a significantly enhanced therapy efficacy, exhibiting great potential in biological applications for GSH associated disease monitoring and clinical diagnostics. 4


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Scheme 1. Schematic illustration of synthesis of CS-AuNPs-DOX and its response to GSH (A). Schematic diagram of multifunctional CS-AuNPs-DOX for cell imaging (B).

EXPERIMENTAL SECTION Chemicals

and

Materials.

Hydrogen

tetrachloroaurate

(III)

tetrahydrate

(HAuCl4·3H2O) and 2-iminothiolane were purchased from Aladdin reagent (Shanghai) co., Ltd. Doxorubicin hydrochloride (DOX·HCl), N-succinimidyl 3-(2-pyridyldithio) propionate

(SPDP),

glutathione

(GSH),

dimethyl

sulfoxide

(DMSO),

methoxy-PEG-thiol (mPEG-SH), and cysteamine hydrochloride (2-amino-ethanethiol, CS) were purchased from Shanghai Sangon Biotechnology. Cervix carcinoma (HeLa cells), breast cancer cells (MCF-7), glioma cell (U-87), Human Umbilical Vein 5



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Endothelial Cells (HUVEC) and tongue squamous cancer cells (CAL-27) were obtained from FDCC (Shanghai, China). RPMI 1640 cell culture medium and fetal bovine serum were purchased from Invitrogen (Gibco). The Annexin V-FITC apoptosis detection kit and Spectra/Por dialysis membrane with an MW cutoff of 10 kDa were purchased from Shanghai Sangon Biotechnology. Fluorescence

measurements

were

performed

on

a

Hitachi

F-4600

spectrofluorimeter (Tokyo, Japan) with a scan rate of 1200 nm/min. UV−vis spectra were recorded with a UH5300 spectrometer. Cellular imaging analysis laser scanning confocal microscopy was performed using Leica SP8 Point Scanning Confocal (Heidelberg, Germany). Cell apoptosis analysis was performed by using BECKMAN flow cytometric (Miami, America). The mean particle size and the zeta potential were determined by using Malvern Zetasizer Nano ZS (Malvern, United Kingdom). Transmission electron microscopy (TEM) images were carried out with JEOL 2100F transmission electron microscope (Electronics Corporation, Japan). Functional groups of CS-AuNPs were detected by Fourier transform infrared spectroscopy (FT-IR) (Verona, Madison, WI, USA). Synthesis of CS-AuNPs. CS-AuNPs were prepared according to the previously reported protocols.37 Briefly, 100 µL of 0.21 M cysteamine (CS) hydrochloride and 10 mL of 1.40 mM HAuCl4·3H2O were mixed in a 100 mL glass vial. The mixture was stirred for 20 min at room temperature in the dark. Then, 200 µL of freshly prepared NaBH4 solution (1 mM) was added slowly into the above aqueous solution under vigorous stirring, and the mixture was further stirred for 12 h. The resulted wine-red 6


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solution was filtered by dialysis in a MWCO 10 kDa chamber overnight to remove excessive cysteamine hydrochloride and HAuCl4. Preparation of Thiolated Doxorubicin. Thiolated doxorubicin (DOX-SH) was created by interaction of DOX (0.75 mg, 1.30×10-6 mol) with 2-iminothiolane (1.00 mg, 7.30×10-6 mol) for 2h in PBS (pH = 6.0).38 Preparations of CS-AuNPs-DOX Complexes. Firstly, CS-AuNPs (8.40×10-8 M, 1 mL) reacted with mPEG-SH (8×10-4 M, 1 µL) and SPDP (3.2×10-2 M, 10 µL) in PBS (pH = 6.0) under slow stir for 4 h at room temperature, followed by dialysis in a MWCO 10 kDa chamber overnight. Then, DOX-SH was incubated with CS-AuNPs-SPDP to

obtain

the

crude

product

of

CS-AuNPs-SPDP-DOX

(CS-AuNPs-DOX for short). The solution was purified by dialysis for 24 h and stored in a refrigerator at 4 °C. CS-AuNPs-DOX for GSH detection. For detection of GSH, GSH of different concentrations were applied to CS-AuNPs-DOX complexes respectively for 4 h at 25 o

C. The reaction solutions were diluted to 1.00 mL by pH 6.0 PBS buffer (10 mM)

solution. Then, the final production was put into the 2 mL calibrated tubes to centrifuge at 5590 g for 15 min and the fluorescence spectra of their supernatants were detected. Confocal Fluorescence Imaging of Intracellular Drug Release. Firstly, HeLa cells were seeded into Petri dishes and cultured for 24 h in RPMI 1640 medium. Then the cells were rinsed with PBS solution for three times. 600 µL of CS-AuNPs-DOX storage solution was added into the Petridish and cultured with HeLa cells at 37 °C. 7



After 2 h, the cells were rinsed with PBS buffer before imaging. The cells were examined by confocal laser scanning microscopy with 488 nm excitation, and the fluorescence from 570 to 600 nm was collected. Apoptosis Analysis by Flow Cytometry. Firstly, HeLa cells were incubated with PBS, free DOX, CS-AuNPs, CS-AuNPs-SPDP and CS-AuNPs-SPDP-DOX complexes respectively at 37 °C. After 2 h, cells were rinsed with ice-cold PBS solution, harvested through trypsin (without EDTA), and centrifuged at 559 g for 2 min. After being rinsed with PBS solution for three times, the cells were dispersed in 100 µL binding buffer, followed by the addition of 5 µL annexin V-FITC and 5 µL PI. After incubation for 15 min, the cells were centrifuged at 559 g for 2 min. Finally, HeLa cells were diluted to 1.0 mL by binding buffer and analyzed by flow cytometry within 4 h. Antitumor Effect Assay In Vivo. Female Balb/c nude mice weighing 18–22 g, obtained from Jiangsu Keygen Biotech Co., Ltd., were housed under aseptic conditions in a small animal isolator. HeLa cells were harvested and resuspended in 40 µL PBS. Then, these HeLa cells (1×106 cells/well) were subcutaneously injected into the back of each female Balb/c mouse. The mice were randomly grouped (n = 2) when the tumor nodules grew to a volume of 100 mm3, and treated with 100 µL PBS and CS-AuNPs-DOX complexes respectively after 3 weeks of post implantation. They were treated with a 488 nm laser for 1, 2, 3, 4, 6 and 8 h. Body weight and tumor sizes were measured every day in 4 weeks.

8


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RESULTS AND DISCUSSION Characterization of CS-AuNPs and CS-AuNPs-DOX. The multifunctional CS-AuNPs-DOX nanoprobes were accomplished mainly by three steps, and CS and SPDP were utilized for further modification with anti-cancer drug, DOX. Firstly, CS-AuNPs were synthesized by reducing HAuCl4 with NaBH4, and CS was capped onto the surfaces of AuNPs through strong Au–S bonds. The FT-IR spectrum (Figure 1C) was applied to characterize CS-AuNPs, that stretching of N−H bond was located at 3400-3500 cm−1 and the C−H bond at 2850-3000 cm−1 could be assigned to the ethyl group of thioethylamine. These results confirmed the CS-AuNPs structure as we envisaged. According to the transmission electron microscopy (TEM) image (Figure 1A), the size distribution of CS-AuNPs was uniformand the particle size was approximately 13 nm, which was consistent with its hydrate particle diameter obtained by dynamic light scattering (DLS, Figure S1A). Bathochromic-shift of absorption band from 520 nm (curve a, Figure S1D) to 525 nm (curve b, Figure S1D) and a sharp stretch of C=O bond at 1702 cm-1 in FT-IR spectrum (Figure 1C) were observed upon the introduction of SPDP, indicating that CS-AuNPs-SPDP was synthesized successfully. When DOX was covalently bound to CS-AuNPs-SPDP, the absorption of CS-AuNPs-DOX (curve c, Figure S1D) showed a characteristic DOX peak at 480 nm, confirming that CS-AuNPs-DOX has been successfully prepared. Moreover, the thickness of the capping layer increased upon the functionalization by SPDP and DOX (Figure 1B), and the hydrate diameters of CS-AuNPs-SPDP and CS-AuNPs-DOX were measured to be 15 nm and 18 nm 9



respectively (Figure S1B and S1C). In addition, the surface charge of CS-AuNPs, CS-AuNPs-SPDP and CS-AuNPs-DOX were determined by zeta potential measurements, as shown in Figure 1D. As CS-AuNPs were modified with SPDP, the zeta potential decreased gradually from +31.8 mV to +12.5 mV because of the introduction of negative-charged SPDP, and further modification by positive-charged DOX contributed to the increase of zeta potential to +29 mV. These results also supported that DOX-SH was bound with CS-AuNPs-SPDP by disulfide bonds. The positive-charged CS-AuNPs-DOX probe suggested its further usage in live cells.

Figure 1. TEM images of CS-AuNPs before (A) and after functionalization with DOX (B).

FT-IR spectra analysis of CS-AuNPs, CS-AuNPs-SPDP and CS-AuNPs-DOX (C). Zeta potential of CS-AuNPs, CS-AuNPs-SPDP and CS-AuNPs-DOX (D).

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In Vitro Release Study in the Presence of GSH. The sensing conditions in vitro were evaluated in terms of pH, temperature and reaction time. Absorption of CS-AuNPs remained stable within pH range of 4.5-6.5 which centered at 520 nm. The CS-AuNPs aggregated when pH was raised to 7.0, leading to broadened absorption peak with bathochromic shift to 590 nm (Figure S2A). Stability of CS-AuNPs-DOX was verified from time/temperature-dependent fluorescence spectra (Figure S2C and S2D). Upon the introduction of GSH, the probe CS-AuNPs-DOX produced a significant fluorescence response during the whole incubation time of 6 h, and the maximum fluorescence intensity appeared after incubation with GSH for 4 h (Figure S2C). The quenching fluorescence afterward was ascribed to the self-aggregation of CS-AuNPs-DOX. The probe remained active within the temperature range of 10-40 oC, and showed better reactivity at 25°C (Figure S2D). On the other hand, the probe was nonfluorescent under the experimental conditions, providing a background-free condition for the detection. DOX-SH exhibited red emission with a maximum fluorescence at 595 nm (red curve), which was completely quenched upon its bonding with CS-AuNPs-SPDP (black curve), as shown in Figure 2A. The quenching procedure was rationally attributed to Fluorescence Resonance Energy Transfer (FRET) from DOX to AuNPs, referring to the well overlap of AuNPs absorption and DOX-SH emission in Figure 2B. The red fluorescence from DOX-SH was effectively restored as the addition of GSH, because the disulfide bonds bridging DOX and AuNPs could be cleaved and DOX-SH was released subsequently. Figure 2C showed the fluorescence spectral 11



response of CS-AuNPs-DOX to different concentrations of GSH, which featured remarkable enhancement of fluorescence intensity. Additionally, there was a good linearity within the concentrations of GSH of 5.0×10-7-1.0×10-4 M (R2 = 0.9947), offering a limit of detection as low as 5×10-7 M.

Figure 2. Fluorescence spectra of DOX-SH (red curve) and CS-AuNPs-DOX in the presence (blue curve) or absence (black curve) of GSH. Inset: photographs of the corresponding solutions under 365 nm UV lamp (from left to right: DOX-SH, CS-AuNPs-DOX and CS-AuNPs-DOX+GSH) (A). Absorption of CS-AuNPs (black curve) and fluorescence of DOX-SH (red curve) under excitation of 488 nm (B). Fluorescence spectra of CS-AuNPs-DOX with different concentrations of GSH (from bottom to top: 0, 1×10-8, 5×10-8, 1×10-7, 5×10-7, 1×10-6, 5×10-6, 1×10-5, 5×10-5, 1×10-4, 5×10-4 and 1×10-3 M). Inset: plots of FR/FR0 at 595 nm as a function of GSH concentration (C). Linearity curve corresponding to FR/FR0 at 595 nm versus concentration of GSH increasing from 5.0×10-7 to 1.0×10-4 M (D).

In order to further confirm that the fluorescence recovery of DOX arose from the break of S-S bonds by GSH rather than other factors, a control probe AuNPs-DOX (without S-S bonds) was synthesized. The fluorescence changes of AuNPs-DOX with increasing concentration of GSH were measured under the same conditions as 12


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CS-AuNPs-DOX, and the experimental results are shown in Figure S4. As can be clearly seen from the Figure, the additional GSH made no difference to the fluorescence of AuNPs-DOX, indicating that GSH of low concentration (＜10-3 M) cannot replace the DOX-SH at the AuNPs surface. These results show that the fluorescence enhancement of CS-AuNPs-DOX caused by GSH is due to the cleavage of the S-S bonds and the subsequent release of red emissive DOX-SH. Selectivity of the CS-AuNPs-DOX System Furthermore, to assess the specificity of CS-AuNPs-DOX probe for detection of GSH, influence of potential interferences involving metal ions (Mg2+, Mn2+, etc.), amino acids (Lys, etc.), saccharides (glucose, etc.) and other biomolecules (Lyso and ATP) were studied under the same conditions. As shown in Figure S5B, fluorescence of CS-AuNPs-DOX stayed constant in the presence of the aforementioned interferences, indicating that it possessed superior selective response to GSH over other potential interferences (Figure S5B). The detection outputs could be retained for at least 2 days with little loss in intensity, and the slight decrease in 9 days originated from self-assembly of AuNPs, as illustrated in Figure S5A. The ultra-sensitive and selective probe was thus expected to be applied for intracellular detection and imaging. Real-Time Study of Cell Imaging and DOX-SH Release in Living Cells. Confocal laser scanning microscopic (CLSM) was utilized to monitor the cell imaging and drug release procedure of CS-AuNPs-DOX in live cells. For comparison, HeLa cells were treated with PBS solution, CS-AuNPs, CS-AuNPs-SPDP and 13



CS-AuNPs-DOX probe for 2 h respectively. As shown in Figure S6, cells cultured with PBS, CS-AuNPs or CS-AuNPs-SPDP displayed no signals, whereas cells incubated with CS-AuNPs-DOX responded remarkably in red channel, suggesting that CS-AuNPs-DOX could enter the cells and release DOX-SH as indicators. Response in red channel was attributed to intrinsic fluorescence of DOX-SH, which demonstrated visual response of CS-AuNPs-DOX to GSH in live cells and its potential application for cancer therapy. To further monitor the time-dependent drug release process of CS-AuNPs-DOX, CLSM images of HeLa cells were acquired at different incubation time intervals (Figure 3). Signals in red channel appeared after HeLa cells were cultured with CS-AuNPs-DOX for 2 h, indicative of the entrance of CS-AuNPs-DOX and the release of DOX-SH triggered by intracellular GSH (Scheme 1). The fluorescence intensity was enhanced gradually during the further observation in 6 h, resulting from the progressive emission activation of DOX-SH. Meanwhile, the cells at prolonged incubation time (Figure 3, 4-6 h) shrank obviously, some of which even burst. The morphological changes were related to cell apoptosis caused by the anti-cancer drug, DOX-SH, which was released continuously in tumor cells. These results verified that the membrane-permeable probe CS-AuNPs-DOX could serve as a promising candidate for both real-time detection of intracellular GSH and target cell apoptosis considering its intrinsic pro-apoptotic.

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Figure 3. Real-time fluorescence imaging of HeLa cells treated with CS-AuNPs-DOX of 600 µL. The scale bar is 20 µm.

Efficiency of CS-AuNPs-DOX with Multifold Cancer Cells General applicability of CS-AuNPs-DOX was investigated in the other tumor cells. HeLa, MCF-7, CAL-27 and U-87 cells were treated with CS-AuNPs-DOX for 2 h, and CLSM was employed to study the intracellular imaging. As shown in Figure 4, bright red emission was also available in the observed window, suggestive of the cellular internalization and intracellular distribution of CS-AuNPs-DOX. Additionally, DOX-SH in cancer cells was concentrated mainly in the nuclear, which was extremely brighter than the cytoplasm regions. This clear distribution was assigned to DOX 15



property of nuclear targeting. For comparison, normal human cells, HUVEC, were also introduced, and negligible signal could be captured. The well-performed discrimination of normal cells from tumor ones by cell imaging suggested the feasibility of CS-AuNPs-DOX for selective activation in cancer cells with over expressed GSH.

Figure4. Confocal microscopy images of HeLa, MCF-7, CAL-27, U-87 and HUVEC cells (from top to bottom) incubated with CS-AuNPs-DOX for 2 h. From left to right: images represent bright-field, DOX and merge channel fluorescence imaging.

Flow Cytometric Analysis of Apoptosis To evaluate the apoptosis feasibility of the well-designed probe, flow cytometry detection was therefore carried out by Annex in V-FITC/PI double staining assay. 16


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HeLa cells were pretreated with CS-AuNPs-DOX for 1, 2, 6, 12, 24 and 48 h respectively, and notable apoptosis of HeLa cells in 2 h was triggered with apoptotic rate of 19.68%, as displayed in Figure 5. Extent of cell apoptosis caused by the as-prepared CS-AuNPs-DOX gained a steady promotion from 10.94% to 37.75% along with the increasing incubation time in 24 h. After HeLa cells were incubated with the probe for 48 h, 53.20% of cells were in the apoptotic stage, which was relatively high according to Figure 5F.

Figure5. Cell apoptosis analysis. Annexin V-FITC/PI double staining flow cytometric analysis of HeLa cells (A-F) incubated with the CS-AuNPs-DOX system for 1, 2, 6, 12, 24 and 48 h.

Cytotoxicity Assay of Different Nanoparticles To confirm the pro-apoptosis capability of different component of the probe, the cytotoxicity assays of CS-AuNPs, CS-AuNPs-SPDP and DOX against HeLa cells were studied, as shown in Figure 6. The existence of apoptosis cells after cultured with CS-AuNPs and CS-AuNPs-SPDP were determined to be around 10%, which was close to the controlled experiment treated with PBS solution. The relatively low 17



apoptosis

ratio

was

rationalized

by

unavoidable

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mechanical

damage

or

gene-controlled normal apoptosis during the operation. HeLa cells with the treatment of DOX showed higher apoptosis rate of 25.99%, because DOX has been used extensively as an anti-cancer drug.39 CS-AuNPs-DOX probe (equivalent to 4.88×10-6 M free DOX according to the intensity of fluorescence) performed better in the pro-apoptosis procedure, as evidenced by an obvious enhancement in apoptosis rate upto 47.5%. These results suggested that CS-AuNPs-DOX showed stronger toxicity for cancer cells compared with free DOX. Meanwhile, there is a gradual enhancement of the PE-A fluorescence in HeLa cell supon incubation time (Figure S8), corresponding to the increasing release of DOX-SH from the probe. It is noteworthy that the nano-carrier, CS-AuNPs-SPDP, was practically non-toxic according to cell viability of above 90% during the treatment for 48 h (Figure S7). This result further proved that cell apoptosis was induced by DOX-SH upon the stimulation by intracellular GSH of high level. Thus, the in vitro assays claimed that CS-AuNPs exerted great influence on target drug delivery and cell apoptosis by improving the cell uptake efficiency.21

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Figure6. Analysis of cell apoptosis for HeLa cells treated with PBS solution (A), CS-AuNPs (B), CS-AuNPs-SPDP (C), DOX (D) and CS-AuNPs-DOX (E) for 2 h. The cytotoxicity assay histogram of different complexes (F).

Antitumor Effect Assay in Vivo Under an overall consideration of future therapy and drug discovery, the tumor inhibition capacity of the CS-AuNPs-modified drug in vivo was also evaluated. PBS and CS-AuNPs-DOX solutions (the same as mentioned previously) were administered by a single intratumoral injection into mice bearing subcutaneously implanted tumors. Hence, target drug delivery was thereby evaluated by tracing fluorescence images of the cancer sites in 8 h. Differentiation of tumor part from surrounding normal tissue was observed in 1 h through fluorescence images with clear boundaries, which was contributed from the released DOX-SH, as illustrated in Figure 7A. The fast localization proved that CS-AuNPs-DOX was active enough for monitoring target tumor. The fluorescent region of the cancer site increased rapidly and reached a maximum at 2 h post injection (p.i.), which was maintained for another1 h p.i. In the further 5 h p.i., fluorescence of the cancer site reduced gradually, arising from the 19



process of metabolism along with blood circulation (Figure 7). The fluorescence represented the level of released DOX-SH, and the long lasting fluorescence indicated a continuous treatment of the drug. Additionally, the tumor size and body weight after treatment with PBS solution and the CS-AuNPs-DOX system were monitored synchronously (Figure S9). In contrast to the saline control, the mouse treated with CS-AuNPs-DOX system hardly showed any weight loss (Figure S9A) under consideration of its pronounced tumoricidal effect, suggestive of its biocompatibility for organism with little toxicity. At the same time, the therapeutic effect was evaluated by recording the tumor size at regular intervals for 30 days (Figure S9B), and the tumor size of mouse treated with CS-AuNPs-DOX system was smaller comparing with the saline control (Figure 7B). These in vivo results verified our initial expectation for optimized drug therapy by inhibiting tumor growth with little toxicity to the body.

Figure7. Fluorescence images of HeLa tumor-bearing mice exposed to the laser (488 nm) after injection of the CS-AuNPs-DOX system in vivo (A). Images of tumors removed from the back of mice after injection of PBS solution and CS-AuNPs-DOX system at 30 days (B).

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CONCLUSIONS In conclusion, a versatile fluorescence lighting-on probe for cell fluorescence imaging and target drug delivery has been successfully developed based on an intriguing FRET-dependent quenching mechanism between red-emissive DOX and AuNPs, succeeding in a background free detection pattern. Covalent disulfide linkage for bridging DOX and AuNPs could be cleaved by GSH, releasing red emissive DOX-SH as an indicator of GSH level. Moreover, the probe possesses relatively high selectivity and sensitivity for GSH, providing a limit of detection as low as 5×10-7 M. This well-designed system govern improved cell internalization efficiency compared with free DOX, and were generally applicable for signaling cancer cells with intracellular GSH of high level by visual luminescence change. Notably, a significant achievement in pro-apoptotic procedure in contrast with original DOX has been made, as evidenced by overall studies of morphology observation and flow cytometric analysis. Extensive investigations of this system in vivo further convinced its good performance in view of fast localization, efficiency for inhibiting tumor cell growth and little side-effect during the treatment. We envisage that this AuNPs probe puts forward a feasible strategy for drug modification with convenient preparation and good biocompatibility, which is rather meaningful for exploiting apoptosis-targeted drug and cancer precision chemotherapy.

ASSOCIATED CONTENT Supporting Information 21



DLS date and absorption spectra of CS-AuNPs, CS-AuNPs-SPDP and CS-AuNPs-DOX; condition optimization of the nanoprobe for GSH sensing and drug release; excitation and emission spectra of DOX; stability of CS-AuNPs-DOX before and after treated with GSH; selectivity of CS-AuNPs-DOX for GSH sensing; feasibility of cell imaging and DOX release; cytotoxicity of CS-AuNPs-SPDP against HUVEC cells; flow cytometry measurements in HeLa cells upon incubated with CS-AuNPs-DOX system; the weight changes of tumor-bearing mice treated without or with CS-AuNPs-DOX system.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (P. Zhang) [email protected] (C. F. Ding) ORCID Peng Zhang, 0000-0002-4495-6771; Cai-feng Ding, 0000-0002-5281-0158. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Natural Science Foundation of China (21375070, 21422504), the Fund Project for Shangdong Key R&D Program (2017GGX20121), and the support from Key Laboratory of 22


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Spectrochemical Analysis & Instrumentation (Xiamen University), Ministry of Education (SCAI1702, SCAI1703).

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Multifunctional Gold Nanoparticles-Based Fluorescence Resonance

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