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TAT-modified gold nanoparticle carrier with enhanced anticancer activity and size effect on overcoming multidrug resistance Rui-Hui Wang, Jie Bai, Jun Deng, Chen-Jie Fang, and Xiaoyuan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15200 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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ACS Applied Materials & Interfaces

TAT-Modified Gold Nanoparticle Carrier with Enhanced Anticancer Activity and Size Effect on Overcoming Multidrug Resistance Rui-Hui Wang,† Jie Bai,† Jun Deng,§ Chen-Jie Fang,*,† and Xiaoyuan Chen*,‡

†

School of Pharmaceutical Sciences, Capital Medical University, Beijing, 100069, China.

‡

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical

Imaging and Bioengineering, National Institutes of Health, Bethesda, 20892, USA. §

Core Facilities Center, Capital Medical University, Beijing, 100069, China.

KEYWORDS：：AuNPs, TAT, anthracene derivatives, anti-proliferation, size effect on MDR

ABSTRACT: Highly efficient targeted delivery is crucial for successful anticancer chemotherapy. In this study, we developed a drug delivery system ANS–TAT–AuNP that loads anticancer molecule 2-(9-anthracenylmethylene)-hydrazinecarbothioamide (ANS) via conjugation with cell-penetrating peptide TAT modified AuNPs. The in vitro study showed that the IC50 value of ANS–TAT–AuNPs3.8nm reduced by 11.28- (24 h) and 12.64-fold (48 h) after incubation with liver hepatocellular carcinoma HepG2 cells compared to that of free ANS, suggesting that TAT modified AuNPs could enhance the anti-proliferative activity of ANS. Also, ANS–TAT–AuNPs showed a size effect on overcoming multidrug resistance (MDR). The potential of ANS–TAT–AuNPs in overcoming MDR was assessed with MCF-7/ADR drug-resistant cell line, the drug resistance index (DRI) of which was extremely high (> 190). The DRI of ANS–TAT–AuNPs22.1nm decreased dramatically to 1.48 (24 h) and 1 ACS Paragon Plus Environment


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2.20 (48 h), while that of ANS–TAT–AuNPs3.8nm decreased to 7.64 (24 h) and 7.77 (48 h), indicating that ANS–TAT–AuNPs22.1nm could treat extremely resistant MCF-7/ADR cancer cells as drug sensitive ones. The data suggest that the larger AuNPs had more profound effect on overcoming MDR, which could effectively prevent drug efflux due to their size being much larger than that of p-glycoprotein channel (9∼25 Å).

1. INTRODUCTION

In cancer chemotherapy, both the limited therapeutic efficacy of chemotherapeutic agents and cell resistance to multiple anticancer drugs (multidrug resistance, MDR) are serious clinical problems. Especially, MDR accounts for approximately 90% of chemotherapy failure in patients with metastatic cancer.1-3 The most well documented mechanism of MDR involves drug efflux which is mediated by ATP-binding cassette (ABC) transporters, typically p-glycoprotein (P-gp).4-6 The anticancer drug efflux is remarkably increased in the MDR cancer cells that overexpress P-gp, leaving the MDR cells unhurt and giving rise to a revitalization. Therefore, overcoming the increased drug efflux is a great challenge for cancer chemotherapy.

The current strategies of overcoming MDR include using an inhibitor of drug efflux pump to circumvent the efflux by P-gp and using targeted drug delivery system (DDS),7,8 and the latter is highly promising for successful cancer chemotherapy, due to efficient cellular delivery to cancer cells and thus reduced adverse effects of chemotherapeutic agents.9,10 As a potential alternative to virus and lipid, nanoparticles such as gold, silica, carbon, and iron 2 ACS Paragon Plus Environment

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oxide have stirred much interest, as these nanoparticles are relatively non-toxic and promise for controlled delivery. However, cellular transfer efficiency with these unmodified nanoparticles is relatively low, which stirred new effort to improve the delivery through surface modification and functionalization. In recent years, various natural and artificial peptides capable of targeting cancer cells have been discovered, of which short cationic and/or amphipathic cell-penetrating peptides (CPPs) received great attention due to their intrinsic ability to enter cells and mediate uptake of various cargos such as drugs, plasmid DNA (pDNA), and small interfering RNA (siRNA).11-13 In our present work, a peptide CYRGRKKRRQRRR containing cell-penetrating domain RKKRRQRRR is anchored onto gold nanoparticles (AuNPs) via the SH group of cysteine, as RKKRRQRRR is a basic domain of trans-activator of transcription (TAT),14,15 in which the arginine-rich motif derived from the protein HIV-116 functions as protein transduction domain, penetrating cell membranes in a manner of adsorptive endocytosis.17 AuNPs with a number of virtues such as ease of synthesis and size control, chemical stability, unique optical properties including well-characterized surface plasmon resonance (SPR), and excellent biocompatibility,18-20

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make them great candidates for biomedical applications such as biosensing,21 bioimaging,22 photothermal

therapy,23,24

drug

delivery25,26

and

in

vitro

diagnostics

(IVD).27

2-(9-Anthracenylmethylene)-hydrazinecarbothioamide (ANS)28 which showed anticancer activity in vitro was selected and connected directly to TAT modified AuNPs for the purpose of improving the anti-proliferative activity of ANS against both sensitive and resistant cancer cells (Scheme 1).

2. MATERIALS AND METHODS

All chemicals and reagents were of analytical grade or of the highest purity available and were used without further purification. Ultrapure water was used throughout the experiments. HAuCl4·3H2O (≥49.0% Au basis), NaBH4 (98%), and sodium citrate were purchased from Sigma-Aldrich (USA). CYRGRKKRRQRRR (95%) was purchased from GL Biochem Ltd. (Shanghai, China).

2.1 Preparation of ANS–TAT–AuNPs

ANS was synthesized according to our previous work.29,30 AuNPs of two different sizes (3.8 and 22.1 nm) were prepared with a modified typical procedure using sodium borohydride (NaBH4) and sodium citrate as reducing agents. For AuNPs3.8nm,31 freshly prepared ice-cold NaBH4 solution (0.1 M, 6 mL) was added into a mixture of HAuCl4 (0.25 mM) and sodium citrate (0.25 mM) in water (200 mL), and then the solution was stirred at room temperature for 2 h. For AuNPs22.1nm,32 an aqueous solution of HAuCl4 (0.23 mM, 192 mL) was brought to reflux while stirring, and then 10 mL of 1% trisodium citrate solution was added quickly, 4 ACS Paragon Plus Environment

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the solution was refluxed for 2 h, and then cooled to room temperature. For ANS-AuNPs3.8nm, 15 mL of freshly prepared ANS (0.2 mg/mL in DMSO) was added dropwise to 197 mL of AuNP solution and stirred for 2 h. For ANS–TAT–AuNPs3.8nm, 1 mL of TAT (1 mM in H2O) was add to 197 mL AuNPs solution and stirred for 1 min. Into this solution, 15 mL of freshly prepared ANS solution (0.2 mg/mL in DMSO) was added dropwise and stirred for another 2 h. To maintain the same molar ratio of TAT to AuNPs33 in the fabrication of ANS–TAT– AuNPs22.1nm, 43.95 µL of TAT (0.1 mM in H2O) was add to 202 mL AuNPs solution and stirred for 1 min, then 10 mL of ANS solution (0.05 mg/mL in DMSO) followed by another 5 mL of ANS solution (0.2 mg/mL in DMSO) were added dropwise and stirred for 2 h. Afterwards, the samples were sealed and stored in the dark for overnight without disturbance, followed by centrifugation and washing. Then, the sediment was re-dispersed in the mixture of DMSO and water.

The transmission electron microscopy was carried out with JEM-2100 (JEOL, Japan). The concentration of Au was measured with ICP-OES (Varian, USA), and the concentration of ANS was determined with UV-Vis standard curve method at 390nm (UV-2600 spectrophotometer, Shimadzu, Japan).

2.2 Cell culture

Hepatocellular carcinoma HepG2 cells were incubated in DMEM medium (Gibco, USA), sensitive breast cancer cells MCF-7 and drug resistant cancer cells MCF-7/ADR in RPMI 1640 medium (Hyclone, USA), supplemented with 10% (v/v) fetal bovine serum (ExCell 5 ACS Paragon Plus Environment


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Biology, Inc. Shanghai, China), 1% antibiotics (Solarbio, Beijing, China) and cultured at 37 °C in an atmosphere of 5% CO2.

2.3 Cellular uptake

The cellular uptake was assessed with confocal laser scanning microscopy (CLSM)34 and flow cytometry (FCM) (Ex: 405 nm, Em: 500 nm). For CLSM, HepG2 cells were seeded in 35 mm glass bottom dishes (4 × 104 cells/well) for 24 h. Then, 2 mL/well of free ANS, ANS– AuNPs, and ANS–TAT–AuNPs (with equivalent ANS concentration = 14.0 µM) were replaced on the confocal dishes. After 24 h incubation, the cells were washed with PBS solution, and digital monochromatic images were acquired using Leica confocal microscope (TCSSP8, Leica Application Suite X Software). The 3D intensity images were acquired with Image J software.35 For FCM, HepG2 cells (2 × 105 cells/well) were seeded in 6-well plates and grown for 24 h. The cells were then treated with 2 mL of free ANS, ANS–AuNPs, and ANS–TAT–AuNPs (with equivalent ANS concentration of 14.0 µM) for 6, 12, and 24 h. Thereafter, the cells were washed and trypsinized, and then the cellular uptake was analysed using BD LSR Fortessa Flow cytometer (BD FACS Diva Software).

2.4 Evaluation of cell viability

The effect of AuNPs, TAT–AuNPs, ANS–AuNPs, ANS–TAT–AuNPs, and DOX on cell viability was assessed using standard 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) analysis. The cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After 6, 24, and 48 h incubation with different drug, the medium was discarded 6 ACS Paragon Plus Environment

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and the cells were washed twice with PBS, and then 100 µL fresh medium and 20 µL 0.5 mg/mL MTT solution were added. After 4 h incubation, MTT solution was discarded and 100 µL DMSO was added per well. The optical density was measured at 570 nm on PerkinElmer EnSpire 2300 Multilabel Plate Reader.

2.5 Raman microscopy and SERS spectroscopy

Raman spectra and Raman mapping images were recorded at LabRAM HR Evolution spectrometer (Horiba, USA) using 632.8 nm He-Ne laser source, and the system was calibrated with a silicon semiconductor using the Raman peak at 520 cm−1. LabSpec 6-Horiba Scientific software was used to control the system and to acquire all data.

For experiments, MCF-7 and MCF-7/ADR cells were seeded in 6-well plates containing a coverslip at a density of 4 × 104 cells/well for 24 h, then 13.1 µM ANS–TAT–AuNPs22.1nm was replaced. After 9 h incubation, the cell culture medium was removed and adhered cells were washed with PBS buffer, then fixed with 4% paraformaldehyde solution for 20 min at room temperature and washed with PBS buffer after fixation. The coverslip containing the fixed cells was removed from 6-well plates after fixation/washing and mounted to a standard glass microscope slide for data collection.

3. RESULTS AND DISCUSSION

3.1 Characterization of AuNPs and conjugation of ANS to AuNPs



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Figure 1. Transmission electron microscopic (TEM) image and size distribution of ANS– TAT–AuNPs3.8nm (A and B) and ANS–TAT–AuNPs22.1nm (C and D).

Mono-dispersed colloidal AuNPs3.8nm and AuNPs22.1nm were fabricated with the reduction of Au(III) by sodium borohydride and sodium citrate, respectively. The average sizes of AuNPs obtained were 3.8 ± 0.7 and 22.1 ± 3.6 nm by counting randomly 200 particles on the TEM images (Figure S1). ANS–AuNPs (Figure S2) and ANS–TAT–AuNPs (Figure 1) with different sizes were further fabricated with 3.8 and 22.1 nm AuNPs (thereafter the subscript digital represents the size of the AuNP used). The average sizes of ANS–TAT–AuNPs obtained were 3.9 ± 0.7 and 22.5 ± 1.9 nm by counting randomly 200 particles on the TEM images (Figure 1). The formation of ANS–AuNPs was monitored with UV–visible spectroscopy (Figure 2) and 1HNMR spectroscopy (Figure S3). With increased amount of ANS added to AuNPs3.8nm, the absorption of ANS at 390 nm increased whereas a SPR band 8 ACS Paragon Plus Environment

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Figure 2. The spectra profile of AuNPs3.8nm with addition of ANS (0.2 mg/mL) (A) and the spectra profile of TAT modified AuNPs3.8nm with addition of ANS (0.2 mg/mL) (B).

Figure 3. Raman spectra of solid ANS and solutions of ANS, ANS–AuNPs3.8nm, and ANS–AuNPs22.1nm excited by 632.8 nm laser.

of AuNPs at 512 nm red-shifted. The intensity of the SPR decreased and peak broadened, indicating the interactions between ANS and AuNPs. Moreover, Raman spectra of ANS and ANS–AuNPs were recorded to characterize the chelating model between ANS and AuNPs (Figure 3). For both ANS–AuNPs3.8nm and ANS–AuNPs22.1nm, the band at 1250 cm−1 9 ACS Paragon Plus Environment


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appeared in the Raman spectra, which is assigned to S–H bending vibration (δSH).36-38 Also, the bands at 1074 cm−1 (νC=N, ρNH2) and 1347 cm−1 (ρNH2, νC=N) appeared.38-41 These data indicate that ANS chelates with AuNP through S atom of S–H and N atom of C=N in an enol form (Scheme 1).

3.2 Biological assays

Figure 4. HepG2 cell viability after 24 h incubation with AuNPs3.8nm and AuNPs22.1nm at different concentrations.

To study the delivery efficiency of parental AuNPs and TAT-modified AuNPs, the standard MTT assay was carried out to assess the cellular viability of HepG2 cells. Free ANS and clinically used doxorubicin (DOX) were set as positive controls. The toxicity of AuNPs3.8nm and AuNPs22.1nm was first evaluated with MTT assay (Figure 4). The cellular viability of HepG2 cells is around 90% after 24 h incubation at up to 100 µg/mL, suggesting a low toxicity of AuNPs. Free ANS showed moderate potency to inhibit cell growth, which is 10 ACS Paragon Plus Environment

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Figure 5. In vitro cellular inhibition rate after incubation of HepG2 cells with DOX, ANS, AuNPs, TAT–AuNPs, ANS–AuNPs, and ANS–TAT–AuNPs for 6 h (A), 24 h (B), and 48 h (C). The horizontal axis indicates equivalent DOX/ANS concentration. The size of AuNPs is 3.8 ± 0.7 nm.

concentration- and incubation time-dependent (Figure 5). The highest inhibition rate is not more than 50% even at the highest concentration tested and 48 h incubation time. In contrast, the anticancer activity is significantly enhanced after ANS conjugation to AuNPs and TAT– AuNPs. It is noted that the anti-proliferative activity is even higher than DOX after 6 h incubation, with the inhibition rate of ANS–TAT–AuNPs3.8nm reaching 20 to 40% whereas that of DOX is around 20%. The data imply an efficient delivery at an initial stage due to quick intracellular delivery mediated by TAT.42,43 Like ANS, both ANS–AuNPs3.8nm and ANS–TAT–AuNPs3.8nm were capable to inhibit the cellular growth in a concentration- and time-dependent fashion after prolonged incubation. The inhibition rate increases over time and reaches almost 100% as the concentration and time increase, which is comparable to DOX. The data suggest a high efficiency of the conjugation of ANS with AuNPs3.8nm and TAT–AuNPs3.8nm for the purpose of improving the anticancer activity of ANS. The IC50 values (the concentration at 50% cellular inhibition) of DOX, ANS, ANS–AuNPs3.8nm, and 11 ACS Paragon Plus Environment


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ANS–TAT–AuNPs3.8nm are reflective of this result (Table 1). The IC50 values of DOX against HepG2 cells are 3.42 ± 0.75 µM (24 h) and 0.57 ± 0.18 µM (48 h), while those of ANS are 77.02 ± 1.69 µM (24 h) and 39.18 ± 2.21 µM (48 h). After conjugation with AuNPs, the IC50 values of ANS contained in ANS–AuNPs3.8nm and ANS–TAT–AuNPs3.8nm are 23.98 ± 1.98 µM (24 h), 9.17 ± 2.28 µM (48 h) and 6.83 ± 1.28 µM (24 h), 3.10 ± 0.52 µM (48 h), respectively. In comparison with free ANS, IC50 values of ANS contained in ANS– AuNPs3.8nm and ANS–TAT–AuNPs3.8nm were reduced by 3.21- (24 h) and 4.27-fold (48 h), 11.28- (24 h) and 12.64-fold (48 h), respectively. It suggests a significant enhancement of anticancer activity of ANS through conjugation with AuNPs and TAT–AuNPs.

Table 1. The IC50 values of DOX, ANS, ANS–AuNPs3.8nm and ANS–TAT–AuNPs3.8nm after incubation with HepG2 cells for 24 h and 48 h. IC50 (µM) Time DOX

ANS

ANS–AuNPs3.8nm

ANS–TAT–AuNPs3.8nm

24 h

3.42 ± 0.75

77.02 ± 1.69

23.98 ± 1.98

6.83 ± 1.28

48 h

0.57 ± 0.18

39.18 ± 2.21

9.17 ± 2.28

3.10 ± 0.52

LCSM was used to trace the cellular uptake of ANS–AuNPs3.8nm and ANS–TAT– AuNPs3.8nm. Before the measurement, the quenching effect of AuNPs3.8nm and AuNPs22.1nm on the fluorescence of ANS was first examined44,45 (Figure S4). Due to the more severe quenching effect, the fluorescence intensity in the 22.1 nm nanoparticles is lower than that in the 3.8 nm nanoparticles, so that only the 3.8 nm nanoparticles were used in LCSM


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Figure 6. Confocal laser scanning images of 14.0 µM ANS, ANS–AuNPs3.8nm, and ANS–TAT–AuNPs3.8nm after 24 h incubation with HepG2 cells. The images from left to right correspond to ANS fluorescence (λex = 405nm, λem = 500 nm), bright field, overlay of the above images and the 3D fluorescence intensity profile of ANS.

experiments in order to acquire reliable fluorescent signals. For HepG2 cells incubated with equivalent concentration of ANS in each formulation and incubated for the same time, it is obvious that the fluorescence intensity from ANS in the ANS–TAT–AuNPs3.8nm group is much stronger than that in free ANS and ANS–AuNPs3.8nm group (Figure 6). The ANS– TAT–AuNPs3.8nm group showed the brightest fluorescence, followed by ANS–AuNPs3.8nm group. These results verify that the cellular uptake of ANS can be enhanced remarkably with assistance of the cell-penetrating peptide TAT on the AuNPs surface. It suggests that TAT modified AuNPs improve significantly the intracellular delivery of ANS, which was further 13 ACS Paragon Plus Environment


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confirmed by flow cytometry study (Figure 7). The MFI in the ANS–TAT–AuNPs3.8nm group is the highest among the control, ANS, ANS–AuNPs3.8nm, and ANS–TAT–AuNPs3.8nm groups, which is almost 5-fold stronger than that in ANS–AuNPs3.8nm group after 24 h incubation. The data suggest an effective improvement of cellular uptake after TAT modification.

Figure 7. Flow cytometric histograms of HepG2 cells incubated with free ANS, ANS– AuNPs3.8nm, and ANS–TAT–AuNPs3.8nm for 6 h (A), 12 h (B), and 24 h (C), and mean fluorescence intensity (MFI) in the cytoplasm of the HepG2 cells (D). ANS concentration: 14.0 µM.

MDR is a major barrier to the successful chemotherapy, the mechanism of which is mainly related to drug efflux from cancer cells mediated by P-gp. Many human cancers 14 ACS Paragon Plus Environment

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overexpress P-gp, which exports drugs from the cells, reduces an effective intracellular drug concentration, and thus decreases drug sensitivity. However, it is worth noting that macromolecules such as some kinds of proteins remain effective against MDR, because the protein molecules are too large to be pumped out by the P-gp. The theoretic and crystallography studies have revealed that molecular size of P-gp is ∼160 Å long and 45 × 65 Å wide, with the core consisting of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs).46-48 The residues in transmembrane segments form a funnel shaped drug-binding domain, which is narrow at the cytoplasmic side, wide at the extracellular side and 9∼25 Å in the middle (Figure 8A).48 Due to the size of the drug-binding domain, the efflux function of P-gp is therefore strictly subjected to the size of its substrates.

Figure 8. The P-gp model (PDB ID 3G60) (A) and drug resistance index (DRI) of DOX and different size of ANS–TAT–AuNPs after 24 h and 48 h (B) calculated via MTT assay over MCF-7 and MCF-7/ADR cell lines.

Inspired by this size-exclusion effect, that is, too-big-to-be-pumped-out, we fabricated large AuNPs22.1nm to deliver ANS, in comparison with the overcoming MDR efficiency of



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small AuNPs3.8nm against the MDR cells. Breast cancer is one of the most frequently diagnosed malignancy and is the leading cause of cancer-related death in females worldwide. MCF-7 is a breast cancer cell line, and MCF-7/ADR is adriamycin (ADR) resistant, derived from the parental MCF-7 with increasing concentrations of the anthracycline antibiotic ADR treatment.49 This MCF-7/ADR cell line is widely used as a stable MDR experiment model because of its high expression level of P-gp.50,51 Therefore, DOX-resistant cell line MCF-7/ADR was selected to evaluate the efficiency of ANS–TAT–AuNPs to overcome MDR. Through MTT assay, IC50 values of ANS–TAT–AuNPs of 3.8 and 22.1 nm nanoparticles against the sensitive MCF-7 breast cancer cells and DOX-resistant MCF-7/ADR cells were evaluated and thereby the DRI were calculated and listed in Table 2. The DRI value suggests a level of drug-resistance of MCF-7/ADR cell in comparison to its parental sensitive cell MCF-7.52 According to the DRI value, cancer cells can be classified into three categories: drug-sensitive one with DRI ranging from 0 to 2, moderate drug-resistant one with DRI from 2 to 10, and highly drug-resistant one with DRI higher than 10.52,53 As listed in Table 2, the DRI value in the DOX treated breast cancer cells is 190.02 for 24 h incubation and 606.67 for 48 h incubation, manifesting an extremely high resistance of the cancer cells we used in the MTT assay. For the ANS–TAT–AuNPs3.8nm group, the IC50 value of ANS against sensitive MCF-7 cells is 2.01 ± 0.26 µM for 24 h and 1.28 ± 0.09 µM for 48 h incubation, comparable to that of DOX, while that against resistant MCF-7/ADR cells is 15.35 ± 0.93 (24 h) and 9.94 ± 0.38 µM (48 h), which is 12.50- (24 h) and 12.82-fold (48 h) decrease over that of DOX. The DRI values are 7.64 and 7.77, indicating that ANS– 16 ACS Paragon Plus Environment

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TAT–AuNPs3.8nm treated the extremely resistant MCF-7/ADR as moderately drug-resistant cancer cells. Likewise, the IC50 values of ANS in ANS–TAT–AuNPs22.1nm against sensitive MCF-7 cells are 13.11 ± 1.18 (24 h) and 5.91 ± 0.72 µM (48 h), while those against resistant MCF-7/ADR cells are 19.37 ± 0.84 (24 h) and 13.00 ± 1.01 µM (48 h), which are 9.91- (24 h) and 9.80-fold (48 h) decrease compared with that of DOX. The DRI values are 1.48 and 2.20 (Figure 8B), indicating that ANS–TAT–AuNPs22.1nm treated the extremely resistant MCF-7/ADR as drug sensitive cancer cells. Therefore, it is concluded that both ANS–TAT– AuNPs3.8nm and ANS–TAT–AuNPs22.1nm show great potential to overcome MDR, with ANS– TAT–AuNPs22.1nm being more efficient in overcoming MDR. This is attributed to the size-exclusion effect of P-gp. ANS–TAT–AuNPs22.1nm is much larger than ANS–TAT– AuNPs3.8nm, thus it is unmatchable to the binding site of P-gp, whilst ANS–TAT–AuNPs3.8nm has a size that matches well the binding site of P-gp. Definitely, it is necessary to compare more different sized AuNPs to identify a critical size with the most efficiency of overcoming MDR.

Table 2. The IC50 values and DRI of different sizes of ANS–TAT–AuNPs after incubation with MCF-7 and MCF-7/ADR cells for 24 h and 48 h. Drug

IC50 (µM)

Time

DRI

MCF-7

MCF-7/ADR

24 h

1.01 ± 0.28

191.92 ± 15.70

190.02

48 h

0.21 ± 0.02

127.40 ± 9.99

606.67

24 h

2.01 ± 0.26

15.35 ± 0.93

7.64

48 h

1.28 ± 0.09

9.94 ± 0.38

7.77

24 h

13.11 ± 1.18

19.37 ± 0.84

1.48

48 h

5.91 ± 0.72

13.00 ± 1.01

2.20

DOX





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Figure 9. Incubation MCF-7 cells with 13.1 µM ANS–TAT–AuNPs22.1nm for 9h. (A) SERS map acquired by the intensity at 1250 cm−1. (B) Bright field image of MCF-7 cells. (C) The intensity distribution of 1250 cm−1 peak at different z-depths of the point in (B). (D) SERS spectra taken from different z-depths of the point in (B). The “-” sign indicates the inside of the intracellular compartment from the starting positions.

Although the cellular uptake efficiency improvement by TAT modified AuNPs3.8nm has been demonstrated in HepG2 cells with intracellular ANS fluorescence measurement (Figures 6 and 7), TAT-assisted cellular uptake was further confirmed by the surface enhanced Raman scattering (SERS) effect of ANS–AuNPs22.1nm. This is because larger AuNPs usually show a strong and reliable SERS signal.40 SERS spectra and maps from MCF-7 and MCF-7/ADR


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cells incubated with ANS–TAT–AuNPs22.1nm are shown in Figures 9 and 10, respectively. The 2D Raman maps in Figures 9A and 10A show the intensity of the characteristic ANS

Figure 10. Incubation of MCF-7/ADR cells with 13.1 µM ANS–TAT–AuNPs22.1nm for 9 h. (A) SERS map acquired by the intensity at 1250 cm−1. (B) Bright field image of MCF-7/ADR cells. (C) The intensity distribution of 1250 cm−1 peak at different z-depths of the point in (B). (D) SERS spectra taken from different z-depths of the point in (B). The “-” sign indicates the inside of the intracellular compartment from the starting positions.

peak at 1250 cm–1. After 9 h of incubation, both the SERS spectra (Figures 9C, D and 10C, D) and SERS map collected from MCF-7 and MCF-7/ADR cells incubated with ANS–TAT– AuNPs22.1nm suggest that cellular uptake has progressed, resulting in great accumulation of the nanoparticles throughout the cell interior and extensive ANS signals can be seen 19 ACS Paragon Plus Environment


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throughout the cells in the SERS maps. In addition, the intensity of signals from ANS showed a depth-dependence as screening along z-depth at the green point in Figures 9B and 10B. Combination of the Raman spectra and mapping suggests an effective cellular uptake of ANS–TAT–AuNPs22.1nm by MCF-7 and MCF-7/ADR cells.

4. CONCLUSION

The AuNPs and cell-penetrating peptide TAT modified AuNPs with sizes of 3.8 and 22.1 nm were fabricated to deliver an anticancer molecule ANS. The conjugation of ANS with AuNPs and TAT–AuNPs can highly improve drug delivery, and thus cytotoxicity of ANS against cancer cells HepG2, MCF-7, and MCF-7/ADR are remarkably enhanced. With the assistance of TAT, both ANS–TAT–AuNPs3.8nm and ANS–TAT–AuNPs22.1nm showed enhanced delivery efficiency and more effective tumor cell killing effect. Considering the size of binding site in P-gp, the larger ANS–TAT–AuNPs22.1nm is more favourable to overcome MDR, which can treat an extremely resistant cancer cell line MCF-7/ADR (DRI high than 190) as drug sensitive cancer cells. Although further efforts are necessary to reveal the mechanism of the nanoparticle size effect against MDR, the present result suggests that large nanoparticles could serve as a feasible method for MDR cancer treatment, which shed light on the nanopharmaceutical strategy to overcome MDR.

■ ASSOCIATED CONTENT

Supporting Information.


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The Supporting Information [TEM, 1H NMR, fluorescence quench effect of AuNPs, Raman, UV-Vis spectra, confocal image, and in vitro cellular inhibition results] is available free of charge on the ACS Publications website at http://pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Author *E-mail：[email protected] *E-mail：[email protected]

Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENT The authors thank the NSFC (21571133, 21171120), Natural Science Foundation of Beijing Municipality (7132020), and the Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH) for financial support. The authors also thank Dr. Yilin Lu for his nice support in the Raman spectroscopy.

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