Enhanced Peptide Delivery into Cells by Using the Synergistic Effects

Apr 17, 2015 - In addition, the fluorescence of the peptide conjugate at effective doses was well-distributed in the intracellular of different cell l...
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Enhanced Peptide Delivery into Cells by Using the Synergistic Effects of a Cell-Penetrating Peptide and a Chemical Drug to Alter Cell Permeability Jie-Lan Ma,† Hu Wang,‡ Yan-Lin Wang,† Yong-Huang Luo,*,§ and Chang-Bai Liu*,† †

The Institute of Molecular Biology and ‡Medical School, China Three Gorges University, Yichang 443002, China § Chongqing Engineering Technology Research Centre of Veterinary Drug, College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400715, China S Supporting Information *

ABSTRACT: Cell-penetrating peptides (CPPs) are short, often hydrophilic peptides that can deliver many kinds of molecules into cells and that are likely to serve as a useful tool of future biotherapeutics. However, CPPs application is limited because of insufficient transduction efficiency and unpredictable cellular localization. Here, we investigated the enhancement of 1,2-benzisothiazolin-3-one (BIT) on the uptake of a synthetic fluorescent TAT and a TAT-conjugated green fluorescent protein (GFP) or pro-apoptotic peptide KLA and evaluated its toxicity in various cell lines. Our results showed that BIT pretreatment can enhance the penetration efficiency of TAT and its fusion peptide. In addition, the fluorescence of the peptide conjugate at effective doses was well-distributed in the intracellular of different cell lines without membrane perforation or detectable cytotoxicity. The internalization of the peptides was serum-dependent and temperatureindependent. These findings imply that BIT may serve as a newly found delivery enhancer that is suitable for improving the penetration of CPPs. KEYWORDS: cell-penetrating peptides (CPPs), TAT, 1,2-benzisothiazolin-3-one (BIT), internalization, GFP

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Tat protein that is an essential viral transcription factor),4−8 penetratin,3 transportan,9 polyarginine,10 and MPG (fusion peptide of HIV-1 gp41 and SV40 large T antigen).11 CPPs have been successfully used to deliver biomolecules to target a variety of diseases at the laboratory level as well as in clinical trials.12,13 Intracellular delivery of various biologically active molecules, such as proteins and peptides,12−14 antisense DNA, peptide nucleic acids,15 plasmid DNA,16 liposomes,5,17 and iron beads,18 have been improved by CPPs. Delivery was evident with a certain degree of efficiency and very little cytotoxicity. Moreover, CPP is a safe and effective tool for permitting access of several agents to organelles in vitro with promising results.19 As the first identified and the most studied, TAT shows clear potential to deliver drugs into different cells; however, the transduction efficiency of TAT is often insufficient for therapeutic application. CPP-mediated biologically active molecules are trapped within endocytic vesicles.20 Some researchers have observed that most of the TAT labeled with fluorescence or TAT fusion proteins is in punctate vesicles, not in the cytosol or nucleus.21,22 Thus, many strategies have now

INTRODUCTION Efficient internalization of potential therapeutic agents (nonreceptor agonists or antagonists) into cells is critical for the desired therapeutic effect. However, the selective permeability of the plasma membrane, which prohibits the cellular internalization of small hydrophilic molecules and macromolecules, poses a significant challenge to the use of macromolecules (proteins, DNA, and RNA) in clinical therapy.1 Many approaches of macromolecules introduction have been developed, including viral (lentiviruses, retroviruses, etc.) and nonviral (bioballistics, liposomes, etc.) delivery vectors. Nonetheless, these strategies still have cytotoxicity, immunogenicity, and complex manipulation, which have limited the use of macromolecule entry for routine therapeutic purposes. The discovery of cell-penetrating peptides (CPPs) that have a greater ability to penetrate into the intracellular space has created new opportunities for delivery of biologically active cargos. CPPs, also known as protein transduction domains (PTDs), are a class of short peptides (less than 30 amino acids) entering into cells by penetrating cell membranes. Generally, CPPs are partly hydrophobic and/or polybasic peptides with positive charge at physiological pH.2 Since the first report of CPPs in 1994,3 a number of CPPs have been well characterized, e.g., TAT peptide (a basic peptide, residues 48−60 of HIV-1 © XXXX American Chemical Society

Received: December 16, 2014 Revised: April 13, 2015 Accepted: April 17, 2015

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Figure 1. FITC-labeled peptide uptake in Caski cells pretreated with the indicated concentrations of BIT. Caski cells were pretreated with different concentrations of BIT for 1 h, washed, and then incubated with 5 μM FITC-labeled peptide for 1 h. The fluorescence intensity in cell lysates was measured at 494/518 nm. (A) Fluorescence quantification of FITC-labeled peptide uptake in Caski cells pretreated with concentrations of BIT from 0.04 to 5.20 mM. (B) Peptide−FITC distribution in Caski cells with pretreatment or without pretreatment observed using fluorescence microscopy. (C) Prolonging trypsinization time did not reduce intracellular fluorescence intensity. After pretreatment with BIT followed by incubation with TAT−FITC, the cells were extensively trypsinized for 2 or 10 min before harvest. The fluorescence intensity from cells was measured at 494/518 nm. Data of fluorescence intensity are shown as the mean ± SD. All the experiments were done three times independently. Scale bars = 50 μm.

CPPs before clinical application. Previously, we have found that DMSO effectively promotes the uptake of TAT.27 Here, we investigate the effect of another common solvent, benzisothiazolinone (1,2-benzisothiazolin-3-one, BIT) on the penetrating efficiency of TAT and TAT−protein conjugates. As a preservative of industrial settings, BIT was used in waterbased technochemical products,28,29 such as cutting oils,

emerged that enable the escape of CPP−cargo conjugates from endosomal entrapment.23 Chemical agents, including chloroquine,24 calcium,25 and sucrose,26 have been used to overcome the above limitations. However, the enhancement of CPP penetrating efficiency by these agents is still limited. Thus, it is necessary to find agents with lower cytotoxicity to enhance the penetrating efficiency of B

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Figure 2. Fluorescence distribution and intensity in different cells pretreated with 0.65 mM BIT with different peptide concentrations. (A) Cultured HepG2 and A549 cell lines were pretreated with BIT for 1 h, washed, and then incubated with 5 μM FITC-labeled peptides for 1 h. Fluorescence distribution in different cell lines was observed using fluorescence microscopy. (B) Fluorescence intensity in the cell lysates was measured at 494/518 nm. Pretreatment with 100 μM chloroquine was used as the positive control. Data are shown as the mean ± SD of three independent experiments. (C) Quantification of fluorescence uptake into the cytosol of Caski cells pretreated with 0.65 mM BIT followed by incubation with FITC-labeled peptides at concentrations ranging from 2.5 to 10 μM. Data are shown as the mean ± SD of three independent experiments. Scale bars = 50 μm.

cleaning agents, polishes, pigments, plasticizers,29 and cosmetics.30

The results showed that BIT increased the transduction potential in a concentration-dependent manner (Figure 1B), thus indirectly proving that BIT can efficiently enhance TAT entry into cells. In the subsequent experiments, we selected 0.65 mM BIT as the most efficacious concentration. To rule out that the FITC-labeled peptides were surfacebound, we pretreated with BIT, incubated with the FITClabeled peptides, and then subjeced cells to 2 or 10 min trypsinization at 37 °C before harvest. There was no different in fluorescence intensities between 2 and 10 min trypsinization of the cells before lysis (Figure 1C). We also analyzed the effect of exposure time on fluorescence observation (Figure S1); the result show that fluorescence intensity in the 1/30 s group was

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RESULTS Uptake of TAT−FITC by Cultured Cells Is Enhanced by Pretreatment with 0.65 mM BIT. Pretreatment of Caski cells with BIT produced a concentration-dependent increase in TAT−FITC uptake (Figure 1A). Fluorescence microscopy indicated that uptake of TAT−FITC in untreated cells was very weak. Caski cells that were pretreated with BIT showed a uniform distribution of TAT−FITC throughout the cytosol and nucleus, and no fluorescence was observed in the NCO−FITC (NCO, HIV-1 Tat residues 38 to 48) negative control group. C

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Figure 3. Effect of serum presence and incubation temperature on cellular uptake of TAT−FITC. (A) Fluorescence intensity in lysates from Caski cells pretreated with BIT in serum-free and serum-containing medium was measured at 494/518 nm. Data are shown as the mean ± SD of three independent experiments. (B) Fluorescence intensity in lysates from Caski cells pretreated with BIT at 4 and 37 °C was measured at 494/518 nm. Data are shown as the mean ± SD of three independent experiments.

Figure 4. Enhancement of TAT−GFP fusion protein transduction by 0.65 mM BIT pretreatment. TAT−GFP fusion protein uptake into ECV-304 cells was observed using fluorescence microscopy. ECV-304 cells were pretreated with 0.65 mM BIT for 1 h, washed twice with fresh serum-free medium, and then incubated with 5 μM TAT−GFP fusion protein for 3 h. GFP localization in cells was observed using fluorescence microscopy. BIT pretreatment leads to significant enhancement of TAT−GFP uptake. Pretreatment with 100 μM chloroquine was used as a positive control. Scale bars = 50 μm.

permeation-enhancing effect would not remain after BIT treatment for 1 h and then waiting for various times before addition of TAT−cargo (Figure S2A). Comparsion between

weaker than that in the 1/3 s group. To show the penetration more clearly and naturally, we selected an exposure time of 1/ 30 s in all fluorescence observations. Moreover, the D

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Molecular Pharmaceutics DMSO and BIT pretreatment was also analyzed on the same systems; as shown in Figure S3, TAT penetration efficiency enhancement by BIT is similar to that of DMSO. The enhancing effect of BIT was also observed in HepG2A and 549 cells (Figures 2A and S4), and there were minor differences in TAT−FITC uptake between the cell lines. Pretreatment of the cells with 100 μM chloroquine (positive control) was used (Figure 2B). At 0.65 mM BIT, penetrating efficiency improved as the TAT concentration increased from 2.5 to 10 μM (Figure 2C). These results suggested that the penetrating efficiency of TAT is significantly enhanced by BIT pretreatment compared with chloroquine pretreatment. Although various CPPs have cytotoxicity at high concentrations, they are relatively nontoxic at low concentrations.31 To balance toxicity and efficacy, a 5 μM concentration of TAT− FITC was used for subsequent experiments. Serum, but Not Temperature, Affects TAT Internalization Efficiency. After pretreatment with BIT, Caski cells were incubated with TAT−FITC in serum-free or serum medium. The fluorescence microscopy results indicated that TAT−FITC was well-distributed throughout the Caski cells in serum-free medium, but less fluorescence was observed in the culture medium contained serum (data not shown). Fluorescence quantitation analysis indicated that the internalization of TAT−FITC was decreased in serum-containing culture media compared to that of serum-free culture media (Figure 3A). When Caski cells were treated with TAT−FITC at different temperatures, there was no difference in the distribution of fluorescence between cells incubated at 4 and 37 °C (data not shown); as is shown in Figure 3B, fluorescence quantitative analysis showed that TAT internalization was not affected by the low temperature (4 °C). Although we know that TAT internalizaiton was temperature-dependent in previous research, we still do not know whether a small molecule such as BIT pretreatment may change the pattern of TAT internalizaiton. The results revealed that the internalization of TAT was independent of the incubation temperature and energy after BIT treatment and implied that TAT−FITC is likely to penetrate into cells with a endocytosis-independent internalizaiton manner (Figure S2B). BIT Enhances Cellular Uptake of TAT−GFP Fusion Protein. To assess the penetration of TAT conjugated to macromolecules after BIT pretreatment, the subcellular localization of TAT−GFP fusion protein (Fusion protein were purified according our previous research27) with or without BIT pretreatment was investigated. As shown in Figures 4 and S5, the uptake of TAT−GFP fusion protein in 0.65 mM BIT pretreatment group were higher than in those cells that were not pretreated with BIT. Fluorescent was distributed in the nucleus as well as in the cytosol of the cells, whereas those cells that were pretreated with chloroquine showed little fluorescence in the cytosol, which is consistent with the FITClabeled TAT peptide experiments’ result (Figure 1B). TAT−KLA-Induced Cell Apoptosis Was Enhanced by BIT. To study whether TAT−cargo bioactivity was interfered with by BIT, a TAT-conjugated version of pro-apoptotic peptide KLA, TAT−KLA, was used to induce Caski cell apoptosis. Results from western blotting and TUNEL analysis showed that BIT increased TAT−KLA enty into the cells and then apoptosis induction in Caski cells (Figures 5A and S6A). We also detected the apoptotic relative protein, PARP (Figures 5B and S6B). These findings indicated that both TAT−KLA

Figure 5. Caski cell apoptosis induced by TAT−KLA fusion peptide was enhanced by BIT pretreatment. (A) Detection of TAT−KLA fusion peptide induced Caski cell apoptosis by using indirect TUNEL labeling and western blotting assay. Caski cells were incubated for 4 h with 5 μM TAT−KLA, then fixed with 4% paraformaldehyde in PBS. TUNEL staining was carried out following the protocol of Situ Cell Death Detection Kit. (B) Poly(ADP-ribose) polymerase (PARP) is a well-known substrate for caspase-3 cleavage during apoptosis, and cleaved PARP was detected by western blotting. Scale bars = 50 μm.

penetration and the biological activity to induce cell apoptosis were enhanced by BIT. BIT Enhances TAT Penetrating Efficiency without Cell Membrane Perforation or Cytotoxicity. To explore the mechanism of BIT penetration enhancement, a hemolysis assay was used, which showed that under physiological conditions BIT treatment damages the plasma membrane weakly (Figure 6A). This suggested that the enhancement of cell membrane penetration by BIT was not caused by the induction of pores on the cell membrane. Although previous research has shown that the membrane was damaged by BIT treatment, the BIT treatment should be kept for 24 h. Active transport and pore formation have been ruled out; the mechanism needs to be explored in further studies. The growth of different cells after BIT treatment was evaluated using an MTT assay following incubation of the cells with various concentrations of BIT for 2 h. Treatment of cells with a final concentration of BIT up to 1.30 mM for 2 h showed almost no repressive effect on cell growth (Figure 6B,C). The cell viability was also analyzed by PI staining (Figure 6D), which showed that the cells are still alive E

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Figure 6. Hemolysis of RBCs and cell viability analysis. (A) RBCs were incubated with BIT at various concentrations for 1 h at 37 °C. The amount of hemoglobin released from RBCs was measured at 541 nm. Low concentrations of hemoglobin were released at 0.65 mM BIT compared with those of the 0.1% Triton X-100 positive control. (B) Different cell types were pretreated with BIT at various concentrations for 2 h at 37 °C, washed three times with fresh serum-free medium, and incubated in fresh medium for 24 h. Cell viability measurement was carried out according to the MTT kit manual. (C) Caski cells were incubated with 0.65 mM BIT for 2 h at 37 °C, then cultured in fresh medium for 0, 24, 48, and 72 h after washing three times. Viability of Caski cell was assessed using the same procedure as in B. (D) Cell viability was analyzed by PI staining after BIT pretreatment for 1 h and peptide addition for 1 h (n = 3, carried out in three independent experiments; mean ± SD).

the precise mechanism by which BIT enhances CPP penetration as well as the safety considerations for using BIT still need to be investigated. CPPs have already served as useful tools in promoting the cellular internalization of biomolecules, and they have considerably broadened the spectrum of biotherapeutic applications in vitro and in vivo for several diseases.37

after BIT pretreatment. These results suggested that there is an undetectable cytotoxicity with ≤0.65 mM BIT treatment

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DISCUSSION CPPs are useful tools in a wide variety of biological applications with the potential to alleviate the problems caused by poor membrane permeability of biomolecules and to increase their therapeutic applications, and the feasibility of CPP-linked macromolecules as therapeutic agents have been tested in recent clinicial trials.32 Although some studies have already shown how to enhance the uptake of TAT into cells,24−26 the applicability of CPPs is still limited. Here we have introduced BIT, a novel agent that could increase the efficiency of TAT penetration of the cell membrane. This work indicates that BIT allows the rapid and efficient internalization of TAT and TAT conjugates by cells via a temperature-independent mechanism without detectable toxicity at an effective concentration. Endocytosis is an energy-dependent pathway that is suppressed by low temperatures;33 our data supports the hypothesis that CPPs or small molecules attached to CPPs translocate in a direct and nonendocytic manner.34,35 Although the exact mechanisms of TAT uptake remain unclear,36 the current results show that serum could have negative effects on the internalization of TAT. Hemolytic and MTT assays showed that BIT treatment did not lyse the cell membrane and had no effect on cell viability at effective concentrations, which demonstrates that BIT has the potential to enhance the penetration of TAT and TAT fusion proteins into cells. BIT can markedly improve the penetration of TAT and conjugates into cells without membrane perforation. However,

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MATERIAL AND METHODS

Chemicals, Reagents, and Cells. 1,2-Benziso-thiazolin-3one (BIT) was purchased from Fluka Corporation (Fluka, USA); Caski and Hela (cervical carcinoma cell lines), HepG2 (hepatocellular carcinoma cell line), A549 (human nonsmall cell lung cancer cell), and ECV-304 (human endothelial cell line) cells were cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA). Peptide Internalization. FITC-labeled peptides (TAT− KLA: YGRKKRRQRRRKGGGSKLAKLAKKLAKLAK, TAT: YGRKKRRQRRRK, and NCO, a nonsense peptide: KALGISYGRKK) were synthesized by SBS Genetech (Beijing, China), then purified using reversed-phase analytical HPLC to >99% purity, diluted to 500 μM in PBS, and stored at −20 °C for later use. The internalization of NCO−FITC and TAT−FITC was observed using fluorescence microscopy (Nikon, Japan) with a band-pass filter. Caski, HepG2, or A549 cells were seeded into plates (12-well, Greiner, Germany) at a density of 5 × 105 cells/well, and then cultivated to semiconfluence in RPMI-1640 medium in a humidified air atmosphere containing 5% CO2 for 24 h at 37 °C. Cells were washed twice with fresh serum-free medium and incubated with various concentrations

F

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Molecular Pharmaceutics of BIT supplemented with serum-free medium for 1 h at 37 °C in 5% CO2. Subsequently, 5 μM FITC-labeled peptide was added, and the cells were incubated for a further 1 h at 37 °C in 5% CO2. The cells were then washed twice with 0.1 M PBS, and the fluorescence was observed. The intracellular fluorescence intensity of FITC-labeled peptide under the conditions of BIT pretreatment was quantitatively analyzed by multimode spectrophotometry. Briefly, after the incubation of FITC-labeled peptide using the same procedure as above, cells were washed twice with PBS, lysed by the addition of 300 μL of 0.1 M NaOH, and incubated at room temperature for 10 min. The cell lysate was subsequently centrifuged (14 000 g for 5 min), and the fluorescence intensity of the supernatant was measured in a Multimode Microplate Reader (Tecan 2000, Mannedorf, Switzerland) at 494/518 nm. The total cellular protein concentration was determined using the Bradford protein assay (Bio-Rad, US). The fluorescence of the cellular uptake was normalized by cellular protein. To rule out the possibility that FITC-labeled peptides were surface-bound rather than inside of the cells, after the treatment of TAT, trypsinization of cells was also carried out using a 0.05% trypsin-EDTA solution (Invitrogen, US) for 10 min at 37 °C before lysis. All experiments were carried out in triplicate. Pretreatment of the cells with 100 μM chloroquine for 1 h was used as a positive control. Internalization of the TAT−GFP fusion protein was also observed following the protocol above. When the cells were 70% confluent, the culture medium was discarded and replaced with fresh serum-free medium containing the indicated concentrations of BIT, and the cells were then incubated for 1 h at 37 °C in 5% CO2. The ECV-304 cells were washed three times with fresh serum-free medium; then, TAT−GFP was added to the serum-free medium to a final concentration of 5 μM. The cells were incubated for 3 h at 37 °C in 5% CO2. TAT−GFP-treated cells were washed three times with PBS, and the fluorescence in cells was observed using fluorescence microscopy. Effect of Serum and Temperature on Cellular Uptake. Cellular uptake method also followed above procedures. To assess the effect of serum on the internalization of TAT−FITC, we carried out a revised procedure in which seeded cells were pretreated with 0.65 mM BIT in complete culture medium (with 10% FBS) for 1 h at 37 °C in 5% CO2. Next, 5 μM TAT−FITC was added, and the cells were incubated for another 1 h at 37 °C with 5% CO2. Cells were washed twice with PBS, and cellular uptake was observed under fluorescent microscopy or determined using multimode spectrophotometry at 494/518 nm. To determine whether the entry of TAT− FITC into the cells was energy-dependent in the Caski cell line, two incubation temperatures (4 and 37 °C) were tested. For 4 °C culturing, briefly, Caski cells were seeded onto a 6-well plate and cultured at 37 °C in 5% CO2 in RPMI-1640 containing 10% FBS. Then, the cells were incubated at 4 °C for 1 h, then for 1 h with 0.65 mM of BIT, followed by adding 5 μM FITClabeled peptide, and the cells were incubated for a further 1 h at 4 °C. The cells were then washed with 0.1 M PBS twice, and the fluorescence was observed under fluorescent microscopy or determined using multimode spectrophotometry at 494/518 nm. Hemolysis Assay. To rule out the possibility that BIT enhanced TAT penetration by perforating the plasma membrane, hemolysis assays were employed to determine

whether BIT could perforate the red blood cell (RBC) membrane. Whole blood was collected from BALB/c mice, RBCs separated; then, collected RBCs were diluted to 2 × 108 cells/ml in 0.1 M PBS with different BIT concentrations (from 0.32 to 2.60 mM) or 0.1% Triton X-100 (Sigma) as a positive control. The cells were then incubated for 1 h at 37 °C. The maximum release of hemoglobin was detected by incubation of the RBCs in water under the same conditions. The optical density (OD) at 414 nm was collected for hemolysis evaluation. The OD value of the cells that were untreated, treated with the indicated concentration of BIT, or treated with 0.1% Triton X100 is indicated by ODB, ODT, and ODM, respectively, and the cell toxicity was calculated by toxicity = 100 × ((ODT − ODB)/(ODM − ODB))%. Analysis of Cell Viability Using MTT Assay. HepG2, Caski, and A549 cells were seeded to a density of 4 × 104 cells per well in 96-well plates and incubated in a humidified incubator for 24 h at 37 °C in 5% CO2. Cells were washed and then incubated with different concentrations of BIT in serumfree medium. Cells were incubated for 2 h at 37 °C in 5% CO2, washed, and then incubated for 24 h with fresh medium containing serum. Cell viability was assessed using an MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-razolium bromide] (Sigma-Aldrich) assay. Briefly, 20 μL of 5.5 mg/mL MTT in serum-free media was added at a final concentration of 0.5 mg/mL. The cells were then incubated for 4 h at 37 °C in 5% CO2. The plates were centrifuged at 1000 g for 5 min before the supernatant was removed and 100 μL DMSO was added. The samples were finally incubated at 37 °C for 30 min and then measured at 550 nm. TUNEL Analysis of Cell Apoptosis Induced by TAT− KLA Fusion Peptide. Cell apoptosis induced by TAT−KLA, a proapoptotic peptide KLA fused to the C terminus of TAT, was analyzed by Indirect TUNEL (the terminal deoxynucleotidyl transferase dUTP nick end labeling) labeling assay using alkaline phosphatase labeling In Situ Cell Death Detection Kit (Roche Applied Science, Germany). Briefly, after the incubation of BIT using the same procedure as above, the Caski cells were incubated for 4 h with 5 μM TAT−KLA fusion peptide, washed twice with PBS, then fixed with 4% paraformaldehyde in PBS, and subjected to TUNEL staining, followed by visualization under a light microscope according to the instructions of the TUNEL kit. Western Blotting Assay. Caski cells were plated in 24-well plates and grown to 80% confluence, incubated with TAT− KLA (5 μM) conjugate at 37 °C for 4 h, and washed twice with PBS. The cells were collected and lysed in RIPA lysis buffer (Beyotime) on ice and then boiled with loading buffer (50 mM Tris−HCl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 0.2 mg/mL bromophenol blue) for 10 min. Protein concentration of the sample was quantitated by the Bradford protein assay; protein extracts were separated by 10% SDS-PAGE and then transferred to nitrocellulose. PARP cleavage was verified using monoclonal anti-PARP antibodies (Cell Signaling Technology, 1:1000 dilution) and secondary HRP-labeled goat antimouse antibodies (Sigma, 1:5000 dilution). β-actin was used as the internal loading control. Signals were visualized by enhanced chemiluminescence (ECL) and a UV Products Imaging system (Fuji). Flow Cytometric Analysis. Caski cells were cultured to 80% confluence in a 6-well plate using RPMI-1640 medium containing 10% FBS; then, the cells were washed twice with 0.1 G

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M PBS and incubated with 0.65 mM BIT for 1 h in serum-free culture media prior to addition of FITC-labeled peptides with a final concentration of 5 μM in a serum-free medium. After another 1 h incubation, the cells were washed twice with PBS and treated with 0.05% trypsin solution to detach adherent cells and to remove cell-surface-bound peptide. Trypsin treatment was halted by addition of 2 mL of fresh media supplemented with 10% FBS; then, cells were collected by centrifugation (1500 g, 3 min, 4 °C), washed twice, and resuspended in PBS to a final concentration of 106 cells/mL. Subsequently, each cell suspension was treated with propidium iodide (PI) (Invitrogen, US) (0.1 mg/mL) to stain dead cells and kept on ice until analysis. A total of 1000 events were recorded on a FACS Caliber flow cytometer (Beckman Coulter, Fullerton, CA, USA). Statistical Analysis. The statistical analysis was carried out on SPSS software. Student’s t test was used for the statistical analysis, and p < 0.05 was taken as the level of significance.

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ASSOCIATED CONTENT

* Supporting Information S

Additional figures showing the effect of exposure time on fluourescence observation, BIT and endocytosis inhibitor effects, effect of BIT and DMSO on fluorescence, distribution of FITC label, fluorescence quantification after BIT pretreatment, and apoptotic rates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*The Institute of Molecular Biology, China Three Gorges University, 8 Daxuelu Road, Yichang 443002, China. Tel.: +86 717 639 7179. E-mail: [email protected]. *Tel.: +86-23-13883808688. Fax: +86-23-68251048. E-mail: [email protected]. Author Contributions

J.-L.M. and H.W. contributed equally to this work. J.-L.M. and H.W. prepared experiments and wrote the main manuscript; C.-B.L., Y.-L.W., and Y.-H.L. conceived and designed the experiments. All authors reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank John R. Hood, BSc (Hons), PhD (GlaxoSmithKline, Stevenage) for critical reading of the manuscript and for the helpful suggestions. We are thankful for the financial support of the Nature Science Foundation of Hubei Province (no. 2010CDB10705) to C.-B.L. and CSTC (No. 2009CB1010) to Y.-H.L., and FSTEHU (T201203) to Y.-L.W.

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DOI: 10.1021/mp500838r Mol. Pharmaceutics XXXX, XXX, XXX−XXX