Protease-Responsive Prodrug with Aggregation-Induced Emission

Subscriber access provided by Northern Illinois University

Article

A Protease-Responsive Prodrug with AIE Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells Yong Cheng, Fujian Huang, Xuehong Min, Pengcheng Gao, Tianchi Zhang, Xinchun Li, Bi-Feng Liu, Yuning Hong, Xiaoding Lou, and Fan Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02833 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 11, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Protease-Responsive Prodrug with AIE Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells Yong Cheng,a,b Fujian Huang,a Xuehong Min,a Pengcheng Gao,a Tianchi Zhang,a Xinchun Li,a Bifeng Liu,b Yuning Hong,c Xiaoding Lou*a,d and Fan Xia*a,b,d a

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China. b National Engineering Research Center for Nanomedicine, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China c School of Chemistry, The University of Melbourne, Parkville Victoria 3010, Australia d Shenzhen Institute of Huazhong University of Science & Technology, Shenzhen 518000, PR China ABSTRACT: Controlled drug delivery and real-time tracking of drug release in cancer cells are essential for cancer therapy. Herein, we report a protease-responsive prodrug (DOX-FCPPs-PyTPE, DFP) with aggregation-induced emission (AIE) characteristic, for controlled drug delivery and precise tracking of drug release in living cells. DFP consists of three components: AIE-active tetraphenylethene (TPE) derivative PyTPE, functionalized cell penetrating peptides (FCPPs) containing a cell penetrating peptide (CPP) and a short protease-responsive peptide (LGLAG) that can be selectively cleaved by a cancer-related enzyme matrix metalloproteinase-2 (MMP-2), and therapeutic unit (doxorubicin, DOX). Without MMP-2, this prodrug cannot go inside the cell. In the presence of MMP-2, DFP can be cleaved into two parts. One is cell penetrating peptides (CPPs) linked DOX, which can easily interact with cell membrane and then go inside the cell with the help of CPPs. Another is the PyTPE modified peptide which will selfaggregate because of the hydrophobic interaction and turn on the yellow fluorescence of PyTPE. The appearance of the yellow fluorescence indicates the release of the therapeutic unit to the cells. The selective delivery of the drug to the MMP-2 positive cells was also confirmed by using the intrinsic red fluorescence of DOX. Our result suggests a new and promising method for controlled drug delivery and real-time tracking of drug release in MMP-2 over-expression cells.

To minimize the side effect of chemotherapy, rapid delivery of anti-tumour drugs with high selectivity has attracted extensive attention.1-6 Strategies such as targeting delivery,7,8 controlled release systems,9-11 combination therapy,12,13 and transmembrane transport14-16 have been reported to improve the drug delivery. Along with the drug delivery, real-time drug release tracking is also essential. A number of imaging techniques have been applied in this aspect, such as magnetic resonance imaging (MRI),17,18 positron emission tomography (PET)19 and fluorescence imaging.20-26 Among them, fluorescence imaging has aroused great interest because of its rapid signal acquisition and high detection sensitivity and therefore has been widely used in the biochemical and immune analysis. However, many of the conventional fluorescence imaging dyes have been limited by the notorious aggregation-caused quenching (ACQ) effect. The fluorescence intensity of conventional dyes decreases with the increasing dye concentration, and is almost completely quenched in the solid state. In order to solve this problem, fluorescent molecules with the opposite aggregation-induced emission (AIE) characteristics is emerging in recent years.27-31 AIE is a novel photophysical phenomenon discovered by Tang’s group in 2001.32 Propeller-shaped AIE fluorogens are non-emissive in good solvent but become highly fluorescent upon aggregate formation.28,30 As one of the typical AIE fluorogens, tetraphenylethylene (TPE) and its derivatives have

been widely used in many fields spanning from optoelectronic devices,33 fluorescent bioprobes34 and as fluorescence imaging platform.35 The high emission efficiency and strong photobleaching resistance property make them promising bioimaging materials. Incorporating AIE fluorogen with peptide with specific sequence could enable specific targeting and functions. Liu and co-workers have synthesized a TPE derivative with a caspase-specific peptide for monitoring cell apoptosis and drug screening.36 The change of the hydrophobicity upon cleavage of the peptide enables the tuning of the TPE fluorescence. Later on, they have improved the apoptosis probe with longer wavelength emission and better live cell permeability.37-39 On the other hand, Wei and co-workers have conjugated TPE with a heparin specific binding peptide AG73 to afford a “turn on” probe for heparin detection.40 In terms of drug delivery, Liang’s group linked TPE and anti-cancer drug, doxorubicin (DOX), with a pH-responsive linker. They utilized the dual-color fluorescent property of the probe-inspired nanoprodrug to reveal drug release with spatiotemporal resolution.41 These prior studies have demonstrated the feasibility of using AIE-peptide conjugates as a cargo for drug delivery and tracking of the release process. The efficiency of the probe or prodrug to penetrate into cells and release the drug is the most critical step for disease surveillance and treatment. A system that can not only deliver the drug in a controllable and high-

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Schematic illustration shows that probe DFP for rapid drug delivery and drug release tracking in MMP-2 overexpression living cells. (a) DFP is specifically cleaved by the MMP-2. (b1 & c1) CPPs could rapid adhere and penetrate the cell membrane to translocate linked cell-impermeable cargoes into living cells. (b2 & c2) PyTPE part self-assemble with yellow fluorescence according to the AIE mechanism and slowly enter into living cells. (d) The rapid delivery and tracking process of DFP. (e) The drug kills cancer cells.

Page 2 of 8

functionalized TPE derivative (PyTPE) and the unnatural alkyne amino acid on the C-terminus of the LALAG peptide. In the absence of MMP-2, DFP is highly water-soluble mainly due to the arginine residues, and only the red fluorescence from DOX can be observed. After being cleaved by MMP-2, DFP is divided into a hydrophilic part DOXCRRRRRRRR-RPLG and a hydrophobic part LAGPraPyTPE. According to the aggregation-induced emission (AIE) characteristic, strong yellow fluorescence from PyTPE aggregation via hydrophobic interaction will appear and thus enable the tracking of the drug delivery process in living cells. On the other hand, after removing the PyTPE part, the CPPfunctionalized DOX could rapidly enter MMP-2 overexpression cells. With the aid of specific protease-responsive property, DFP could distinguish different cells with the different fluorescence intensity and exhibit higher cytotoxicity towards the MMP-2 over-expressed cells.

EXPERIMENTAL SECTION

efficient manner but also enable to achieve the tracking of this process will be highly desirable and still challenging. Herein, we synthesized an efficient cell-permeable and matrix metalloproteinase-2 (MMP-2) protease-responsive prodrug (DOX-FCPPs-PyTPE, DFP) with aggregation-induced emission (AIE) characteristic. DFP involves three segments (Scheme 1 and Scheme S1). (1) A maleimide-functionalized DOX (DOX-MHS). DOX is a broad-spectrum anti-cancer drug that has been widely used in the treatment of different types of tumours. (2) A functionalized cell penetrating peptides (FCPPs, CRRRRRRRRRPLGLAGPra). Specifically, FCPPs consists of five important domains in terms of function: (i) a cysteine residue (C), which is able to form chemical coupling with cargo through a metal free thiol–maleimide “click” reaction;42 (ii) a hydrophilic arginine-rich motif containing nine arginine residues (RRRRRRRRR), which enhances the water solubility of DFP to reduce the aggregation level of AIE fluorogens, helps DOX to penetrate the cell membrane and translocate the linked cell-impermeable cargoes into live cells.43-46 (iii) a proline residue (P), which improves the flexibility and the integrity of the probe;47 (iv) a hydrophobic and protease-responsive domain (LGLAG), which could be cleaved specifically between LG and LAG by MMP-2.48,49 MMP-2 is overexpressed in certain cells and has a profound impact on cells metabolism.50-52 LGLAG sequence which has been joint used with cell-permeable peptides,53-58 block copolymers59-63 and fluorescence probes64 for drug delivery, protein detection and biomedical imaging (v) a propargylglycine residue (Pra), which is able to form chemical coupling with cargo through a copper-catalyzed azide-alkyne “click” reaction.65 The last part of our probe is (3) an azide-functionalized TPE derivative (PyTPE). Taking advantage of the AIE effect,66 PyTPE has been utilized to control and track the release of drug in MMP-2 over-expression cells. Generally speaking, these three components were covalently linked through the Michael addition (thiol–maleimide “click” reaction) between maleimide-functionalized DOX and the cysteine residue on the N-terminus of the cell-penetrating polyarginine peptide as well as a copper catalysed click reaction between azide-

Materials and methods. Sodium ascorbate, doxorubicin, N, N-diisopropylethylamine (DIPEA), tris-(2-carboxyethyl)phosphine (TCEP), copper bromide were purchased from Aladdin. 6-Maleimidohexanoic acid N-hydroxysuccini-mide ester was purchased from Ark Pharm Inc. FCPPs was designed by ourselves and customized from GL Biochem Ltd. (Shanghai, China). p-(Acetoxymercuri) aniline was purchased from GenMed Scientifics Inc. Matrix metalloproteinase-2 (MMP-2) was purchased from R&D Systems Inc. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. Breast cancer cells (MCF-7), human lung fibroblast cells (HLF), fibrosarcoma cells (HT-1080) were purchased from Boshide (Wuhan, China). Bladder cancer cells (E-J) and cervical cancer cells (HeLa) were gained from Xiangya Central Experiment Laboratory. All other reagents were obtained from commercial sources and used without further purification. 1 H and 13C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer using chlroform-d (CDCl3-d) as solvent and tetramethylsilane (TMS) as internal reference. Splitting patterns are reported as s (single), d (doublet), t (triplet) and m (multiplet). High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF II mass spectrometer system operating in a MALDI-TOF mode. High performance liquid chromatography (HPLC) was performed by using Agilent 1000 for analytical HPLC and Wufeng 100 for semipreparative HPLC. The sample was dissolved in water solution or acetonitrile, applied on a Kromasil C18 column (10 µm, 250 × 4.6 mm) from Teknokroma, and eluted at 1 mL/min with a 55 min gradient from 20% to 95% solvent B, where solvent A is water (0.1% TFA solution) and solvent B is acetonitrile (0.1% TFA solution). All products were purified by HPLC to reach purity of 95%. UV-Vis absorption spectra were taken on an Agilent Cary 60 UV/Visible Spectrometer. All fluorescence measurements were performed on an Agilent Cary Eclipse Fluorescence Spectrophotometer. Confocal laser scanning microscopy images were obtained on a Fluoview FV1200 confocal laser scanning microscope (Olympus). MTT assay was obtained on an Infinite M200 PRO Microplate Reader (Tecan Austria). The synthesis and Characterization of DFP. For further details, please see Supporting Information (SI)


Page 3 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cell Culture. MCF-7 cells, HeLa cells and E-J cells were cultured in 1640 (GIBCO) medium with 10% fetal calf serum (FBS) and 1% penicillin streptomycin (PS, 10000 IU penicillin and 1000 µg/mL streptomycin, Multicell) in a culture flask at 37 °C in a humidified atmosphere containing 5% CO2. HLF cells were cultured in minimum essential medium (MEM). DFP is cleaved by MMP-2. The MMP-2 proenzyme needed to be activated by 4-aminophenylmercuric acetate (APMA) before enzyme-responded test usage. APMA (2.5 mM) and MMP-2 (5µg) were added into 50 mM Tris-HCl solution. The solution was incubated at 37 oC for 2 h. Then, activated MMP2 was used to incubate with the DFP at 37 °C. The PL spectra were measured in the range from 450 to 750 nm (λex= 405 nm). Incubation Living Cells with Probe or Drug. For confocal laser scanning microscopy imaging, cells were seeded into cell culture dishes at a density of 2.0 × 104 in growth medium (1640 or MEM supplemented with 10% FBS, 200 ml). After an overnight incubation, the cells were washed with phosphate-buffered saline (PBS, pH 7.2~7.4) for three times. A solution of the indicated probe or drug in PBS was then added, and the cells were incubated in a 5% CO2 atmosphere at 37 oC for further usage. The supernatant was then discarded, the cells were washed gently twice with PBS and immersed in growth medium prior to optical imaging. Confocal laser scanning microscopy (CLSM) imaging. The fluorescence signals were detected by using a Fluoview FV1200 confocal laser scanning microscope (Olympus), equipped with a 60/1.42 numerical aperture oil-immersion objective lens. A 405 nm laser was chosen for the excitation of PyTPE and the emission was collected at 495–575 nm. A 488 nm laser was chosen for the excitation of DOX and the emission was collected at 595-675 nm. Real-time confocal imaging was performed by the incubation system for microscopes (Tokai Hit, Japan). All fluorescence images were analysed with FV10-ASW V4.0 Image software (Olympus). Threedimensional map of cells were used 3D rendering by Imaris (Andor-Bitplane, Zurich) Cytotoxicity assay. The cytotoxic potential of PyTPE, DOX and DFP was assessed using the MTT assay. MCF-7 cells, HeLa cells and E-J cells were respectively treated with PyTPE (0.5 µM, 1.0 µM and 2.0 µM), DOX (0.5 µM, 1.0 µM and 2.0 µM) and DFP (0.5 µM, 1.0 µM and 2.0 µM) for 24 h in triplicate in a 96-well plate. The absorbance of MTT at 570 nm was recorded by the Infinite M200 PRO microplate reader.

temperature. The structure of DOX-FCPPs was confirmed by HRMS (Figure 1c and S6). Finally, DFP was synthesized by treating PyTPE with DOX-FCPPs in the presence of CuBr/sodium ascorbate in a DMSO/water mixture (v/v=1:1) at room temperature. From the high resolution mass spectra in positive mode (Figure 1b and S7), we observed strong peak (at 1133.6110) which was attributed to [M+3H]3+ ion (calculated: 1133.6086); strong peak (at 850.4632) which was attributed to [M+4H]4+ ion (calculated: 850.4584); strong peak (at 680.5713) which was attributed to [M+5H]5+ ion (calculated: 680.5683). All the products were purified by HPLC shown in Figure 1b and S8.

Figure 1. (a) Structure of DFP. From left to right: maleimidefunctionalized DOX; cysteine residue; hydrophilic arginine-rich motif containing nine arginine residues (RRRRRRRRR); hydrophobic domain (LGLAG); proline residue (P); propargylglycine residue (Pra); AIE-active tetraphenylethene (TPE) derivative PyTPE. (b) High performance liquid chromatography (HPLC) results and schematic illustrations of DFP, FCPPs, DOX and PyTPE. (c) High resolution mass spectra (HRMS) of DFP, DOXFCPPs and FCPPs.

RESULTS AND DISCUSSION Synthesis of DFP. We synthesized DFP probe (Figure 1a) through three main steps as described in Scheme S1. The detailed synthesis and characterization of intermediates were shown in the supporting information. Firstly, DOX was modified with 6-maleimidohexanoic acid to form DOX-MHS. The structural characterization of DOX-MHS was confirmed by 1 H-NMR, 13C-NMR spectra and HRMS (electrospray ionisation, ESI) (Figure S1-3). PyTPE was prepared according to the procedures in literatures and confirmed by HPLC and HRMS (Figure 1a and S4). The FCPPs was designed by us and synthesized by GL Biochem Ltd. (Shanghai, China). The HPLC and HRMS of FCPPs were shown in Figure 1b, 1c and S5. Then, in presence of TCEP (prevent thioalcohol from forming disulfide bond), DOX-MHS and FCPPs reacted with each other to obtain DOX-FCPPs in PBS buffer (pH=7.4) at room

Figure 2. (a) Schematic illustration of DFP that is cleaved by MMP-2. (b) UV-Vis absorption spectra of DOX, PyTPE and DFP in DMSO/water (v/v = 1:199). (c) Photoluminescence (PL) spectra of PyTPE, DFP and DOX in DMSO/water mixture (v/v = 1:199). (d) PL spectra of DFP treated with different concentrations of MMP-2 (0, 10, 20, 50, 100 nM) for 30 min. (e) PL spectra of DFP incubated with different time in 100 nM MMP-2 (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min). [PyTPE] = [DFP] = 10 µM; λex = 405 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DFP Cleaving by MMP-2 in Solution. To validate that the fluorescence signal of our probe is regulated by MMP-2 (Figure 2a), UV-vis absorption spectra were checked. As shown in Figure 2, DOX and PyTPE displayed absorption maxima at 488 and 405 nm, respectively, while DFP possesses two distinct absorption peaks at both 405 and 488 nm (Figure 2b). We then chose 405 nm as excitation wavelength for the PyTPE unit in DFP in the following experiments. The photoluminescence (PL) spectra of PyTPE had strong fluorescence signal from 500 to 650 nm in a mixture of DMSO/water (v/v = 1:199) at room temperature (Figure 2c, yellow). As 405 nm was not the optimal excitation wavelength for DOX, DOX showed only a weak fluorescence signal at 595nm (Figure 2c, red). In the absence of MMP-2, DFP is highly water-soluble and only has a weak fluorescence signal at 595 nm in solutions upon excited at 405 nm (Figure 2c, pink). When DFP was incubated with MMP-2 (10 nM in 50 mM Tris-HCl buffer) at 37 oC for 30 min, a new peak at 565 nm of the emission spectrum occurs (Figure 2d, green), indicating that some of DFP has been cleaved by MMP-2. With the increase of MMP-2 concentration (from 10 to 100 nM), the fluorescence intensity of DFP at 565 nm is gradually enhanced, suggesting the increased amount of PyTPE aggregates are formed in solution. This result indicates that we can quantify MMP-2 based on the fluorescence signal changes (Figure 2d). Subsequently, the kinetic studies were conducted by incubating MMP-2 (100 nM) with DFP (10 µM) at 37 °C, and then the fluorescence changes were recorded over time (Figure 2e). Finally, the product of DFP cleaved by MMP-2 was proved by HRMS as predicted (Figure S9). In Situ Confocal Imaging of Living Cells Incubated with DFP. As the fluorescence of DFP is corresponding to the level of MMP-2 in solution, we further investigated the potential of using this probe for live cell imaging of MMP-2 activity. Breast cancer cell (MCF-7) with overexpressed MMP-2 was

Figure 3. Confocal laser scanning microscopy (CLSM) images of MCF-7 cells incubated with PyTPE (a, 3.0 µM), DOX (b, 3.0 µM) and DFP (c, 3.0 µM) for 1 h, respectively. Channel 1= PyTPE fluorescence, Excitation wavelength: 405 nm, emission collected: 495-575 nm; Channel 2 = DOX fluorescence, Excitation wavelength: 488 nm, emission collected: 595-675 nm. The scale bar is 20 µm.

chosen as MMP-2-positive cancer cell.67,68 MCF-7 cells were incubated with different concentrations of DFP (0.5 µM, 1.0

Page 4 of 8

µM, 3.0 µM, 5.0 µM and 10.0 µM) and their fluorescence signals were monitored under confocal laser scanning microscope. The yellow channel was under excitation at 405 nm with a 495–575 nm band-pass filter while the red channel was excited at 488 nm with a 595–675 nm band-pass filter. Upon incubation with DFP for 30 min, both the yellow and red fluorescence intensity in MCF-7 cells increased gradually as the concentration increased and the fluorescence signal mainly distributed in the membrane and cytoplasm (Figure S10-S11).

Figure 4. Real-time CLSM images showing the responsive progress of MCF-7 cells treated with DFP (3.0 µM) during150 min. Insets are the magnified views of single cell chosen in CLSM imaging. Channel 1 = PyTPE fluorescence, Excitation wavelength: 405 nm, emission collected: 495-575 nm; Channel 2 = DOX fluorescence, Excitation wavelength: 488 nm, emission collected: 595-675 nm. The scale bars represent 20 µm (white) and 5 µm (blue), respectively. A 3D surface projection of Z-stack images for MCF-7 cell treated with DFP clearly showed the distribution of the red and yellow signals in cells (Figure S12). These results demonstrated that DFP can be used as a cellular MMP-2 activity probe and we thus chose 3.0 µM as optimized concentration for further evaluation. Figure 3 showed CLSM images of MCF-7 cell after incubation with PyTPE, DOX and DFP for 30 min, respectively. As can be seen from Figure 3c, the fluorescence in channel 1 and channel 2 colocalized well with the yellow and red fluorescence than the cells stained with PyTPE and DOX only, respectively (Figure S13). It is worth noting that the fluorescence signals inside the cells are from the red fluorescent DOX and the yellow fluorescent PyTPE, confirming that DFP was specifically cleaved. Real-time and long-term tracking of drug release. To explore whether DFP can be used for tracking the intracellular distribution of anti-cancer drugs, real-time imaging experiments were performed (Figure 4). MCF-7 cells were incubated with DFP (3.0 µM) at 37 °C and then in-situ monitored with CLSM to obtain real-time fluorescence images under the physiological conditions. In the beginning, both channel 1 (PyTPE) and channel 2 (DOX) remained dark, which indicated the low background of DFP in the cell culture medium. Further observation revealed the gradual increase of the fluorescence signals along with the cellular MMP-2 induced cleavage progress. In yellow fluorescence channel, nearly no fluorescence signal was observed within the first 15 min. From 15 min onward, the yellow fluorescence intensity in the PyTPE channel was gradually enhanced and reached around 95-fold on plasma membrane after MCF-7 cells were incubated with DFP for 150 min (Figure S14). In contrast to the yellow fluorescence (channel 1) which was triggered on after 15-30 min incubation, the red fluorescence signal (channel 2) could be clearly


Page 5 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Live-cell imaging of (a) MCF-7 cells; (b) HeLa cells; (c) E-J cells; (d) HLF cells after incubation with 3.0 µM DFP for 30 min and (e) the average fluorescence intensity in the same area. [DFP] = 3 µM; Channel 1 = PyTPE fluorescence, Excitation wavelength: 405 nm, emission collected: 495-575 nm; Channel 2 = DOX fluorescence, Excitation wavelength: 488 nm, emission collected: 595–675 nm. The scale bar is 20 µm.

Figure 5. Real-time confocal imaging of MCF-7 cells treated with DOX (a, 1.0 µM) and DFP (b, 1.0 µM) in 70 min. Excitation wavelength: 488 nm, emission collected: 595-675 nm. The scale bar is 10 µm. observed in a much shorter time (within 15 min) (Figure S14). This is because after cleavage, the PyTPE moiety would aggregate first before the yellow fluorescence could be observed, while the red fluorescence distributing in the cytoplasmic region could be readily discerned once DFP was cleaved by the enzyme MMP-2. Compared with DOX itself, DFP could deliver the segment of DOX into MMP-2 positive cells more rapidly. For timedependent experiments, As shown in Figure 5, MCF-7 cells were incubated with either 1.0 µM DOX or 1.0 µM DFP under the same conditions for time-dependent experiments. The cells treated with DOX started to show a few spots of red fluorescence after 10 min incubation and the signals slowly increased for about 50 min (Figure 5a). On the other hand, the red fluorescence from DFP appeared within 5 min after incubation with DFP (Figure 5b), indicating a faster delivery process of DFP than DOX only. Because of the positive charge of guanidinium groups, DPF tended to form electrostatic interaction with negatively-charged functional groups in cell membrane surface. 69 Since multiple pathways exist for CPPs to enter cells, numerous studies have now shown that the CPPs with cargoes are still able to penetrate into cells after inhibition of endocytic pathways. The aforementioned studies revealed the specific and efficient cellular penetration ability of the DFP. However, after 80 min incubation with free DOX, the red fluorescence obvious appealed in the nucleus and became stronger (Figure S15). The nucleus localization was also confirmed by the three-dimensional map of MCF-7 cells for incubation with 1.0 µM DOX in 2h (Figure S16). After examining the fluorescence responsiveness of PyTPE, DOX and DFP in vitro, we evaluated their cytotoxicity. Three different kinds of cancer

cells were incubated with PyTPE, DOX and DFP at various concentrations (0.5, 1.0, and 2.0 µm) for 24h under the standard culture conditions. The metabolic viability was investigated by the widely used MTT assays. According to the cellviability results, PyTPE shows negligible toxicity to different cell lines (Figure S17). While DOX and DFP were cytotoxic toward MCF-7, HeLa and E-J cells after 24 h treatment. Distinguish different cell lines by DFP. With the specific protease-responsive property, DFP may distinguish the relative concentration of MMP-2 in the different cell lines. MCF-7 and HeLa cells are MMP-2 over-expressed cell lines, 53, 58while the E-J cells and HLF cells are MMP-2 low-expressed ones. 52 As can be seen from Figure 6, the yellow and red fluorescence intensities related to MMP-2 over-expressed cells were much higher than that of MMP-2 low-expressed cells. The activity and concentration of MMP-2 are different between various cell lines and so as the inside and outside of cells. According to the literature, 61, 67 the concentrations of MMP-2 are expressed in the following order: MCF-7~HeLa>E-J>HLF, which is nearly the same with ours.

CONCLUSION In summary, we have synthesized an efficient cellpermeable and protease-responsive prodrug (DFP) with AIE characteristic for specific and rapid drug delivery as well as drug release tracking in living cells. As a linker, FCPPs can combine two cargoes together through different “click” reaction in mild conditions with high yield. Moreover, FCPPs plays an important role in adjusting the fluorescence of two dyes (PyTPE and DOX) by MMP-2 cleavage and accelerating drug delivery process. DFP was highly water-soluble and only had a weak fluorescence at 595 nm in solutions but exhibited two fluorescence signals at 565 nm and 595 nm when cleaved by MMP-2. It enables monitoring of MMP-2 activities in different cell lines with distinct variation. Additionally, DFP could rapid deliver drugs into cells, showing high toxicity for MMP-2 over-expression living cell. We develop a useful system to study the relationship between drugs, fluorescent molecules and proteins in the chemical and biological fields. It may



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

provide an innovative method to get further understand the rapid drug delivery process and accomplish accurate diagnosis and efficient therapy for disease.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website Experimental procedures, structural characterization data, and additional figures and schemes.

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by National Basic Research Program of China (973 Program, 2015CB932600, 2016YFF0100800, and 2013CB933000), the National Natural Science Foundation of China (21525523, 21375042, 21574048, and 21405054), the Special Fund for Strategic New Industry Development of Shenzhen, China (Grant No. JCYJ20150616144425376) and 1000 Young Talent (to F.X.). Y.H. thanks the support of McKenzie Fellowship from The University of Melbourne.

REFERENCES (1) Shewach, D. S.; Kuchta, R. D. Chem. Rev. 2009, 109, 28592861. (2) Mahato, R.; Tai, W.; Cheng, K. Adv. Drug. Deliv. Rev. 2011, 63, 659-670. (3) Jana, A.; Devi, K. S. P.; Maiti, T. K.; Singh N. D. P. J. Am. Chem. Soc. 2012, 134, 7656-7659. (4) Yuan, Y.; Kwok, R. T. K.; Zhang, R.; Tang, B. Z.; Liu, B. Chem. Commun. 2014, 50, 11465-11468. (5) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. J. Am. Chem. Soc. 2014, 136, 3579-3588. (6) Li, S.; Liu, L.; Jia, H.; Qiu, W.; Rong, L.; Cheng, H.; Zhang, X. Chem. Commun., 2014, 50, 11852-11855. (7) Rajendran, L.; Knölker, H.; Simons, K. Nat. Rev. Drug. Discov., 2010, 9, 29-42. (8) Deng, C.; Jiang, Y.; Cheng, R.; Meng, F.; Zhong, Z. Nano Today 2012, 7, 467-480. (9) Huang, C.; Yang, G.; Ha, Q.; Meng, J.; Wang, S. Adv. Mater. 2015, 27, 310-313. (10) Wong, P. T.; Choi, S. K. Chem. Rev. 2015, 115, 3388-3432. (11) Karimi, M.; Ghasemi, A.; Zangabad, P. S.; Rahighi, R.; Basri, S. M. M.; Mirshekari, H.; Amiri, M.; Pishabad, Z. S.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A. R.; Haghani, L.; Bahrami, S.; Hamblin, M. R. Chem. Soc. Rev. 2016, 45, 1457-1501. (12) Mignani, S.; Bryszewska, M.; Maculewicz, B. K.; Zablocka, M.; Majoral, J. Biomacromolecules 2015, 16, 1-27. (13) Jang, B.; Kwon, H.; Katila, P.; Lee, S. J.; Lee, H. Adv. Drug. Deliver. Rev. 2016, 98, 113-133. (14) Fonseca, S. B.; Pereira, M. P.; Kelley, S. O. Adv. Drug. Deliver. Rev. 2009, 61, 953-964. (15) Krol, S.; Macrez, R.; Docagne, F.; Defer, G.; Laurent, S.; Rahman, M.; Hajipour, M. J.; Kehoe, P. G.; Mahmoudi, M. Chem. Rev. 2013, 113, 1877-1903. (16) Buchheit, C. L.; Weigel, K. J.; Schafer, Z. T. Nat. Rev. Cancer 2014, 14, 632-641. (17) DeCharms, R. C. Nat. Rev. Neurosci. 2008, 9, 720-729. (18) Werner, E. J.; Datta, A.; Jocher, C. J.; Raymond, K. N. Angew. Chem. Int. Ed. 2008, 47, 8568-8580.

Page 6 of 8

(19) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Chem. Rev. 2008, 108, 1501-1516. (20) Pazos, E.; Vázquez, O.; Mascareñas, J. L.; Vázquez, M. E. Chem. Soc. Rev. 2009, 38, 3348-3359. (21) Deng, R.; Tang, L.; Tian, Q.; Wang, Y.; Lin, L.; Li, J. H. Angew. Chem. Inter. Ed. 2014, 53, 2389-2393. (22) Wang, Y.; Tang, L.; Li, Z.; Lin, Y.; Li, J. H. Nat. Protoc. 2014, 9, 1944-1955. (23) Kowada, T.; Maeda, H.; Kikuchi, K. Chem. Soc. Rev. 2015, 44, 4953-4972. (24) Tang, L.; Wang, Y. and Li, J. H. Chem. Soc. Rev. 2015, 44, 6954-6980. (25) He, Y.; Kang, Z.; Li, Q.; Tsang, C. H. A.; Fan, C. H.; Lee, S. Angew. Chem, Int. Ed. 2009, 48, 128-132. (26) Wang, J.; Wei, Y.; Hu, X.; Fang, Y.; Li, X.; Liu, J.; Wang, S.; Yuan, Q. J. Am. Chem. Soc. 2015, 137, 10576-10584. (27) Li, Z. Sci. Bull. 2010, 55, 2924-2925. (28) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Soc. Rev. 2011, 40, 5361. (29) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Acc. Chem. Res. 2013, 46, 2441-2453. (30) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Chem. Rev. 2015, 115, 11718-11940. (31) Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. Chem. Soc. Rev. 2015, 44, 4228-4238. (32) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Tang, B. Z.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D. Chem. Commun. 2001, 18, 1740-1741. (33) Chen, D.; Feng, X.; Gu, S.; Tong, B.; Shi, J.; Zhi, J.; Dong, Y. Sci. Bull. 2013, 58, 2728-2732. (34) Wang, Z.; Chen, S.; Lam, J. W. Y.; Qin, W.; Kwok, R. T. K.; Xie, N.; Hu, Q.; Tang, B. Z. J. Am. Chem. Soc. 2013, 135, 8238-8245. (35) Li, J.; Li, Y.; Chan, C. Y. K.; Kwok, R. T. K.; Li, H.; Zrazhevskiy, P.; Gao, X.; Sun, J. Z.; Qin, A.; Tang, B. Z. Angew. Chem. Int. Ed. 2014, 53, 13518-13522. (36) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2012, 134, 17972-17981. (37) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Sci. Rep. 2013, 3, 1150. (38) Shi, H.; Zhao, N.; Ding, D.; Liang, J.; Tang, B. Z.; Liu, B. Org. Biomol. Chem. 2013, 11, 7289-7296. (39) Liang, J.; Feng, G.; Kwok, R. T. K.; Ding, D.; Tang, B. Z.; Liu, B. Sci. China. Chem. 2016, 59, 53-61. (40) Ding, Y.; Shi, L.; Wei, H. Chem. Sci. 2015, 6, 6361-6366. (41) Xue, X.; Jin, S.; Zhang, C.; Yang, K.; Huo, S.; Chen, F.; Zou, G.; Liang, X. ACS Nano 2015, 9, 2729-2739. (42) Iha, R. K.; Wooley, K. L.; Nyström, A. M.; Burke, D. J.; Kade, M. J.; Hawker, C. J. Chem. Rev., 2009, 109, 5620-5686. (43) Sakai, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 1434814356. (44) Delehanty, J. B.; Bradburne, C. E.; Susumu, K.; Boeneman, K.; Mei, B. C.; Farrell, D.; Canosa, J. B. B.; Dawson, P. E.; Mattoussi, H.; Medintz, I. L. J. Am. Chem. Soc. 2011, 133, 10482-10489. (45) Fu, J.; Yu, C.; Li, L.; Yao, S. Q. J. Am. Chem. Soc. 2015, 137, 12153-12160. (46) Ji, T.; Ding, Y.; Zhao, Y.; Wang, J.; Qin, H.; Liu, X.; Lang, J.; Zhao, R.; Zhang, Y.; Shi, J.; Tao, N.; Qin, Z.; Nie, G. Adv. Mater. 2015, 27, 1865-1873. (47) Morris, M. C.; Depollier, J.; Heitz, F.; Divita, G. Nat. Biotechnol. 2006, 19, 1173-1176. (48) Fessler, L. I.; Duncan, K. G.; Fessler, J. H.; Salo, T.; Karl, T. J. Biol. Chem. 1984, 259, 9783-9789. (49) Sirkka, L. H.; Karl, T. J. Biol. Chem. 1988, 263, 19488-19493. (50) Brooks, P. C.; Mblad, S. S.; Sanders, L. C.; Schalscha, T. L. v.; Aimes, R. T.; Stevenson, W. G. S.; Quigley, J. P.; Cheresh, D. A. Cell 1996, 85, 683-693. (51) Nagase, Jr. H.; Woessne, J. F. J. Biol. Chem. 1999, 24, 2149121494. (52) Kessenbrock, K.; Plaks, V.; Werb, Z. Cell 2010, 141, 52-67.


Page 7 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(53) Jiang, T.; Olson, E. S.; Nguyen, Q. T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1786717872. (54) Levi, J.; Kothapalli, S. R.; Ma, T.; Hartman, K.; Yakub, B. T. K.; Gambhir, S. S. J. Am. Chem. Soc. 2010, 132, 11264-11269. (55) Olson, E. S.; Jiang, T.; Aguilera, T. A.; Nguyen, Q. T.; Ellies, L. G.; Scadeng, M.; Tsien, R. Y. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4311-4316. (56) Zhu, L.; Wang, T.; Perche, F.; Taigind, A.; Torchilin, V. P. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17047-17052. (57) Duijnhoven, S. M. J.; Robillard, M. S.; Hermann, S.; Kuhlmann, M. T.; Schäfers, M.; Nicolay, K.; Grüll, H. Mol. Pharmaceutics. 2014, 11, 1415-1423. (58) Han, K.; Wang, S.; Lei, Q.; Zhu, J.; Zhang, X. ACS Nano 2015, 9, 10268-10277. (59) Zhang, J.; Yuan, Z.; Wang, Y.; Chen, W.; Luo, G.; Cheng, S.; Zhuo, R.; Zhang, X. J. Am. Chem. Soc. 2013, 135, 5068-5073. (60) Chien, M.; Thompson, M. P.; Barback, C. V.; Ku, T.; Hall, D. J.; Gianneschi, N. C. Adv. Mater. 2013, 25, 3599-3604. (61) Qin, S.; Feng, J.; Rong, L.; Jia, H.; Chen, S.; Liu, X.; Luo, G.; Zhuo, R.; Zhang, X. Small 2014, 10, 599-608. (62) Chen, W.; Luo, G.; Lei, Q.; Jia, H.; Hong, S.; Wang, Q.; Zhuo, R.; Zhang, X. Chem. Commun. 2015, 51, 465-468. (63) Peng, Z.; Kopeček, J. J. Am. Chem. Soc. 2015, 137, 67266729. (64) Myochin, T.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. J. Am. Chem. Soc. 2012, 134, 13730-13737. (65) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem. Rev. 2013, 113, 4905-4979. (66) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Adv. Mater. 2014, 26, 5429-5479. (67) Egeblad, M.; Werb, Z. Nat. Rev. Cancer 2002, 2, 161-174. (68) Nezza, L. A. D.; Misajon, A.; Zhang, J.; Jobling, T.; Quinn, M. A.; Östör, A. G.; Nie, G.; Lopata, A.; Salamonsen, L. A. Cancer 2002, 94, 1466-1475. (69) Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug. Deliver. Rev. 2008, 60, 452-472.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only


Page 8 of 8

Protease-Responsive Prodrug with Aggregation-Induced Emission

Recommend Documents