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Magainin II modified polydiacetylene micelles for cancer therapy† Danling Yang, Rongfeng Zou, Yu Zhu, Ben Liu, Defan Yao, Juanjuan Jiang, Junchen Wu* and He Tian* Polydiacetylene (PDA) micelles have been widely used to deliver anticancer drugs in the treatment of a variety of tumours and for imaging living cells. In this study, we developed an effective strategy to directly conjugate magainin II (MGN-II) to the surface of PDA micelles using a fluorescent dye. These stable and well-defined PDA micelles had high cytotoxicity in cancer cell lines, and were able to reduce the tumour size in mice. The modified PDA micelles improved the anticancer effects of MGN-II in the A549 cell line
Received 1st August 2014, Accepted 1st October 2014 DOI: 10.1039/c4nr04405c www.rsc.org/nanoscale
only at a concentration of 16.0 μg mL−1 (IC50). In addition, following irradiation with UV light at 254 nm, the PDA micelles gave rise to an energy transfer from the fluorescent dye to the backbone of PDA micelles to enhance the imaging of living cells. Our results demonstrate that modified PDA micelles can not only be used in the treatment of tumors in vitro and in vivo in a simple and directed way, but also offer a new platform for designing functional liposomes to act as anticancer agents.
Introduction In the past few decades, nanomaterials have been widely used in biomedicine, especially for specific anticancer treatments.1–9 For most cancer patients, therapeutic options are
Key Labs for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China. E-mail: [email protected], [email protected]; Fax: (+86)-21-6425-2288; Tel: (+86)-21-6425-3674 † Electronic supplementary information (ESI) available: Detailed synthetic procedures of hemicyanine dye and supplementary figures. See DOI: 10.1039/ c4nr04405c
Danling Yang
Danling Yang was born in China in 1989. She received her BSc. degree from the College of Chemical Science and Engineering from the Qingdao University in 2012, and is currently pursuing her MSc. degree under the supervision of Associate Professor Junchen Wu at the East China University of Science & Technology (ECUST). Her research focuses on the development of anti-cancer drugs and fluorescent sensors.
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mainly limited to surgery, radiation and chemotherapy.10–14 However, these methods have significant disadvantages, including low efficacy and high toxicity, resulting in damage to normal tissues and the immune system and even normal physiological functions. Thus, the development of new treatments is urgently needed as alternatives to conventional methods. It is well known that polydiacetylene (PDA) is a conjugated polymer15 and PDA liposomes have attracted increasing interest in the effort at improving the effectiveness of cancer treatments. Polymerized PDA micelles, which act as nanocarriers, have relatively good stability, can solubilize hydrophobic drug molecules and can increase drug loading and tumor uptake.16–19 PDA liposomes are currently a powerful tool and
Rongfeng Zou received his BSc. degree in Fine Chemistry from the Nanjing Forest University in 2012. He joined ECUST for his MSc. degree after graduation. His research interests are in metal-triggered self-assembly of peptide systems.
Rongfeng Zou
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have been used as biosensors and chemosensors based on their optical responses.15,20–22 Functionalizing PDA liposomes with different molecules can enhance their utility as biosensor materials through color and fluorescent changes to detect chemically and biologically interesting molecules including viruses, proteins, surfactants and enzymes.23–30 In our previous study, PDA liposomes were developed for use as a fluorescent turn-on sensor to detect bacterial polysaccharides.31 More importantly, as pioneered by Eric Doris, PDA nanomaterials also have a significant effect in cancer therapy when loaded with a hydrophobic anticancer drug and a fluorescent dye for drug delivery and in vivo imaging.16–19,32–34 PDA vesicles as an anticancer drug carrier are capable of controlled drug release and have enhanced physicochemical stability.16,35–38 For example, the amphiphilic peptides of PDA liposomes have been demonstrated to respond to external stimuli such as smart materials,30,39–41 which can adhere to cells41 and specifically bind to cancer cells.30 These studies have provided a good foundation for the application of PDA liposomes in the diagnosis of cancer and in drug delivery systems for cancer therapy. Thus, we develop PDA liposomes with a cationic antimicrobial peptide (AMP) to directly treat tumours without the need for additional vectors to facilitate uptake in cancer cells. Recently, AMPs have been extensively studied, and were shown to exhibit marked cytotoxicity against cancer cells.42,43 The AMPs with positive charges interact with negative charges on the surface of cancer cells and then disrupt the cancer cell membranes.44 It is worth noting that a known magainin II (MGN-II) has been isolated from the skin of the African clawed frog Xenopus laevis,44,45 and was reported to be selectively cytotoxic in hematopoietic and solid tumors.46,47 The ionophoric peptide has an α-helical structure which enables it to target the net negative charge on cancer cell membranes where it can form ion-permeable channels in the membrane, thus giving rise to irreversible cytolysis of cancer cells.44,47 Moreover, the main advantage of MGN-II is its selectivity for tumor cells compared to normal cells, and it is toxic to cancer cells at a concentration lower than that required to lyse peripheral blood lymphocytes, erythrocytes, and normal fibroblasts.44,47
In order to use MGN-II as a potential drug for cancer treatment, it is necessary to increase its selective toxicity against cancer cells. The poor potency of MGN-II is most likely attributed to the cell membrane binding ability. Thus, MGN-II analogues with increased positive charges and a hydrophobic nature exhibit enhanced membrane binding affinity48 and cytotoxicity.47,49 However, peptide liposomes have a high affinity for lipid bilayers, increasing their ability to disrupt the cell membrane by anchoring their hydrophobic tails into cell membranes.50 The peptide itself may not be enough to kill cancer cells due to its low binding affinity and cytotoxicity to cancer cells; thus multiple peptide aggregations may be an effective method of increasing binding affinity.41,51,52 For example, amphiphilic peptide micelles can not only increase the concentration of peptides by taking advantage of increasing positive charges and hydrophobicity, but also protect the peptides from degradation,53,54 prolonging the time in vivo and stimulating antigen-specific humoral immune responses relative to the peptides alone.55,56 In this work, we report MGN-II modified PDA micelles which exhibited excellent cancer therapeutic efficacy both in vitro and in vivo (Fig. 1). The liposomes of AMPs with 10,12tricosadiyonic acid (DA) were polymerized by UV irradiation at 254 nm leading to energy transfer from the fluorescent dye to the backbone of PDA, which effectively enhanced the fluorescence quantum yield for living cell imaging. Moreover, the PDA micelles increased the number of charges and the hydrophobic tails effectively bound to cancer cells, further increasing the local concentration of MGN-II, thereby killing multiple cancer cells at low concentrations. The polymerized micelles connected by covalent bonds were more stable when compared with monomers both in vivo and in vitro.
Yu Zhu received her BSc. degree from the College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University in 2012, and is currently pursuing her MSc. degree under the supervision of Associate Professor Junchen Wu at ECUST. Her research focuses on antibacterial peptides.
Ben Liu received his BSc. degree from the College of Chemistry and Environmental Engineering, Wuhan Polytechnic University in 2012, and is currently pursuing his MSc. degree at ECUST. His research focuses on the development of anticancer peptides.
Yu Zhu
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Experimental Materials All solvents were dried and distilled under argon before use. Anhydrous dimethylformamide (DMF) and dichloromethane
Ben Liu
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Fig. 1
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Scheme showing the structure (a) and applications (b) of MGN-II-PDA micelles.
using a Millipore filtration system. All other reagents and solvents were purchased from commercial sources and used directly without further purification.
(DCM) were distilled over CaH2 and CaCl2, respectively, and kept anhydrous with 4 Å molecular sieves. 2-Methylbenzothiazole and methyl iodide (CH3I), phosphorus oxychloride (POCl3), N-(2-cyanoethyl)-N-ethylaniline, Pd(PPh3)4 and 10,12tricosadiyonic acid (96%) were obtained from Aladdin Chemistry Co., Ltd (Shanghai, China); N,N-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIEA) and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) were purchased from Suzhou Highfine Biotech Co. Ltd, China. Fmoc-protected amino acids were purchased from GL Biochem (Shanghai) Ltd, China. Ultrapure water was obtained
MGN-II-DA was synthesized using a standard Fmoc method. The monomer was synthesized on Rink amide resin (640 μmol g−1, 250 μmol, 1.0 eq.) using a CEM Liberty one microwaveassisted peptide synthesizer. Amino acid couplings were performed after the Fmoc protected group was removed by reacting with piperidine (20%) and then washing with DMF seven
Defan Yao received his bachelor’s degree in 2012 from Chemistry and Chemical Engineering Department of Jiangsu University. Subsequently he joined the group of He Tian at ECUST for his PhD. His research focuses on fluorescent sensor and molecular recognition.
Juanjuan Jiang received her bachelor’s degree from the College of Chemical Engineering and Life Sciences from Chaohu University in 2012, and is currently pursuing her master’s degree in ECUST. Her recent research focuses on the development of antibacterial peptide.
Defan Yao
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Synthesis of MGN-II-DA
Juanjuan Jiang
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times. The subsequent amino acids were attached under microwave irradiation conditions (35 W, 55 ± 5 °C, 20 min) according to the SPPS protocol described: Fmoc-protected amino acid (0.75 mmol, 3.0 equiv.), PyBOP (0.75 mmol, 3.0 equiv.) and N-methyl-morpholine (2.0 mmol, 8.0 equiv.) in DMF (8.0 mL). At the end of the chain, coupling by Fmoc-Lys(Alloc)-OH occurred and the side chain of the lysine residue was protected by Alloc to selectively deprotect specific attachment of hemicyanine dye (for details, see ESI†).57,58 When all the amino acids were attached, the crude product was transferred into a glass peptide synthesis vessel and the Alloc group was then removed by adding Pd(PPh3)4 (0.05 mmol, 0.2 eq.) and PhSiH3 (12 mmol, 48 eq.) in DMF for 30 min under argon conditions. After the Alloc was deprotected, the product was coupled with hemicyanine dye. After the reaction was complete, followed by Fmoc deprotection, the product was reacted with 10,12-tricosadiyonic acid. After completion of peptide synthesis, the final product was cleaved from the solid support in a mixture of TFA–H2O–triisopropylsilane (95 : 2.5 : 2.5) for 3 h. The product was a dark red solid. After removing TFA, the peptide was precipitated three times using cold diethyl ether and centrifuged at 10 000 r min−1 for approximately 10 min. The crude peptide was purified by HPLC on a RP18-column using H2O–CH3CN (with 0.1% TFA) as the eluent. The solid was then dissolved in water, acidified with hydrochloric acid (0.1 N) and lyophilized. This step was repeated three times. The product was identified by MALDI-TOF using α-cyano-4hydroxycinnamic acid (CHCA) as the matrix.
either used freshly prepared or stored in the fridge at 4.0 °C until use.
Preparation of MGN-II-PDA
Atomic force microscopy (AFM)
First, MGN-II-DA was dissolved in TBS (50 mM Tris and 50 mM NaCl, pH 7.4, 25 °C) at a final concentration of 1 mM and then transferred into a quartz cuvette using UV irradiation at 254 nm (approximately 0.2 mW cm−2) for about 90 min to provide a stock solution of MGN-II-PDA. This solution was diluted to the desired concentrations and the solution was
Images were recorded by Veeco/DI atomic force microscopy. MGN-II-PDA was dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C). A drop of the 20 μM MGN-II-PDA solution was placed on freshly cleaved mica for 30 s and allowed to dry at room temperature. The sample was then analyzed in tapping mode.
Dr Wu received his Ph.D. degree from Fudan Universiy in 2009. After graduation, he went to work in the University of DuisburgEssen with the support of the Alexander von Humboldt Foundation. He became an associate professor of the Institute of Fine Chemicals at ECUST in 2011. His current research interests include antitumor drugs of polypeptide, enzyme inhibitors based on peptides, and self-assembly and recognition of biomolecules.
Prof. He Tian received his Ph.D. degree from ECUST in 1989. Then, Dr Tian worked on organic laser dyes at Siegen University, Germany and on organic photochromic copolymers at the Max Planck Institute for Polymer Research in Mainz, Germany in 2000. Both studies were supported by the Alexander von Humboldt Foundation. In 1994, Dr Tian became a full professor and director of the Institute of He Tian Fine Chemicals at ECUST. In 2011, he was selected as a member of the Chinese Academy of Science. His current research interests center on the development of interdisciplinary materials science.
Junchen Wu
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Fluorescence and UV-vis experiments Fluorescence emission spectra were recorded using a Fluoromax-4 spectrofluorometer and UV-vis absorption spectra were recorded on a Cary 100 UV-visible spectrophotometer with a slit width of 5 nm at 25 °C for both excitation and emission. The data were collected at 1 nm increments with a 0.1 s integration time. All spectra were corrected for intensity using the manufacturer-supplied correction factors and corrected for background fluorescence and absorption by subtracting the value obtained for a blank scan of the buffer system. Absolute fluorescence quantum yield was determined using an FLS-980stm spectrometer (Edinburgh Instruments Ltd, UK). All samples were dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) at a concentration of 20 μM. CD spectroscopy The CD spectra of MGN-II-DA and MGN-II-PDA were recorded on a JASCO J-815 CD spectrometer at 25 °C. The spectra were scanned in a quartz optical cell of 1 cm path length and the sample was dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) at a concentration of 20 μM. Data were collected at 1 nm increments with a 0.1 s integration period from 250 to 190 nm at each wavelength.
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Dynamic light scattering (DLS) study The average particle size was measured using a Nano-ZS (Malvern Instruments). MGN-II-PDA (20 μM) was measured in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C). The solution was prepared according to a routine method without filtration.
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Cell culture A549 lung carcinoma, KB epidermal carcinoma and MCF-7 breast adenocarcinoma cells were purchased from Shanghai Bogoo Biotech Co., Ltd, China. These cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), 50 U mL−1 penicillin and 50 μg mL−1 streptomycin (P/S, HyClone). The cell cultures were maintained at 37 °C under humidified conditions of 95% air and 5% CO2 in the culture medium. The culture medium was changed every two days to maintain exponential growth of the cells. Cells were passaged using 0.05% Trypsin/EDTA (Sigma) when they reached 80–90% confluence and seeded for the experiments. Cytotoxicity assay The cytotoxicity of MGN-II-PDA against cancer cells was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, A549 lung carcinoma, KB epidermal carcinoma and MCF-7 breast adenocarcinoma cells in complete medium were seeded into 96-well plates at a density of 10 000 cells per well and cultured overnight at 37 °C under 5% CO2 before treatment. After the complete medium was removed, the MGN-II-PDA solution (100 μL per well) at concentrations of 8, 16, 32 and 64 μg mL−1 in RPMI-1640 was added to the wells, and the cells were incubated for 24 h at 37 °C under 5% CO2. Subsequently, 10 µL of MTT solution (5 mg mL−1 in PBS) was added to the medium and the cells were incubated at 37 °C for 4 h. The resulting formazan crystals were solubilized using 100 µL of DMSO after the growth medium was removed and the quantity was determined colorimetrically using a Synergy H4 Hybrid Microplate reader (Biotek, USA) at 490 nm. Cell viability (%) was calculated using the following formula: Cell viability = (mean absorbance value of the treatment group-blank/mean absorbance value of the control-blank) × 100. Furthermore, IC50 values were obtained by plotting the signal responses (OD490) against the logarithm of analyte concentrations using Origin software (Origin 8.0). The 4-parameter logistic equation y = A2 + (A1 − A2)/[1 + (x/x0)p] was used for curve fitting in the whole concentration range, where A1 is the maximum signal at no analyte, A2 is the minimum signal at infinite concentration, p is the curve slope at the inflection point, and x0 is the IC50 (analyte concentration causing a 50% inhibition of the maximum response, a measure of immunoassay detectability).59 Confocal microscopic imaging of cells Cells were seeded in a glass-bottomed dish. After overnight culture, the cells were incubated with MGN-II-PDA in RPMI-1640 medium at a final concentration of 16 μg mL−1 for
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30 min at 37 °C. The cells were then washed three times with PBS to remove MGN-II-PDA. Then, the cells were stained with 10 μg mL−1 DAPI in RPMI-1640 medium for 15 min. Cell images were obtained with a confocal laser scanning microscope (CLSM), Nikon A1 (Japan), at an excitation wavelength of 488 nm and the emission was collected at 600–650 nm. Flow cytometry The cell uptake of MGN-II-PDA was measured by flow cytometry. A549, KB and MCF-7 cells were seeded into six-well plates (1 mL cell suspension per well) at a density of 1 × 106 cells mL−1, and allowed to attach overnight before treatment. MGN-II-PDA was dispersed in RPMI-1640 medium at a concentration of 10 μg mL−1. The cells were treated with the MGN-II-PDA solution and incubated for 30 min at 4 °C and 37 °C. Cells incubated in RPMI-1640 medium alone were set as the control. The medium was then removed and the cells were washed three times with cold PBS. The cells were harvested using 0.05% (w/v) trypsin/0.02% (w/v) EDTA, and centrifuged at 1000 rpm for 5 min. The harvested cells were then resuspended in PBS. Each cell with MGN-II-PDA was quickly analyzed on a Millipore Guava flow cytometer (Billerica, MA, USA) using a 488 nm argon laser for excitation and the emitted 628 nm fluorescence for detection. Animals and tumor implantation All animal experiments were performed in agreement with the guidelines of the Institutional Animal Care and Use Committee of East China University of Science and Technology and conformed to the guide for the care and use of laboratory animals. A549 cells were washed with PBS, and harvested using trypsin-EDTA. After centrifugation, the cell pellets were resuspended in PBS and adjusted to a concentration of 1 × 107 cells mL−1. The suspended cells in 0.2 mL PBS were implanted into the right axilla of six-week-old (approximately 20 g) male BALB/c nude mice (Shanghai Slac Laboratory Animal Co. Ltd, China). In vivo antitumor studies When the tumors reached a mean volume of 50 mm3 after inoculation of A549 cells, the mice were randomly separated into two treatment groups (n = 5 per group): (1) negative control (PBS); (2) MGN-II-PDA at 5 mg kg−1 in 0.2 mL PBS. The two groups received intravenous injections via the tail vein every 2 days for a total of 5 cycles. During therapy, the tumor size and body weight were measured every two days. Tumors were measured individually using a Vernier caliper. Tumor volume was calculated using the following formula: tumor volume = length × width2 × 0.5. The mice were sacrificed 8 days after the 5th treatment according to institutional guidelines. Tumors were resected, photographed, weighed, fixed in formalin and then embedded in paraffin. The therapeutic efficacy of the treatment was evaluated by the tumor-inhibition rate (TIR). This was calculated using the following equation: TIR = 100% × (mean tumor weight of control group − mean tumor weight of experimental group)/mean tumor weight of
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control group. One of the tumor-bearing mice was sacrificed right after 4 h injection of MGN-II-PDA (0.2 mL, 2 mg mL−1) via the tail vein, and tumor and other normal organs were isolated and subjected to Carestream In Vivo FX-PRO imaging (Carestream Health, Inc.).
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Histological examination of tumor tissues Tumors from control and treated mice were collected and fixed in neutral formalin. The samples were dehydrated and embedded in paraffin. Tumor sections 3.0 μm thick were dewaxed with xylene, hydrated consecutively with 100%, 95%, 85%, and 75% ethanol and distilled water, and stained with hematoxylin and eosin (H&E) for histological examination.
Results and discussion Synthesis and characterization of MGN-II modified PDA micelles MGN-II (sequence: SNMIEGTFAKGFKKASHLFKGIG) has potential as a novel antitumor agent. However, the IC50 value of MGN-II in A549 cells reached 110 µg mL−1.49 In order to improve the potency against anticancer cells, the multivalent strategy of MGN-II micelles was used in this study. Thus, MGN-II micelles were synthesized by aqueous self-assembly of amphiphilic monomers containing the hydrophobic C25 alkane chain (10,12-tricosadiyonic acid, DA) and a hydrophilic head group (MGN-II sequence). In addition, in order to track the distribution of MGN-II in the cell lines, the fluorophore (hemicyanine dye) was introduced and attached to the side of lysine. MGN-II-DA was synthesized on Rink amide resin by a Fmoc solid-phase method, and purified to 97.0% purity by HPLC with a yield of 3.1% after cleavage and deprotection, and identified by MALDI-TOF (Fig. S1 and S2†). It is well known that DA containing a diyne group can be polymerized via a topochemical 1,4-mechanism under UV irradiation at 254 nm. The functionalized PDA micelles can not only generate multiple MGN-II in the micelles, but also increase the local concentration of micelles in the blood. To further confirm the formation of PDA micelles, the time-dependent absorption (Fig. 2a and 2b) and fluorescent spectra (Fig. 2c and 2d) were used to monitor the polymerization process of monomers (20 µM) in TBS buffer. For self-assembly of PDA micelles, the amphiphilic MGN-II-DA solution was sonicated for approximately 30 min. The solution was then subjected to UV irradiation at 254 nm for about 90 min and the pH was adjusted to 7.4. As shown in Fig. 1a–1d, on UV irradiation of MGN-II-DA in TBS buffer at 254 nm, the absorption bands gradually decreased at approximately 500 nm during the 90 min irradiation period. In addition, the fluorescent emission at 600 nm increased as a function of UV irradiation time. In general, we can achieve a non-fluorescent ‘blue form’ of the PDA liposomes, which changes to a weakly fluorescent ‘red form’. However, in this case, irradiation of the highly fluorescent self-assembled monomer based on the fluorophore
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(hemicyanine dye) enhanced the fluorescence of the backbone of PDA. This enhancement was caused by energy transfer from the hemicyanine fluorophore (emission maximum at 600 nm) to the polymer backbone (absorption maximum at 600 nm).27,60–62 Moreover, the fluorescence quantum yield (Φ) of MGN-II-PDA increased approximately 3-fold compared to the monomer of MGN-II-DA from 1.7% to 5.2% in TBS, which strongly suggested that the energy transfer to the backbone of PDA liposomes was from the fluorescent dye (Fig. 2e). On the basis of the above results, we further evaluated whether the polymerization of MGN-II-DA impacted the secondary structure of MGN-II (α-helix) in TBS buffer after UV irradiation at 254 nm using circular dichroic (CD) spectra (Fig. 2f ). The MGN-II conjugated to DA and hemicyanine dye still maintained an α-helix-rich CD pattern with two negative Cotton effects at 218 and 228 nm, in accordance with its crystal structure extracted from the Protein Data Bank (PDB: 2MAG).63 This suggests that the secondary structure of MGN-II is partially perturbed due to the long alkane chain and fluorescent dye. Following UV irradiation at 254 nm for approximately 90 min, the CD spectra of PDA micelles changed from an α-helical-rich profile to a β-sheet-rich profile, as suggested by the emergence of a new CD band at 212 nm. This change in conformation may be attributed to the polymerized perturbation under UV irradiation at 254 nm. In addition, the structure of the polymerized micelles was further characterized by atomic force microscopy (AFM) and dynamic light scattering (DLS). The AFM image of MGN-II-PDA clearly shows these PDA micelles with a diameter of approximately 50–100 nm which is consistent with the DLS results (Fig. 3a and 3b). In addition, zeta-potential measurements showed that the surface of MGN-II-PDA was positively charged at a zeta-potential of 17.53 ± 0.57 mV. In vitro efficacy of MGN-II-PDA in cancer cell lines After obtaining the PDA micelles, we determined the MGN-II-PDA-induced membrane permeabilization and the mechanism of uptake in cancer cell lines. Thus, confocal laser scanning microscopy (CLSM) was used to monitor the cellular uptake of MGN-II-PDA micelles in A549, KB and MCF-7 cell lines. Subsequently, the three cells types were incubated in RMPI-1640 medium at 37 °C, and a co-localization experiment of MGN-II-PDA micelles with DAPI (the nuclear counterstain) was carried out. The blue and red signals in the cell images were from DAPI (Fig. 4a, 4d and 4g) and MGN-II-PDA micelles (Fig. 4b, 4e and 4h), respectively. The overlay images demonstrated that MGN-II-PDA micelles were distributed in both the cytoplasm and the membrane in A549 (Fig. 4c), KB (Fig. 4f ) and MCF-7 cells (Fig. 4i). These results showed that MGN-II-PDA was localized in the membrane and the cytoplasm. Moreover, the time-lapse images indicated that the MGN-II-PDA micelles rapidly accumulated in the cytoplasm and reached the summit for about 20 min (Fig. S3†). To further explore the membrane and cytoplasm entry pathway of MGN-II-PDA micelles, the uptake ratio of
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Fig. 2 (a) Absorption and (b) the change in absorption intensity at 500 nm of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 M NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min; (c) Fluorescence emission spectra (λex = 492 nm) and (d) the change in fluorescence intensity at 600 nm of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 M NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min; (e) and (f ) show fluorescence quantum yield and CD spectra of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for approximately 90 min. The data are presented as mean ± SD (n = 5).
MGN-II-PDA micelles was investigated in the three cell lines under different temperature conditions by flow cytometry. As shown in Fig. 5a and 5b, the uptake ratios were not significantly different at 4 °C and 37 °C in A549 cells following incubation with MGN-II-PDA for 30 min. The uptake ratio of MGN-II-PDA reached 92.9% at 4 °C and 99.1% at 37 °C. These
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results indicated that temperature did not affect the cellular uptake by A549 cells. The uptake ratio of MGN-II-PDA in MCF-7 cells increased from 71.6% at 4 °C to 91.4% at 37 °C (Fig. 5e and 5f ). However, the uptake ratio of MGN-II-PDA in KB cells increased significantly from 37.0% at 4 °C to 94.5% at 37 °C. This is not surprising as cell activity is usually inhibited
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Fig. 3 AFM image (a) of MGN-II-DA (20.0 μM) in ultrapure water and DLS (b) of MGN-II-DA (20.0 μM) in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 90 min.
Fig. 4 Confocal laser scanning microscopy (CLSM) images illustrating the intracellular distribution of MGN-II-PDA (red) and DAPI (blue) in (a–c) A549, (d–f ) KB and (g–i) MCF-7 cells. The cells were stained with (a, d, g) 10 μg mL−1 DAPI for 15 min (Channel 1: excitation: 405 nm, emission collected: 425–475 nm), (b, e, h) 16 μg mL−1 MGN-II-PDA for 30 min (Channel 2: excitation: 488 nm, emission collected: 600–650 nm), and (c, f, i) overlay of (a) and (b), (d) and (e), (g) and (h).
at low temperature. These findings suggest that energy plays a key role in MGN-II-PDA crossing the plasma membrane and eventually reaching the cytoplasm in KB cells. Interestingly, with a further increase in temperature to 37 °C, the uptake ratio of MGN-II-PDA micelles was increased in the membrane
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and cytoplasm regions. As seen from the uptake ratios of MGN-II-PDA in the cells, the function of MGN-II-PDA micelles was unaffected, further demonstrating that temperature promoted the cellular uptake of MGN-II-PDA. It is also possible that the rigid membranes were less favourable for transloca-
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Fig. 5 Cellular uptake of MGN-II-PDA by flow cytometry analysis. The cells were treated with 10 μg mL−1 MGN-II-PDA for 30 min at 4 °C and 37 °C. (a) and (b) show A549 cells treated with MGN-II-PDA at 4 °C and 37 °C; (c) and (d) show KB cells treated with MGN-II-PDA at 4 °C and 37 °C; (e) and (f ) show MCF-7 cells treated with MGN-II-PDA at 4 °C and 37 °C.
Fig. 6 Dose–response curves for MTT toxicity assays (a) for MGN-II-PDA in A549 cells, MCF-7 cells and KB cells. (b) shows the IC50 values of the three cancer cell lines. A549 cells, MCF-7 cells, and KB cells were seeded at 1 × 104 cells per well on a 96-well plate, and then treated with MGN-II-PDA at concentrations of 8, 16, 32 and 64 μg mL−1 in RPMI-1640 medium for 24 h. The data are presented as mean ± SD (n = 5).
tion. Although we cannot fully exclude internalization by endocytosis, we suggest that the uptake was due to a cell-penetrating process by MGN-II-PDA micelles in these cancer cell lines. Thus, these results demonstrate that electrostatic attractions of cationic peptides to the cell membranes44 and the endocytotic pathway are mainly responsible for cellular uptake.64–66 To investigate the cytotoxicity of MGN-II-PDA in cancer cell lines, mammalian cell lines A549, MCF-7 and KB were treated with MGN-II-PDA. The three cell lines were incubated with various concentrations of MGN-II-PDA in RMPI-1640 medium
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for 24 h at 37 °C, and the cytotoxicity of MGN-II-PDA was assessed by the MTT assay. The relative viability of cells at different concentrations of MGN-II-PDA was confirmed spectrophotometrically. The results are summarized in Fig. 6 and are represented by the mean viability ± standard deviation (SD) of three independent experiments, each of which was performed in triplicate. Fig. 6b shows that the IC50 value of MGN-II-PDA in A549, KB and MCF-7 cells was 16.0, 15.7, and 23.9 μg mL−1, respectively. Compared to the literature, the IC50 value in A549 cells found in this study was improved approximately 7-fold.49 The viability of A549, MCF-7 and KB cells was
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that the MGN-II-PDA micelles are biocompatible in vitro (Fig. S4†). In vivo antitumor activity Encouraged by these results, we determined whether MGN-II-PDA could effectively suppress tumor growth in vivo. The therapeutic efficacy of MGN-II-PDA was subsequently assessed using a murine xenograft model (Fig. 7). The mice
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reduced by more than 99% after 24 h incubation with 64 μg mL−1 MGN-II-PDA, which strongly suggested that MGN-II-PDA had effective and broad spectrum antitumor activities enabling positively charged peptides to bind to the negatively charged membrane surface to disrupt lipid membranes. To further evaluate the biocompatibility of MGN-II-PDA, cell viabilities towards the normal cell line (L929) were over 75% after a 24 h incubation with 32 μg mL−1 of MGN-II-PDA, whereas the cancer cell viabilities were less than 30%. This result indicates
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Fig. 7 MGN-II-PDA blocks tumor growth in vivo. A549 xenograft mice were treated with MGN-II-PDA at a dose of 5 mg kg−1 every two days by intravenous injection via the tail vein. The results are summarized as tumor volumes (a), body weight changes (b) and the tumor-inhibition rate (TIR) (c) of mice exposed to PBS and MGN-II-PDA. (d) Photograph of dissected tumors from the two groups of mice. (e) The fluorescence images of excised mouse organs after 4 h injection of MGN-II-PDA (0.2 mL, 2 mg mL−1) via the tail vein (f ) and (g) represent histological features of A549 tumors from the mice treated with PBS and MGN-II-PDA (scale bar is 20 μm).
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treated with MGN-II-PDA showed significant inhibition of A549 tumor growth compared to the PBS negative group (Fig. 7a and 7c). After the mice were sacrificed, the tumors were dissected and weighed, and the tumor-inhibition rate (TIR) of MGN-II-PDA in A549 tumors was calculated (Fig. 7c). In the presence of MGN-II-PDA the TIR was 54.4% ( p < 0.05). In addition, tumor images corresponded with the TIR data (Fig. 7d). The mouse body weight showed little change throughout the study (Fig. 7b) and no gross abnormalities were noted in any of the organs or tissues at the time of necropsy, indicating that MGN-II-PDA did not cause severe systemic side effects. To characterize the distribution of MGN-II-PDA in vivo, the fluorescence of tumor and other normal organs was obtained by Carestream imaging (Fig. 7e). The MGN-II-PDA was markedly accumulated in the stomach and tumor. In contrast, little or no detectable level of MGN-II-PDA could be observed in the kidney, heart, lungs, spleen, liver and brain. Additionally, the A549 xenograft tumors were fixed and prepared for histological analysis. In the control group (PBS treated), the tumor tissue sections were composed of tightly packed tumor cells interspersed with various amounts of stroma (Fig. 7f ). However, dead tumor cells were rarely observed after treatment with MGN-II-PDA (Fig. 7g). The histological features of tumors showed significant differences following treatment with the micelles, which significantly inhibited tumor growth compared to the PBS negative group.
Conclusions In summary, we have developed novel amphiphilic MGN-II-PDA liposomes that self-assemble and polymerize into micelles, act as an antitumor agent under UV irradiation at 254 nm, and have a broad spectrum of cytotoxic activity against cancer cells. Moreover, the monomer (MGN-II-DA) can be polymerized by UV irradiation at 254 nm and causes energy transfer from the fluorescent dye to the backbone of MGN-II-PDA micelles, which effectively enhanced the fluorescence quantum yield for imaging living cells. Flow cytometry demonstrated that the MGN-II-PDA internalization pathway may be a cell-penetrating process. MGN-II-PDA also showed significant in vivo antitumor activity without reducing the body weight of treated animals and with few side effects, and is thus a promising and extremely valuable approach in the design of peptide-based anticancer agents.
Conflict of interest The authors declare no competing financial interest.
Acknowledgements This work was supported by grants from the National Basic Research 973 Program (2013CB733700) and the Fundamental
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Research Funds for the Central Universities (WJ1213007), the Program of Shanghai Pujiang (K100-2-1275) and the Innovation Program of Shanghai Municipal Education Commission (J100-2-13104) for financial support.
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Magainin II modified polydiacetylene micelles for cancer therapy† Danling Yang, Rongfeng Zou, Yu Zhu, Ben Liu, Defan Yao, Juanjuan Jiang, Junchen Wu* and He Tian* Polydiacetylene (PDA) micelles have been widely used to deliver anticancer drugs in the treatment of a variety of tumours and for imaging living cells. In this study, we developed an effective strategy to directly conjugate magainin II (MGN-II) to the surface of PDA micelles using a fluorescent dye. These stable and well-defined PDA micelles had high cytotoxicity in cancer cell lines, and were able to reduce the tumour size in mice. The modified PDA micelles improved the anticancer effects of MGN-II in the A549 cell line
Received 1st August 2014, Accepted 1st October 2014 DOI: 10.1039/c4nr04405c www.rsc.org/nanoscale
only at a concentration of 16.0 μg mL−1 (IC50). In addition, following irradiation with UV light at 254 nm, the PDA micelles gave rise to an energy transfer from the fluorescent dye to the backbone of PDA micelles to enhance the imaging of living cells. Our results demonstrate that modified PDA micelles can not only be used in the treatment of tumors in vitro and in vivo in a simple and directed way, but also offer a new platform for designing functional liposomes to act as anticancer agents.
Introduction In the past few decades, nanomaterials have been widely used in biomedicine, especially for specific anticancer treatments.1–9 For most cancer patients, therapeutic options are
Key Labs for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China. E-mail: [email protected], [email protected]; Fax: (+86)-21-6425-2288; Tel: (+86)-21-6425-3674 † Electronic supplementary information (ESI) available: Detailed synthetic procedures of hemicyanine dye and supplementary figures. See DOI: 10.1039/ c4nr04405c
Danling Yang
Danling Yang was born in China in 1989. She received her BSc. degree from the College of Chemical Science and Engineering from the Qingdao University in 2012, and is currently pursuing her MSc. degree under the supervision of Associate Professor Junchen Wu at the East China University of Science & Technology (ECUST). Her research focuses on the development of anti-cancer drugs and fluorescent sensors.
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mainly limited to surgery, radiation and chemotherapy.10–14 However, these methods have significant disadvantages, including low efficacy and high toxicity, resulting in damage to normal tissues and the immune system and even normal physiological functions. Thus, the development of new treatments is urgently needed as alternatives to conventional methods. It is well known that polydiacetylene (PDA) is a conjugated polymer15 and PDA liposomes have attracted increasing interest in the effort at improving the effectiveness of cancer treatments. Polymerized PDA micelles, which act as nanocarriers, have relatively good stability, can solubilize hydrophobic drug molecules and can increase drug loading and tumor uptake.16–19 PDA liposomes are currently a powerful tool and
Rongfeng Zou received his BSc. degree in Fine Chemistry from the Nanjing Forest University in 2012. He joined ECUST for his MSc. degree after graduation. His research interests are in metal-triggered self-assembly of peptide systems.
Rongfeng Zou
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have been used as biosensors and chemosensors based on their optical responses.15,20–22 Functionalizing PDA liposomes with different molecules can enhance their utility as biosensor materials through color and fluorescent changes to detect chemically and biologically interesting molecules including viruses, proteins, surfactants and enzymes.23–30 In our previous study, PDA liposomes were developed for use as a fluorescent turn-on sensor to detect bacterial polysaccharides.31 More importantly, as pioneered by Eric Doris, PDA nanomaterials also have a significant effect in cancer therapy when loaded with a hydrophobic anticancer drug and a fluorescent dye for drug delivery and in vivo imaging.16–19,32–34 PDA vesicles as an anticancer drug carrier are capable of controlled drug release and have enhanced physicochemical stability.16,35–38 For example, the amphiphilic peptides of PDA liposomes have been demonstrated to respond to external stimuli such as smart materials,30,39–41 which can adhere to cells41 and specifically bind to cancer cells.30 These studies have provided a good foundation for the application of PDA liposomes in the diagnosis of cancer and in drug delivery systems for cancer therapy. Thus, we develop PDA liposomes with a cationic antimicrobial peptide (AMP) to directly treat tumours without the need for additional vectors to facilitate uptake in cancer cells. Recently, AMPs have been extensively studied, and were shown to exhibit marked cytotoxicity against cancer cells.42,43 The AMPs with positive charges interact with negative charges on the surface of cancer cells and then disrupt the cancer cell membranes.44 It is worth noting that a known magainin II (MGN-II) has been isolated from the skin of the African clawed frog Xenopus laevis,44,45 and was reported to be selectively cytotoxic in hematopoietic and solid tumors.46,47 The ionophoric peptide has an α-helical structure which enables it to target the net negative charge on cancer cell membranes where it can form ion-permeable channels in the membrane, thus giving rise to irreversible cytolysis of cancer cells.44,47 Moreover, the main advantage of MGN-II is its selectivity for tumor cells compared to normal cells, and it is toxic to cancer cells at a concentration lower than that required to lyse peripheral blood lymphocytes, erythrocytes, and normal fibroblasts.44,47
In order to use MGN-II as a potential drug for cancer treatment, it is necessary to increase its selective toxicity against cancer cells. The poor potency of MGN-II is most likely attributed to the cell membrane binding ability. Thus, MGN-II analogues with increased positive charges and a hydrophobic nature exhibit enhanced membrane binding affinity48 and cytotoxicity.47,49 However, peptide liposomes have a high affinity for lipid bilayers, increasing their ability to disrupt the cell membrane by anchoring their hydrophobic tails into cell membranes.50 The peptide itself may not be enough to kill cancer cells due to its low binding affinity and cytotoxicity to cancer cells; thus multiple peptide aggregations may be an effective method of increasing binding affinity.41,51,52 For example, amphiphilic peptide micelles can not only increase the concentration of peptides by taking advantage of increasing positive charges and hydrophobicity, but also protect the peptides from degradation,53,54 prolonging the time in vivo and stimulating antigen-specific humoral immune responses relative to the peptides alone.55,56 In this work, we report MGN-II modified PDA micelles which exhibited excellent cancer therapeutic efficacy both in vitro and in vivo (Fig. 1). The liposomes of AMPs with 10,12tricosadiyonic acid (DA) were polymerized by UV irradiation at 254 nm leading to energy transfer from the fluorescent dye to the backbone of PDA, which effectively enhanced the fluorescence quantum yield for living cell imaging. Moreover, the PDA micelles increased the number of charges and the hydrophobic tails effectively bound to cancer cells, further increasing the local concentration of MGN-II, thereby killing multiple cancer cells at low concentrations. The polymerized micelles connected by covalent bonds were more stable when compared with monomers both in vivo and in vitro.
Yu Zhu received her BSc. degree from the College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University in 2012, and is currently pursuing her MSc. degree under the supervision of Associate Professor Junchen Wu at ECUST. Her research focuses on antibacterial peptides.
Ben Liu received his BSc. degree from the College of Chemistry and Environmental Engineering, Wuhan Polytechnic University in 2012, and is currently pursuing his MSc. degree at ECUST. His research focuses on the development of anticancer peptides.
Yu Zhu
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Experimental Materials All solvents were dried and distilled under argon before use. Anhydrous dimethylformamide (DMF) and dichloromethane
Ben Liu
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Fig. 1
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Scheme showing the structure (a) and applications (b) of MGN-II-PDA micelles.
using a Millipore filtration system. All other reagents and solvents were purchased from commercial sources and used directly without further purification.
(DCM) were distilled over CaH2 and CaCl2, respectively, and kept anhydrous with 4 Å molecular sieves. 2-Methylbenzothiazole and methyl iodide (CH3I), phosphorus oxychloride (POCl3), N-(2-cyanoethyl)-N-ethylaniline, Pd(PPh3)4 and 10,12tricosadiyonic acid (96%) were obtained from Aladdin Chemistry Co., Ltd (Shanghai, China); N,N-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIEA) and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) were purchased from Suzhou Highfine Biotech Co. Ltd, China. Fmoc-protected amino acids were purchased from GL Biochem (Shanghai) Ltd, China. Ultrapure water was obtained
MGN-II-DA was synthesized using a standard Fmoc method. The monomer was synthesized on Rink amide resin (640 μmol g−1, 250 μmol, 1.0 eq.) using a CEM Liberty one microwaveassisted peptide synthesizer. Amino acid couplings were performed after the Fmoc protected group was removed by reacting with piperidine (20%) and then washing with DMF seven
Defan Yao received his bachelor’s degree in 2012 from Chemistry and Chemical Engineering Department of Jiangsu University. Subsequently he joined the group of He Tian at ECUST for his PhD. His research focuses on fluorescent sensor and molecular recognition.
Juanjuan Jiang received her bachelor’s degree from the College of Chemical Engineering and Life Sciences from Chaohu University in 2012, and is currently pursuing her master’s degree in ECUST. Her recent research focuses on the development of antibacterial peptide.
Defan Yao
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Synthesis of MGN-II-DA
Juanjuan Jiang
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times. The subsequent amino acids were attached under microwave irradiation conditions (35 W, 55 ± 5 °C, 20 min) according to the SPPS protocol described: Fmoc-protected amino acid (0.75 mmol, 3.0 equiv.), PyBOP (0.75 mmol, 3.0 equiv.) and N-methyl-morpholine (2.0 mmol, 8.0 equiv.) in DMF (8.0 mL). At the end of the chain, coupling by Fmoc-Lys(Alloc)-OH occurred and the side chain of the lysine residue was protected by Alloc to selectively deprotect specific attachment of hemicyanine dye (for details, see ESI†).57,58 When all the amino acids were attached, the crude product was transferred into a glass peptide synthesis vessel and the Alloc group was then removed by adding Pd(PPh3)4 (0.05 mmol, 0.2 eq.) and PhSiH3 (12 mmol, 48 eq.) in DMF for 30 min under argon conditions. After the Alloc was deprotected, the product was coupled with hemicyanine dye. After the reaction was complete, followed by Fmoc deprotection, the product was reacted with 10,12-tricosadiyonic acid. After completion of peptide synthesis, the final product was cleaved from the solid support in a mixture of TFA–H2O–triisopropylsilane (95 : 2.5 : 2.5) for 3 h. The product was a dark red solid. After removing TFA, the peptide was precipitated three times using cold diethyl ether and centrifuged at 10 000 r min−1 for approximately 10 min. The crude peptide was purified by HPLC on a RP18-column using H2O–CH3CN (with 0.1% TFA) as the eluent. The solid was then dissolved in water, acidified with hydrochloric acid (0.1 N) and lyophilized. This step was repeated three times. The product was identified by MALDI-TOF using α-cyano-4hydroxycinnamic acid (CHCA) as the matrix.
either used freshly prepared or stored in the fridge at 4.0 °C until use.
Preparation of MGN-II-PDA
Atomic force microscopy (AFM)
First, MGN-II-DA was dissolved in TBS (50 mM Tris and 50 mM NaCl, pH 7.4, 25 °C) at a final concentration of 1 mM and then transferred into a quartz cuvette using UV irradiation at 254 nm (approximately 0.2 mW cm−2) for about 90 min to provide a stock solution of MGN-II-PDA. This solution was diluted to the desired concentrations and the solution was
Images were recorded by Veeco/DI atomic force microscopy. MGN-II-PDA was dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C). A drop of the 20 μM MGN-II-PDA solution was placed on freshly cleaved mica for 30 s and allowed to dry at room temperature. The sample was then analyzed in tapping mode.
Dr Wu received his Ph.D. degree from Fudan Universiy in 2009. After graduation, he went to work in the University of DuisburgEssen with the support of the Alexander von Humboldt Foundation. He became an associate professor of the Institute of Fine Chemicals at ECUST in 2011. His current research interests include antitumor drugs of polypeptide, enzyme inhibitors based on peptides, and self-assembly and recognition of biomolecules.
Prof. He Tian received his Ph.D. degree from ECUST in 1989. Then, Dr Tian worked on organic laser dyes at Siegen University, Germany and on organic photochromic copolymers at the Max Planck Institute for Polymer Research in Mainz, Germany in 2000. Both studies were supported by the Alexander von Humboldt Foundation. In 1994, Dr Tian became a full professor and director of the Institute of He Tian Fine Chemicals at ECUST. In 2011, he was selected as a member of the Chinese Academy of Science. His current research interests center on the development of interdisciplinary materials science.
Junchen Wu
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Fluorescence and UV-vis experiments Fluorescence emission spectra were recorded using a Fluoromax-4 spectrofluorometer and UV-vis absorption spectra were recorded on a Cary 100 UV-visible spectrophotometer with a slit width of 5 nm at 25 °C for both excitation and emission. The data were collected at 1 nm increments with a 0.1 s integration time. All spectra were corrected for intensity using the manufacturer-supplied correction factors and corrected for background fluorescence and absorption by subtracting the value obtained for a blank scan of the buffer system. Absolute fluorescence quantum yield was determined using an FLS-980stm spectrometer (Edinburgh Instruments Ltd, UK). All samples were dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) at a concentration of 20 μM. CD spectroscopy The CD spectra of MGN-II-DA and MGN-II-PDA were recorded on a JASCO J-815 CD spectrometer at 25 °C. The spectra were scanned in a quartz optical cell of 1 cm path length and the sample was dissolved in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) at a concentration of 20 μM. Data were collected at 1 nm increments with a 0.1 s integration period from 250 to 190 nm at each wavelength.
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Dynamic light scattering (DLS) study The average particle size was measured using a Nano-ZS (Malvern Instruments). MGN-II-PDA (20 μM) was measured in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C). The solution was prepared according to a routine method without filtration.
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Cell culture A549 lung carcinoma, KB epidermal carcinoma and MCF-7 breast adenocarcinoma cells were purchased from Shanghai Bogoo Biotech Co., Ltd, China. These cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), 50 U mL−1 penicillin and 50 μg mL−1 streptomycin (P/S, HyClone). The cell cultures were maintained at 37 °C under humidified conditions of 95% air and 5% CO2 in the culture medium. The culture medium was changed every two days to maintain exponential growth of the cells. Cells were passaged using 0.05% Trypsin/EDTA (Sigma) when they reached 80–90% confluence and seeded for the experiments. Cytotoxicity assay The cytotoxicity of MGN-II-PDA against cancer cells was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, A549 lung carcinoma, KB epidermal carcinoma and MCF-7 breast adenocarcinoma cells in complete medium were seeded into 96-well plates at a density of 10 000 cells per well and cultured overnight at 37 °C under 5% CO2 before treatment. After the complete medium was removed, the MGN-II-PDA solution (100 μL per well) at concentrations of 8, 16, 32 and 64 μg mL−1 in RPMI-1640 was added to the wells, and the cells were incubated for 24 h at 37 °C under 5% CO2. Subsequently, 10 µL of MTT solution (5 mg mL−1 in PBS) was added to the medium and the cells were incubated at 37 °C for 4 h. The resulting formazan crystals were solubilized using 100 µL of DMSO after the growth medium was removed and the quantity was determined colorimetrically using a Synergy H4 Hybrid Microplate reader (Biotek, USA) at 490 nm. Cell viability (%) was calculated using the following formula: Cell viability = (mean absorbance value of the treatment group-blank/mean absorbance value of the control-blank) × 100. Furthermore, IC50 values were obtained by plotting the signal responses (OD490) against the logarithm of analyte concentrations using Origin software (Origin 8.0). The 4-parameter logistic equation y = A2 + (A1 − A2)/[1 + (x/x0)p] was used for curve fitting in the whole concentration range, where A1 is the maximum signal at no analyte, A2 is the minimum signal at infinite concentration, p is the curve slope at the inflection point, and x0 is the IC50 (analyte concentration causing a 50% inhibition of the maximum response, a measure of immunoassay detectability).59 Confocal microscopic imaging of cells Cells were seeded in a glass-bottomed dish. After overnight culture, the cells were incubated with MGN-II-PDA in RPMI-1640 medium at a final concentration of 16 μg mL−1 for
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30 min at 37 °C. The cells were then washed three times with PBS to remove MGN-II-PDA. Then, the cells were stained with 10 μg mL−1 DAPI in RPMI-1640 medium for 15 min. Cell images were obtained with a confocal laser scanning microscope (CLSM), Nikon A1 (Japan), at an excitation wavelength of 488 nm and the emission was collected at 600–650 nm. Flow cytometry The cell uptake of MGN-II-PDA was measured by flow cytometry. A549, KB and MCF-7 cells were seeded into six-well plates (1 mL cell suspension per well) at a density of 1 × 106 cells mL−1, and allowed to attach overnight before treatment. MGN-II-PDA was dispersed in RPMI-1640 medium at a concentration of 10 μg mL−1. The cells were treated with the MGN-II-PDA solution and incubated for 30 min at 4 °C and 37 °C. Cells incubated in RPMI-1640 medium alone were set as the control. The medium was then removed and the cells were washed three times with cold PBS. The cells were harvested using 0.05% (w/v) trypsin/0.02% (w/v) EDTA, and centrifuged at 1000 rpm for 5 min. The harvested cells were then resuspended in PBS. Each cell with MGN-II-PDA was quickly analyzed on a Millipore Guava flow cytometer (Billerica, MA, USA) using a 488 nm argon laser for excitation and the emitted 628 nm fluorescence for detection. Animals and tumor implantation All animal experiments were performed in agreement with the guidelines of the Institutional Animal Care and Use Committee of East China University of Science and Technology and conformed to the guide for the care and use of laboratory animals. A549 cells were washed with PBS, and harvested using trypsin-EDTA. After centrifugation, the cell pellets were resuspended in PBS and adjusted to a concentration of 1 × 107 cells mL−1. The suspended cells in 0.2 mL PBS were implanted into the right axilla of six-week-old (approximately 20 g) male BALB/c nude mice (Shanghai Slac Laboratory Animal Co. Ltd, China). In vivo antitumor studies When the tumors reached a mean volume of 50 mm3 after inoculation of A549 cells, the mice were randomly separated into two treatment groups (n = 5 per group): (1) negative control (PBS); (2) MGN-II-PDA at 5 mg kg−1 in 0.2 mL PBS. The two groups received intravenous injections via the tail vein every 2 days for a total of 5 cycles. During therapy, the tumor size and body weight were measured every two days. Tumors were measured individually using a Vernier caliper. Tumor volume was calculated using the following formula: tumor volume = length × width2 × 0.5. The mice were sacrificed 8 days after the 5th treatment according to institutional guidelines. Tumors were resected, photographed, weighed, fixed in formalin and then embedded in paraffin. The therapeutic efficacy of the treatment was evaluated by the tumor-inhibition rate (TIR). This was calculated using the following equation: TIR = 100% × (mean tumor weight of control group − mean tumor weight of experimental group)/mean tumor weight of
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control group. One of the tumor-bearing mice was sacrificed right after 4 h injection of MGN-II-PDA (0.2 mL, 2 mg mL−1) via the tail vein, and tumor and other normal organs were isolated and subjected to Carestream In Vivo FX-PRO imaging (Carestream Health, Inc.).
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Histological examination of tumor tissues Tumors from control and treated mice were collected and fixed in neutral formalin. The samples were dehydrated and embedded in paraffin. Tumor sections 3.0 μm thick were dewaxed with xylene, hydrated consecutively with 100%, 95%, 85%, and 75% ethanol and distilled water, and stained with hematoxylin and eosin (H&E) for histological examination.
Results and discussion Synthesis and characterization of MGN-II modified PDA micelles MGN-II (sequence: SNMIEGTFAKGFKKASHLFKGIG) has potential as a novel antitumor agent. However, the IC50 value of MGN-II in A549 cells reached 110 µg mL−1.49 In order to improve the potency against anticancer cells, the multivalent strategy of MGN-II micelles was used in this study. Thus, MGN-II micelles were synthesized by aqueous self-assembly of amphiphilic monomers containing the hydrophobic C25 alkane chain (10,12-tricosadiyonic acid, DA) and a hydrophilic head group (MGN-II sequence). In addition, in order to track the distribution of MGN-II in the cell lines, the fluorophore (hemicyanine dye) was introduced and attached to the side of lysine. MGN-II-DA was synthesized on Rink amide resin by a Fmoc solid-phase method, and purified to 97.0% purity by HPLC with a yield of 3.1% after cleavage and deprotection, and identified by MALDI-TOF (Fig. S1 and S2†). It is well known that DA containing a diyne group can be polymerized via a topochemical 1,4-mechanism under UV irradiation at 254 nm. The functionalized PDA micelles can not only generate multiple MGN-II in the micelles, but also increase the local concentration of micelles in the blood. To further confirm the formation of PDA micelles, the time-dependent absorption (Fig. 2a and 2b) and fluorescent spectra (Fig. 2c and 2d) were used to monitor the polymerization process of monomers (20 µM) in TBS buffer. For self-assembly of PDA micelles, the amphiphilic MGN-II-DA solution was sonicated for approximately 30 min. The solution was then subjected to UV irradiation at 254 nm for about 90 min and the pH was adjusted to 7.4. As shown in Fig. 1a–1d, on UV irradiation of MGN-II-DA in TBS buffer at 254 nm, the absorption bands gradually decreased at approximately 500 nm during the 90 min irradiation period. In addition, the fluorescent emission at 600 nm increased as a function of UV irradiation time. In general, we can achieve a non-fluorescent ‘blue form’ of the PDA liposomes, which changes to a weakly fluorescent ‘red form’. However, in this case, irradiation of the highly fluorescent self-assembled monomer based on the fluorophore
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(hemicyanine dye) enhanced the fluorescence of the backbone of PDA. This enhancement was caused by energy transfer from the hemicyanine fluorophore (emission maximum at 600 nm) to the polymer backbone (absorption maximum at 600 nm).27,60–62 Moreover, the fluorescence quantum yield (Φ) of MGN-II-PDA increased approximately 3-fold compared to the monomer of MGN-II-DA from 1.7% to 5.2% in TBS, which strongly suggested that the energy transfer to the backbone of PDA liposomes was from the fluorescent dye (Fig. 2e). On the basis of the above results, we further evaluated whether the polymerization of MGN-II-DA impacted the secondary structure of MGN-II (α-helix) in TBS buffer after UV irradiation at 254 nm using circular dichroic (CD) spectra (Fig. 2f ). The MGN-II conjugated to DA and hemicyanine dye still maintained an α-helix-rich CD pattern with two negative Cotton effects at 218 and 228 nm, in accordance with its crystal structure extracted from the Protein Data Bank (PDB: 2MAG).63 This suggests that the secondary structure of MGN-II is partially perturbed due to the long alkane chain and fluorescent dye. Following UV irradiation at 254 nm for approximately 90 min, the CD spectra of PDA micelles changed from an α-helical-rich profile to a β-sheet-rich profile, as suggested by the emergence of a new CD band at 212 nm. This change in conformation may be attributed to the polymerized perturbation under UV irradiation at 254 nm. In addition, the structure of the polymerized micelles was further characterized by atomic force microscopy (AFM) and dynamic light scattering (DLS). The AFM image of MGN-II-PDA clearly shows these PDA micelles with a diameter of approximately 50–100 nm which is consistent with the DLS results (Fig. 3a and 3b). In addition, zeta-potential measurements showed that the surface of MGN-II-PDA was positively charged at a zeta-potential of 17.53 ± 0.57 mV. In vitro efficacy of MGN-II-PDA in cancer cell lines After obtaining the PDA micelles, we determined the MGN-II-PDA-induced membrane permeabilization and the mechanism of uptake in cancer cell lines. Thus, confocal laser scanning microscopy (CLSM) was used to monitor the cellular uptake of MGN-II-PDA micelles in A549, KB and MCF-7 cell lines. Subsequently, the three cells types were incubated in RMPI-1640 medium at 37 °C, and a co-localization experiment of MGN-II-PDA micelles with DAPI (the nuclear counterstain) was carried out. The blue and red signals in the cell images were from DAPI (Fig. 4a, 4d and 4g) and MGN-II-PDA micelles (Fig. 4b, 4e and 4h), respectively. The overlay images demonstrated that MGN-II-PDA micelles were distributed in both the cytoplasm and the membrane in A549 (Fig. 4c), KB (Fig. 4f ) and MCF-7 cells (Fig. 4i). These results showed that MGN-II-PDA was localized in the membrane and the cytoplasm. Moreover, the time-lapse images indicated that the MGN-II-PDA micelles rapidly accumulated in the cytoplasm and reached the summit for about 20 min (Fig. S3†). To further explore the membrane and cytoplasm entry pathway of MGN-II-PDA micelles, the uptake ratio of
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Fig. 2 (a) Absorption and (b) the change in absorption intensity at 500 nm of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 M NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min; (c) Fluorescence emission spectra (λex = 492 nm) and (d) the change in fluorescence intensity at 600 nm of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 M NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min; (e) and (f ) show fluorescence quantum yield and CD spectra of MGN-II-DA (20 µM) in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for approximately 90 min. The data are presented as mean ± SD (n = 5).
MGN-II-PDA micelles was investigated in the three cell lines under different temperature conditions by flow cytometry. As shown in Fig. 5a and 5b, the uptake ratios were not significantly different at 4 °C and 37 °C in A549 cells following incubation with MGN-II-PDA for 30 min. The uptake ratio of MGN-II-PDA reached 92.9% at 4 °C and 99.1% at 37 °C. These
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results indicated that temperature did not affect the cellular uptake by A549 cells. The uptake ratio of MGN-II-PDA in MCF-7 cells increased from 71.6% at 4 °C to 91.4% at 37 °C (Fig. 5e and 5f ). However, the uptake ratio of MGN-II-PDA in KB cells increased significantly from 37.0% at 4 °C to 94.5% at 37 °C. This is not surprising as cell activity is usually inhibited
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Fig. 3 AFM image (a) of MGN-II-DA (20.0 μM) in ultrapure water and DLS (b) of MGN-II-DA (20.0 μM) in TBS (50 mM Tris, 50 mM NaCl, pH 7.4, 25 °C) after irradiation with UV light (254 nm, 0.2 mW cm−2) for 90 min.
Fig. 4 Confocal laser scanning microscopy (CLSM) images illustrating the intracellular distribution of MGN-II-PDA (red) and DAPI (blue) in (a–c) A549, (d–f ) KB and (g–i) MCF-7 cells. The cells were stained with (a, d, g) 10 μg mL−1 DAPI for 15 min (Channel 1: excitation: 405 nm, emission collected: 425–475 nm), (b, e, h) 16 μg mL−1 MGN-II-PDA for 30 min (Channel 2: excitation: 488 nm, emission collected: 600–650 nm), and (c, f, i) overlay of (a) and (b), (d) and (e), (g) and (h).
at low temperature. These findings suggest that energy plays a key role in MGN-II-PDA crossing the plasma membrane and eventually reaching the cytoplasm in KB cells. Interestingly, with a further increase in temperature to 37 °C, the uptake ratio of MGN-II-PDA micelles was increased in the membrane
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and cytoplasm regions. As seen from the uptake ratios of MGN-II-PDA in the cells, the function of MGN-II-PDA micelles was unaffected, further demonstrating that temperature promoted the cellular uptake of MGN-II-PDA. It is also possible that the rigid membranes were less favourable for transloca-
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Fig. 5 Cellular uptake of MGN-II-PDA by flow cytometry analysis. The cells were treated with 10 μg mL−1 MGN-II-PDA for 30 min at 4 °C and 37 °C. (a) and (b) show A549 cells treated with MGN-II-PDA at 4 °C and 37 °C; (c) and (d) show KB cells treated with MGN-II-PDA at 4 °C and 37 °C; (e) and (f ) show MCF-7 cells treated with MGN-II-PDA at 4 °C and 37 °C.
Fig. 6 Dose–response curves for MTT toxicity assays (a) for MGN-II-PDA in A549 cells, MCF-7 cells and KB cells. (b) shows the IC50 values of the three cancer cell lines. A549 cells, MCF-7 cells, and KB cells were seeded at 1 × 104 cells per well on a 96-well plate, and then treated with MGN-II-PDA at concentrations of 8, 16, 32 and 64 μg mL−1 in RPMI-1640 medium for 24 h. The data are presented as mean ± SD (n = 5).
tion. Although we cannot fully exclude internalization by endocytosis, we suggest that the uptake was due to a cell-penetrating process by MGN-II-PDA micelles in these cancer cell lines. Thus, these results demonstrate that electrostatic attractions of cationic peptides to the cell membranes44 and the endocytotic pathway are mainly responsible for cellular uptake.64–66 To investigate the cytotoxicity of MGN-II-PDA in cancer cell lines, mammalian cell lines A549, MCF-7 and KB were treated with MGN-II-PDA. The three cell lines were incubated with various concentrations of MGN-II-PDA in RMPI-1640 medium
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for 24 h at 37 °C, and the cytotoxicity of MGN-II-PDA was assessed by the MTT assay. The relative viability of cells at different concentrations of MGN-II-PDA was confirmed spectrophotometrically. The results are summarized in Fig. 6 and are represented by the mean viability ± standard deviation (SD) of three independent experiments, each of which was performed in triplicate. Fig. 6b shows that the IC50 value of MGN-II-PDA in A549, KB and MCF-7 cells was 16.0, 15.7, and 23.9 μg mL−1, respectively. Compared to the literature, the IC50 value in A549 cells found in this study was improved approximately 7-fold.49 The viability of A549, MCF-7 and KB cells was
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that the MGN-II-PDA micelles are biocompatible in vitro (Fig. S4†). In vivo antitumor activity Encouraged by these results, we determined whether MGN-II-PDA could effectively suppress tumor growth in vivo. The therapeutic efficacy of MGN-II-PDA was subsequently assessed using a murine xenograft model (Fig. 7). The mice
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reduced by more than 99% after 24 h incubation with 64 μg mL−1 MGN-II-PDA, which strongly suggested that MGN-II-PDA had effective and broad spectrum antitumor activities enabling positively charged peptides to bind to the negatively charged membrane surface to disrupt lipid membranes. To further evaluate the biocompatibility of MGN-II-PDA, cell viabilities towards the normal cell line (L929) were over 75% after a 24 h incubation with 32 μg mL−1 of MGN-II-PDA, whereas the cancer cell viabilities were less than 30%. This result indicates
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Fig. 7 MGN-II-PDA blocks tumor growth in vivo. A549 xenograft mice were treated with MGN-II-PDA at a dose of 5 mg kg−1 every two days by intravenous injection via the tail vein. The results are summarized as tumor volumes (a), body weight changes (b) and the tumor-inhibition rate (TIR) (c) of mice exposed to PBS and MGN-II-PDA. (d) Photograph of dissected tumors from the two groups of mice. (e) The fluorescence images of excised mouse organs after 4 h injection of MGN-II-PDA (0.2 mL, 2 mg mL−1) via the tail vein (f ) and (g) represent histological features of A549 tumors from the mice treated with PBS and MGN-II-PDA (scale bar is 20 μm).
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treated with MGN-II-PDA showed significant inhibition of A549 tumor growth compared to the PBS negative group (Fig. 7a and 7c). After the mice were sacrificed, the tumors were dissected and weighed, and the tumor-inhibition rate (TIR) of MGN-II-PDA in A549 tumors was calculated (Fig. 7c). In the presence of MGN-II-PDA the TIR was 54.4% ( p < 0.05). In addition, tumor images corresponded with the TIR data (Fig. 7d). The mouse body weight showed little change throughout the study (Fig. 7b) and no gross abnormalities were noted in any of the organs or tissues at the time of necropsy, indicating that MGN-II-PDA did not cause severe systemic side effects. To characterize the distribution of MGN-II-PDA in vivo, the fluorescence of tumor and other normal organs was obtained by Carestream imaging (Fig. 7e). The MGN-II-PDA was markedly accumulated in the stomach and tumor. In contrast, little or no detectable level of MGN-II-PDA could be observed in the kidney, heart, lungs, spleen, liver and brain. Additionally, the A549 xenograft tumors were fixed and prepared for histological analysis. In the control group (PBS treated), the tumor tissue sections were composed of tightly packed tumor cells interspersed with various amounts of stroma (Fig. 7f ). However, dead tumor cells were rarely observed after treatment with MGN-II-PDA (Fig. 7g). The histological features of tumors showed significant differences following treatment with the micelles, which significantly inhibited tumor growth compared to the PBS negative group.
Conclusions In summary, we have developed novel amphiphilic MGN-II-PDA liposomes that self-assemble and polymerize into micelles, act as an antitumor agent under UV irradiation at 254 nm, and have a broad spectrum of cytotoxic activity against cancer cells. Moreover, the monomer (MGN-II-DA) can be polymerized by UV irradiation at 254 nm and causes energy transfer from the fluorescent dye to the backbone of MGN-II-PDA micelles, which effectively enhanced the fluorescence quantum yield for imaging living cells. Flow cytometry demonstrated that the MGN-II-PDA internalization pathway may be a cell-penetrating process. MGN-II-PDA also showed significant in vivo antitumor activity without reducing the body weight of treated animals and with few side effects, and is thus a promising and extremely valuable approach in the design of peptide-based anticancer agents.
Conflict of interest The authors declare no competing financial interest.
Acknowledgements This work was supported by grants from the National Basic Research 973 Program (2013CB733700) and the Fundamental
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Research Funds for the Central Universities (WJ1213007), the Program of Shanghai Pujiang (K100-2-1275) and the Innovation Program of Shanghai Municipal Education Commission (J100-2-13104) for financial support.
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