Article pubs.acs.org/Biomac
Folate and Heptamethine Cyanine Modified Chitosan-Based Nanotheranostics for Tumor Targeted Near-Infrared Fluorescence Imaging and Photodynamic Therapy Yingying Zhang,†,‡ Tingting Lv,†,‡ Huijuan Zhang,†,‡ Xiaodong Xie,†,‡ Ziying Li,†,‡ Haijun Chen,‡ and Yu Gao*,†,‡ †
Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis, State Key Laboratory of Photocatalysis on Energy and Environment and ‡Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350108, China S Supporting Information *
ABSTRACT: Folate (FA) and heptamethine cyanine (Cy7)modified chitosan (CF7) was synthesized by click chemistry and its self-assembled nanoparticles (CF7Ns) were developed for tumor-specific imaging and photodynamic therapy. The characterization spectrum confirmed CF7 had a good FA and Cy7 conjugation efficacy. The diameter of CF7Ns measured by DLS was about 291.6 nm, and the morphology observed with AFM showed filamentous clusters of particles. The results of targeting ability of CF7Ns demonstrated enhanced targeting behaviors of CF7Ns compared with non-FA-modified nanoparticles C7Ns in FA receptor-positive HeLa cells. The cytotoxicity and cell apoptosis assay showed that CF7Ns under near-infrared light irradiation led to more apoptotic cell death in HeLa cells to improve the therapeutic efficacy. The mechanisms of the photodynamic effects of CF7Ns were demonstrated through measurement of intracellular reactive oxygen species and the apoptosis-related cytokines. These results suggested that CF7Ns are promising tumor targeting carriers for simultaneous fluorescence imaging and photodynamic therapy.
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INTRODUCTION Cancer is one of the most serious life-threatening diseases worldwide. Operation, radiotherapy, and chemotherapy are the main cancer treatment alternatives at present. However, the clinical outcomes of radiotherapy and chemotherapy were impeded by the lack of tumor selectivity and severe toxicity.1 Photodynamic therapy (PDT) is a noninvasive cancer therapy modality which allows the local creation of singlet oxygen or other free radicals by the activation of photosensitizers (PSs) via a specific frequency of light in the visible or near-infrared (NIR) region, thus selectively destroying tumor cells in the targeted tumor tissues.2 Hence, PDT could be a promising approach for cancer treatment with comparable effectiveness to that of standard therapies without the corresponding side effects. Although PSs used in clinic for PDT have been shown to be both effective and safe in the treatment of a variety of human cancers,3 they still suffered from the drawback of poor selectivity in tumors with low PDT selectivity indexes.4 PSs by systemic administration could distribute all over the body especially the skin. Most patients received PDT treatment exhibit cutaneous photosensitivity which might last for up to 6−8 weeks, such that they have to stay away from the sunlight to avoid getting sunburned.5,6 Therefore, it is greatly important to develop a kind of PSs which can target and precisely accumulate in tumor cells, without affecting other normal cells. © XXXX American Chemical Society
One of the most efficient ways to achieve precise tumor accumulation is the use of tumor-targeted drug delivery systems (TDDSs) with the help of nanomaterials.7 Because of their small physical sizes, nanomaterials could passively accumulate in tumor sites by enhanced permeability and retention (EPR) effects.8 To obtain active targeting characteristics, nanoparticles could be functionalized with ligands, such as peptides, antibodies, antibody fragments, and nucleic acids.9 Folic acid (FA) is one of the most used ligands for recognition of folate receptors (FRs)10 which are highly expressed on a variety of cancers.11 FA-modified nanoparticles for FR targeting could sharply enhance the specificity, efficacy, and bioavailability of the loaded drugs or contrast agents in drug delivery or tumorimaging applications.12 In consideration of the multilevel complexities and variability of cancers, cancer treatment is moving toward personalized medicine for individual patients. Multifunctional nanoparticles which enable a combination of two or more functions, such as cancer imaging and therapy, could facilitate the development of personalized medicine for tailored cancer treatment. NIR fluorescence imaging has been used in cancer diagnostics due to Received: March 30, 2017 Revised: June 6, 2017 Published: June 19, 2017 A
DOI: 10.1021/acs.biomac.7b00466 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules Scheme 1. Synthesis of ALK-F and ALK-Cy7a
a
Reagents and conditions. (A) Synthesis of ALK-F; (a) FA, DCC, NHS, DMSO, 18 h; (b) propargylamine, DIPEA, 48 h. (B): Synthesis of ALKCy7; (a) phenylhydrazine, 3-methyl-2-butanone, HAc, 100 °C, 3 h; (b) MeI, PhMe, 92 °C, overnight; (c) sodium acetate, AA, 105 °C, 6 h; (d) piperazine, ACN, 65 °C, overnight; (e) propiolic acid, EDCI, DMAP, DCM, in the dark at room temperature, overnight.
chemical modification.21 To make regioselective modification of Cs, some N-substituted derivatives were synthesized as precursors for subsequent modification due to its solubility in common aprotic polar solvents. N-(2-phthaloylation) chitosan is regarded as one of the most popular precursors for synthesis of novel Cs derivatives with excellent solubility and bioactivities, especially for the synthesis of substituents at the C6-OH position.22,23 As a widely used coupling technology with many advantages of high stereospecificity, high yield, easy separation of products, and mild reaction conditions with benign solvents, copper-catalyzed azide−alkyne cycloaddition (click chemistry) had been used to synthesize various C-6 substituent Cs derivatives based on the N-(2-phthaloylation) Cs precursor.24,25 We have interest in developing a new Cs-based nanotheranostics including folate as a tumor targeting ligand and Cy7 derivative used both as an NIR fluorescent reporter and a PS for PDT therapy. In this work, Cs was first modified with azide groups at C-6 position. Next the alkylated FA derivative (ALK-F) and the alkylated Cy7 derivative (ALK-Cy7) were interacted with Cs derivative bearing azide groups by click chemistry to obtain a new polymer derivative, named as CF7. The CF7 self-assembled nanoparticles (CF7Ns) were prepared and the physiochemical properties of CF7Ns were investigated. Then the in vitro drug release behavior of CF7Ns was characterized. Finally, the tumor targeting efficiency and the PDT therapy efficacy of CF7Ns were studied in HeLa and HepG2 cancer cells.
its distinct advantages including the convenient use in cell labeling, no ionic radiation effect, low cost of the instrumentation, and especially, the deep tissue penetration and negligible background autofluorescence for in vivo imaging applications.13 Heptamethine cyanines (Cy7s) are fluorescent dyes being used as biological molecular markers in imaging and other biological analyses.14 They have a strong adsorption peak in the NIR region with minimal scattering and low tissue absorption.11 In addition, Cy7s can not only be selected as a kind of PSs for photothermal therapy (PTT), but also can generate cytotoxic reactive oxygen species (ROS) for PDT under NIR irradiation. Among the Cy7s, indocyanine green (ICG) is the only optical reagent approved by the Food and Drug Administration for use in human studies. Because of the lack of group for additional functionalization of ICG, a great deal of study on functionalization of the cyclohexenyl ring in the Cy7 dyes have been done to develop probes for NIR imaging applications.15 Because of the fragile structure of NIR dyes, only the physical incorporation not the chemical conjugation of Cy7s into nanoparticles have been reported to facilitate tumor targeted NIR fluorescence imaging.16,17 Among currently available nanoparticle platforms, chitosan (Cs), a natural polysaccharide, is a very attractive biomaterial utilized widely in biological and pharmaceutical field such as bioimaging18 and drug delivery19 for its high yield, low cost, high biocompatibility, biodegradability, and low toxicity.20 However, Cs is only soluble in water containing organic acids. The insolubility in organic solvents extremely limits its B
DOI: 10.1021/acs.biomac.7b00466 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules Scheme 2. Synthesis of CF7a
Reagents and conditions. (a) Phthalic anhydride, DMF, 120 °C, 0.5 h; (b) NBS, TPP, NMP, 80 °C, 2 h; (c) sodium azide, NMP, 80 °C, 4 h; (d) ALK-Cy7, ALK-F, CuSO4, sodium ascorbate, DMSO, 50 °C, 72 h.
a
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solvent, and the yellow solid was precipitated. ALK-F was collected by centrifugation as yellow solid26 (Scheme 1A). 1H NMR (400 MHz, DMSO): δ −8.64 (s, 1H), 8.41−8.20 (m, 1H), 8.12−7.89 (m, 1H), 7.77−7.52 (m, 2H), 7.19 (s, 1H), 6.93 (d, J = 5.8 Hz, 1H), 6.61 (dd, J = 38.4, 8.3 Hz, 2H), 4.66−4.38 (m, 2H), 4.36−4.12 (m, 1H), 3.78 (d, J = 40.7 Hz, 2H), 3.47−3.30 (m, 1H), 3.05 (dd, J = 14.6, 11.6 Hz, 1H), 2.94−2.81 (m, 1H), 2.34−2.08 (m, 2H), 2.02 (dd, J = 14.8, 7.1 Hz, 1H), 1.91 (dd, J = 14.5, 7.0 Hz, 1H). ALK-Cy7 was obtained as a deep-blue solid by the previously reported methods27,28(Scheme 1B). 1H NMR (400 MHz, CDCl3): δ −7.81−7.69 (m, 2H), 7.39−7.28 (m, 4H), 7.21−7.13 (m, 2H), 7.08− 7.01 (m, 2H), 6.03−5.90 (m, 2H), 4.27−4.22 (m, 1H), 4.10−4.06 (m, 1H), 4.05−4.00 (m, 1H), 3.96−3.87 (m, 2H), 3.81 (s, 1H), 3.78−3.72 (m, 1H), 3.69−3.66 (m, 1H), 3.63 (s, 4H), 3.61−3.58 (m, 2H), 3.56− 3.53 (m, 1H), 2.06−1.99 (m, 2H), 1.85 (s, 6H), 1.67 (s, 12H). Synthesis of FA-Modified Cs (CF), Cy7-Modified Cs (C7), and Cs Conjugated with Both FA and Cy7 (CF7). CF7 was synthesized as displayed in the Scheme 2. First, Cs was in reaction with phthalic anhydride to obtain N-phthaloyl-chitosan (2). NBS and TPP were reacted with (2) to introduce the bromide groups at the C-6 position to afford 6-bromide-6-deoxy-N-phthaloyl-chitosan (3). 6-Azido-6deoxy-N-phthaloyl−chitosan (Cs−N3, 4) was prepared by replacing the bromide groups with azido groups in accordance with the previously reported methods by Satoh et al.21 Finally, CF7 (5) was synthesized as follows: Cs−N3 (20 mg), ALK-F (10 mg), ALK-Cy7 (10 mg), CuSO4 (2.5 mg predissolved in 100 μL ddH2O), and sodium ascorbate (2 mg predissolved in 100 μL ddH2O) were added to DMSO (5 mL). The solution was stirred in the dark at 50 °C for 3 days. After the reaction was completed, the solution was dialyzed in the ddH2O with dialysis bag (MWCO:14000) for 3 days. The solution was lyophilized to get CF7 as the brown powder. CF and C7 were obtained by reaction of Cs−N3 with ALK-F and ALK-Cy7, respectively, along with the method of CF7. Characterization of ALK-F, ALK-Cy7 and Cs Derivatives. 1H NMR spectra were obtained from a Bruker AVANCE 400 spectrometer (400 MHz). Infrared spectra were analyzed using a
EXPERIMENTAL SECTION
Materials. Cs (Mw = 60 K, deacetylation degree ≥90.0%) was purchased from BoAo Biotechnologies Co., Ltd. (Shanghai, China). Propargylamine, phenylhydrazine, 3-methyl-2-butanone, acetic acid (HAc), methyl iodide (MeI), methylbenzene (PhMe), acetic anhydride (AA), piperazine, acetonitrile (ACN), propiolic acid, 4dimethylaminopyridine (DMAP), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDCI), phthalic anhydride, dicyclohexylcarbodiimide (DCC), dichloromethane (DCM), N-hydroxysuccinimide (NHS), N,N-diisopropylethylamine (DIPEA), N-bromosuccinimide (NBS), triphenylphosphine (TPP), dimethylformamide (DMF), Nmethyl-2-pyrrolidone, copper sulfate (CuSO4), sodium azide, and FA were bought from Sinopharm Chemical Reagent Co., Ltd. (China). Lysozyme was got from Genview Co., Ltd. (USA). Cy7 derivative was synthesized according to a previously reported method.11 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich. Phosphate buffered saline (PBS), RNase A, and trypsin-EDTA were purchased from Gibco-BRL (Burlington, ON, Canada). The RPMI 1640 medium, dulbeccos modified eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technologies GmbH (Darmstadt, Germany). The rabbit antihuman Survivin, p21, and GAPDH antibodies were purchased from Wanlei bio Co., Ltd. (Shenyang, China). The secondary antibody goat antirabbit IgG horseradish peroxidase conjugate was obtained from Promega. Synthesis of ALK-F and ALK-Cy7. ALK-F and ALK-Cy7 were synthesized as displayed in the Scheme 1. FA (1 g, 2.27 mmol, 1 equiv) was added to DMSO (16.7 mL) and ultrasound dissolved. Afterward, DCC (560 mg, 2.27 mmol, 1 equiv) and NHS (314 mg, 2.27 mmol, 1eq) were added in. The solution was stirred in the dark at the room temperature for 18 h. After collecting the filtrate by suction filtration, propargylamine (207 mg, 2.84 mmol, 1.25 equiv) and DIPEA (367 mg, 3.76 mmol, 1.66 equiv) were put in the filtrate. The solution was stirred in the dark at the room temperature for 48 h. The reaction solution was poured in ether/acetone (v/v, 7/3) mixed C
DOI: 10.1021/acs.biomac.7b00466 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
1.4 μg/mL of Cy7. After incubation for 2 h, the cells were washed three times with PBS (pH = 7.4). Then DAPI (10 μg/mL) was added to stain cell nucleus for 15 min. In the end, the cells were fixed with 4.0% (w/v) paraformaldehyde for 15 min at room temperature in the dark. The confocal images were taken by Leica confocal microscope (Leica TCS SP8, Germany). Negative controls were used for each experiment to minimize the background color. In Vitro Cytotoxicity Studies. The cytotoxicity of CF7Ns was determined by the MTT assay. To evaluate the efficacy of PDT therapy with different power densities, cells were exposed to a NIR irradiation with power densities of 1.0, 2.0, and 2.5 W/cm2 for 1, 5, and 10 min, respectively. HeLa and HepG2 cells were separately seeded in a 96-well plate at a density of 8000 cells per well and cultured for 24 h. Then cells were treated with Cy7, C7Ns, or CF7Ns with different concentrations (0.07, 0.35, 0.7, 1.4, 2.8 μg/mL of Cy7) with or without exposition to a NIR irradiation by an infrared lamp with a power density of 2.0 W/cm2 for 5 min. Control cells were exposed to 0.1% DMSO. After incubation for 24 and 48 h, cells were washed three times with PBS and then 150 μL of MTT solution was added. The cells were incubated for 4 h at 37 °C. After the medium was removed, 150 μL of DMSO was added to the well and incubated for 10 min. The absorbance of the solution was measured at 570 nm by TECAN Infinite F200 Microplate Reader with six replicates analyzed at each dose. Cell Cycle Analysis. HeLa cells were seeded on the six-well plates and incubated with Cy7, C7Ns, and CF7Ns at concentration equivalent to 1.4 μg/mL of Cy7 with exposition to a NIR irradiation (2.0 W/cm2, 5 min) at 37 °C for 48 h. Cells without treatment were used as control. After incubation, cells were collected, washed twice with PBS, fixed with 70% cold ethanol, and stored at 4 °C overnight. Cells were centrifuged again and washed with cold PBS. The cellular DNA was stained with fluorescent solution (1% Triton X-100, 0.01% RNase A, and 0.05% PI) for 30 min at 37 °C in darkness. The DNA content was measured by flow cytometry, and the percentage of cells in each phase of the cell cycle was evaluated using the ModFit software. Ten-thousand events were recorded for each sample. All experiments were performed three times. Cells Apoptosis Assay. Cells Apoptosis was detected by using an Annexin V-FITC/PI Staining Kit. HeLa cells were placed into six-well plates at a density of 5 × 105 cells per well. Cells were incubated with Cy7, C7Ns, and CF7Ns at concentration equivalent to 1.4 μg/mL of Cy7 with or without exposition to a NIR irradiation (2.0 W/cm2, 5 min) for 48 h. The cells were harvested, suspended with 500 μL of binding buffer, and stained with 5 μL of Annexin V-FITC and PI following the manufacturer’s instructions. After reaction at room temperature in the dark for 15 min, all the samples were immediately detected by using flow cytometry. Ten-thousand events were recorded for each sample. All experiments were performed three times. Measurement of Intracellular ROS. Intracellular ROS generation was detected by reactive oxygen species assay kit (Beyotime Biotechnology, Haimen, China). HeLa cells were seeded in six-well plate and cultured for 24 h, followed by treatment with Cy7, C7Ns, and CF7Ns at concentration equivalent to 1.4 μg/mL of Cy7 for 24 h. Then cells were loaded with DCFH-DA dye (10 μM) in serum-free DMEM medium for 20 min at 37 °C. After washing three times with PBS, the cells were irradiated with NIR lamp for 5 min. DCF fluorescence intensity was detected using the flow cytometry to quantify the levels of ROS. Western Blot Analysis. HeLa cells (pretreated with 20 ng/mL IL1β for 4 h) were seeded on the six-well plates and incubated with Cy7, C7Ns, and CF7Ns at concentration equivalent to 1.4 μg/mL of Cy7 with an exposure to NIR irradiation at 37 °C for 24 h. Cells were collected and the cell lysates were prepared by treating RIPA lysis buffer. Then equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by blocking solution and incubated with antibodies against Survivin, p21 and GAPDH overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The target proteins were detected by Super Signal West Pico chemiluminescent substrate kit and ChemiDoc XRS system. The
FTIR spectrometer (Intelligent, Nicolet 360, USA) with KBr pellet. The samples were scanned from 500 to 4000 cm−1. UV−vis spectra were acquired on a Quawell Q5000 microvolume UV−vis Spectrophotometer. Fluorescence spectra were achieved by a FS5 spectrofluorometer (Edinburgh Instruments, UK). The amount of FA in CF7 was quantified spectrophotometrically by absorbance measurement at 280 nm by using the calibration curve of standard solutions of FA in DMSO. The Cy7 conjugation efficiencies were determined by using the calibration curve of standard solutions of ALK-Cy7 in DMSO through fluorescent measurements of Cy7 (λex = 633 nm). Preparation and Characterization of CF7Ns. CF7Ns was prepared through self-assembly as follows. CF7 (1 mg) was dissolved in DMSO (1 mL), and the solution was added into ddH2O drop by drop and stirred. Then the DMSO was removed by dialysis. The excess solvent could be evaporated to obtain the appropriate concentration of CF7Ns. C7 self-assembled nanoparticles (C7Ns) and CF self-assembled nanoparticles (CFNs) were obtained according to the same method. Fluorescence measurements were conducted with an excitation wavelength of 633 nm to scan the respective emission profiles. The mean particle size, zeta potential, and polydispersity index (PDI) were measured by the dynamic light scanning (DLS) using a Zetasizer Nano ZS Particle Size and Zeta Potential Analyzer (Malvern Instruments Ltd., UK). The morphology of CF7Ns was observed using atomic force microscopy (AFM) on a Multimode 8 AFM series (Bruker, USA) in tapping/AC mode. The scanning electron microscopy (SEM) micrographs were obtained by NOVA NANOSEM 230 (FEI, USA). The critical micelle concentration (CMC) of CF7 is detected using FITC as a fluorescence probe. CF7 dissolved in DMSO were diluted in ddH2O to various concentrations, and then 1 μg/mL FITC in methanol was added. The fluorescence intensity at λem = 519 nm was monitored using a Hitachi F-7000 fluorescence spectrometer with a quartz fluorescence cell at λex = 490 nm. Stability Study. The stability of CF7Ns was studied in DMEM or DMEM containing 10% FBS for 1 week at 25 °C. At certain time points (1, 3, 5, and 7 days), samples were collected to monitor the changes in particle size. In Vitro Drug Release of CF7Ns. In vitro release of Cy7 dye from CF7Ns was performed using the dialysis bag diffusion technique with lysozyme (1.25 mg/mL)-contained 40% ethanol-PBS as dissolution medium. One mL of CF7Ns prepared from 5 mg of CF7 was diluted with 1 mL dissolution medium. The suspension was then put into a pretreated dialysis bag. The samples were incubated with 20 mL 40% ethanol-lysozyme-PBS solution (pH = 5.5 or 7.4) at 37 °C in a lightsealed condition. At predetermined time intervals, samples were taken out and the concentration of the released Cy7 dye was determined based on a calibration curve of standard solutions of free ALK-Cy7 in dissolution medium using the fluorescence spectra method as described above. The release experiments were carried out in triplicates and the results were expressed as mean ± standard deviation (SD). Cell Culture. Human cervical cancer HeLa cells and human liver cancer HepG2 cells were purchased from Cell Resource Center of Shanghai Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). HeLa cells were cultured in DMEM culture medium mixed with 10% fetal bovine serum (FBS) and 100 U/ mL penicillin and 100 μg mL−1 streptomycin. HepG2 cells were grown in RPMI 1640 medium containing 10% FBS, 0.1 mg/mL streptomycin and 100 U/mL penicillin. Cells were cultured at 37 °C in a humidified incubator of 5% CO2. Cellular Uptake of CF7Ns. HeLa and HepG2 cells were seeded in a six-well plate with 1.5 mL medium and cultured for 24 h. Afterward cells were incubated with C7Ns and CF7Ns with or without free FA (4 mg/mL) for 2 h at 37 °C. Then cells were collected and subjected to flow cytometry (Becton Dickinson FACSAriaIII cell sorter). Tenthousand events were recorded for each sample. The data were processed by FlowJo software. Confocal Fluorescence Microscopy Studies. HeLa and HepG2 cells were seeded in a 24-well plate and cultured for 24 h, followed by incubation with Cy7, C7Ns, and CF7Ns at concentration equivalent to D
DOI: 10.1021/acs.biomac.7b00466 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 1. Spectroscopic characterization of ALK-F and ALK-Cy7. (A) UV−vis spectra of ALK-F and ALK-Cy7 dissolved in DMSO. (B) UV−vis spectra of different concentrations of ALK-F: (1) 0.0028 mg/mL, (2) 0.0056 mg/mL, (3) 0.0112 mg/mL, (4) 0.014 mg/mL, (5) 0.0168 mg/mL, (6) 0.0196 mg/mL. (C) Fluorescence emission spectra of ALK-F and ALK-Cy7 dissolved in DMSO at λex = 633 nm. (D) Fluorescence emission spectra of different concentrations of ALK-Cy7. expression of proteins of interest was analyzed with Image Lab analysis software. Statistical Analysis. All data were expressed as the mean ± SD for at least three separate experiments. Statistical analysis was performed using the Student’s t-test. The differences were considered significant for p value
