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Mitochondria-Targeting Polydopamine Nanoparticles To Deliver Doxorubicin for Overcoming Drug Resistance Wen-Qing Li,† Zhigang Wang,† Sijie Hao, Hongzhang He, Yuan Wan, Chuandong Zhu, Li-Ping Sun, Gong Cheng, and Si-Yang Zheng* Department of Biomedical Engineering and Material Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *
ABSTRACT: Mitochondria play a critical role in diverse cellular processes, such as energy production and apoptosis regulation. The mitochondria-targeted drug delivery is becoming a potential novel strategy for overcoming drug resistance in cancer therapy. Herein, we synthesize nature-inspired dopamine-derived polydopamine (PDA) nanoparticles. Using triphenylphosphonium (TPP) as the mitochondrial penetration molecule to improve the target efficiency, we synthesize poly(ethylene glycol) (PEG)-modified PDA (PDA-PEG) and TPP-functionalized PEGmodified PDA (PDA-PEG-TPP) nanoparticles. Then anticancer drug doxorubicin (DOX) was loaded on PDA-PEG and PDA-PEG-TPP (PDA-PEG-DOX and PDA-PEG-TPP-DOX) nanoparticles, which are apt to deliver DOX to cell nuclei and mitochondria, respectively. To mimic the repeated anticancer drug treatment in clinical cases, we repeatedly treated the MDAMD-231 cancer cells for a long time using DOX-loaded nanoparticles and find that the mitochondria targeting PDA-PEG-TPP-DOX has higher potential to overcome the drug resistance than the regular delivery nanoparticles PDA-PEG-DOX. These results indicate the promising potential of applying PDA-PEG-TPP-DOX nanoparticles in mitochondria-targeted drug delivery to overcome the drug resistance in long-time anticancer chemotherapy. KEYWORDS: drug resistance, mitochondria-targeted, polydopamine nanoparticles, doxorubicin, triphenylphosphonium (TPP)
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INTRODUCTION Multidrug resistance (MDR) is a huge challenge in cancer therapy, which causes failure of many chemotherapeutic drugs in clinical treatment.1,2Drug resistance is a multifactorial process with several different intrinsic and acquired mechanisms. Intrinsic resistance is a pre-existing resistance that exists prior to exposure to a given drug. In contrast, acquired resistance usually begins to appear after tumors are exposed to the drug for a period of time, although the tumors are initially sensitive to the drug. Drug resistance is a manifestation of cancers. The somatic mutations and genomic plasticity associated with cancer are the foundation of drug resistance.3,4 Cancer heterogeneity and cancer stem cells explain the fact that every cancer expresses a different set of drug-resistance genes.5−8 Drug resistance generally arises from overexpression of drug efflux ABC transporters, stress response proteins, or antiapoptotic factors within cancer cells after repetitive treatment with chemotherapeutic agents.9−12 In basic research, various elegant strategies have been developed trying to overcome drug resistance in cancer chemotherapy by using nanotechnology-based drug delivery systems,13−16 such as liposomes, polymer conjugates, dendrimers, carbon-based, and metal nanoparticles.17,18 Despite these explorations, current nanoscale drug delivery systems aiming to improve drug resistance still suffer from shortcomings including laborious preparation procedure, unstable structures, potential cytotoxicity, and unsatisfied combating drug-resistance performance. © 2017 American Chemical Society
Therefore, exploring new strategies to overcoming drug resistance for the clinical cure is an arduous and necessary work. As the powerhouse inside the cells, mitochondria have been associated with the process of carcinogenesis due to their vital roles in energy production, apoptosis, signaling processes, cell cycle, and cell differentiation.19 Previous reports investigated multiple mechanisms of drug resistance related to abnormal mitochondrial activities. Dysfunctional mitochondria may contain mtDNA mutations that conferred drug resistance.20 Abnormal mitochondria can have a high reactive oxygen species (ROS) level that instigates drug resistance.21,22 Also, a small subpopulation of slow-cycling cells has been isolated from different tumors, which is endowed with tumorigenic potential and multidrug resistance.22,23 These cells are defective in mitochondrial respiration due to their dominant glycolytic metabolism. Recently, mitochondria-targeted drug delivery systems have attracted intensive attention. Several nanostructures with mitochondria-targeted properties have been developed and achieved promising anticancer effects in cancer chemotherapy.19,24−33 Encouragingly, research that explores the mitochondria-targeted nanoparticle as a potential drug delivery system to overcome drug resistance has been reported. Wang and co-workers synthesized TPGS2k/PLGA/SN-38 nanoReceived: January 31, 2017 Accepted: May 8, 2017 Published: May 8, 2017 16793
DOI: 10.1021/acsami.7b01540 ACS Appl. Mater. Interfaces 2017, 9, 16793−16802
Research Article
ACS Applied Materials & Interfaces particles that overcame multidrug resistance by interfering mitochondria of human alveolar adenocarcinoma cells.34 Wang, Yin, and co-workers reported folate-mediated mitochondriatarget doxorubicin-polyrotaxane nanoparticles to overcome drug resistance in MCF-7 cells and MCF-7/Adr cells.35 However, those studies have mainly focused on investigating the anticancer drug-resistance effect of those mitochondriatargeted nanoparticles under short-time treatment using drugresistant cell lines. As we known, in clinical settings of cancer treatment, drug resistance often occurred within cancer cells after long-time repetitive treatment with chemotherapeutic agents. Normally, some cancer cells are killed by traditional chemotherapy drugs during the initial treatment, but some other tumor cells gain resistance against the original drugs as the therapy continues, which severely hampers the anticancer effect and leads to cancer recurrence.11,36 Since mitochondria are unlikely to generate drug resistance due to the lack of nucleotide excision repair,37 we hypothesized that mitochondria-targeted delivery of anticancer drug might overcome drug resistance in long-term treatment. In this study, polydopamine (PDA), a nature-inspired melanin-like synthetic polymer derived from dopamine and widely used in various biomedical applications,38 was employed as the delivery vehicle because of its unique merits such as facile synthesis, easy modification, excellent biocompatibility, and high drug loading capacity.39−42 Furthermore, previous studies also demonstrated that dopamine induces a direct modification on the subunits of complex I and complex III of the electron transport chain, which leads to damage of mitochondrial respiration and mitochondrial depolarization.43,44 To facilitate the penetration of nanoparticles through the potential barrier of the mitochondrial intermembrane and improve the mitochondrial targeting efficiency, triphenylphosphonium (TPP), a mitochondrial targeting moiety with high lipophilicity, served as the targeting moiety.45,46 Herein, we synthesized poly(ethylene glycol) (PEG)-modified PDA (PDA-PEG) and TPPfunctionalized PEG-modified PDA (PDA-PEG-TPP) nanoparticles. Moreover, doxorubicin (DOX) has been a widely used anticancer drug, which could cause cancer cell death by inhibiting DNA topoisomerase II, an enzyme that is both in mitochondria and in nuclei of human cells. Free DOX will enter the nuclei and interact with DNA topoisomerase II. Unfortunately, when challenged with a cell line that overexpresses a common efflux pump (e.g., P-glycoprotein efflux pumps), part of the DOX released from nontargeting delivery vehicles could be pumped out of the cell before they reach the nuclei to interact with DNA topoisomerase II. However, the targeted delivery of DOX to mitochondria may provide a means to limit drug efflux and improve chemotherapy efficiency based on mitochondrial-targeted DNA topoisomerase II poison.47 Thus, the DOX was then loaded onto the PDAPEG and PDA-PEG-TPP. We reason that most of DOX will be delivered to nuclear DNA by PDA-PEG-DOX and mitochondrial DNA (mtDNA) and by PDA-PEG-TPP-DOX (Scheme 1). Aiming to mimic the repeated anticancer drug treatment in clinical cases, we treated the MDA-MD-231 cancer cells repeatedly for a long time using DOX-loaded nanoparticles. Then the combating drug-resistance performance of these two delivery systems (i.e., PDA-PEG and PDA-PEG-TPP) was investigated, and the results demonstrated that mitochondriatargeted PDA-PEG-TPP showed better potential in reducing drug resistance.
Scheme 1. Schematic Diagram of PDA-PEG-DOX and PDAPEG-TPP-DOX Syntheses (A) and Mitochondria-Targeted Subcellular Drug Delivery (B)a
a
Most of PDA-PEG-TPP-DOX mainly delivered and released DOX to mitochondria, whereas most of PDA-PEG-DOX delivered and released DOX to the nucleus.
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EXPERIMENTAL SECTION
Materials. Polyoxyethylene (5) nonylphenyl ether (Igepal CO-520, Mn = 441), cyclohexane (anhydrous, 99.5%), dopamine hydrochloride (DA·HCl, 99%), ammonium hydroxide (ACS, 28.0−30.0% NH3 by weight), and (3-carboxypropyl) triphenylphosphonium bromide (98%) were obtained from Sigma-Aldrich Co. (St. Louis, MO). 1Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 97%), and doxorubicin hydrochloride (DOX·HCl) were obtained from Alfa Aesar (Ward Hill, USA). 4Arm polyethylene glycol amine (4Arm-PEG-NH2MW, 10 kDa) was purchased from Laysan Bio, Inc. (Alabama). All reagents were used without further purification. Milli-Q water (18.2 MO·cm) was used in all experiments. In the present work, we use DOX to indicate the doxorubicin hydrochloride. General Characterization. Transmission electron microscopy (TEM) images were acquired on a FEI Tecnai F20 microscope. Zeta potential and size of the nanoparticles were measured on a Malvern Zetasizer. Also, the Gatan software was used to manually measure the diameters of PDA NPs for the nanoparticle diameter statistics (n = 300). The UV−vis spectrum was measured using a PerkinElmer Lambda 950 UV/vis/NIR spectrophotometer. An FTIR spectrometer (Bruker Vertex V70) was used to record the Fourier transform infrared (FTIR) spectra. CCK-8 assay was performed using a TecanInfinite M200 Pro microplate reader. Preparation of PDA-PEG-DOX and PDA-PEG-TPP-DOX. The small PDA nanoparticles were synthesized based on a reported method.48 Briefly, Igepal CO-520 (0.65 mL) was added into cyclohexane (10 mL), the mixture was stirred for 10 min, and then ammonium hydroxide (80 μL, 28 wt % in water) was added into the 16794
DOI: 10.1021/acsami.7b01540 ACS Appl. Mater. Interfaces 2017, 9, 16793−16802
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(200 μL) and transferred to a 96-well plate. The fluorescence intensity was recorded at an emission wavelength of 585 nm by using a microplate reader with an excitation wavelength at 482 nm. Cytotoxicity Assay. MDA-MB-231 cells were cultured in a cell culture incubator. Cells were seeded in a 96-well plate with 1 × 104 cells per well. After incubation for 24 h, the cells were treated with various concentrations of PDA-PEG-DOX and PDA-PEG-TPP-DOX NPs and incubated for another 24 or 72 h. The cells were then washed once with PBS, and then new medium containing CCK-8 was added into cells. After 4 h incubation, the UV absorbance at λ = 450 nm was measured using a microplate reader. The percentage of cell viability was calculated based on UV absorbance changes. The experiments were repeated 3 times for statistical analysis. Mitochondrial Membrane Potential Measurement. The mitochondrial membrane potential measurement was performed by using a TMRE mitochondrial membrane potential assay kit (Abcam). Briefly, the MDA-MB-231 cells were seeded in a 96-well plate with 1 × 104 cells each well. After incubation for 24 h, the cells were treated with 5 μg/mL PDA-PEG-DOX and PDA-PEG-TPP-DOX NPs and incubated for another 24 h. TMRE with a final concentration of 200 nM was then added to cells in the medium, and cells were further incubated at 37 °C for 20 min. The cells were then washed once with PBS, and then the fluorescence intensity of TMRE was recorded at an emission wavelength of 575 nm by using a microplate reader at an excitation wavelength of 549 nm. The images of stained cells were captured by fluorescence microscopy. The FCCP-treated cells were used as positive control. Long-Time Treatment with PDA-PEG-DOX and PDA-PEGTPP-DOX NPs. As we know, drug resistance was induced within cancer cells by long-time repetitive treatment with chemotherapeutic agents (such as DOX) in clinical settings of cancer treatment. Considering the repeated drug treatment in clinical cases, here we performed long-term repeated treatment on cancer cells using PDAPEG-DOX and PDA-PEG-TPP-DOX nanoparticles. MDA-MB-231 cells were treated with 0.1 μg/mL PDA-PEG-DOX for 48 h as one treatment cycle, and the treatment was repeated for four cycles. On the other hand, MDA-MB-231 cells were also treated with 0.1 μg/mL PDA-PEG-TPP-DOX for 48 h as one treatment cycle; the treatment was also repeated four times. Then the cells were treated with PDAPEG-DOX and PDA-PEG-TPP-DOX for another 24 h. Western Blot. The cells were also lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1 mM EDTA) with complete EDTA-free protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride, and 5 mM phosphatase inhibitor “sodium orthovanadate”. Protein lysates were resolved by SDSPAGE, transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Bio-Rad), immunoblotted with antibodies, and visualized by ECL (Bio-Rad). Protein levels were normalized to GAPDH. The primary antibodies (mouse monoclonal anti-GAPDH (Abcamab8245), rabbit monoclonal anti-Caspase-3 (Abcamab32351), mouse monoclonal anti-PCNA (Abcamab29)) and secondary antibodies (antirabbit and antimouse IgG (Fc) AP conjugate) were used for immunoblotting. Statistical Analysis. Student’s t test was applied to examine the differences among variables. Data were shown as mean ± SD. *p values ≤ 0.05 are considered to be statistically significant.
above mixture followed by ultrasonic treatment for 20 min. After stirring for another 30 min, DA·HCl aqueous solutions (25 wt %, 7.5 μL) were injected into the reaction mixture. After 24 h at 20 °C, the NPs were precipitated using ethanol, collected by centrifugation, and washed with ethanol and water. Finally, the polydopamine nanoparticles were resuspended in ethanol for future use. For the synthesis of PDA-PEG NPs, PDA NPs (5 mg) were resuspended in DI water (10 mL) under sonication, and then this NPs solution mixed with 4branched-PEG-NH2 (10 mg) under vigorous stirring for 24 h to obtain PDA-PEG NPs. In the modification of PDAPEG NPs with the TPP ligand, the synthesized PDA-PEG NPs (2.5 mg) were resuspended in DI water (1 mL); then TPP (10 mg/mL, 42 μL), EDC·HCl (2 mg) and Sulfo-NHS (2 mg) were added successively to resuspended PDA-PEG NPs solution. The mixture was stirred for 24 h, and the PDA-PEG-TPP NPs were collected by centrifugal ultrafiltration (MWCO10kD). The amount of TPP modified onto the nanoparticles was calculated by deducting the TPP in the supernatant from initial TPP according to the UV−vis standard curve of TPP (Figure S3b). To load DOX onto the PDA-PEG and PDA-PEG-TPP NPs, PDAPEG and PDA-PEG-TPP NPs (2.5 mg) were suspended in 1 mL of DI water with DOX (0.5 mg), and the mixtures were incubated for 12 h; then the products were ultrafiltration centrifuged (MWCO10kD) and the PDA-PEG-DOX and PDA-PEG-TPP-DOX NPs obtained. The amount of loaded DOX onto the nanoparticles is calculated by deducting the DOX in the supernatant from initial DOX according to the standard UV−vis curve of DOX (Figure S3a). To investigate the in vitro DOX release from the nanoparticles, DOX-loaded nanoparticles with determined DOX amount were suspended in 5 mL of release buffer (pH 5.0 acetate buffer or pH 7.4 PBS) at 37 °C with gentle shaking. At determined time points, 1 mL of suspension was ultrafiltration centrifuged (MWCO10kD) and 500 μL of supernatant was collected for UV−vis analysis. The remaining supernatant and nanoparticles as well as 500 μL of fresh buffer were added back to the initial suspension. Cell Culture. MDA-MB-231 cells were cultured in DMEM with high glucose, L-glutamine, 10% fetal bovine serum (FBS, Thomas Scientific), 1% nonessential amino acids, and 1% penicillin/ streptomycin (Invitrogen) and without phenol red. The culture condition was maintained in a humidified atmosphere containing 5% CO2 under 37 °C. Confocal Microscopy. MDA-MB-231 cells were seeded on 35 mm cell culture dishes in 3 × 105 cells per dish and cultured in DMEM for 24 h. The cells were then treated with PDA-PEG-DOX and PDAPEG-TPP-DOX NPs for 12 h. Then the cells in dishes were washed with PBS three times. The cells were then stained Mito Tracker Green (50 nM) and Lyso-Tracker Green (50 nM) for mitochondria and lysosomes, respectively, in culture media for 45 min. After staining, the cells were washed with PBS three times and fixed with the treatment of 10% formalin at 4 °C for 15 min. Lastly, the fixed cells were washed with PBS three times before taking images by confocal microscopy. Mitochondria Isolation and Fluorescence Intensity Analysis. Isolation of mitochondria from MDA-MB-231 cells was conducted by using a mitochondria isolation kit (Thermo Fisher). Briefly, around 1 × 107 MDA-MB-231 cells were cultured in a flask T-75. After treating the cells with 1 μg/mL PDA-PEG-DOX and PDA-PEG-TPP-DOX for 12 h, the cells were harvested. Cells were pelleted by centrifuging the cell suspension in a 2.0 mL centrifuge tube at 850g for 2 min. Pelleted cells were treated with a cell rupturing reagent A (800 μL) under vortex at a medium speed for 5 s and incubated on ice for 2 min. After that the mitochondrial isolation reagent B (10 μL) was added into the pelleted cells, and the mixtures were further vortexed at a maximum speed for 5 s. After incubating the mixtures in tubes on ice for 5 min, the isolation reagent C (800 μL) was added into the mixture, and the mixtures in tubes were inverted several times. After that the mixtures in tubes were centrifuged at 700g at 4 °C for 10 min. The supernatant was transferred to a new tube (2.0 mL). The mitochondria were pelleted by centrifuging the supernatant in a tube at 12 000g at 4 °C for 15 min. The pelleted mitochondria were washed two times with PBS. This mitochondria-containing pellet was resuspended in PBS
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RESULTS AND DISCUSSION Preparation and Characterization of PDA, PDA-PEG, and PDA-PEG-TPP. The PDA were synthesized according to a previously reported reverse microemulsion method.39,48 The synthesized nanoparticles were further characterized by transmission electron microscopy (TEM). PDA nanoparticles are in spherical morphology with a narrow size distribution (Figure 1A). The average diameter of the PDA is around 28 nm, based on the manual statistics of 300 nanoparticles using Gatan software (Figure 1B), which is consistent with the DLS particle 16795
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after modification of PEG and TPP, the PDA-PEG and PDAPEG-TPP were not as uniform as pristine PDA. However, as these two modified NPs showed very similar morphology and size, the homogeneity change was believed to have an insignificant effect on the later results. Furthermore, the cell viability test showed the free TPP and PDA-PEG-TPP have no significant cytotoxicity (Figure S4), indicating synthesized nanoparticles can be used as a safe drug carrier in the following studies. DOX is an anticancer drug that has been widely used in a number of cancer types such as leukemia, lymphoma, carcinoma, etc. Herein, we employed DOX·HCl as an anticancer drug model throughout this study for our PDAbased mitochondria-targeted delivery system. We reasoned that DOX with its aromatic ring would be effectively loaded on the surface of PDA NPs through the hydrophobic interaction, π−π stacking, and electrostatic attraction.39 To verify this and measure the loading efficiency, 2.5 mg of PDA-PEG and 2.5 mg PDA-PEG-TPP were incubated with 0.5 mg of DOX in DI water overnight. After DOX loading, unbound DOX was completely removed by centrifugal ultrafiltration and the supernatant was retained. The loading efficiency of DOX was determined according to the UV−vis absorption standard curve of DOX with various concentrations of DOX at 482 nm (Figure S3a). The DOX loading capacity is determined to be 0.475 and 0.125 mg for 2.5 mg of PDA-PEG and PDA-PEGTPP, respectively, based on the UV−vis absorption intensities changes in the supernatant. The electronic repulsion between the positive DOX and the highly positive TPP ligand on the surface of PDA-PEG-TPP may result in the lower loading capacity of DOX on the PDA-PEG-TPP. Despite the difference in DOX loading capacities, these two drug-loaded nanoparticles have similar pH-sensitive drug release profiles (Figure S5), i.e., the DOX molecules on both nanoparticles showed higher release in pH 5.0 compared to pH 7.4, but there was no significant difference between the release profile of the two types of nanoparticles in each pH.53,54 In the following sections, the concentrations of PDA-PEG-DOX and PDA-PEG-TPPDOX are expressed as the DOX concentration unless indicated specifically. Short Time Treatment by PDA-PEG and PDA-PEGTPP. The free DOX enter the cytoplasm and then diffuse to the nucleus and bind to the nuclear DNA, which leads to cell apoptosis.55,56 Differently, PDA-PEG-TPP-DOX is functionalized with mitochondrial targeting ligand TPP, which facilitates
Figure 1. Characterization of PDA, PDA-PEG, and PDA-PEG-TPP. (A) TEM images of PDA and PDA-PEG-TPP. (B) Average diameter of the PDA is around 28 nm, based on manual measurement of 300 nanoparticles using the Gatan software of the TEM system. (C) Zeta potential of PDA, PDA-PEG, and PDA-PEG-TPP in H2O.
size (Figure S1a). The small particle size is favorable for these nanoparticles to enter the mitochondria.29 Although PDA can be well suspended in water, their colloidal stability in physiological saline is not good enough. Therefore, PDA were further functionalized with amine-terminated PEG via the Michael addition and/or Schiff base reaction to improve their colloidal stability.49 The stability test indicated that the stability of the PDA-PEG was significantly improved in PBS and DMEM (Figure S2) compared with pristine PDA, which could be due to the modified PEG. The characteristic FTIR spectral peak of 1100 cm−1 (C−O−C stretching) also verified the PEG modification (Figure S1b).50−52To obtain PDA-PEG-TPP NPs, TPP ligand was then modified onto the PDA-PEG, which could be proved by the new Ph-P band at 1439 cm−1 (Figure S1b). It should be noted that after the TPP modification, the nanoparticles still showed excellent stability in PBS and DMEM (Figure S2), which is expected to benefit the nanoparticle delivery in body fluids. Zeta potential changes of PDA, PDA-PEG, and PDA-PEG-TPP in water further verified that the PDA NPs were successfully modified with amineterminated PEG and TPP (Figure 1C). As shown in Figure 1A,
Figure 2. Confocal microscopy images of MDA-MB-231 cells treated with PDA-PEG-DOX and PDA-PEG-TPP-DOX. DOX emits red fluorescence. Mitochondria were stained by 50 nM Mito-Tracker Green emit green fluorescence. Cell nuclei were stained with 10 ng/mL DAPI (blue fluorescence) for 10 min. 16796
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PDA-PEG-TPP-DOX targeting delivered DOX to the mitochondria efficiently. Previous reports demonstrate that loss of mitochondrial membrane potential (Δψm) is an early event in mitochondriatriggered apoptosis, and mtDNA expression alterations are closely associated with the mitochondrial membrane potential.11,57,58 To investigate mitochondria dysfunction caused by NPs, we therefore studied intracellular mitochondrial membrane potential changes caused by both PDA-PEG-DOX and PDA-PEG-TPP-DOX NPs with a short time treatment (24 h). Tetramethylrhodamineethyl ester (TMRE) mitochondrial membrane potential assay kit (Abcam) was used to measure the mitochondrial potential. In this kit, TMRE stains mitochondria with active membrane potential. Since mitochondrial membrane potential and TMRE staining could be removed after cells were treated with carbonylcyanide 4(trifluoromethoxy) phenylhydrazone (FCCP),58 we used FCCP-treated cells as a positive control. As shown in Figure 4, different levels of decrease in mitochondrial membrane
nanoparticle penetration through the mitochondria membrane. We reasoned that DOX-loaded PDA-PEG-TPP would effectively deliver DOX to mitochondria, and the released free DOX would bind to mtDNA and trigger the apoptosis of cancer cells. To evaluate the mitochondrial targeting effect of PDA-PEG-TPP-DOX, we incubated human breast carcinoma cells (MDA-MB-231) with PDA-PEG-DOX and PDA-PEGTPP-DOX for 12 h, respectively. The cells were further stained with mitochondria-selective probe Mito-Tracker Green and imaged by confocal laser scanning microscopy. DOX and MitoTracker Green emit red and green fluorescence, respectively. The merged image of red fluorescence and green fluorescence shows most of the PDA-PEG-DOX are accumulated in the cell nucleus (Figure 2). However, the yellow fluorescence in the cytoplasm of the cells treated with PDA-PEG-TPP-DOX indicates the successful mitochondria-targeted delivery of DOX with PDA-PEG-TPP-DOX (Figure 2). Furthermore, a confocal laser microscopy study was carried out in order to investigate the interaction between lysosomes and the PDAPEG-DOX and PDA-PEG-TPP-DOX. The lysosomes were stained by Lyso-Tracker Green emitting green fluorescence.The merged images of red and green fluorescence show the PDAPEG-DOX are more accumulated in lysosomes than PDAPEG-TPP-DOX after 4 and 8 h treatment (Figure S6). Such a relatively quick escape of PDA-PEG-TPP-DOX from lysosomes is probably a result of the good lipophilicity of the TPP targeting ligand, which leads to the quick interaction between PDA-PEG-TPP-DOX and mitochondria. To further investigate the mitochondrial targeting efficiency of the PDA-PEG-TPP-DOX, we measured the red fluorescence intensity of DOX in the mitochondria from PDA-PEG-DOXand PDA-PEG-TPP-DOX-treated MDA-MB-231 cells, respectively. After incubation of MDA-MB-231 cells with PDA-PEGDOX and PDA-PEG-TPP-DOX for 12 h, the mitochondria were isolated by using a mitochondria isolation kit (Thermo Fisher). Then the isolated mitochondria fractions were washed by phosphate-buffered saline (PBS) once before measurements to eliminate fluorescence interference from free nanoparticles. As shown in Figure 3, the red fluorescence intensity of DOX in the isolated mitochondria fraction of the PDA-PEG-TPP-DOXtreated group is about three times higher than that of the PDAPEG-DOX-treated group. The results above suggest that the
Figure 4. Mitochondrial membrane potential Δψm of MDA-MB-231 cells treated with 5 μg/mL PDA-PEG-DOX and PDA-PEG-TPP-DOX for 24 h. PBS treatment as control group; FCCP treatment as positive control. Δψm was measured by fluorescence intensity after cells were stained with TMRE. Data are represented as mean ± SD (n = 3). *: p < 0.05. **: p < 0.001 (two-tailed Student’s t test).
potential were observed when cells were treated with PDAPEG-DOX and PDA-PEG-TPP-DOX NPs. However, the decrease of mitochondrial membrane potential in the PDAPEG-TPP-DOX NPs-treated cells is more significant than that of the PDA-PEG-DOX NPs treatment (Figure 4 and Supporting Information Figure S7). These results suggested that mitochondria were damaged significantly by mitochondriatargeted PDA-PEG-TPP-DOX than PDA-PEG-DOX. We also investigated the cytotoxicity of both PDA-PEGDOX and PDA-PEG-TPP-DOX with short time treatment (24 h) at different DOX concentration using Cell Counting Kit-8 (CCK-8). The result showed that the cell viability in the PDAPEG-TPP-DOX treatment group was significantly lower than that of the PDA-PEG-DOX treatment with DOX concentration at 1 μg/mL (Figure 5A), while no significant differences in cell viabilities were observed at other DOX concentrations (0.5 and 5 μg/mL). It is reported that the cell viability was usually determined by cell death and cell proliferation.59 Therefore, we
Figure 3. Red fluorescence intensities of DOX in mitochondria isolated from MDA-MB-231 cells treated with 1 μg/mL of PDA-PEGDOX and PDA-PEG-TPP-DOX. PBS was used as the negative control. Data are represented as mean ± SD (n = 3). 16797
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investigated a cell apoptosis marker pro-Caspase 3 and a cell proliferation marker proliferating cell nuclear antigen (PCNA) expression level changes after treatment with both PDA-PEGDOX and PDA-PEG-TPP-DOX for a short time (24 h). As shown in Figure 5B, Western blot result shows that there is no obvious difference in pro-Caspase 3 expression level between PDA-PEG-DOX and PDA-PEG-TPP-DOX treatment, although we observed that the pro-Caspase 3 expression level in the control group was higher than that of both the PDAPEG-DOX and the PDA-PEG-TPP-DOX treatment group. Similarly, in terms of the expression level of PCNA, no obvious difference was observed between all groups. Those results suggested that both PDA-PEG-DOX and PDA-PEG-TPP-DOX with short-term treatment (24 h) caused cell apoptosis, but there was no obvious difference in cell apoptosis between these two groups. Long Time Treatment by PDA-PEG and PDA-PEG-TPP. Drug resistance is a huge challenge in cancer treatment, which results from repetitive treatment with chemotherapeutic agents. Cancer cells usually were induced to adapt to and resist the chemotherapeutic drug after a few treatment cycles.11,36 Some elegant drug delivery strategies have been developed to reverse some drug-resistance cell lines. However, those strategies did not overcome drug resistance in clinical settings due to the complexity of the drug-resistance mechanisms. Herein, we investigated whether long-term treatment with mitochondriatargeted PDA-PEG-TPP-DOX could overcome anticancer drug resistance. Initially we treated the MDA-MB-231 cells with PDA-PEGDOX or PDA-PEG-TPP-DOX containing 0.1 μg/mL DOX for 48 h, and this treatment was repeated for 4 cycles. Then the cells were further treated with PDA-PEG-DOX and PDA-PEGTPP-DOX containing 5 μg/mL DOX. As shown in Figure 6, the mitochondrial membrane potential of cells treated by the PDA-PEG-TPP-DOX group and the PDA-PEG-DOX group decreased compared with the control group, while the reduction of the PDA-PEG-TPP-DOX group was more significant than that of the PDA-PEG-DOX group (Figure 6, Supporting Information Figures S8 and S9). This result indicated that the mitochondrial membrane of cells was damaged after long-term treatment with both PDA-PEGDOX and PDA-PEG-TPP-DOX. However, PDA-PEG-TPPDOX caused more severe damage to the mitochondrial
Figure 5. Cytotoxicity of PDA-PEG-DOX and PDA-PEG-TPP-DOX treatment for 24 h. (A) Cell viability assay of MDA-MB-231 cells treated with different concentrations of PDA-PEG-DOX and PDAPEG-TPP-DOX for 24 h. PBS was used as negative control. Data are represented as mean ± SD (n = 3). *: p < 0.05 (two-tailed Student’s t test). Purple dashed line represents the average cell viability of 75% treated with 1 μg/mL PDA-PEG-DOX, and purple dotted line represents the average cell viability of 60% treated with 5 μg/mL PDAPEG-DOX. (B) Western blot assay for Caspase 3 and PCNA in MDAMB-231 cells treated with different concentrations of PDA-PEG-DOX and PDA-PEG-TPP-DOXfor 24 h. PBS served as negative control, and GAPDH as an inner control.
Figure 6. Mitochondrial membrane potential of MDA-MB-231 cells with a long-term treatment of NPs. MDA-MB-231 cells were treated with 0.1 μg/mLPDA-PEG-DOX (left) and PDA-PEG-TPP-DOX (right) NPs for 48 h and repeated 4 cycles. Then the cells were treated with 5 μg/mL PDAPEG-DOX and PDA-PEG-TPP-DOX for another 24 h. Data are represented as mean ± SD (n = 3). *: p < 0.05 (two-tailed Student’s t test). 16798
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ACS Applied Materials & Interfaces
Figure 7. Cytotoxicity of MDA-MB-231 cells treated with PDA-PEG-DOX and PDA-PEG-TPP-DOX for a long time (48 h × 4 cycles). (A) MDAMB-231 was treated with 0.1 μg/mL PDA-PEG-DOX (left) and PDA-PEG-TPP-DOX (right) NPs for 48 h with 4 cycles. Then the cells were treated with different concentrations of PDA-PEG-DOX and PDA-PEG-TPP-DOX for another 24 h. Treatment with PBS served as control. Data represent mean ± SD (n = 3). *: p < 0.05 (two-tailed Student’s t test). Purple dashed line represents the average cell viability of 75% treated with 1 μg/mL PDA-PEG-DOX in Figure 5A, and purple dotted line represents the average cell viability of 60% treated with 5 μg/mL PDA-PEG-DOX in Figure 5A. (B) Western blot assay for Caspase 3 and PCNA in MDA-MB-231 cells treated with 0.1 μg/mL PDA-PEG-DOX (left) and PDA-PEGTPP-DOX (right) for 8 days, followed by treatment with different concentrations of PDA-PEG-DOX and PDA-PEG-TPP-DOX NPs for another 24 h. PBS treatments after pretreatment by PDA-PEG-DOX or PDA-PEG-TPP-DOX NPs were used as controls. GAPDH is an inner control.
DOX and PDA-PEG-TPP-DOX (Figure S10). To evaluate the short-term and long-term treatment effects, we calculated that solid purple lines in Figure 7A would represent 75% cell viability of the cells treated with 1 μg/mL PDA-PEG-DOX for short time (Figure 5A) and purple dash lines represented the 60% cell viability of the cells treated with 5 μg/mL PDA-PEGDOX for short time (Figure 5A). We observed that after treatment with 0.1 μg/mL PDA-PEG-DOX for 48 h × 4 cycles, the cell viabilities of cells with 0.5 and 1 μg/mL PDA-PEGDOX and PDA-PEG-TPP-DOX treatment were higher than 75% (black line), while the cell viabilities of cells with 5 μg/mL PDA-PEG-DOX and PDA-PEG-TPP-DOX were a little below 60%. Those results suggested that cells might be drug resistant after long-term PDA-PEG-DOX treatment. However, after treatment with 0.1 μg/mL PDA-PEG-TPP-DOX for 48 h × 4 cycles, the cell viability in cells treated with 1 μg/mL PDAPEG-TPP-DOX were lower than 75% and the cell viability in cells treated with 5 μg/mL PDA-PEG-DOX and PDA-PEGTPP-DOX were both lower than 60%. The results indicated
membrane of cells than that of PDA-PEG-DOX, which suggested that PDA-PEG-TPP-DOX had better efficiency in long-term DOX treatment. We further investigated the long-term cytotoxicity of PDAPEG-DOX and PDA-PEG-TPP-DOX using CCK-8 assay. After treatment with 0.1 μg/mL PDA-PEG-DOX for 48 h × 4 cycles, the cells were incubated with different concentrations of PDAPEG-DOX and PDA-PEG-TPP-DOX for another 24 h, while PBS treatment served as a control. The result shows that the cell viability in cells with the PDA-PEG-TPP-DOX treatment is just a little lower than that of PDA-PEG-DOX treatment (Figure 7A). However, after long-term treatment with 0.1 μg/ mL PDA-PEG-TPP-DOX, cell viability in the cells treated with 1 and 5 μg/mL PDA-PEG-TPP-DOX NPs is significantly lower than those of the PDA-PEG-DOX treatment counterparts (Figure 7A). Similar but more profound cytotoxicity differences were observed when the cells were incubated with different concentrations of PDA-PEG-DOX and PDA-PEG-TPP-DOX a total of 72 h after pretreatment with 0.1 μg/mL PDA-PEG16799
DOI: 10.1021/acsami.7b01540 ACS Appl. Mater. Interfaces 2017, 9, 16793−16802
Research Article
ACS Applied Materials & Interfaces that PDA-PEG-TPP-DOX with mitochondria-targeted delivery could reduce drug resistance. Additionally, we investigated the expression level changes of cell death marker pro-Caspase 3 and cell proliferation marker PCNA. As shown in Figure 7B, after treatment with 0.1 μg/mL PDA-PEG-DOX or 0.1 μg/mL PDA-PEG-TPP-DOX for 48 h × 4 cycles, the pro-Caspase 3 expression level decreased in both the PDA-PEG-TPP-DOX and the PDA-PEG-DOX treatment groups compared with the control group. Meanwhile, the proCaspase 3 expressions were lower in 1 and 5 μg/mL PDA-PEGTPP-DOX than that of PDA-PEG- DOX. However, no obvious difference of PCNA expression level was observed between all treated groups with the control group. The results are consistent with the cell viability assay obtained from longterm NPs treatment, together indicating that the mitochondriatargeted PDA-PEG-TPP-DOX reduce long-term drug resistance.
Si-Yang Zheng: 0000-0002-0616-030X Author Contributions †
W.L. and Z.W. contributed equally to this work.
Funding
We appreciate the National Cancer Institute of the National Institutes of Health for funding this work (Award Number DP2CA174508). Notes
The authors declare no competing financial interest.
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CONCLUSION In conclusion, we synthesized drug delivery nanoparticle PDAPEG and mitochondria-targeted drug delivery nanoparticle PDA-PEG-TPP in which TPP was employed as mitochondrial targeting ligand. Anticancer drug DOX has been successfully loaded onto the two drug delivery systems. The colocalization experiments show that PDA-PEG-TPP could deliver DOX into mitochondria by using PDA-PEG-TPP. PDA-PEG-TPP-DOX with a short-term treatment plan showed higher toxicity toward MDA-MD-231 cells than PDA-PEG-DOX. Meanwhile, longterm repetitive treatment with mitochondria-targeted PDAPEG-TPP-DOX could contribute to overcome drug resistance, while long-term repetitive treatment with traditional PDAPEG-DOX did not. This work demonstrated that the mitochondria-targeted polydopamine nanoparticles loaded with anticancer drug could potentially overcome drug resistance, which would further improve the chemotherapeutic efficacy and patient outcome in cancer.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01540. DLS of PDA and FTIR spectra of PDA, PDA-PEG, and PDA-PEG-TPP; suspensions of PDA, PDA-PEG, and PDA-PEG-TPP in PBS and DMEM; UV−vis absorption standard curve of DOX and TPP; cell viabilityof with different concentration of TPP and PDA-TPP for 24 h; accumulative release profiles of DOX in 0.1 M pH 5.0 acetate buffer and pH 7.4 PBS; confoal micrscopy images of the PDA-PEG-DOX and PDA-PEG-TPP-DOX in lysosomes; mitochondrial membranepotential assay images for 24 h; mitochondrial membranepotential assay images with PDA-PEG-DOX for a long time; mitochondrial membranepotential assay images with PDA-PEG-TPP-DOX for a long time; cell viability of long time treated with PDA-PEG-DOX and PDA-PEGTPP-DOX then for another 72 h treatment (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: (814) 865-8090. E-mail:
[email protected]. ORCID
Gong Cheng: 0000-0002-2217-6408 16800
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