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Evaluation of in vitro toxicity of polymeric micelles to human endothelial cells under different conditions
Chemico-Biological Interactions 263 (2017) 46e54
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Evaluation of in vitro toxicity of polymeric micelles to human endothelial cells under different conditions Fang Liu a, Haikang Huang b, Yu Gong a, Juan Li a, Xuefei Zhang b, *, Yi Cao a, ** a Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry of Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University, Xiangtan 411105, PR China b Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry of Education, Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province and Key Laboratory of Polymeric Materials & Application Technology of Hunan Province, College of Chemistry, Xiangtan University, Xiangtan 411105, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 October 2016 Received in revised form 16 December 2016 Accepted 22 December 2016 Available online 23 December 2016
Polymeric micelles have been extensively studied in the area of antitumor therapy, and more recently explored as nanocarriers for atherosclerosis. These applications of polymeric micelles in biomedicine will increase their contact with human blood vessels. However, few studies have considered the interactions between polymeric micelles and endothelial cells, especially in a complex system. This study used human umbilical vein endothelial cells (HUVECs) as an in vitro model for endothelial cells to investigate the toxic effects of methoxy-poly(ethylene glycol)-poly(D,L-lactide) (MPEG-PLA) based micelles. In addition, an endoplasmic reticulum stress inducer thapsigargin (TG), and a pro-atherogenic stimulus palmitate (PA), were used to co-expose HUVECs to further mimic the responses of diseased endothelial cells to micelle exposure. Overall, up to 200 mg/mL micelles did not significantly induce cytotoxicity, reactive oxygen species (ROS), release of inflammatory mediators in terms of interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), or adhesion of THP-1 monocytes to HUVECs. TG and PA significantly induced cytotoxicity and THP-1 adhesion as well as modestly promoted the release of IL-6, but did not affect ROS or release of sVCAM-1 and IL-8. Co-exposure of micelles did not significantly enhance the effects of TG and PA to HUVECs, and ANOVA analysis indicated no interaction between concentrations of micelles and the presence of TG/PA. Taken together, these data indicated that micelles are not toxic to HUVECs under different conditions in vitro. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Micelles Methoxy-poly(ethylene glycol)-poly(D,Llactide) (MPEG-PLA) Human umbilical vein endothelial cells (HUVECs) Thapsigargin (TG) Palmitate (PA)
1. Introduction With their unique advantages, a number of engineered nanoparticles (NPs) has been produced for biomedical uses, but their potential adverse effects should be carefully assessed to ensure the safe use [1]. Polymeric micelles, formed by amphiphilic copolymers with a hydrophilic shell and a hydrophobic core, are among one of the most commonly produced engineered NPs and have been extensively studied for anticancer therapy [2,3]. Recently, it was also shown that polymeric micelles may be used for atherosclerosis imaging and therapy [4,5]. As a consequence, the applications of polymeric micelles in biomedicine will inevitably increase their contact with human blood vessels, but few studies have concerned
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.cbi.2016.12.014 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
their side effects to relevant cells in the circulation system [6]. One of such relevant cells that should be used to understand NP-blood vessel contact is endothelial cell (EC), as suggested by a recent review [6]. The ECs cover the surface area and regulate the blood vessel tone, thrombogenicity, homeostasis, monocytes recruitment, and hormone transport [7]. Furthermore, they also serve as the first contact cells with engineered NPs when the NPs enter the blood [6]. During the early development of atherosclerosis, inflammatory and oxidative stimuli activate ECs, which express adhesion molecules and release inflammatory mediators. Circulating monocytes then adhere to the activated ECs, differentiate into macrophages, engulf excessive lipids and finally lead to plaque progression [7,8]. Whether polymeric micelles will induce adverse effects to ECs needs to be carefully assessed to ensure their safe use in nanomedicine. This should be done not only in ‘healthy’ ECs, but also diseased ECs, as the response of ECs to NPs could be different due to the diversity of EC types [6]. There may be also a need to assess the
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toxicity of polymeric micelles to ECs in a complex system where the nutrients are present, as the nutrients may also define the nanotoxicological responses [9,10]. The present study investigated the toxicity of micelles to human vein ECs (HUVECs) under different conditions. The HUVECs were used because they have been extensively used as an in vitro model for human ECs to understand the interactions of engineered NPs with blood vessels [6]. The empty micelles without any associated drugs were used because a recent study showed that drug-loaded micelles may induce drug-related hematological toxicity [11]. The cytotoxicity was determined by three different methods, namely water soluble tetrazolium-1 (WST-1), neutral red uptake and lactate dehydrogenase (LDH) assay. Oxidative stress was indicated by the measurement of intracellular reactive oxygen species (ROS). The inflammatory response was indicated by the measurement of release of inflammatory mediators, including interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), as well as the adhesion of THP-1 monocytes to HUVECs. Meanwhile, to induce the injury of HUVECs and mimic the response of ECs under atherosclerotic conditions, thapsigargin (TG) or palmitate (PA) was used to co-expose HUVECs. TG is a classical endoplasmic reticulum (ER) stress inducer, and elevated ER stress has been suggested to play a pivotal role in the pathology of atherosclerosis [12,13]. A recent study showed that ZnO NPs could induce ER stress to HUVECs, which may be related with the cardiovascular effects [14]. PA is one of the basic fatty acids present in human blood. Elevated circulating PA may induce endothelial dysfunction and thus links excessive nutrients to the development of atherosclerosis [13,15]. A recent study showed that the presence of PA may enhance the vascular health effects of multi-walled carbon nanotubes to HUVECs in vitro, which indicated an interaction between saturated fatty acids and NPs [16]. 2. Materials and methods 2.1. Cell culture HUVECs (passage 1; purchased from ScienCell Research Laboratories, Carlsbad, CA) were cultured in supplemented endothelial medium and used at passage 3e6 as our previously described [17]. For each experiment, 1 104/well (on 96-well plates) or 4 104/ well (on 24-well plates) HUVECs were seeded on 0.2% gelatin solution (ScienCell Research Laboratories, Carlsbad, CA) pre-coated plates and grown for 2 days prior to exposure. THP-1 monocytes (ATCC) were cultured in RPMI 1640 medium (sodium pyruvate and HEPES added; Gibco, USA) supplemented with 10% FBS and 1% penicillin-streptomycin (P/S) solution in a CO2 incubator at 37 C. The cells were used within 3 month to keep their best characteristics. 2.2. Preparation of polymeric micelles The amphiphilic copolymer methoxy-poly(ethylene glycol)poly(D,L-lactide) (MPEG-PLA) was synthesized as our previously described with slight modifications [18]. Briefly, 0.5 g lactide monomer (LA) was reacted with 1.0 g methoxy-poly(ethylene glycol) (MPEG) in 1 mL CH2Cl2 under nitrogen gas condition, catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-CH2Cl2 ([M]/ [Cat] ¼ 100). Here we used a non-metallic reagent TBD as the catalyst to reduce the possible metallic contaminations in micelles [19,20]. After 48 h reaction under room temperature, the polymer/ CH2Cl2 solution was precipitated by absolute ether, followed by vacuum drying to remove CH2Cl2. To make the micelle solution, MPEG-PLA was dissolved in acetone, followed by drop wise addition of the MPEG-PLA acetone solution into MilliQ water during
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slowly constant stirring. The cloudy micelle solution was further constantly stirred for 0.5 h, and then rotary evaporated overnight under room temperature to remove acetone. Finally, the stock solution of micelles was made as 2 mg/mL in MilliQ water. The critical micelle concentration (CMC) of the polymeric micelles was determined by using a fluorescent probe pyrene. A stock solution of pyrene was made as 6 mg/mL in acetone. A working solution was then prepared by adding 0.1 mL of pyrene solution into 1000 mL MilliQ water with constant stirring for 2 h, after which the working solution was placed at room temperature for 2 days to allow the acetone to evaporate. Micelles in MilliQ water were diluted to a series of concentrations from 0.0001 mg/mL to 1 mg/mL and mixed with equal concentrations of pyrene solution (final volume 10 mL). Micelleepyrene solutions were briefly sonicated in ultrasonic water bath for 15 min and then placed for 12 h under room temperature to allow partition of the pyrene into the micelles. Emission was done from 230 to 360 nm, with 392 nm as the excitation wavelength. The maximum absorption of pyrene shifted from 335.91 to 339.05 nm on micelle formation. The ratio of absorption of encapsulated pyrene (339.05 nm) to pyrene in water (335.91 nm) was plotted as the logarithm of polymer concentrations. The inflection point of the curve was taken as the CMC. 2.3. Characterizations 1
H nuclear magnetic resonance (NMR) spectra of the synthesized MPEG-PLA were measured by a Bruker AV-400 NMR spectrometer at room temperature, with CDCl3 as solvent. The molecular weight of MPEG-PLA was measured by gel permeation chromatography (GPC) using a PL-GPC 120 instrument (UK), with tetrahydrofuran (THF) as eluent at a flow rate of 1.0 mL/min. The hydrodynamic size distribution of the polymeric micelles was measured using 1 mg/mL particles suspended in MilliQ water by Zetasizer Nano ZS90 (Malvern, UK). The hydrodynamic size was measured for three times, and mean ± S.D. (standard deviation) was calculated. The stability of the micelles solution was tested as changes of hydrodynamic size distribution after stored at room temperature for 10 and 20 days. The micelles were used within 20 days for all the experiments in this study as they slowly precipitated after 20-day storage at room temperature. 2.4. Cytotoxicity The cytotoxicity was measured by using WST-1, neutral red uptake and LDH kits according to manufacturer's instruction (Beyotime, China). WST-1 reagent can indicate the mitochondrial activity as it could be converted to a yellow formazan by mitochondria in living cells. For the assay, HUVECs seeded on 24-well plates were exposed to 0 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL and 200 mg/mL micelles, with or without the presence of 500 nM TG or 200 mM PA (from Sigma-Aldrich, USA). After 24 h exposure, the cells were rinsed once with Hank's solution, and then incubated with 10% WST-1 reagent for 2 h. The absorbance was read at 450 nm with 690 nm as reference by an ELISA reader (Synergy HT, BioTek, USA). Neutral red is a dye which could be incorporated into intact lysosomes of living cells. For the assay, HUVECs on 24-well plates were incubated with various concentrations of micelles with or without the presence of TG or PA for 24 h as indicated above, rinsed and then incubated with 10% neutral red for 2 h. After rinsed once again, the neutral red incorporated into lysosomes was dissolved in the lysis solution provided by the kit, and the absorbance was read at 540 nm with 690 nm as reference. LDH is an enzyme in cytoplasm, which could be released into extracellular fluid with the break of membrane. The LDH assay was
F. Liu et al. / Chemico-Biological Interactions 263 (2017) 46e54 Fig. 1. Characteristics of the synthesized MPEG-PLA and the micelles based on the MPEG-PLA. 1A, 1H NMR spectra of the synthesized MPEG-PLA. 1B, the GPC measurement of MPEG5000 and MPEG-PLA. Data for Fig. 1A and B were based on a single run. 1C, the hydrodynamic size distribution of micelles. The hydrodynamic diameters of micelles did not significantly change after storage for 20 days. Data for Fig. 1C were representatives from three measurement based on a single run.
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done in parallel with WST-1 assay. Briefly, after exposure the supernatant from WST-1 assay was collected, and 60 mL supernatant from each sample was mixed with 120 mL LDH reaction buffer provided by the kit for 30 min. The absorbance was read at 490 nm with 690 nm as reference. To induce a 100% of LDH release, the positive control cells were incubated with 10% lysis buffer 1 h before the assay and the activity of LDH was measured as indicated above. 2.5. Analysis of reactive oxygen species ROS was estimated by using 20 ,7’-dichlorofluorescein diacetate (DCFH-DA) as previously described [21]. DCFH-DA is cell permeable which could be oxidized to a fluorescent probe by the reaction with a variety of ROS inside the cells. Briefly, HUVECs seeded on black 96-well plates were exposed to various concentrations of micelles with or without the presence of 500 nM TG or 200 mM PA for 3 h. After exposure, the cells were rinsed once with Hanks solution, and then stained by 10 mM DCFH-DA in serum free medium for 30 min. After rinsed again with Hanks solution, the fluorescence was read at ex 485 ± 20 nm and em 528 ± 20 nm by an ELISA reader. To further validate the method, HUVECs were also exposed to H2O2 from 0.0625% to 1% for 3 h, followed by the measurement of ROS as described above. 2.6. Inflammatory response The supernatant from the exposed HUVECs was collected and stored at 80 C before analysis. Inflammatory mediators IL-6, IL-8 and sVCAM-1 was measured by ELISA kits according to manufacturer's instruction (Neobioscience Technology Co., Ltd., China). Some of the samples were diluted for the measurement. 2.7. Monocyte adhesion The adhesion of THP-1 monocytes to HUVECs was done as previously described [16]. Briefly, HUVECs on black 96-well plates were exposed to various concentrations of micelles with or without the presence of 500 nM TG or 200 mM PA for 24 h. Before the end of the exposure, THP-1 monocytes were labeled with 10 mM CellTracker™ Green CMFDA (5-Chloromethylfluorescein Diacetate, Invitrogen, Carlsbad, CA) for 30 min in RPMI 1640 medium. The free probe was removed by centrifuge, and 5 104/well labeled THP1 cells were incubated with the exposed HUVECs for 1 h for adhesion. The doubling time for THP-1 cells was measured to be 31 h [22], therefore it is expected that during 1 h incubation there was minimal proliferation of THP-1 monocytes [16]. After that, the unbound THP-1 cells were washed away, and the fluorescence was read at ex 485 ± 20 nm and em 528 ± 20 nm by an ELISA reader. 2.8. Statistics All the data were expressed as mean ± S.D. (standard deviation) of means of at least three independent experiments. Two-way ANOVA (concentrations and treatment as categorical factors) followed by Tukey HSD test using R 3.2.2; p value < 0.05 was considered to be statistically significant. 3. Results 3.1. Characteristics of MPEG-PLA and micelles 1 H NMR spectra of the synthesized MPEG-PLA is shown in Fig. 1A dA (3.4 ppm) and dBþC (3.66 ppm) belong to methyl group and methylene group of MPEG, respectively. dD (3.84 ppm) and dE
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Fig. 2. Cytotoxicity as measured by WST-1 (2A), neutral red uptake (2B) and LDH (2C) assay. HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, and WST-1, neutral red uptake and LDH assay were used to determine cytotoxicity. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.01, compared with untreated cells.
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Fig. 3. Oxidative stress as indicated by intracellular ROS. 3A, HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 3 h, and intracellular ROS was measured by a fluorescent probe. 3B, HUVECs were exposed to H2O2 from 0.0625% to 1%, followed by DCFH assay to indicate intracellular ROS. Data were expressed as mean ± S.D. (standard deviation) of means of three (3A) or four (3B) independent experiments. *, p < 0.05, ANOVA.
(4.31 ppm) belong to the two methylene group near LA. dF/H (5.19/ 5.21) belong to the two methyne group of LA, whereas dG/I (1.54/ 1.61) belong to the methyl group of LA. 1H NMR analysis indicated the successful formation of MPEG-PLA product. According to the GPC measurement (Fig. 1B), the Mn of MPEG-PLA was calculated as 7785, and PDI was calculated as 1.058 (Mw of the MPEG-PLA is 8242). The CMC was calculated as 3.3 mg/mL, and the concentrations used in this study were all above CMC. The size distribution of micelles based on the MPEG-PLA is shown in Fig. 1C. The micelles had hydrodynamic diameters 20 ± 1 nm, which were not significantly changed after storage for 20 days. After that the micelle solution slowly precipitated, which was not further used for this study. 3.2. Cytotoxicity As illustrated in Fig. 2, WST-1 assay showed significantly decreased viability of HUVECs by the treatment of TG or PA (p < 0.01), whereas ANOVA analysis indicated no dose-dependent effect of micelles up to 200 mg/mL (p > 0.05). For neutral red uptake assay, TG only induced an insignificant decrease in neutral red uptake (p > 0.05). The treatment of TG reduced neutral red uptake to 80.6% (95% CI: 56.3%e105%). PA significantly decreased neutral red uptake (p < 0.01) and the uptake was reduced to 46.6% (95% CI: 22.3%e71%). There was no dose-dependent effect of micelles as revealed by neutral red uptake assay (p > 0.05). The cytotoxicity was further measured by LDH assay to indicate the membrane integrity and result is shown in Fig. 2C. Exposure to TG did not significantly affect LDH release (p > 0.05), whereas PA exposure significantly induced LDH release (p < 0.01). Again, there was no dose-dependent effect of micelles (p > 0.05). 3.3. Analysis of reactive oxygen species The exposure to micelles up to 200 mg/mL did not significantly affect the intracellular ROS (p > 0.05; Fig. 3A). TG or PA treatment did not significantly affect ROS (p > 0.05), and ANOVA analysis showed no interaction between concentrations of micelles and TG/ PA treatment on ROS (p > 0.05). As a positive control, 3 h exposure of HUVECs to H2O2 dose-dependently increased ROS, and there was statistically significantly increased ROS after exposure to 0.5% (p < 0.05) and 1% (p < 0.01) H2O2, respectively, which indicated the
confidence of the assay (Fig. 3B). 3.4. The release of inflammatory mediators Fig. 4 shows the release of inflammatory mediators including IL6 (4A), IL-8 (4B) and sVCAM-1 (4C). For IL-6, micelles alone did not significantly affect the release of IL-6 (p > 0.05). 500 nM TG treatment did not significantly affect IL-6 release either (p > 0.05). With the presence of TG, 200 mg/mL micelle resulted in a modest increase of IL-6, but the increase was not statistically different compared with control (p > 0.05). There was a single factor effect by 200 mM PA treatment (p < 0.01, ANOVA); however, Tukey HSD posthoc analysis showed that IL-6 release was not significantly elevated by micelles with the presence of PA compared with control (p > 0.05). In addition, ANOVA test indicated no significant interaction between concentrations of micelles and TG/PA treatment (p ¼ 0.08). For IL-8 and sVCAM-1, micelles did not significantly affect the release with or without the presence of TG/PA (p > 0.05). 3.5. Monocyte adhesion As shown in Fig. 5, when TG or PA was absent, all the concentrations of micelles did not significantly promote the adhesion of THP-1 monocytes to HUVECs (p > 0.05). TG or PA treatment significantly promoted the adhesion to a comparable level (ca. 50% increase to control; p < 0.01). With the presence of TG, all the concentrations of micelles significantly induced the adhesion compared with control (p < 0.01). Similarly, with the presence of PA, 12.5 mg/mL (p < 0.01), 25 mg/mL (p < 0.01), 50 mg/mL (p < 0.01) and 100 mg/mL (p < 0.05) micelles significantly promoted adhesion compared with control. ANOVA test showed that there is no interaction between concentrations of micelles and TG/PA treatment (p > 0.05). 4. Discussions In this study, the effects of micelles on key events of the early development of atherosclerosis, namely endothelial viability, oxidative stress, inflammation and monocyte adhesion [7,8], was studied in vitro to indicate the safety of micelles. In addition, the ER stress inducer TG, and a saturated fatty acid PA, were used to coexpose HUVECs in order to mimic the responses of human ECs to
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Fig. 4. Release of inflammatory mediators interleukin-6 (IL-6, 4A), IL-8 (4B) and soluble vascular cell adhesion molecule (sVCAM-1, 4C). HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, and ELISA was used to measure the inflammatory mediators in supernatants. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.01, single factor effect, ANOVA.
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Fig. 5. THP-1 monocytes adhesion to HUVECs. HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, followed by 1 h adhesion assay. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.05, compared with untreated cells.
polymeric micelles under disease-like conditions. The results showed that the MPEG-PLA based micelles with or without the presence of TG/PA induced little to no adverse effects to HUVECs in vitro, which indicated a relatively high biocompatibility of the micelles to ECs even in a complex system. We used three different methods to measure the cytotoxicity, which may reflect the mitochondrial viability (WST-1), lysosomal dysfunction (neutral red uptake) and membrane integrity (LDH). The results showed that exposure to polymeric micelles up to 200 mg/mL did not significantly induce cytotoxicity as assessed by the three independent assays (p > 0.05; Fig. 2). To induce the injury of HUVECs, TG and PA were used to co-expose the cells. TG could induce ER stress, and prolonged ER stress can induce the apoptosis of cells [23,24]. High concentrations of PA could also induce the apoptosis of cells by the induction of ER stress [25] and ROS [26]. Consistent with the previous reports, in this study we also found increased cytotoxicity by TG and PA treatment (Fig. 2). Here we used cytotoxic PA (200 mM) because our recent studies showed combined effects of NPs and PA at cytotoxic concentrations. For example, 50 mM PA (cytotoxic), but not 10 mM PA (non-cytotoxic), enhanced the toxicity of ZnO NPs to lysosomes in macrophages [27]. In another study, a combined toxicity in HUVECs was observed by using ZnO NPs and 200 mM PA but not non-cytotoxic lipopolysaccharide [28]. Therefore we used TG/PA at cytotoxic concentrations to investigate the possible combined effects in this study. The cytotoxicity of PA appears to be higher than that of TG because PA showed cytotoxicity as assessed by three different methods, whereas TG only significantly decreased cellular viability by WST-1 assay, but not by neutral red or LDH assay. In our recent study, we also found that 1 mM TG significantly decreased mitochondrial viability without an effect on LDH release [17]. A previous study showed that carbon black NP exposure induced cytotoxicity to THP1 monocytes, THP-1 macrophages and HUVECs as indicated by WST-1 assay, whereas LDH release was only significantly increased in the exposed THP-1 monocytes [29]. Therefore, WST-1 assay may be more sensitive to indicate cytotoxicity, whereas LDH assay may reflect severely cellular damage, e.g., membrane break and necrosis. Atherosclerosis is a chronic disease closely associated with
oxidative stress and inflammation. Inhibition of oxidative stress and inflammation has been proposed as a strategy for nanomedicine to prevent atheroprogression [5], whereas some solid NPs have been shown to induce vascular health effects by inducing oxidative stress and inflammation [30e32]. For this consideration, the micelles for atherosclerosis treatment should not induce oxidative stress and inflammatory response. In this study, up to 200 mg/mL micelles did not induce ROS (Fig. 3A) or release of inflammatory mediators including IL-6, IL-8 and sVCAM-1 (Fig. 4). To further induce an atherosclerosis-like condition, the ER stress inducer TG was used, as ER stress has been suggested to be involved in atherosclerosis [12,13]. In contrast to our expectation, the treatment of TG did not affect ROS or release of inflammatory mediators either (p > 0.05). A recent study showed that 1 mM TG, but not 0.5 mM TG, significantly induced ER stress in HUVECs [33]. However, in our recent study, 1 mM TG did not affect inflammatory mediators neither, whereas it was too toxic to cells [17], therefore we used a relatively low concentration of TG to see if micelles could exacerbate the toxic effects of TG. One possible explanation to the unaltered inflammatory response by TG treatment is that inflammatory input is an upstream to ER stress, rather than a result of ER stress, as revealed by a recent study [34]. With the presence of TG, 200 mg/mL micelles induced a modest increase of IL-6, but this increase was not statistically significant (p > 0.05, Fig. 4A), which indicated little to no effects of micelles on the inflammation with the presence of TG. PA is a pro-atherogenic stimulus, and a recent study showed the presence of PA may enhance the adverse effects of carbon nanotubes to HUVECs [16]. The normal concentration of PA in blood is approximately 100 mM, which may be elevated in the context of metabolic diseases including atherosclerosis [35]. 100 mM PA could induce the activation of ECs in vitro, but HUVECs were shown to be more resistant to PA treatment compared with other types of ECs [36]. Indeed, it has been shown that exposure of HUVECs to 500 mM PA significantly induced ROS and inflammatory response [37]. However, in our preliminary experiments, 500 mM PA was shown to be too toxic to HUVECs. Therefore in this study we used a relatively low concentration of PA (200 mM) to see if the presence of
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various concentrations of polymeric micelles will enhance the toxic effects of PA to HUVECs. PA at 200 mM showed cytotoxicity to HUVECs (Fig. 2), but there was unaltered ROS (Fig. 3A), IL-8 and sVCAM-1, although IL-6 was modestly increased (Fig. 4). It may be possible that PA can only significantly induce oxidative stress and inflammatory responses to HUVECs at highly cytotoxic concentrations (e.g., 500 mM). Nevertheless, micelles did not significantly affect ROS or release of inflammatory mediators in the presence of PA, which indicated that micelles did not accelerate the toxic effects of PA. Monocyte adhesion to activated endothelial cells and the subsequent differentiation of monocytes into macrophages is a hallmark of atherosclerosis [8]. Recent studies successfully reduced atherosclerotic plaque sizes and increased plaque stability by using NPs that could inhibit monocyte recruitment [38,39]. Therefore, a safe nanocarrier commonly used in nanomedicine should not promote monocyte adhesion. To this end we estimated the THP-1 adhesion to HUVECs that were exposed to various concentrations of micelles with or without the presence of TG or PA. THP-1 monocytes were used because they were among one of the most used in vitro models for human monocytes as they keep many important monocytic characteristics [40]. Moreover, some studies showed that the adhesion of THP-1 monocytes to ECs was similar to that of primary cells, which indicated that THP-1 cells could be used as a reliable model to study monocyte-ECs interactions [40e42]. As there is no significant increase of sVCAM-1 release by TG or PA treatment (Fig. 4C), the elevated adhesion is probably mediated by adhesion molecule independent pathway as suggested by a previous study [43]. However, this hypothesis is not further tested in this study. There was a modest increase of IL-6 release especially by PA treatment (Fig. 4A), but a recent study showed that IL-6 was not associated with THP-1 adhesion to HUVECs [16]. Nevertheless, the micelles used in this study did not significantly promote monocyte adhesion to HUVECs, which indicated that they were relatively safe to the human endothelial cells in vitro. In this study we assessed the in vitro toxicity of MPEG-PLA based micelles at concentrations from 12.5 mg/mL up to 200 mg/mL. These concentrations were used according to a recent report which used pitavastatin loaded micelles at 0.1 mg to treat atherosclerosis in mice [39]. Assuming the blood volume of mice as 1e2 mL and an equal distribution of micelles in circulation, 0.1 mg micelles may give a concentration of 50 mg/mL to 100 mg/mL in mice. Under in vitro conditions, the authors used drug loaded micelles up to 36.7 mg/mL to inhibit the chemotaxis of THP-1 monocytes [39]. The concentrations of micelles used in this study were within the ranges used in the previous report and may be likely to happen in real life when using the micelles as vehicles to deliver drugs for atherosclerosis treatment. Collectively, this study showed little to no adverse effects of MPEG-PLA based micelles on HUVECs with or without the presence of TG/PA treatment, therefore they were not toxic to human ECs in vitro. In perspective, we are developing drug-loaded NPs based on this type of micelles for atherosclerosis therapy. Conflict of interest The authors have no conflict of interest. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (51273169, 51473141), Xiangtan University grant (15XZX19) and Xiangtan University start-up grant (15QDZ47).
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Loft, Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar-endothelial co-cultures, Nanotoxicology (2016 Mar 12) 235e244. [17] Y. Ji, M. Zhu, Y. Gong, H. Tang, J. Li, Y. Cao, Thermoresponsive polymers with lower critical solution temperature- or upper critical solution temperaturetype phase behaviour do not induce toxicity to human endothelial cells, Basic Clin. Pharmacol. Toxicol. (In press 2016), http://dx.doi.org/10.1111/ bcpt.12643. [18] X. Zhang, Y. Li, X. Chen, X. Wang, X. Xu, Q. Liang, et al., Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate, Biomaterials 26 (14) (2005 May) 2121e2128. [19] G. Ma, D. Li, J. Wang, X. Zhang, H. Tang, Methoxy-Poly(ethylene glycol)-blockPoly(epsilon-caprolactone) bearing pendant aldehyde groups as pHresponsive drug delivery carrier, Aust. J. Chem. 66 (12) (2013) 1576e1583. [20] J. Wang, H. Sun, D. Li, J. Yuan, X. Zhang, H. Tang, Water-soluble poly(epsiloncaprolactone)-paclitaxel prodrugs toward an efficient drug delivery system, Aust. J. Chem. 68 (7) (2015) 1136e1143. [21] Y. Cao, K. Jantzen, A.C. Gouveia, A. Skovmand, M. Roursgaard, S. Loft, et al., Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro, Environ. Toxicol. Pharmacol. 40 (1) (2015 Jul) 164e171. [22] Y. Cao, N.R. Jacobsen, P.H. Danielsen, A.G. Lenz, T. Stoeger, S. Loft, et al., Vascular effects of multiwalled carbon nanotubes in dyslipidemic ApoE-/mice and cultured endothelial cells, Toxicol. Sci. 138 (1) (2014 Mar) 104e116. [23] F. Engin, G.S. Hotamisligil, Restoring endoplasmic reticulum function by chemical chaperones: an emerging therapeutic approach for metabolic diseases, Diabetes Obes. Metab. 12 (2010 Oct) 108e115. [24] A.I. Placido, C.R. Oliveira, P.I. Moreira, C.M. 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Evaluation of in vitro toxicity of polymeric micelles to human endothelial cells under different conditions Fang Liu a, Haikang Huang b, Yu Gong a, Juan Li a, Xuefei Zhang b, *, Yi Cao a, ** a Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry of Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University, Xiangtan 411105, PR China b Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry of Education, Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province and Key Laboratory of Polymeric Materials & Application Technology of Hunan Province, College of Chemistry, Xiangtan University, Xiangtan 411105, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 October 2016 Received in revised form 16 December 2016 Accepted 22 December 2016 Available online 23 December 2016
Polymeric micelles have been extensively studied in the area of antitumor therapy, and more recently explored as nanocarriers for atherosclerosis. These applications of polymeric micelles in biomedicine will increase their contact with human blood vessels. However, few studies have considered the interactions between polymeric micelles and endothelial cells, especially in a complex system. This study used human umbilical vein endothelial cells (HUVECs) as an in vitro model for endothelial cells to investigate the toxic effects of methoxy-poly(ethylene glycol)-poly(D,L-lactide) (MPEG-PLA) based micelles. In addition, an endoplasmic reticulum stress inducer thapsigargin (TG), and a pro-atherogenic stimulus palmitate (PA), were used to co-expose HUVECs to further mimic the responses of diseased endothelial cells to micelle exposure. Overall, up to 200 mg/mL micelles did not significantly induce cytotoxicity, reactive oxygen species (ROS), release of inflammatory mediators in terms of interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), or adhesion of THP-1 monocytes to HUVECs. TG and PA significantly induced cytotoxicity and THP-1 adhesion as well as modestly promoted the release of IL-6, but did not affect ROS or release of sVCAM-1 and IL-8. Co-exposure of micelles did not significantly enhance the effects of TG and PA to HUVECs, and ANOVA analysis indicated no interaction between concentrations of micelles and the presence of TG/PA. Taken together, these data indicated that micelles are not toxic to HUVECs under different conditions in vitro. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Micelles Methoxy-poly(ethylene glycol)-poly(D,Llactide) (MPEG-PLA) Human umbilical vein endothelial cells (HUVECs) Thapsigargin (TG) Palmitate (PA)
1. Introduction With their unique advantages, a number of engineered nanoparticles (NPs) has been produced for biomedical uses, but their potential adverse effects should be carefully assessed to ensure the safe use [1]. Polymeric micelles, formed by amphiphilic copolymers with a hydrophilic shell and a hydrophobic core, are among one of the most commonly produced engineered NPs and have been extensively studied for anticancer therapy [2,3]. Recently, it was also shown that polymeric micelles may be used for atherosclerosis imaging and therapy [4,5]. As a consequence, the applications of polymeric micelles in biomedicine will inevitably increase their contact with human blood vessels, but few studies have concerned
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.cbi.2016.12.014 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
their side effects to relevant cells in the circulation system [6]. One of such relevant cells that should be used to understand NP-blood vessel contact is endothelial cell (EC), as suggested by a recent review [6]. The ECs cover the surface area and regulate the blood vessel tone, thrombogenicity, homeostasis, monocytes recruitment, and hormone transport [7]. Furthermore, they also serve as the first contact cells with engineered NPs when the NPs enter the blood [6]. During the early development of atherosclerosis, inflammatory and oxidative stimuli activate ECs, which express adhesion molecules and release inflammatory mediators. Circulating monocytes then adhere to the activated ECs, differentiate into macrophages, engulf excessive lipids and finally lead to plaque progression [7,8]. Whether polymeric micelles will induce adverse effects to ECs needs to be carefully assessed to ensure their safe use in nanomedicine. This should be done not only in ‘healthy’ ECs, but also diseased ECs, as the response of ECs to NPs could be different due to the diversity of EC types [6]. There may be also a need to assess the
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toxicity of polymeric micelles to ECs in a complex system where the nutrients are present, as the nutrients may also define the nanotoxicological responses [9,10]. The present study investigated the toxicity of micelles to human vein ECs (HUVECs) under different conditions. The HUVECs were used because they have been extensively used as an in vitro model for human ECs to understand the interactions of engineered NPs with blood vessels [6]. The empty micelles without any associated drugs were used because a recent study showed that drug-loaded micelles may induce drug-related hematological toxicity [11]. The cytotoxicity was determined by three different methods, namely water soluble tetrazolium-1 (WST-1), neutral red uptake and lactate dehydrogenase (LDH) assay. Oxidative stress was indicated by the measurement of intracellular reactive oxygen species (ROS). The inflammatory response was indicated by the measurement of release of inflammatory mediators, including interleukin 6 (IL-6), IL-8 and soluble vascular cell adhesion molecule 1 (sVCAM-1), as well as the adhesion of THP-1 monocytes to HUVECs. Meanwhile, to induce the injury of HUVECs and mimic the response of ECs under atherosclerotic conditions, thapsigargin (TG) or palmitate (PA) was used to co-expose HUVECs. TG is a classical endoplasmic reticulum (ER) stress inducer, and elevated ER stress has been suggested to play a pivotal role in the pathology of atherosclerosis [12,13]. A recent study showed that ZnO NPs could induce ER stress to HUVECs, which may be related with the cardiovascular effects [14]. PA is one of the basic fatty acids present in human blood. Elevated circulating PA may induce endothelial dysfunction and thus links excessive nutrients to the development of atherosclerosis [13,15]. A recent study showed that the presence of PA may enhance the vascular health effects of multi-walled carbon nanotubes to HUVECs in vitro, which indicated an interaction between saturated fatty acids and NPs [16]. 2. Materials and methods 2.1. Cell culture HUVECs (passage 1; purchased from ScienCell Research Laboratories, Carlsbad, CA) were cultured in supplemented endothelial medium and used at passage 3e6 as our previously described [17]. For each experiment, 1 104/well (on 96-well plates) or 4 104/ well (on 24-well plates) HUVECs were seeded on 0.2% gelatin solution (ScienCell Research Laboratories, Carlsbad, CA) pre-coated plates and grown for 2 days prior to exposure. THP-1 monocytes (ATCC) were cultured in RPMI 1640 medium (sodium pyruvate and HEPES added; Gibco, USA) supplemented with 10% FBS and 1% penicillin-streptomycin (P/S) solution in a CO2 incubator at 37 C. The cells were used within 3 month to keep their best characteristics. 2.2. Preparation of polymeric micelles The amphiphilic copolymer methoxy-poly(ethylene glycol)poly(D,L-lactide) (MPEG-PLA) was synthesized as our previously described with slight modifications [18]. Briefly, 0.5 g lactide monomer (LA) was reacted with 1.0 g methoxy-poly(ethylene glycol) (MPEG) in 1 mL CH2Cl2 under nitrogen gas condition, catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-CH2Cl2 ([M]/ [Cat] ¼ 100). Here we used a non-metallic reagent TBD as the catalyst to reduce the possible metallic contaminations in micelles [19,20]. After 48 h reaction under room temperature, the polymer/ CH2Cl2 solution was precipitated by absolute ether, followed by vacuum drying to remove CH2Cl2. To make the micelle solution, MPEG-PLA was dissolved in acetone, followed by drop wise addition of the MPEG-PLA acetone solution into MilliQ water during
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slowly constant stirring. The cloudy micelle solution was further constantly stirred for 0.5 h, and then rotary evaporated overnight under room temperature to remove acetone. Finally, the stock solution of micelles was made as 2 mg/mL in MilliQ water. The critical micelle concentration (CMC) of the polymeric micelles was determined by using a fluorescent probe pyrene. A stock solution of pyrene was made as 6 mg/mL in acetone. A working solution was then prepared by adding 0.1 mL of pyrene solution into 1000 mL MilliQ water with constant stirring for 2 h, after which the working solution was placed at room temperature for 2 days to allow the acetone to evaporate. Micelles in MilliQ water were diluted to a series of concentrations from 0.0001 mg/mL to 1 mg/mL and mixed with equal concentrations of pyrene solution (final volume 10 mL). Micelleepyrene solutions were briefly sonicated in ultrasonic water bath for 15 min and then placed for 12 h under room temperature to allow partition of the pyrene into the micelles. Emission was done from 230 to 360 nm, with 392 nm as the excitation wavelength. The maximum absorption of pyrene shifted from 335.91 to 339.05 nm on micelle formation. The ratio of absorption of encapsulated pyrene (339.05 nm) to pyrene in water (335.91 nm) was plotted as the logarithm of polymer concentrations. The inflection point of the curve was taken as the CMC. 2.3. Characterizations 1
H nuclear magnetic resonance (NMR) spectra of the synthesized MPEG-PLA were measured by a Bruker AV-400 NMR spectrometer at room temperature, with CDCl3 as solvent. The molecular weight of MPEG-PLA was measured by gel permeation chromatography (GPC) using a PL-GPC 120 instrument (UK), with tetrahydrofuran (THF) as eluent at a flow rate of 1.0 mL/min. The hydrodynamic size distribution of the polymeric micelles was measured using 1 mg/mL particles suspended in MilliQ water by Zetasizer Nano ZS90 (Malvern, UK). The hydrodynamic size was measured for three times, and mean ± S.D. (standard deviation) was calculated. The stability of the micelles solution was tested as changes of hydrodynamic size distribution after stored at room temperature for 10 and 20 days. The micelles were used within 20 days for all the experiments in this study as they slowly precipitated after 20-day storage at room temperature. 2.4. Cytotoxicity The cytotoxicity was measured by using WST-1, neutral red uptake and LDH kits according to manufacturer's instruction (Beyotime, China). WST-1 reagent can indicate the mitochondrial activity as it could be converted to a yellow formazan by mitochondria in living cells. For the assay, HUVECs seeded on 24-well plates were exposed to 0 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL and 200 mg/mL micelles, with or without the presence of 500 nM TG or 200 mM PA (from Sigma-Aldrich, USA). After 24 h exposure, the cells were rinsed once with Hank's solution, and then incubated with 10% WST-1 reagent for 2 h. The absorbance was read at 450 nm with 690 nm as reference by an ELISA reader (Synergy HT, BioTek, USA). Neutral red is a dye which could be incorporated into intact lysosomes of living cells. For the assay, HUVECs on 24-well plates were incubated with various concentrations of micelles with or without the presence of TG or PA for 24 h as indicated above, rinsed and then incubated with 10% neutral red for 2 h. After rinsed once again, the neutral red incorporated into lysosomes was dissolved in the lysis solution provided by the kit, and the absorbance was read at 540 nm with 690 nm as reference. LDH is an enzyme in cytoplasm, which could be released into extracellular fluid with the break of membrane. The LDH assay was
F. Liu et al. / Chemico-Biological Interactions 263 (2017) 46e54 Fig. 1. Characteristics of the synthesized MPEG-PLA and the micelles based on the MPEG-PLA. 1A, 1H NMR spectra of the synthesized MPEG-PLA. 1B, the GPC measurement of MPEG5000 and MPEG-PLA. Data for Fig. 1A and B were based on a single run. 1C, the hydrodynamic size distribution of micelles. The hydrodynamic diameters of micelles did not significantly change after storage for 20 days. Data for Fig. 1C were representatives from three measurement based on a single run.
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done in parallel with WST-1 assay. Briefly, after exposure the supernatant from WST-1 assay was collected, and 60 mL supernatant from each sample was mixed with 120 mL LDH reaction buffer provided by the kit for 30 min. The absorbance was read at 490 nm with 690 nm as reference. To induce a 100% of LDH release, the positive control cells were incubated with 10% lysis buffer 1 h before the assay and the activity of LDH was measured as indicated above. 2.5. Analysis of reactive oxygen species ROS was estimated by using 20 ,7’-dichlorofluorescein diacetate (DCFH-DA) as previously described [21]. DCFH-DA is cell permeable which could be oxidized to a fluorescent probe by the reaction with a variety of ROS inside the cells. Briefly, HUVECs seeded on black 96-well plates were exposed to various concentrations of micelles with or without the presence of 500 nM TG or 200 mM PA for 3 h. After exposure, the cells were rinsed once with Hanks solution, and then stained by 10 mM DCFH-DA in serum free medium for 30 min. After rinsed again with Hanks solution, the fluorescence was read at ex 485 ± 20 nm and em 528 ± 20 nm by an ELISA reader. To further validate the method, HUVECs were also exposed to H2O2 from 0.0625% to 1% for 3 h, followed by the measurement of ROS as described above. 2.6. Inflammatory response The supernatant from the exposed HUVECs was collected and stored at 80 C before analysis. Inflammatory mediators IL-6, IL-8 and sVCAM-1 was measured by ELISA kits according to manufacturer's instruction (Neobioscience Technology Co., Ltd., China). Some of the samples were diluted for the measurement. 2.7. Monocyte adhesion The adhesion of THP-1 monocytes to HUVECs was done as previously described [16]. Briefly, HUVECs on black 96-well plates were exposed to various concentrations of micelles with or without the presence of 500 nM TG or 200 mM PA for 24 h. Before the end of the exposure, THP-1 monocytes were labeled with 10 mM CellTracker™ Green CMFDA (5-Chloromethylfluorescein Diacetate, Invitrogen, Carlsbad, CA) for 30 min in RPMI 1640 medium. The free probe was removed by centrifuge, and 5 104/well labeled THP1 cells were incubated with the exposed HUVECs for 1 h for adhesion. The doubling time for THP-1 cells was measured to be 31 h [22], therefore it is expected that during 1 h incubation there was minimal proliferation of THP-1 monocytes [16]. After that, the unbound THP-1 cells were washed away, and the fluorescence was read at ex 485 ± 20 nm and em 528 ± 20 nm by an ELISA reader. 2.8. Statistics All the data were expressed as mean ± S.D. (standard deviation) of means of at least three independent experiments. Two-way ANOVA (concentrations and treatment as categorical factors) followed by Tukey HSD test using R 3.2.2; p value < 0.05 was considered to be statistically significant. 3. Results 3.1. Characteristics of MPEG-PLA and micelles 1 H NMR spectra of the synthesized MPEG-PLA is shown in Fig. 1A dA (3.4 ppm) and dBþC (3.66 ppm) belong to methyl group and methylene group of MPEG, respectively. dD (3.84 ppm) and dE
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Fig. 2. Cytotoxicity as measured by WST-1 (2A), neutral red uptake (2B) and LDH (2C) assay. HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, and WST-1, neutral red uptake and LDH assay were used to determine cytotoxicity. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.01, compared with untreated cells.
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Fig. 3. Oxidative stress as indicated by intracellular ROS. 3A, HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 3 h, and intracellular ROS was measured by a fluorescent probe. 3B, HUVECs were exposed to H2O2 from 0.0625% to 1%, followed by DCFH assay to indicate intracellular ROS. Data were expressed as mean ± S.D. (standard deviation) of means of three (3A) or four (3B) independent experiments. *, p < 0.05, ANOVA.
(4.31 ppm) belong to the two methylene group near LA. dF/H (5.19/ 5.21) belong to the two methyne group of LA, whereas dG/I (1.54/ 1.61) belong to the methyl group of LA. 1H NMR analysis indicated the successful formation of MPEG-PLA product. According to the GPC measurement (Fig. 1B), the Mn of MPEG-PLA was calculated as 7785, and PDI was calculated as 1.058 (Mw of the MPEG-PLA is 8242). The CMC was calculated as 3.3 mg/mL, and the concentrations used in this study were all above CMC. The size distribution of micelles based on the MPEG-PLA is shown in Fig. 1C. The micelles had hydrodynamic diameters 20 ± 1 nm, which were not significantly changed after storage for 20 days. After that the micelle solution slowly precipitated, which was not further used for this study. 3.2. Cytotoxicity As illustrated in Fig. 2, WST-1 assay showed significantly decreased viability of HUVECs by the treatment of TG or PA (p < 0.01), whereas ANOVA analysis indicated no dose-dependent effect of micelles up to 200 mg/mL (p > 0.05). For neutral red uptake assay, TG only induced an insignificant decrease in neutral red uptake (p > 0.05). The treatment of TG reduced neutral red uptake to 80.6% (95% CI: 56.3%e105%). PA significantly decreased neutral red uptake (p < 0.01) and the uptake was reduced to 46.6% (95% CI: 22.3%e71%). There was no dose-dependent effect of micelles as revealed by neutral red uptake assay (p > 0.05). The cytotoxicity was further measured by LDH assay to indicate the membrane integrity and result is shown in Fig. 2C. Exposure to TG did not significantly affect LDH release (p > 0.05), whereas PA exposure significantly induced LDH release (p < 0.01). Again, there was no dose-dependent effect of micelles (p > 0.05). 3.3. Analysis of reactive oxygen species The exposure to micelles up to 200 mg/mL did not significantly affect the intracellular ROS (p > 0.05; Fig. 3A). TG or PA treatment did not significantly affect ROS (p > 0.05), and ANOVA analysis showed no interaction between concentrations of micelles and TG/ PA treatment on ROS (p > 0.05). As a positive control, 3 h exposure of HUVECs to H2O2 dose-dependently increased ROS, and there was statistically significantly increased ROS after exposure to 0.5% (p < 0.05) and 1% (p < 0.01) H2O2, respectively, which indicated the
confidence of the assay (Fig. 3B). 3.4. The release of inflammatory mediators Fig. 4 shows the release of inflammatory mediators including IL6 (4A), IL-8 (4B) and sVCAM-1 (4C). For IL-6, micelles alone did not significantly affect the release of IL-6 (p > 0.05). 500 nM TG treatment did not significantly affect IL-6 release either (p > 0.05). With the presence of TG, 200 mg/mL micelle resulted in a modest increase of IL-6, but the increase was not statistically different compared with control (p > 0.05). There was a single factor effect by 200 mM PA treatment (p < 0.01, ANOVA); however, Tukey HSD posthoc analysis showed that IL-6 release was not significantly elevated by micelles with the presence of PA compared with control (p > 0.05). In addition, ANOVA test indicated no significant interaction between concentrations of micelles and TG/PA treatment (p ¼ 0.08). For IL-8 and sVCAM-1, micelles did not significantly affect the release with or without the presence of TG/PA (p > 0.05). 3.5. Monocyte adhesion As shown in Fig. 5, when TG or PA was absent, all the concentrations of micelles did not significantly promote the adhesion of THP-1 monocytes to HUVECs (p > 0.05). TG or PA treatment significantly promoted the adhesion to a comparable level (ca. 50% increase to control; p < 0.01). With the presence of TG, all the concentrations of micelles significantly induced the adhesion compared with control (p < 0.01). Similarly, with the presence of PA, 12.5 mg/mL (p < 0.01), 25 mg/mL (p < 0.01), 50 mg/mL (p < 0.01) and 100 mg/mL (p < 0.05) micelles significantly promoted adhesion compared with control. ANOVA test showed that there is no interaction between concentrations of micelles and TG/PA treatment (p > 0.05). 4. Discussions In this study, the effects of micelles on key events of the early development of atherosclerosis, namely endothelial viability, oxidative stress, inflammation and monocyte adhesion [7,8], was studied in vitro to indicate the safety of micelles. In addition, the ER stress inducer TG, and a saturated fatty acid PA, were used to coexpose HUVECs in order to mimic the responses of human ECs to
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Fig. 4. Release of inflammatory mediators interleukin-6 (IL-6, 4A), IL-8 (4B) and soluble vascular cell adhesion molecule (sVCAM-1, 4C). HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, and ELISA was used to measure the inflammatory mediators in supernatants. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.01, single factor effect, ANOVA.
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Fig. 5. THP-1 monocytes adhesion to HUVECs. HUVECs were exposed to various concentrations of micelles with or without the presence of 500 nM thapsigargin (TG) or 200 mM palmitate (PA) for 24 h, followed by 1 h adhesion assay. Data were expressed as mean ± S.D. (standard deviation) of means of three independent experiments. *p < 0.05, compared with untreated cells.
polymeric micelles under disease-like conditions. The results showed that the MPEG-PLA based micelles with or without the presence of TG/PA induced little to no adverse effects to HUVECs in vitro, which indicated a relatively high biocompatibility of the micelles to ECs even in a complex system. We used three different methods to measure the cytotoxicity, which may reflect the mitochondrial viability (WST-1), lysosomal dysfunction (neutral red uptake) and membrane integrity (LDH). The results showed that exposure to polymeric micelles up to 200 mg/mL did not significantly induce cytotoxicity as assessed by the three independent assays (p > 0.05; Fig. 2). To induce the injury of HUVECs, TG and PA were used to co-expose the cells. TG could induce ER stress, and prolonged ER stress can induce the apoptosis of cells [23,24]. High concentrations of PA could also induce the apoptosis of cells by the induction of ER stress [25] and ROS [26]. Consistent with the previous reports, in this study we also found increased cytotoxicity by TG and PA treatment (Fig. 2). Here we used cytotoxic PA (200 mM) because our recent studies showed combined effects of NPs and PA at cytotoxic concentrations. For example, 50 mM PA (cytotoxic), but not 10 mM PA (non-cytotoxic), enhanced the toxicity of ZnO NPs to lysosomes in macrophages [27]. In another study, a combined toxicity in HUVECs was observed by using ZnO NPs and 200 mM PA but not non-cytotoxic lipopolysaccharide [28]. Therefore we used TG/PA at cytotoxic concentrations to investigate the possible combined effects in this study. The cytotoxicity of PA appears to be higher than that of TG because PA showed cytotoxicity as assessed by three different methods, whereas TG only significantly decreased cellular viability by WST-1 assay, but not by neutral red or LDH assay. In our recent study, we also found that 1 mM TG significantly decreased mitochondrial viability without an effect on LDH release [17]. A previous study showed that carbon black NP exposure induced cytotoxicity to THP1 monocytes, THP-1 macrophages and HUVECs as indicated by WST-1 assay, whereas LDH release was only significantly increased in the exposed THP-1 monocytes [29]. Therefore, WST-1 assay may be more sensitive to indicate cytotoxicity, whereas LDH assay may reflect severely cellular damage, e.g., membrane break and necrosis. Atherosclerosis is a chronic disease closely associated with
oxidative stress and inflammation. Inhibition of oxidative stress and inflammation has been proposed as a strategy for nanomedicine to prevent atheroprogression [5], whereas some solid NPs have been shown to induce vascular health effects by inducing oxidative stress and inflammation [30e32]. For this consideration, the micelles for atherosclerosis treatment should not induce oxidative stress and inflammatory response. In this study, up to 200 mg/mL micelles did not induce ROS (Fig. 3A) or release of inflammatory mediators including IL-6, IL-8 and sVCAM-1 (Fig. 4). To further induce an atherosclerosis-like condition, the ER stress inducer TG was used, as ER stress has been suggested to be involved in atherosclerosis [12,13]. In contrast to our expectation, the treatment of TG did not affect ROS or release of inflammatory mediators either (p > 0.05). A recent study showed that 1 mM TG, but not 0.5 mM TG, significantly induced ER stress in HUVECs [33]. However, in our recent study, 1 mM TG did not affect inflammatory mediators neither, whereas it was too toxic to cells [17], therefore we used a relatively low concentration of TG to see if micelles could exacerbate the toxic effects of TG. One possible explanation to the unaltered inflammatory response by TG treatment is that inflammatory input is an upstream to ER stress, rather than a result of ER stress, as revealed by a recent study [34]. With the presence of TG, 200 mg/mL micelles induced a modest increase of IL-6, but this increase was not statistically significant (p > 0.05, Fig. 4A), which indicated little to no effects of micelles on the inflammation with the presence of TG. PA is a pro-atherogenic stimulus, and a recent study showed the presence of PA may enhance the adverse effects of carbon nanotubes to HUVECs [16]. The normal concentration of PA in blood is approximately 100 mM, which may be elevated in the context of metabolic diseases including atherosclerosis [35]. 100 mM PA could induce the activation of ECs in vitro, but HUVECs were shown to be more resistant to PA treatment compared with other types of ECs [36]. Indeed, it has been shown that exposure of HUVECs to 500 mM PA significantly induced ROS and inflammatory response [37]. However, in our preliminary experiments, 500 mM PA was shown to be too toxic to HUVECs. Therefore in this study we used a relatively low concentration of PA (200 mM) to see if the presence of
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various concentrations of polymeric micelles will enhance the toxic effects of PA to HUVECs. PA at 200 mM showed cytotoxicity to HUVECs (Fig. 2), but there was unaltered ROS (Fig. 3A), IL-8 and sVCAM-1, although IL-6 was modestly increased (Fig. 4). It may be possible that PA can only significantly induce oxidative stress and inflammatory responses to HUVECs at highly cytotoxic concentrations (e.g., 500 mM). Nevertheless, micelles did not significantly affect ROS or release of inflammatory mediators in the presence of PA, which indicated that micelles did not accelerate the toxic effects of PA. Monocyte adhesion to activated endothelial cells and the subsequent differentiation of monocytes into macrophages is a hallmark of atherosclerosis [8]. Recent studies successfully reduced atherosclerotic plaque sizes and increased plaque stability by using NPs that could inhibit monocyte recruitment [38,39]. Therefore, a safe nanocarrier commonly used in nanomedicine should not promote monocyte adhesion. To this end we estimated the THP-1 adhesion to HUVECs that were exposed to various concentrations of micelles with or without the presence of TG or PA. THP-1 monocytes were used because they were among one of the most used in vitro models for human monocytes as they keep many important monocytic characteristics [40]. Moreover, some studies showed that the adhesion of THP-1 monocytes to ECs was similar to that of primary cells, which indicated that THP-1 cells could be used as a reliable model to study monocyte-ECs interactions [40e42]. As there is no significant increase of sVCAM-1 release by TG or PA treatment (Fig. 4C), the elevated adhesion is probably mediated by adhesion molecule independent pathway as suggested by a previous study [43]. However, this hypothesis is not further tested in this study. There was a modest increase of IL-6 release especially by PA treatment (Fig. 4A), but a recent study showed that IL-6 was not associated with THP-1 adhesion to HUVECs [16]. Nevertheless, the micelles used in this study did not significantly promote monocyte adhesion to HUVECs, which indicated that they were relatively safe to the human endothelial cells in vitro. In this study we assessed the in vitro toxicity of MPEG-PLA based micelles at concentrations from 12.5 mg/mL up to 200 mg/mL. These concentrations were used according to a recent report which used pitavastatin loaded micelles at 0.1 mg to treat atherosclerosis in mice [39]. Assuming the blood volume of mice as 1e2 mL and an equal distribution of micelles in circulation, 0.1 mg micelles may give a concentration of 50 mg/mL to 100 mg/mL in mice. Under in vitro conditions, the authors used drug loaded micelles up to 36.7 mg/mL to inhibit the chemotaxis of THP-1 monocytes [39]. The concentrations of micelles used in this study were within the ranges used in the previous report and may be likely to happen in real life when using the micelles as vehicles to deliver drugs for atherosclerosis treatment. Collectively, this study showed little to no adverse effects of MPEG-PLA based micelles on HUVECs with or without the presence of TG/PA treatment, therefore they were not toxic to human ECs in vitro. In perspective, we are developing drug-loaded NPs based on this type of micelles for atherosclerosis therapy. Conflict of interest The authors have no conflict of interest. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (51273169, 51473141), Xiangtan University grant (15XZX19) and Xiangtan University start-up grant (15QDZ47).
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