Subscriber access provided by University of Sussex Library
Article
Targeted Therapy of Atherosclerosis by a Broad-Spectrum Reactive Oxygen Species-Scavenging Nanoparticle with Intrinsic Anti-Inflammatory Activity Yuquan Wang, Lanlan Li, Weibo Zhao, Yin Dou, Huijie An, Hui Tao, Xiaoqiu Xu, Yi Jia, Shan Lu, Jianxiang Zhang, and Houyuan Hu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02037 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Targeted Therapy of Atherosclerosis by a Broad-Spectrum Reactive Oxygen
Species-Scavenging
Nanoparticle
with
Intrinsic
Anti-Inflammatory Activity Yuquan Wang,†,‡,#,ǁ Lanlan Li,‡,ǁ Weibo Zhao,† Yin Dou,‡ Huijie An,‡ Hui Tao,‡ Xiaoqiu Xu,‡ Yi Jia,‡ Shan Lu,‡ Jianxiang Zhang,*,‡ and Houyuan Hu*,†
†
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China
‡
Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038,
China #
Department of Cardiology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan
Province, China
Corresponding authors: Jianxiang Zhang, PhD, Prof. E-mail:
[email protected],
[email protected] Houyuan Hu, PhD, Prof. E-mail:
[email protected] ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 43
KEYWORDS: reactive oxygen species, nanoparticle, atherosclerosis, anti-inflammation, anti-oxidative stress ABSTRACT: Atherosclerosis is a leading cause of vascular diseases worldwide. Whereas anti-oxidative therapy has been considered promising for the treatment of atherosclerosis in view of a critical role of reactive oxygen species (ROS) in the pathogenesis of atherosclerosis, currently available antioxidants showed considerably limited clinical outcomes. Herein we hypothesize that a broad-spectrum ROS-scavenging nanoparticle can serve as an effective therapy for atherosclerosis, taking advantages of its anti-oxidative stress activity and targeting effects. As a proof of concept, a broad-spectrum ROS eliminating material was synthesized by covalently conjugating a superoxide dismutase mimetic agent Tempol and a hydrogen peroxide-eliminating compound of phenylboronic acid pinacol ester onto a cyclic polysaccharide β-cyclodextrin (abbreviated as TPCD). TPCD could be easily processed into a nanoparticle (TPCD NP). The obtained nanotherapy TPCD NP could be efficiently and rapidly internalized by macrophages and vascular smooth muscle cells (VSMCs). TPCD NP significantly attenuated ROS-induced inflammation and cell apoptosis in macrophages, by eliminating overproduced intracellular ROS. Also, TPCD NP effectively inhibited foam cell formation in macrophages and VSMCs, by decreasing internalization of oxidized low-density lipoprotein. After intravenous (i.v.) administration, TPCD NP accumulated in atherosclerotic lesions of apolipoprotein E-deficient (ApoE-/-) mice, by passive targeting through the dysfunctional endothelium and translocation via inflammatory cells. TPCD NP significantly inhibited the development of atherosclerosis in ApoE-/- mice after i.v. delivery. More importantly, therapy with TPCD NP afforded stabilized plaques with less cholesterol crystals, smaller necrotic core, thicker fibrous cap, and lower macrophages and matrix metalloproteinase-9, compared with those treated with control drugs previously developed for anti-atherosclerosis. The therapeutic benefits of TPCD NP were mainly resulted from reduced systemic and local oxidative stress and inflammation as well as decreased inflammatory cell infiltration in atherosclerotic plaques. Preliminary in vivo tests implied that TPCD NP was safe after long-term treatment via i.v. injection. Consequently, TPCD NP can be developed as a potential anti-atherosclerotic nanotherapy.
ACS Paragon Plus Environment
Page 3 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Atherosclerosis, a multifocal and smoldering disease, is the leading cause of death worldwide.1-3 Although the pathogenesis is still not fully clarified, atherosclerosis is generally considered to be a chronic inflammatory disease.4 During atherogenesis, the primary process of inflammation is accompanied by a secondary event of oxidative stress, which has been regarded as oxidative response to inflammation.5 Although low levels of reactive oxygen species (ROS) are required for regulating oxygen homeostasis and cellular signaling,6 high levels of ROS can induce oxidative stress that is closely associated with the pathogenesis of atherosclerosis.5 Low-density lipoprotein (LDL) oxidation is an early event in the development of atherosclerotic lesions. Oxidized LDL (oxLDL) has a number of proatherogenic activities,7-9 such as inducing endothelial dysfunction, promoting foam cell formation, stimulating ROS generation via multiple pathways, and increasing the expression of adhesion molecules and scavenger receptors. In addition, ROS can lead to NFκB activation, cell apoptosis, protein modification, and oxidative damages to other biomolecules.10-12 ROS also disrupt redox-dependent signaling in the vessel wall to promote progress of atherosclerosis,5, 13, 14 involving signal transduction pathways,15 regulatory genes associated with vascular function,14 inflammatory components of atherosclerosis,16 and clearance of apoptotic cells by macrophages.17 In addition to a large body of experimental evidence in animal models,5 this oxidation hypothesis of atherosclerosis has been supported by extensive epidemiological data in humans.18 Therefore, attenuating systemic oxidative stress, preventing vascular oxidative stress, and reducing ROS generation in plaques represent reasonable strategies for the treatment of atherosclerosis.13 In this aspect, various types of antioxidants have been investigated,19 such as vitamins E and C,20 probucol and its derivatives,21 coenzyme Q,22 proanthocyanidin,23 Tempol,24 and NADPH oxidase.25 Although these preclinical studies have substantiated protective effects of antioxidants on atherosclerosis, clinical trials did not afford positive effects.26 To a certain degree, this is resulted from nonspecific distribution, rapid elimination via renal filtration and urinary excretion, wrong dosing regimens, as well as low delivery efficiency and short retention time at atherosclerotic plaques.7, 27 Moreover, the limited ROS-eliminating capability by a single antioxidant also contributes to undesirable outcomes of most existing agents clinically studied in inflammatory diseases.28 ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 43
Consequently, other anti-oxidative stress strategies remain to be developed. Increasing evidence has demonstrated that nanoparticle-based targeting strategies are effective and promising for molecular imaging and therapy of atherosclerosis.29-32 Nanoparticles can target plaques by direct infiltration via the injured endothelium or the dysfunctional neovessels in the adventitia.33-36 In addition, nanoparticles administered via intravenous or intraperitoneal injection can be endocytosed by circulating phagocytes, followed by translocation to atherosclerotic lesions by cellular recruitment and infiltration.37-39 Whereas nanoparticles only serve as vehicles for targeted delivery of different therapeutics to atherosclerotic plaques in most studies,33, 40-44 recent progress also indicated that nanoparticles with intrinsic anti-oxidative and anti-inflammatory activities are promising next-generation therapies for the treatment of atherosclerosis and other inflammatory diseases. For example, nanoparticles assembled from sugar-based amphiphilic polymers were effective to counteract atherosclerosis,45 by competitively blocking uptake of oxidized lipids via macrophage scavenger receptors. Nanotherapies formed by Tempol-containing amphiphilic copolymers could effectively treat drug-induced intestinal inflammation and nonalcoholic steatohepatitis.46,
47
In addition, a
dendrimer-N-acetyl-L-cysteine conjugate remarkably attenuated neuroinflammation in newborn rabbits with cerebral palsy,48 while nanoparticles derived from a biodegradable polymer containing an antioxidant p-hydroxybenzyl alcohol significantly inhibited inflammation in ischemic tissues in mice.49 These preclinical studies have substantiated the effectiveness of nanotherapies based on antioxidant or anti-inflammatory materials. However, the development of these nanotherapies generally need preparation of polymers with complicated chemical structures, while there are considerable challenges for their structural tailoring, reproducible synthesis, and quality control from the viewpoint of large-scale production. In addition, in vivo degradation, metabolism, clearance performance, and the safety profile of these bioactive nanoparticles are still elusive. As a result, unmet demand exists for the development of intrinsically active and translational nanoparticles in targeted therapy of atherosclerosis. Most recently, we demonstrated that functional materials based on a cyclic polysaccharide β-cyclodextrin conjugated with different oxidation-labile units of phenylboronic acid pinacol esters were able to eliminate ACS Paragon Plus Environment
Page 5 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
hydrogen peroxide, thereby displaying anti-oxidative stress and anti-inflammatory activities.50 On the basis of this finding and in view of the limitations of most currently existing antioxidants,28 herein we developed a broad-spectrum ROS-eliminating nanoparticle, which was produced from a β-cyclodextrin material simultaneously linked with Tempol and phenylboronic acid pinacol ester. By effectively scavenging multiple species of reactive oxygen, such as hydrogen peroxide, hydroxyl radical, superoxide anion, and hypochlorite, this newly prepared nanotherapy could attenuate oxidative stress-induced inflammation and cell apoptosis as well as inhibit oxLDL-induced foam cell formation that is closely related to the pathogenesis of atherosclerosis. In vivo therapeutic studies in mice substantiated that this nanotherapy prevented the progression of atherosclerosis and stabilized atherosclerotic plaques by suppressing systemic and local inflammation and oxidative stress, which were realized by targeting inflammatory cells and atheromatous plaques.
RESULTS AND DISCUSSION Design, Preparation, and Characterization of a ROS-Scavenging Material and Nanoparticle. A broad-spectrum ROS-scavenging material TPCD was designed, in which two functional moieties of Tempol (Tpl) and phenylboronic acid pinacol ester (PBAP) were covalently linked onto the β-cyclodextrin (β-CD) scaffold (Figure 1A). Tpl, a free radical scavenger, is frequently used as a superoxide dismutase (SOD)-mimetic agent for the treatment of oxidative stress-related diseases in different animal models.51 Our previous study demonstrated that PBAP can effectively eliminate hydrogen peroxide (H2O2),50 thereby functioning as a catalase-mimetic compound. We hypothesize that the nanoparticle based this material (i.e. TPCD NP) can be used as an effective nanotherapy for targeted treatment of atherosclerosis (Figure 1B). TPCD was synthesized by sequentially conjugating Tpl and PBAP onto β-CD (Figure S1A). The synthesized TPCD was measured by 1H NMR, UV-visible, and Fourier-transform infrared (FT-IR) spectroscopy as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Figure S1B-F), which collectively demonstrated successful preparation of TPCD with expected structure. Calculation based on high-performance liquid chromatography of the hydrolyzed products of TPCD in the presence of an ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 43
excess amount of H2O2 revealed approximately 2 Tpl and 5 PBAP units in each TPCD molecule. As expected, TPCD eliminated H2O2 in a dose-dependent profile after 48 h of incubation (Figure 2A). By 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay,52 we also evaluated the free radical-scavenging capability of TPCD. Similar to the H2O2-eliminating profile, the scavenged DPPH radical was dramatically increased with increase in the TPCD dose (Figure 2B). Using a commercially available kit, elimination of superoxide anion (O2•-) by TPCD was quantified, showing a dose-response pattern after incubation for 40 min at 37°C (Figure 2C). To quantify the hypochlorite (ClO-) elimination capacity of TPCD, we used a luminescent nanoprobe developed in our previous study (Figure S2).53 After incubation at 37°C for 15 min, consumed hypochlorite was proportional to the quantity of TPCD (Figure 2D). Collectively, these results substantiated that TPCD can eliminate a broad spectrum of reactive species. Also, it should be noted that TPCD can be completely hydrolyzed into small-molecule products in the existence of H2O2 (Figure S3). Nanoparticles (NPs) derived from TPCD were produced by a self-assembly/nanoprecipitation method with a small amount of lecithin and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG). Both lecithin and DSPE-PEG are biocompatible materials that are widely employed in approved liposomal formulations.54 For NPs based on this approach, the core is composed of hydrophobic carrier materials, which is encased in a shell comprised of peripherally aligned lecithin and DSPE-PEG molecules (Figure 1A).37, 50, 55 The formation of a monolayer of phospholipids on NPs is driven by hydrophobic interactions between the carrier material and lecithin/DSPE-PEG, which can reduce the surface tension of the hydrophobic core in aqueous solution. The presence of peripheral PEG chains affords good colloidal stability and desirable reconstitution capability for the resulting NPs. Observation by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed spherical morphology for the obtained TPCD NP (Figure 2E-F), with a relatively narrow size distribution. Quantification by dynamic light scattering indicated that the average diameter was 128 ± 1 nm (Figure 2G), with the polydispersity index of 0.258 ± 0.01 and a negative zeta-potential of -24.3 ± 0.7 mV. TEM observation of the phosphotungstic acid-stained sample suggested that TPCD NP displayed a core-shell structure (Figure 2H). Characterization by ACS Paragon Plus Environment
Page 7 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
FT-IR spectrometry revealed the presence of DSPE-PEG in TPCD NP, as manifested by absorption bands at 2854, 723, and 1152 cm-1 that are characteristic for DSPE and PEG components (Figure 2I), respectively. In addition, the 1H NMR spectrum of TPCD NP in CD3OD (a good solvent for both TPCD and DSPE-PEG) showed significant proton signals corresponding to both TPCD and DSPE-PEG (Figure 2J). By contrast, the signals due to TPCD were considerably attenuated when TPCD NP was dispersed in D2O, while DSPE-PEG signals were clearly observed. These results collectively confirmed the core-shell structure of TPCD NP. Due to the ROS-responsive feature of PBAP moieties in TPCD, TPCD NP is considered to be hydrolysable in the presence of ROS. When TPCD NP was incubated in 0.01 M PBS (pH 7.4) with various concentrations of H2O2, an evident time-dependent hydrolysis profile could be found (Figure 2K). Additionally, the hydrolysis rate was considerably accelerated by increasing the H2O2 concentration. This is in line with the previous studies on ROS-responsive β-CD materials with different PBAP groups,50 implying that conjugation of Tpl does not influence the ROS-sensitivity of PBAP units. Consequently, concomitant with elimination of ROS, TPCD NP can be hydrolyzed into water-soluble products, which is beneficial for its excretion. In Vitro Cytotoxicity Studies. Before in vitro biological evaluations, we first examined in vitro cellular toxicity of TPCD NP in different cells related to the pathogenesis of atherosclerosis, including endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages. After incubation with TPCD NP at varied doses for 12 or 24 h, relatively high cell viability values were detected for human umbilical vein endothelial cells (HUVEC) (Figure S4A-B). Even at 512 µg/mL TPCD NP, the percentage of viable cells was still above 60%. These results indicated that TPCD NP itself showed low cytotoxicity in endothelial cells. Also, incubation with different doses of TPCD NP for 12 and 24 h resulted in low cytotoxicity in mouse vascular smooth muscle cells (MOVAS) and RAW264.7 mouse macrophages (Figure S4C-F). Accordingly, these results demonstrated that TPCD NP displayed low cytotoxicity, even at relatively high doses. In Vitro Cellular Uptake of TPCD NP in Macrophages and VSMCs. Macrophages play a critical role in the development of atherosclerosis.56 The recruitment of macrophages has an important effect on site-specific delivery of nanomedicines to atherosclerotic lesions.35 On the other hand, phenotypic switching of VSMCs ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 43
results in less-differentiated forms that can also directly facilitate atherosclerosis.57 Consequently, cellular uptake behaviors of TPCD NP in macrophages and VSMCs were examined. We first investigated cellular internalization and intracellular trafficking of TPCD NP in RAW264.7 cells by confocal microscopy. After treatment with Cy5-labeled TPCD NP (Cy5/TPCD NP) for different periods of time, significant red fluorescence was observed in RAW264.7 cells, exhibiting gradually enhanced intensities with prolonged incubation (Figure 3A and Figure S5). Considerable intracellular fluorescent signals could be observed even after 0.5 h of culture. This indicated that macrophages can rapidly endocytose TPCD NP, in line with the phagocytic capacity of macrophages.58 At various time points, we also found the co-localization of fluorescent signals from Cy5 (red) with LysoTracker (green) that can label late endosomes and lysosomes. This suggested that endocytosed TPCD NP was mainly transported through the endolysosomal pathway. Further, quantification by flow cytometry confirmed the time-dependent cellular uptake profile of Cy5/TPCD NP in macrophages (Figure S6A and Figure 3B). In addition, both fluorescence microscopic and flow cytometric analyses demonstrated a dose-response internalization of TPCD NP by RAW264.7 cells (Figure 3C-D and Figure S6B). Since macrophages in atherosclerotic plaques locate in an inflammatory microenvironment with many pro-inflammatory mediators, we examined whether inflammatory stimulation affects their phagocytic capability to TPCD NP. It was found that internalization of Cy5/TPCD NP in RAW264.7 cells was significantly increased at different time points (Figure S7A-B), when the cells were pre-stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) and for 24 h. Similarly, pre-treatment with IFN-γ and LPS promoted cellular uptake of various doses of TPCD NP by RAW264.7 cells after 2 h of incubation (Figure S7C-D). According to the similar methods, we evaluated cellular uptake profiles of TPCD NP in MOVAS cells by quantification via flow cytometry. In this case, time and dose-dependent uptake behaviors were also observed (Figure S8). Taken together, these results demonstrated that TPCD NP can be effectively endocytosed by macrophages and VSMCs. ROS-Scavenging, Anti-Oxidation, and Anti-Inflammatory Activities of TPCD NP in Macrophages. ACS Paragon Plus Environment
Page 9 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
The generation of ROS is a fundamental biological process in phagocytic leukocytes, which regulates multiple vascular cell functions such as growth and migration of endothelial cells and VSMCs.14 However, when overproduced ROS overwhelm endogenous antioxidant capacity, oxidative stress occurs. Uncontrolled ROS generation and sustained oxidative stress can induce tissue and cell injury that further initiates an inflammatory cycle and results in amplification of oxidative stress.59 Therefore, we examined whether TPCD NP can inhibit the ROS generation in RAW264.7 macrophages. In the model group, RAW264.7 cells were simultaneously treated with LPS and IFN-γ, while cells cultured with medium alone served as the normal control. After stimulation for 4 h and incubation with free medium for additional 2 h, cells in the model group displayed a considerably high level of ROS, as probed by a fluorescent dye 2’,7’-dichlorofluorescin-diacetate (DCF-DA) that emits green fluorescence under oxidative conditions (Figure 3E). By contrast, after the stimulated macrophages were treated with free Tpl or TPCD NP at different doses for 2 h, fluorescent signals of DCF-DA were significantly attenuated, particularly in the case of TPCD NP-treated cells. Further quantitative analysis by flow cytometry also demonstrated that intracellular ROS production in stimulated macrophages could be effectively suppressed by treatment with TPCD NP in a dose-response pattern (Figure S9 and Figure 3F). The ROS-scavenging capability of TPCD NP was comparable to that of the same dose of free Tpl that has been considered to be more permeable to cell membrane.60 Furthermore, we interrogated whether TPCD NP can attenuate inflammatory responses in macrophages. Treatment of RAW264.7 cells with LPS/IFN-γ significantly increased the excretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemoattractant protein-1 (MCP-1) (Figure 3G-I). Pre-incubation with 50 or 100 µg/mL of TPCD NP for 2 h significantly inhibited the expression of these cytokines. By contrast, treatment with the same doses of free Tpl showed no positive effects on IL-1β or only limited effects for TNF-α and MCP-1. Previous findings have showed that ROS can activate multiple signal transduction cascades, which in turn modulate inflammation in atherosclerosis.14 Consequently, our results substantiated that TPCD NP can attenuate inflammation in macrophages by decreasing intracellular ROS production. ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 43
As well documented, excessive ROS produced in cells will cause oxidation of lipids, proteins, and DNA.61 We subsequently examined the protective effects of TPCD NP on ROS-induced oxidative damages to biomolecules by quantifying the intracellular levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) and 8-hydroxy-2’-deoxyguanosine (8-OHdG). 8-iso-PGF2α is a metabolic product of arachidonic acid through nonenzymatic free radical-catalyzed peroxidation, which has been considered as a marker and mediator of lipid peroxidation.62 On the other hand, 8-OHdG is one of major biomarkers of ROS-induced oxidative DNA damage, and it is also the most abundant product of oxidative DNA repair found in humans.63 Consistent with previous findings, stimulation with H2O2 resulted in dramatically increased expression of both 8-iso-PGF2α and 8-OHdG in RAW264.7 cells (Figure 3J-K), while pre-treatment with either Tpl or TPCD NP significantly decreased their levels. Of note, the high dose of TPCD NP showed a better effect than that of the same dose of free Tpl. Consistently, pre-treatment with TPCD NP significantly suppressed H2O2-induced cell apoptosis of RAW264.7 cells (Figure S10 and Figure 3L), in a dose-response pattern. In this case, comparable efficacy was observed for the same doses of Tpl and TPCD NP. Collectively, these results demonstrated that TPCD NP can protect macrophages from ROS-induced oxidative damages to biomolecules and cell apoptosis by reducing intracellular ROS generation. In Vitro Inhibition of Cellular Internalization of oxLDL and Foam Cell Formation by TPCD NP. Macrophage foam cell formation in the intima is a major hallmark of early-stage atherosclerotic lesions, while cellular uptake of oxLDL displays a critical role during this process.64 Recent studies indicated that foam cell formation in VSMCs, which can be induced by modified LDLs such as oxLDL, also contributes to the initiation of atherosclerosis.65 Therefore we investigated the effect of TPCD NP treatment on cellular uptake of oxLDL in both RAW264.7 and MOVAS cells. After RAW264.7 cells were treated with DiI-labeled oxLDL (DiI-oxLDL) for 3 h, significant red fluorescence was observed in the cytoplasm (Figure 4A and Figure S11). Pretreatment of macrophages with free Tpl at either 8 or 16 µg/mL did not reduce intracellular uptake of DiI-oxLDL. By contrast, red fluorescent signals of DiI-oxLDL were notably low after RAW264.7 macrophages were pre-incubated with the same doses of TPCD NP (in terms of the Tpl unit). This result based on fluorescence ACS Paragon Plus Environment
Page 11 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
observation was further confirmed by quantification of intracellular DiI-oxLDL via flow cytometric analysis (Figure S12 and Figure 4B). There were significant differences between TPCD NP and Tpl groups. In addition, a dose-dependent effect was found for TPCD NP. Likewise, pretreatment with TPCD NP effectively inhibited oxLDL uptake in MOVAS cells (Figure 4C-D and Figure S13-14), while the same dose of free Tpl showed no significant effects. Subsequently, we examined whether treatment with TPCD NP is effective to inhibit foam cell formation by attenuating oxLDL internalization. Consistent with previous studies, RAW264.7 macrophages treated with 50 µg/mL oxLDL for 24 h showed considerable intracellular lipid droplets and significant macrophage foam cell formation as illustrated by staining with Oil Red O (ORO) (Figure 4E, the upper panel). Pretreatment with Tpl at either 8 or 16 µg/mL, however, did not significantly reduce the formation of foam cells. In contrast, treatment with the same doses of TPCD NP (corresponding to the content of Tpl) notably suppressed foam cell formation. Quantification of intracellularly deposited ORO supported this microscopic observation (Figure 4F). Likewise, treatment with TPCD NP reduced foam cells derived from oxLDL-stimulated MOVAS cells, while the same dose of free Tpl did not afford significant effects (the lower panel of Figure 4E, Figure 4G). These results agree with the finding on oxLDL cellular uptake in RAW264.7 and MOVAS cells (Figure 4A-D and Figure S11-14). Our results are also in accordance with the previous finding that promoted oxLDL uptake is one of key step toward foam cell formation, regardless of macrophages and VSMCs.66, 67 Together, these results demonstrated that TPCD NP can attenuate the formation of foam cells from macrophages and VSMCs, by reducing cellular internalization of oxLDL. Targeting Atherosclerotic Plaques in Mice by i.v. Injected TPCD NP. The in vivo pharmacokinetic profile of TPCD NP was first studied in mice. After i.v. injection of Cy7.5-labeled TPCD NP (Cy7.5/TPCD NP), fluorescence imaging indicated that Cy7.5/TPCD NP was almost completely cleared from the blood after 24 h (Figure 5A-B). Then we investigated in vivo targeting capability of i.v. administered TPCD NP in apolipoprotein E-deficient (ApoE-/-) mice bearing atherosclerotic plaques. At 3 h after i.v. injection of Cy7.5/TPCD NP, the isolated entire aortas showed significant fluorescence in the aortic arch and abdominal aorta (Figure 5C), which ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 43
are susceptible to form plaques in ApoE-/- mice.68 At 12 h, more significant distribution of Cy7.5/TPCD NP in the aortas was observed (Figure 5C-D). This result suggested that i.v. injected TPCD NP was able to accumulate in atherosclerotic lesions. Besides, we observed the accumulation of Cy7.5/TPCD NP in heart, liver, spleen, lung, and kidneys (Figure S15), with a time-dependent accumulation profile in all these examined main organs. Further immunofluorescence analysis was performed after i.v. administration of Cy5/TPCD NP in ApoE-/mice with established plaques. We found the presence of Cy5/TPCD NP in cryosections of the aortic root, aortic arch, and brachiocephalic artery (Figure 5E). The colocalization of Cy5/TPCD NP and CD68+ macrophages was clearly observed. In line with this result, distribution of Cy5/TPCD NP was also detected in isolated macrophages (Figure 5F). Moreover, the colocalization of Cy5 fluorescence with Ly-6G+ neutrophils could be found in the cryosection of brachiocephalic artery (Figure 5G), while there were no significantly overlapped fluorescent signals in the sections of the aortic root and aortic arch. These results demonstrated that i.v. administered TPCD NP can target atherosclerotic plaques, leading to both cellular and extracellular distribution. In addition to translocation through the damaged endothelial barrier of arteries and extravasation via the leaky neovessels,34, 69 transportation by circulating phagocytes may contribute to the accumulation of TPCD NP in the lesions.37-39 In support of these findings, we detected the distribution of Cy5/TPCD NP in different cells in peripheral blood (Figure 5H), including lymphocytes and neutrophils as well as Ly-6Clow and Ly-6Chigh monocytes, which was collected at 30 min after i.v. administration in C57BL/6 mice. Of note, relatively high Cy5/TPCD NP positive cells were found for neutrophils and Ly-6Chigh monocytes, particularly in the case of neutrophils (Figure 5I). Consequently, translocation by neutrophils and Ly-6Chigh monocytes should have contributed to the lesional accumulation of i.v. delivered TPCD NP. Importantly, these two types of cells are pro-inflammatory cells intimately associated with the pathogenesis of atherosclerosis.56, 70 Treatment of Atherosclerosis by i.v. Delivered TPCD NP in ApoE-/- Mice. On the basis of the above promising results, in vivo therapeutic effects of TPCD NP were examined. According to our preliminary studies, ApoE-/- mice may begin to develop atherosclerotic plaques at artery bifurcations after consuming a high-fat diet for one month, while significant atherosclerotic plaques are formed after 3 months. Consequently, a 2-month ACS Paragon Plus Environment
Page 13 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
treatment was performed after one month of high-fat diet for prophylactic therapy. In this case, the treated mice can be clearly compared with the model mice to show therapeutic effects of the examined anti-atherosclerotic nanotherapy. After receiving Western diet for one month, ApoE-/- mice were randomly assigned into different groups that were treated with various formulations, in combination with Western diet for additional two months (Figure 6A). Besides free Tpl, a small-molecule antioxidant probucol was used as another control drug, which has been demonstrated efficacious for the treatment of atherosclerosis in different animal models.71, 72 However, clinical studies on probucol failed to show positive outcomes, partly resulting from its low accumulation in plaques and multiple adverse effects.21, 73 Different formulations were administered by i.v. injection twice per week. After 2 months, the entire aortas were collected and stained with ORO. The saline group showed high ORO-positive areas (Figure 6B). Treatment with free Tpl did not afford significant effects. By contrast, considerably low ORO-stained areas in aortas were observed for mice treated with probucol or TPCD NP at 50 mg/kg (low dose) and 100 mg/kg (high dose). Of note, high-dose TPCD NP contained the same dose of the Tpl unit as free Tpl. Further quantification indicated that the average plaque area was 24.9 ± 1.3%, 22.5 ± 1.5%, 12.3 ± 1.8%, 9.7 ± 1.0%, and 6.3 ± 0.9% for mice treated with saline, Tpl, probucol, low-dose TPCD NP, and high-dose TPCD NP, respectively (Figure 6C). We found significant differences between the high-dose TPCD NP group and the Tpl or probucol group. Consistent with this result, observation on ORO-stained cryosections from the aortic sinus, aortic arch, and brachiocephalic artery also revealed the most significant anti-atherosclerotic activity for TPCD NP at both low and high doses (Figure 6D-G). Together, these results demonstrated that i.v. delivered TPCD NP can efficaciously attenuate the development of atherosclerosis. Whereas treatment with probucol also showed beneficial results, much better outcomes were achieved by TPCD NP at either a low or high dose. Furthermore, the loads of macrophages and cholesterol crystals in atherosclerotic plaques were determined through immunofluorescence analysis according to the previously reported method.74 Direct observation of the immunofluorescent images of sections based on an aortic sinus showed that both red fluorescent and white reflection signals were considerably decreased after treatment with probucol and TPCD NP (Figure S16A), ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 14 of 43
indicating the decrease of macrophages and cholesterol crystals in plaques. Further quantification implied that TPCD NP treatment most significantly reduced macrophage accumulation and cholesterol crystallization in atherosclerotic lesions (Figure S16B-C), while Tpl alone showed no significant effect. As well documented, the generation of ROS may lead to deficient autophagy, which in turn impairs cholesterol efflux from macrophages, thereby increasing the accumulation of cholesterol crystals.75 In addition, oxidative stress can induce translocation of cholesterol and its derivatives to mitochondria by steroidogenic acute regulatory family proteins, which subsequently causes peroxidative damage and accordingly decreases reverse cholesterol transport in macrophages.76 Accordingly, elimination of ROS can reduce the formation of cholesterol crystals in plaques through multiple pathways. Likewise, analysis of these sections indicated that the plaque area was notably reduced after therapy with probucol and TPCD NP (Figure S16D), in line with the results based on ORO staining (Figure 6D-G). The components of atherosclerotic plaques were also examined by histochemistry analyses of aortic sinus sections. In combination with immunofluorescence and hematoxylin-eosin (H&E) staining (Figure S16A and Figure 7A-B), we found that plaques in the saline group were largely consisted of acellular and lipid-rich necrotic cores. As compared to the saline and Tpl groups, the necrotic core area was significantly low in the probucol and TPCD NP groups. Separate staining with anti-CD68 antibody and anti-matrix metalloproteinase-9 (MMP-9) antibody indicated that TPCD NP treatment effectively reduced the macrophage count and MMP-9 expression in plaques (Figure 7A and 7C-D). Because the area of necrotic cores and the levels of macrophage infiltration and MMP-9 expression are positively related to plaque vulnerability,77 these results demonstrated that TPCD NP therapy could more effectively stabilize atherosclerotic plaques. Concordant with these findings, staining with the Masson’s trichrome method showed a significantly higher content of collagen around plaques of TPCD NP-treated groups, resulting in enhanced fibrous cap thickness (Figure 7A and 7E). Also, staining with α-smooth muscle actin (a-SMA) antibody showed a high accumulation of VSMCs in plaques in the TPCD NP groups (Figure 7A and 7F), while recent studies have evidenced that VSMC proliferation is beneficial throughout atherogenesis.57 Since collagen surrounding plaques and VSMCs have been considered to be closely ACS Paragon Plus Environment
Page 15 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
related to plaque stability, these results, together with the decreased necrotic core area, macrophage, and MMP-9, demonstrated that i.v. treatment with TPCD NP can efficaciously stabilize atherosclerotic plaques. Mechanisms Underlying In Vivo Efficacy of TPCD in ApoE-/- Mice. Preliminary studies were also conducted to address mechanisms responsible for in vivo anti-atherosclerotic activity of TPCD NP. After staining with a fluorescent probe dihydroethidium (DHE), sections of the brachiocephalic artery from ApoE-/mice subjected to different treatments were analyzed by fluorescence observation. While the normal group (i.e. healthy C57BL/6 mice) showed negligible fluorescent signals, the group of saline-treated ApoE-/- mice displayed bright fluorescence (Figure 8A), resulting from reaction of DHE with superoxide anions that yields ethidium with red fluorescence.78 After i.v. treatment with different formulations, observation and quantitative analysis revealed that oxidative stress was significantly attenuated by TPCD NP (Figure 8A-B). In line with this result, the group treated with high-dose TPCD NP displayed the lowest levels of ROS and oxLDL (Figure 8C-D). Accordingly, both systemic and lesional oxidative stress was significantly mitigated by i.v. delivered TPCD NP. In addition, we found the lowest expression of TNF-α and IL-1β (two typical inflammatory cytokines) in the aorta and serum from ApoE-/- mice received TPCD NP treatment, especially at 100 mg/kg (Figure 8E-H), while comparable levels were detected for mice treated with saline and Tpl. These results demonstrated that TPCD NP was able to effectively attenuate systemic oxidative stress and inflammation as well as reduce oxidative stress and inflammation in plaques. As well documented, mouse Ly-6Chigh (Gr1+CCR2+CX3CRlow) monocytes, also called as inflammatory monocytes, are the main precursors of macrophages in atheromata, which can adhere to the activated endothelium and finally become lesional macrophages.79 The aforementioned result showed a high distribution of Cy5/TPCD NP in Ly-6Chigh monocytes (~94%) after i.v. injection in mice (Figure 5H-I). In ApoE-/- mice with atherosclerotic lesions, treatment with i.v. administered TPCD NP notably decreased the count of circulating Ly-6Chigh monocytes (Figure 8I-J). On the other hand, monocyte recruitment to the inflammatory sites is modulated by chemotactic cytokines.80 C-C chemokine receptor type 2 (CCR2) dominates the migration of inflammatory Ly-6Chigh monocytes from the bone marrow into the circulating blood, which is a critical step for ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
macrophage accumulation in the pathogenesis of atherosclerosis.56,
Page 16 of 43
79
Consequently, CCR2+Ly-6Chigh
monocytes play a central role in the initiation of atherosclerosis by exacerbating inflammation and plaque remodeling.81 After treatment with TPCD NP at 100 mg/kg every other day for 3 times, flow cytometric analysis indicated that CCR2+Ly-6Chigh monocytes in the blood of ApoE-/- mice were significantly lower than those of mice treated with saline (Figure 8I and 8K). Our result is consistent with a previous finding that inhibition of the production of CCR2+Ly-6Chigh monocytes decelerated the progression of atherosclerosis.79, 82 Collectively, treatment with TPCD NP can inhibit the generation of inflammatory monocytes and reduce their recruitment to atherosclerotic lesions. These preliminary data implied that the anti-atherosclerotic activity of TPCD NP is mainly due to its capabilities of attenuating systemic and local oxidative stress and inflammation as well as inhibiting the generation and recruitment of pro-inflammatory monocytes. Safety Evaluation During and After Treatment with TPCD NP. Finally, we investigated possible side effects during and after the two-month treatment with TPCD NP. Complete blood count implied that the levels of red blood cell, white blood cell, platelet, and hemoglobin were in normal ranges in various groups (Figure S17A). Also, clinical biochemistry analysis showed no significantly increased levels of alanine aminotransferase, aspartate aminotransferase, blood urea, and serum creatinine, indicating that treatment with different formulations did not significantly affect hepatic and renal functions (Figure S17B-C). As compared to the saline and Tpl groups, the probucol and TPCD NP groups displayed lower levels of total cholesterol and LDL (Figure S17D-E), while no significant changes in triglyceride and high-density lipoprotein were found (Figure S17F-G). Additionally, we examined H&E-stained histological sections of major organs. No distinguishable injuries were observed in sections of all treated groups (Figure S18). Accordingly, all these results suggested that i.v. administration of TPCD NP is safe at the examined dose.
CONCLUSIONS A broad-spectrum ROS-scavenging material was successfully synthesized by covalently conjugating two functional moieties onto β-CD. Thus obtained functional material TPCD was able to effectively scavenge H2O2, ACS Paragon Plus Environment
Page 17 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
radical, superoxide anion, and hypochlorite. Though a facile and well-established nanoprecipitation technique, TPCD was developed into a nanotherapy TPCD NP that could be efficiently and rapidly endocytosed by macrophages and VSMCs. By internalization and eliminating overproduced intracellular ROS, TPCD NP effectively inhibited ROS-induced inflammatory responses and cell apoptosis in macrophages. TPCD NP could also significantly decrease cellular uptake of oxLDL, thereby suppressing foam cell formation in macrophages and VSMCs. By passive targeting, i.v. administered TPCD NP could be accumulated in atherosclerotic plaques. After i.v. delivery, TPCD NP effectively delayed the development of atherosclerosis in ApoE-/- mice. More importantly, treatment with TPCD NP resulted in more stabilized plaques with considerably less cholesterol crystals, smaller necrotic core, thicker fibrous cap, and lower macrophages and MMP-9, compared with those treated with control therapies. These desirable therapeutic outcomes of TPCD NP were realized by reducing systemic and local oxidative stress and inflammation as well as attenuating inflammatory cell infiltration in plaques. Preliminary in vivo examination revealed that TPCD NP was safe for long-term treatment by i.v. administration. Consequently, TPCD NP is a promising anti-atherosclerotic nanotherapy that deserves further development. Moreover, its in vivo activity can be further improved by enhancing plaque targeting efficiency, which may be easily achieved by surface engineering with active targeting moieties. Additionally, combination therapy can be realized by loading this nanotherapy with different drugs currently used for the management of atherosclerotic diseases.
EXPERIMENTAL SECTION Materials. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, (Tempol, referred as Tpl), β-cyclodextrin (β-CD), 2’,7’-dichlorofluorescin-diacetate (DCF-DA), Oil Red O (ORO), lipopolysaccharide (LPS), dihydroethidium
(DHE), Nuclease P1
from
Penicillium
citrinum,
1,1-carbonyldiimidazole (CDI),
4-dimethylaminopyridine (DMAP), anhydrous dimethylformamide (DMF), anhydrous dichloromethane (DCM), anhydrous dimethylsulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Trypan Blue were purchased from Sigma-Aldrich (U.S.A.). 4-(Hydroxymethyl) phenylboronic acid pinacol ester (PBAP) was ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 18 of 43
provided by Acro Organics. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) was obtained from Corden Pharma (Switzerland). Lecithin (from soybean) was purchased from Tokyo Chemical Industry Co, Ltd. (Japan). Cyanine5 NHS ester (Cy5) and cyanine7.5 NHS ester (Cy7.5) were from Lumiprobe (U.S.A.). Penicillin, streptomycin, fetal bovine serum (FBS), and Dulbecco’s
Modified
Eagle's
Medium
(DMEM)
were
purchased
from
Gibco
(U.S.A.).
4’,6-Diamidino-2-phenylindole (DAPI), hematoxylin, 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO), and BeyoAP alkaline phosphatase were purchased from Beyotime (China). LysoTracker Green was purchased from Invitrogen (U.S.A.). Recombinant murine interferon-γ (TNF-γ) was purchased from PeproTech (U.S.A). Human high-oxidized low density lipoprotein (oxLDL) and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI)-labeled oxLDL (DiI-oxLDL) were purchased from Yiyuan Biotechnologies (China). Synthesis of a ROS-Scavenging Material Based on β-Cyclodextrin. A ROS-scavenging material TPCD was synthesized by sequentially conjugating Tpl and PBAP onto β-CD. Tpl-conjugated β-CD (TCD) was first prepared. Specifically, Tpl (1.02 g, 5.9 mmol) was dissolved in 10 mL of anhydrous DCM, and then CDI (1.91 g, 11.8 mmol) was added. After reaction for 45 min, 10 mL of DCM was added into the mixture, followed by washing with 10 mL of deionized water. After further rinsing with saturated NaCl solution three times, the obtained sample was concentrated in vacuum and dried over Na2SO4. Thus obtained CDI-activated Tpl (1.65 g) was dissolved in 20 mL of anhydrous DMSO, then 2.35 g β-CD (2.07 mmol) and 1.13 g DMAP (9.30 mmol) were added under nitrogen. The mixture was stirred at 25°C for 24 h. The final product was precipitated from a mixture solution of methanol and diethyl ether. After centrifugation and lyophilization, a pink powder was obtained. Then PBAP was conjugated onto TCD according to our previously established methods.83 Briefly, PBAP (1.11 g) and CDI (1.53 g) were dissolved in 15 mL of anhydrous DCM, which was stirred at 25°C for 30 min under nitrogen. The organic phase was separately washed with deionized water and saturated NaCl solution, dried over Na2SO4, and then concentrated to obtain CDI-activated PBAP. Subsequently, 1.66 g CDI-activated PBAP, 1.06 g DMAP, and 0.42 g TCD were dissolved in 20 mL of anhydrous DMSO under nitrogen. After ACS Paragon Plus Environment
Page 19 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
reaction at 25°C for 48 h, the product was precipitated from 80 mL of deionized water and collected by centrifugation, followed by thoroughly washing with deionized water and freeze-drying to give a light pink powder. Materials Characterization. 1H NMR spectra were acquired on a spectrometer operating at 600 MHz (DD2, Agilent). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was performed with a Waters Micromass TofSpec-2E spectrometer operated in a linear mode. Fourier-transform infrared (FT-IR) spectra were recorded on a PerkinElmer FT-IR spectrometer (100S). UV-visible spectroscopy was conducted on a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd, China). Preparation of Nanoparticles Based on TPCD. TPCD nanoparticles were prepared by a nanoprecipitation/self-assembly method.83 In brief, 6 mg lecithin and 9 mg DSPE-PEG were dissolved in 0.6 mL of ethanol, which was diluted with 15 mL of deionized water. The obtained mixture was heated to 65°C for 1 h, into which 5 mL of methanol solution containing 50 mg TPCD was added, followed by incubation at 25°C for 2 h. The remaining organic solvent was removed by evaporation under vacuum. Finally, TPCD nanoparticles were harvested after freeze-drying. Through similar procedures, nanoparticles labeled with either Cy5 or Cy7.5 were prepared. Characterization of TPCD Nanoparticles. The size, size distribution profiles, polydispersity index (PDI), and zeta-potential of nanoparticles were quantified using a Malvern Zetasizer NanoZS instrument at 25°C. Transmission electron microscopy (TEM) was performed using a JEM-1400 PLUS microscope (JEOL, Japan). Scanning electron microscopy (SEM) was conducted on a FIB-SEM microscope (Crossbeam 340, Zeiss). Determination of ROS-Scavenging Capability. The ROS-eliminating capability of TPCD NP was examined. To evaluate H2O2-scavenging capacity, different concentrations (from 0, 3.125, 6.25, 12.5, 25, 50, 100, to 200 µg/mL) of TPCD NP were incubated in 2.5 mL of 0.01 M PBS (pH 7.4) containing 500 mM H2O2 for 48 h. Then residual H2O2 was determined by a fluorimetric hydrogen peroxide assay kit (MAK165, Sigma-Aldrich), and eliminated H2O2 was calculated. ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 20 of 43
The free radical scavenging capability was measured using a previously established protocol.84 Briefly, 1 mL of a fresh solution of DPPH• (100 µg/mL) was incubated in 2 mL of methanol containing different concentrations of TPCD (from 0, 15.625, 31.25, 62.5, 125, 250, 500 to 1000 µg/mL) for 30 min at 31°C in the dark. Subsequently, the absorbance at 517 nm was recorded by UV-visible spectroscopy and eliminated DPPH was calculated. In addition, the superoxide anion scavenging capability of TPCD NP was evaluated. To this end, various concentrations of TPCD NP were first incubated with an excess amount of superoxide anion that was produced by the xanthine/xanthine oxidase system. After incubation at 37°C for 40 min, the remaining superoxide anion was quantified by a commercially available test kit (Nanjing Jiancheng Bioengineering Institute, China). The superoxide anion-eliminating capacity of TPCD NP was then calculated. To quantify the hypochlorite (ClO-)-scavenging capability of TPCD, a luminescent nanoprobe (Lu-bCD NP) developed in our previous study was used.53 Specifically, 50 µL of aqueous solution containing 10 mg/mL Lu-bCD NP was reacted with 50 µL of aqueous solutions containing NaClO varying from 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, to 100 mM in a black 96-well plate. Immediately after mixing, luminescence imaging was performed using an IVIS Spectrum system (PerkinElmer, U.S.A.). The standard curve was then established by plotting the chemiluminescence intensity as a function of NaClO concentrations. Subsequently, Lu-bCD NP was employed as the probe for final quantification of the hypochlorite scavenging capability of TPCD. Briefly, 25 µL of aqueous solution containing different concentrations of TPCD (varying from 0, 0.1, 0.2, 0.5, 1, 2, to 5 mg/mL) was incubated with 475 µL of aqueous solution containing 100 mM NaClO at room temperature. After 15 min, 50 µL of the supernatant was reacted with 50 µL of aqueous solution containing 10 mg/mL Lu-bCD NP and immediately imaged by an IVIS Spectrum system. Then the residual ClO- was calculated according to the standard curve. In Vitro Hydrolysis of TPCD NP in the Presence of H2O2. For in vitro hydrolysis tests, 3 mg freshly produced TPCD NP was incubated in 3 mL 0.01 M PBS (pH 7.4) containing H2O2 ranging from 0, 0.01, 0.1, 1.0 to 3.0 mM at 37°C. At predefined time points, the transmittance values of TPCD NP-containing aqueous ACS Paragon Plus Environment
Page 21 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
solutions were measured by an UV-visible spectrophotometer at 500 nm and then the hydrolysis degree was calculated. In Vitro Cytotoxicity Evaluation. A mouse macrophage cell line (RAW264.7), mouse vascular smooth muscle cell line (MOVAS), and human umbilical vein endothelial cell line (HUVEC) were cultured in 96-well plates (1.0×104 cells per well) in 100 µL of DMEM with FBS, penicillin, and streptomycin. Cells were treated with varied doses of TPCD NP. After treatment for different time intervals, cell viability was measured by Cell Counting Kit-8 assay. Observation of Cellular Internalization Profiles of Nanoparticles by Confocal Microscopy. RAW264.7 cells were cultured in 12-well plates (1 × 105 cells per well) in 1 mL of growth medium for 12 h. Cells were treated with 100 ng/mL LPS and 100 IU/mL IFN-γ in free medium for 24 h, and then were incubated with Cy5-labeled TPCD NP at 40 µg/mL. After predetermined time periods, RAW264.7 cells were rinsed and separately stained with LysoTracker Green and DAPI. Subsequently, confocal laser scanning microscopy (CLSM) was performed to acquire fluorescence images. Similarly, cellular internalization was studied at various doses of Cy5/TPCD NP (ranging from 1, 5, 10, 20, 50, to 100 µg/mL) after 2 h of incubation. Flow Cytometric Analysis of Cellular Uptake of Nanoparticles. RAW264.7 cells (2 × 105 cells/well) or MOVAS cells (3 × 105 cells/well) were cultured in 12-well plates for 12 h. Then cells were treated with fresh medium with or without 100 ng/mL LPS and 100 IU/mL IFN-γ for RAW264.7, but without LPS/IFN-γ in the case of MOVAS. After 24 h, cells were incubated with Cy5/TPCD NP at 50 µg/mL. At predefined time points, the cells were harvested for flow cytometric analysis (Accuri C6, BD Biosciences). Using similar methods, internalization profiles at different doses of Cy5/TPCD NP (0, 5, 10, 20, 50, to 100 µg/mL) were investigated after 2 h of incubation. In Vitro Anti-Inflammatory Effects of TPCD NP in Macrophages. Specifically, RAW264.7 cells were seeded in 24-well plates at 1 × 105 cells per well. After 12 h, the normal group was treated with fresh medium, while cells in the model group were stimulated with 100 ng/mL LPS and 100 IU/mL IFN-γ. Other groups were first incubated with various concentrations of TPCD NP or free Tpl for 2 h, and then stimulated with 100 ng/mL ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 22 of 43
LPS and 100 IU/mL IFN-γ for 24 h. Subsequently, typical inflammatory cytokines in the culture supernatant, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) were determined by ELISA (Neobioscience, China), while the levels of total protein were quantified by BCA (Beyotime, China). Intracellular ROS Generation in Macrophages. RAW264.7 cells were cultured in 12-well plates for 12 h. After cells were pretreated with various doses of TPCD NP or free Tpl for 2 h, they were stimulated with LPS (100 ng/mL) and IFN-γ (100 IU/mL). The normal control group was treated with fresh medium, and the model group was stimulated with LPS/IFN-γ without treatment with TPCD NP for 4 h. Subsequently, cells were rinsed and treated with DCFH-DA (10 µM) in serum-free DMEM for 30 min. After washing with PBS and harvested in PBS containing 0.01% Trypan Blue, intracellular fluorescent signals were measured via flow cytometry (Accuri C6, BD Biosciences) and analyzed using FlowJo software. Through similar procedures, the intracellular ROS generation was observed in cell culture dishes by CLSM after probing with DCF-DA. In Vitro Anti-Oxidative Stress Activity of TPCD NP in Macrophages. We detected ROS-induced production of 8-iso-prostaglandin F2α (8-iso-PGF2α) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) in macrophages. Briefly, RAW264.7 cells were first incubated in DMEM for 12 h. Then, cells were pre-incubated with different doses of TPCD NP or free Tpl for 2 h, followed by exposure to 300 µM H2O2 in fresh medium for 24 h. The normal group was treated with free medium alone, and the model group was only stimulated with H2O2. The concentration of 8-iso-PGF2α in the culture medium was determined by ELISA (ADI-900-010, ENZO, Switzerland), while the total protein level was quantified by BCA (Beyotime, China). For detection of 8-OHdG, RAW264.7 cells were first pre-treated with different doses of TPCD NP or free Tpl for 2 h, and then stimulated with 400 µM H2O2 for 12 h. The normal group was incubated in fresh medium, while the model group was only exposed to H2O2. Subsequently, total DNA in the culture supernatants and cell lysates was purified using a DNA extraction kit (51304, QIAGEN, German) and the content was detected. DNA was then digested using nuclease P1 (N8630, Sigma-Aldrich, U.S.A.), and pH was adjusted to 7.5-8.5 using 1 M Tris-HCl (pH 8.5, Beyotime, China). Then alkaline phosphatase at 10 U/mg DNA was added. After ACS Paragon Plus Environment
Page 23 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
incubation at 37°C for 30 min and boiling for 10 min, the level of 8-OHdG in total DNA was detected by an ELISA kit (SKT-120, Stressmarq, Canada) following the manufacturer’s instructions. Anti-Apoptosis Activity of TPCD NP in Macrophages. RAW264.7 cells were incubated with fresh medium containing different doses of TPCD NP or Tpl. After 2 h, the cells were exposed to 400 µM H2O2 in fresh medium for 24 h. Cells in the normal control group were cultured with medium alone. Then cells were collected and stained with FITC Annexin V Apoptosis Detection Kit with PI (Biolegend, U.S.A.), and apoptosis was assayed via flow cytometry. Inhibition of Cellular Uptake of oxLDL by TPCD NP. RAW264.7 or MOVAS cells were incubated in DMEM for 12 h. After stimulation with LPS (100 ng/mL) and IFN-γ (100 IU/mL) for 24 h, RAW264.7 cells were treated with different doses of TPCD NP (50 or 100 µg/mL) or free Tpl (8 or 16 µg/mL), while MOVAS cells were treated with TPCD NP at 40 and 80 µg/mL or Tpl at 6.4 and 12.8 µg/mL. After 2 h, RAW264.7 cells were incubated with 40 µg/mL DiI-oxLDL for 4 h, while MOVAS cells were treated with 20 µg/mL DiI-oxLDL for 3 h. Then cells were washed with 0.5 M HCl in 70% ethanol and harvested. Intracellular fluorescence was measured via flow cytometry (Accuri C6, BD Biosciences). Through similar procedures and after cells were treated with Dil-oxLDL, they were washed with 0.5 M HCl in 70% ethanol. Then cells were stained with DIO (10 µM) and DAPI, and CLSM was performed to acquire fluorescence images. The Effect of TPCD NP Treatment on Foam Cell Formation. After stimulation with LPS (100 ng/mL) and IFN-γ (100 IU/mL) for 24 h, RAW264.7 cells were treated with TPCD NP (50 and 100 µg/mL) or Tpl (8 and 16 µg/mL) for 2 h. Then, cells were incubated with oxLDL at 50 µg/mL for 48 h. The normal control group was treated with fresh medium, and the model group was only stimulated with oxLDL. After washing with 0.5 M HCl in 70% ethanol and fixing with 10% neutral buffered formalin, cells were stained with 0.3% ORO and hematoxylin. Then cells were observed by optical microscopy. Through the similar procedures, the foam cell formation of MOVAS cells was also examined, without stimulation with LPS/IFN-γ. In addition, intracellular ORO was extracted by isopropanol and the ORO concentration was determined by measuring its absorbance at ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 24 of 43
492 nm via UV-visible spectrometry. Animals. Animal care and experiments were conducted in line with the Guide for the Care and Use of Laboratory Animals proposed by National Institutes of Health. All procedures and protocols were approved by the Animal Ethics Committee at Third Military Medical University (Army Medical University). Male C57BL/6 mice (6-8 weeks) were obtained from the Animal Center of the Army Medical University. Male apolipoprotein E-deficient (ApoE-/-) mice (about 6 weeks old) were supplied by the Peking University Health Science Center (China). After acclimatization for 7 days, mice were subjected to different experiments. In Vivo Pharmacokinetic Study of TPCD NP. Using Cy7.5-labeled TPCD NP, we examined the pharmacokinetic profile of TPCD NP after intravenous (i.v.) injection in mice. Cy7.5/TPCD NP was administered to C57BL/6 mice at 100 mg/kg. Mice in the control group were treated with the same volume of saline. At predefined time points, an equal volume of whole blood samples were collected in 96-well black plates. An IVIS spectrum system (Perkin Elmer, U.S.A.) was used to determine the mean fluorescence intensity (MFI) of blood samples. Distribution of TPCD NP in Blood Cells after i.v. Injection. The in vivo cellular distribution of TPCD NP in C57BL/6 mice was examined using a previously established method.43 Briefly, Cy5-labeled TPCD NP was i.v. injected to mice at 100 mg/kg. At 30 min after injection, whole blood was collected. Then cell suspensions were prepared and labeled with different antibodies,37 and cell sorting was performed via flow cytometry (Cytoflex) and analyzed with the FlowJo software. Plaque Targeting and Tissue Distribution of TPCD NP after i.v. Injection in ApoE-/- Mice. Male ApoE-/mice were fed with normal diet containing 1.25% cholesterol and 10% lard for 3 months. Then Cy7.5/TPCD NP was administered by i.v. injection at 100 mg/kg. At 3 and 12 h after administration, mice were perfused with 4% paraformaldehyde in PBS under anesthesia. Subsequently, mice were euthanized, and the aortas and main organs were isolated. Ex vivo imaging was carried out using an IVIS spectrum system and MFI was analyzed by the Living Image 4.5 software. Cellular Distribution of TPCD NP in Aortic Plaques after i.v. Injection in ApoE-/- Mice. After ApoE-/ACS Paragon Plus Environment
Page 25 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
mice were fed with high-fat diet for 3 months, they were i.v. injected with Cy5/TPCD NP at 120 mg/kg. At 4 h post injection, mice were perfused according to the aforementioned procedure. Then mice were euthanized, and the aortic root, aortic arch, and brachiocephalic artery were cut down and the cryosections of 8-µm thickness were prepared. After fixation with pre-cooled acetone at 21°C for 30 min, the slides were blocked and permeabilized with 5% FBS, 5% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.1% saponin in PBS for 60 min at 21°C. They were incubated with 2 µg/mL of rat anti-mouse CD68 antibody (MCA1957, Bio-Rad, U.S.A.) in 10% NGS/PBS in the dark at 4°C for 12 h. Subsequently, the sections were incubated with 1:500 diluted Cy3-labeled goat anti-rat IgG (H+L) (A0507, Beyotime, China) in 10% NGS/PBS at 37°C for 30 min and counterstained with DAPI staining solution (Beyotime, China). The slides were finally observed by CLSM. Additionally, the fragments of aortic tissues were digested in a mixture of collagenase I (450 U/mL), collagenase XI (125 U/mL), DNase I (60 U/mL), and hyaluronidase (60 U/mL) (all from Sigma-Aldrich) at 37°C for 45 min. The obtained suspension was filtered through a 70-µm pore nylon mesh, centrifuged, and seeded in a cell culture dish containing 10% FBS in DMEM. After incubation for 4 h, the cells were washed and the attached cells were treated with rat anti-mouse CD68 antibody (MCA1957, Bio-Rad) and 1:500 diluted Cy3-labeled goat anti-rat IgG (H+L) (A0507, Beyotime, China). CLSM images were acquired after counterstaining with DAPI. Treatment of Atherosclerosis in ApoE-/- Mice with TPCD NP. Fifty ApoE-/- mice were fed with high-fat diet for 3 months. After the first month, they were randomly assigned into five groups (n = 10) and different treatments were conducted for additional 2 months. Mice in the model control group were treated with saline, while animals in the other four groups were i.v. injected with free Tpl at 17.2 mg/kg (i.e. 0.1 mmol/kg), probucol at 5.17 mg/kg (i.e. 1 mmol/kg), a low dose of TPCD NP at 50 mg/kg, and a high dose of TPCD NP at 100 mg/kg. Both Tpl and TPCD NP were dissolved in saline, while probucol was dissolved in saline containing 30% ethanol. TPCD NP at 100 mg/kg contained the same dose of the Tpl unit as that of free Tpl at 17.2 mg/kg. All formulations were i.v. injected twice one week. ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 26 of 43
Quantification of Atherosclerotic Plaques by ORO Staining. After different treatments, ApoE-/- mice were euthanized. The lesion area of the aorta from the left common carotid artery to the iliac bifurcation was evaluated. To this end, the aorta was resected and perfused with 10% neutral buffered formalin for 50 min. The aorta was opened longitudinally and stained with ORO. In addition, serial cross-sections of the aortic root, aortic arch, and brachiocephalic artery were prepared and stained with ORO. Plaque area analysis was carried out with the Image-Pro Plus 6.0 software. Evaluation of Vascular Superoxide Anion Generation by Dihydroethidium Staining. Brachiocephalic artery samples were embedded in Tissue-Tek O.C.T. Compound and immediately 8-µm sections were prepared on a Leica cryostat. A fluorescent dye DHE was used to probe in situ generation of superoxide anion as previously described.85 Briefly, after incubation with 2% Triton X-100 for 10 min at 21°C and then blocking with 5% BSA in PBS, slides were stained with 2 µM DHE in Krebs solution and imaged immediately under fluorescence microscopy. The fluorescent intensities were analyzed with the Image-Pro Plus 6.0 software. Quantification of Macrophages and Cholesterol Crystals in Atherosclerotic Plaques. For analysis of macrophages and cholesterol crystals in plaques, aortic cryosections (5-µm thickness) were fixed with 4% paraformaldehyde, blocked, and permeabilized with 5% FBS, 5% BSA, 10% NGS, and 0.1% saponin in PBS. The slides were incubated at 4°C for 12 h, in the presence of the primary rat anti-mouse CD68 antibody (MCA1957, Bio-Rad) that was diluted to 2 µg/mL in 10% NGS/PBS, followed by incubation with the secondary Cy3-labeled goat anti-rat IgG (H+L) (A0507, Beyotime, China) that was diluted to 1:500 in 10% NGS/PBS. The sections were washed 3 times with 10% NGS/PBS and subsequently counterstained with DAPI. Coverslips were mounted with an anti-fluorescence quenching sealant, and then observed by CLSM. The macrophages in plaques were calculated as the ratio of total macrophage area to total plaque area with the Image-Pro Plus 6.0 software. The cholesterol crystals were detected by laser scanning reflection microscopy.86 Briefly, the detector and the acousto-optical beam splitter were set to allow the detection of reflected laser light. The content of plaque cholesterol crystals was depicted as a ratio of the total crystal reflection area to total plaque area by the Image-Pro Plus 6.0 software. ACS Paragon Plus Environment
Page 27 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Histology and Immunohistochemistry. After fixing in 10% neutral buffered formalin, the aortic sinus sections (4-6 µm thickness) in paraffin were prepared, which were separately stained with hematoxylin-eosin (H&E) and Masson’s trichrome. For immunohistochemistry analysis, 4-µm sections of aortic tissues were incubated with antibodies to CD68, matrix metalloproteinase-9 (MMP-9), and α-smooth muscle actin (α-SMA), respectively. Semiquantitative analysis of histological images was performed with the Image-Pro Plus 6.0 software. Besides, the sections of major organs were stained by H&E. Determination of Inflammatory Cytokines in the Aortic Tissue and Serum. After different treatments, the aortas were excised. Pieces of aortic tissues were homogenized in saline and centrifuged. The supernatant was collected and the levels of typical inflammatory cytokines including TNF-α and IL-1β were quantified by ELISA (Neobioscience, China). The total protein tests were performed by BCA (Beyotime, China). Similarly, the serum levels of TNF-α and IL-1β were determined. Quantification of oxLDL and H2O2 in the Serum. After different treatments, blood samples were collected. The serum levels of oxLDL were determined using an ELISA kit (Cusabio, China). The serum levels of H2O2 were quantified by using a fluorimetric hydrogen peroxide assay kit (Sigma-Aldrich, U.S.A.). Complete Blood Count and Clinical Biochemistry Analyses. The collected blood samples were analyzed for routine blood tests of red blood cell (RBC), white blood cell (WBC), platelet (PLT), and hemoglobin (HGB) (Sysmex KX-21, Sysmex Co., Japan). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (SCr), and blood urea nitrogen (BUN) were also measured (Roche Cobas C501, Roche Co., Switzerland). The Effect of TPCD NP on Inflammatory Monocytes in the Blood from ApoE-/- Mice. We examined the effect of i.v. administered TPCD NP on the population of inflammatory monocytes in the blood of ApoE-/- mice according to the previously reported methods.43 Male ApoE-/- mice fed with high-fat diet for 3 months were i.v. injected with TPCD NP at 100 mg/kg once two days for 3 times. Mice in the control group only received saline. At 12 h after the last injection, whole blood samples were collected and lysed with a RBC lysis buffer (Tiangen Biotech, China). Then, cell suspensions were labeled with different antibodies,37, 43 and flow cytometric analysis ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 28 of 43
was performed to quantify the levels of inflammatory monocytes. Statistical Analysis. All data are expressed as the mean ± standard error (SE). Statistical analyses were conducted by SPSS 20.0 using the one-way ANOVA test for experiments with multiple groups, while a two-tailed, unpaired t-test was used for data with two groups. Statistical significance was considered at p < 0.05.
ACS Paragon Plus Environment
Page 29 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
ASSOCIATED CONTENT Supporting Information Supporting Information Available: Supplementary results including Figure S1 to Figure S18. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail:
[email protected];
[email protected]. *E-mail:
[email protected]. Author Contributions ǁ
These authors contributed equally (Y.Q.W. and L.L.L.).
ACKNOWLEDGMENTS This study was supported by the Innovation Program for Key Technologies of Southwest Hospital (No. SWH2016ZDCX1016), the Science and Technology Innovation Program in Military Medicine of Southwest Hospital (No. SWH2016LHYS-05), the National Natural Science Foundation of China (Nos. 81770433 & 81701832), and the Graduate Student Research Innovation Project of Chongqing (to L.L.L.).
REFERENCES (1) Libby, P.; Bornfeldt, K. E.; Tall, A. R. Atherosclerosis: Successes, Surprises, and Future Challenges. Circ. Res. 2016, 118, 531-534. (2) Benjamin, E. J.; Blaha, M. J.; Chiuve, S. E.; Cushman, M.; Das, S. R.; Deo, R.; de Ferranti, S. D.; Floyd, J.; Fornage, M.; Gillespie, C.; Isasi, C. R.; Jimenez, M. C.; Jordan, L. C.; Judd, S. E.; Lackland, D.; Lichtman, J. H.; Lisabeth, L.; Liu, S.; Longenecker, C. T.; Mackey, R. H., et al. Heart Disease and Stroke Statistics-2017 Update: A Report from the American Heart Association. Circulation 2017, 135, e146-e603. (3) Hansson, G. K. Inflammation, Atherosclerosis, and Coronary Artery Disease. N. Engl. J. Med. 2005, 352, 1685-1695. (4) Swirski, F. K.; Nahrendorf, M. Leukocyte Behavior in Atherosclerosis, Myocardial Infarction, and Heart Failure. Science 2013, 339, 161-166. (5) Stocker, R.; Keaney, J. F., Jr. Role of Oxidative Modifications in Atherosclerosis. Physiol. Rev. 2004, 84, 1381-1478. (6) Sena, L. A.; Chandel, N. S. Physiological Roles of Mitochondrial Reactive Oxygen Species. Mol. Cell 2012, 48, 158-167. (7) Levitan, I.; Volkov, S.; Subbaiah, P. V. Oxidized Ldl: Diversity, Patterns of Recognition, and Pathophysiology. Antioxid. Redox Signal. 2010, 13, 39-75. ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 30 of 43
(8) Kita, T.; Kume, N.; Minami, M.; Hayashida, K.; Murayama, T.; Sano, H.; Moriwaki, H.; Kataoka, H.; Nishi, E.; Horiuchi, H.; Arai, H.; Yokode, M. Role of Oxidized Ldl in Atherosclerosis. Ann. N. Y. Acad. Sci. 2001, 947, 199-205. (9) Maiolino, G.; Rossitto, G.; Caielli, P.; Bisogni, V.; Rossi, G. P.; Calo, L. A. The Role of Oxidized Low-Density Lipoproteins in Atherosclerosis: The Myths and the Facts. Mediators Inflamm. 2013, 2013, 714653. (10) Gloire, G.; Legrand-Poels, S.; Piette, J. Nf-Kappab Activation by Reactive Oxygen Species: Fifteen Years Later. Biochem. Pharmacol. 2006, 72, 1493-1505. (11) Mittal, M.; Siddiqui, M. R.; Tran, K.; Reddy, S. P.; Malik, A. B. Reactive Oxygen Species in Inflammation and Tissue Injury. Antioxid. Redox Signal. 2014, 20, 1126-1167. (12) Grimsrud, P. A.; Xie, H.; Griffin, T. J.; Bernlohr, D. A. Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes. J. Biol. Chem. 2008, 283, 21837-21841. (13) Forstermann, U.; Xia, N.; Li, H. Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis. Circ. Res. 2017, 120, 713-735. (14) Munzel, T.; Camici, G. G.; Maack, C.; Bonetti, N. R.; Fuster, V.; Kovacic, J. C. Impact of Oxidative Stress on the Heart and Vasculature: Part 2 of a 3-Part Series. J. Am. Coll. Cardiol. 2017, 70, 212-229. (15) Griendling, K. K.; Sorescu, D.; Lassegue, B.; Ushio-Fukai, M. Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role in Vascular Physiology and Pathophysiology. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 2175-2183. (16) Li, H.; Horke, S.; Forstermann, U. Vascular Oxidative Stress, Nitric Oxide and Atherosclerosis. Atherosclerosis 2014, 237, 208-219. (17) Wang, Y.; Subramanian, M.; Yurdagul, A., Jr.; Barbosa-Lorenzi, V. C.; Cai, B.; de Juan-Sanz, J.; Ryan, T. A.; Nomura, M.; Maxfield, F. R.; Tabas, I. Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages. Cell 2017, 171, 331-345. (18) Witztum, J. L. The Oxidation Hypothesis of Atherosclerosis. Lancet 1994, 344, 793-795. (19) Gotto, A. M. Antioxidants, Statins, and Atherosclerosis. J. Am. Coll. Cardiol. 2003, 41, 1205-1210. (20) Salonen, R. M.; Nyyssonen, K.; Kaikkonen, J.; Porkkala-Sarataho, E.; Voutilainen, S.; Rissanen, T. H.; Tuomainen, T. P.; Valkonen, V. P.; Ristonmaa, U.; Lakka, H. M.; Vanharanta, M.; Salonen, J. T.; Poulsen, H. E. Six-Year Effect of Combined Vitamin C and E Supplementation on Atherosclerotic Progression: The Antioxidant Supplementation in Atherosclerosis Prevention (Asap) Study. Circulation 2003, 107, 947-953. (21) Zimetbaum, P.; Eder, H.; Frishman, W. Probucol: Pharmacology and Clinical Application. J. Clin. Pharmacol. 1990, 30, 3-9. (22) Thomas, S. R.; Witting, P. K.; Stocker, R. A Role for Reduced Coenzyme Q in Atherosclerosis? BioFactors 1999, 9, 207-224. (23) Yamakoshi, J.; Kataoka, S.; Koga, T.; Ariga, T. Proanthocyanidin-Rich Extract from Grape Seeds Attenuates the Development of Aortic Atherosclerosis in Cholesterol-Fed Rabbits. Atherosclerosis 1999, 142, 139-149. (24) Kim, C. H.; Mitchell, J. B.; Bursill, C. A.; Sowers, A. L.; Thetford, A.; Cook, J. A.; van Reyk, D. M.; Davies, M. J. The Nitroxide Radical Tempol Prevents Obesity, Hyperlipidaemia, Elevation of Inflammatory Cytokines, and Modulates Atherosclerotic Plaque Composition in Apoe-/- Mice. Atherosclerosis 2015, 240, 234-241. (25) Langbein, H.; Brunssen, C.; Hofmann, A.; Cimalla, P.; Brux, M.; Bornstein, S. R.; Deussen, A.; Koch, E.; Morawietz, H. Nadph Oxidase 4 Protects against Development of Endothelial Dysfunction and Atherosclerosis in Ldl Receptor Deficient Mice. Eur. Heart J. 2016, 37, 1753-1761. ACS Paragon Plus Environment
Page 31 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
(26) Sugamura, K.; Keaney, J. F., Jr. Reactive Oxygen Species in Cardiovascular Disease. Free Radic. Biol. Med. 2011, 51, 978-992. (27) Kornfeld, O. S.; Hwang, S.; Disatnik, M. H.; Chen, C. H.; Qvit, N.; Mochly-Rosen, D. Mitochondrial Reactive Oxygen Species at the Heart of the Matter: New Therapeutic Approaches for Cardiovascular Diseases. Circ. Res. 2015, 116, 1783-1799. (28) Mecocci, P.; Polidori, M. C. Antioxidant Clinical Trials in Mild Cognitive Impairment and Alzheimer's Disease. Biochim. Biophys. Acta 2012, 1822, 631-638. (29) Alaarg, A.; Perez-Medina, C.; Metselaar, J. M.; Nahrendorf, M.; Fayad, Z. A.; Storm, G.; Mulder, W. J. M. Applying Nanomedicine in Maladaptive Inflammation and Angiogenesis. Adv. Drug Deliv. Rev. 2017, 119, 143-158. (30) Chung, E. J.; Tirrell, M. Recent Advances in Targeted, Self-Assembling Nanoparticles to Address Vascular Damage Due to Atherosclerosis. Adv. Healthc. Mater. 2015, 4, 2408-2422. (31) Dormont, F.; Varna, M.; Couvreur, P. Nanoplumbers: Biomaterials to Fight Cardiovascular Diseases. Mater. Today 2018, 21, 122-143. (32) Mulder, W. J.; Jaffer, F. A.; Fayad, Z. A.; Nahrendorf, M. Imaging and Nanomedicine in Inflammatory Atherosclerosis. Sci. Transl. Med. 2014, 6, 239sr231. (33) Tang, J.; Lobatto, M. E.; Hassing, L.; van der Staay, S.; van Rijs, S. M.; Calcagno, C.; Braza, M. S.; Baxter, S.; Fay, F.; Sanchez-Gaytan, B. L.; Duivenvoorden, R.; Sager, H.; Astudillo, Y. M.; Leong, W.; Ramachandran, S.; Storm, G.; Perez-Medina, C.; Reiner, T.; Cormode, D. P.; Strijkers, G. J., et al. Inhibiting Macrophage Proliferation Suppresses Atherosclerotic Plaque Inflammation. Sci. Adv. 2015, 1, e1400223. (34) Lobatto, M. E.; Calcagno, C.; Millon, A.; Senders, M. L.; Fay, F.; Robson, P. M.; Ramachandran, S.; Binderup, T.; Paridaans, M. P. M.; Sensarn, S.; Rogalla, S.; Gordon, R. E.; Cardoso, L.; Storm, G.; Metselaar, J. M.; Contag, C. H.; Stroes, E. S. G.; Fayad, Z. A.; Mulder, W. J. M. Atherosclerotic Plaque Targeting Mechanism of Long-Circulating Nanoparticles Established by Multimodal Imaging. ACS Nano 2015, 9, 1837-1847. (35) Lobatto, M. E.; Fuster, V.; Fayad, Z. A.; Mulder, W. J. Perspectives and Opportunities for Nanomedicine in the Management of Atherosclerosis. Nat. Rev. Drug. Discov. 2011, 10, 835-852. (36) Wei, X.; Ying, M.; Dehaini, D.; Su, Y.; Kroll, A. V.; Zhou, J.; Gao, W.; Fang, R. H.; Chien, S.; Zhang, L. Nanoparticle Functionalization with Platelet Membrane Enables Multifactored Biological Targeting and Detection of Atherosclerosis. ACS Nano 2018, 12, 109-116. (37) Dou, Y.; Chen, Y.; Zhang, X.; Xu, X.; Chen, Y.; Guo, J.; Zhang, D.; Wang, R.; Li, X.; Zhang, J. Non-Proinflammatory and Responsive Nanoplatforms for Targeted Treatment of Atherosclerosis. Biomaterials 2017, 143, 93-108. (38) Flögel, U.; Ding, Z.; Hardung, H.; Jander, S.; Reichmann, G.; Jacoby, C.; Schubert, R.; Schrader, J. In Vivo Monitoring of Inflammation after Cardiac and Cerebral Ischemia by Fluorine Magnetic Resonance Imaging. Circulation 2008, 118, 140-148. (39) Katsuki, S.; Matoba, T.; Nakashiro, S.; Sato, K.; Koga, J.-i.; Nakano, K.; Nakano, Y.; Egusa, S.; Sunagawa, K.; Egashira, K. Nanoparticle-Mediated Delivery of Pitavastatin Inhibits Atherosclerotic Plaque Destabilization/Rupture in Mice by Regulating the Recruitment of Inflammatory Monocytes. Circulation 2014, 129, 896-906. (40) Kheirolomoom, A.; Kim, C. W.; Seo, J. W.; Kumar, S.; Son, D. J.; Gagnon, M. K.; Ingham, E. S.; Ferrara, K. W.; Jo, H. Multifunctional Nanoparticles Facilitate Molecular Targeting and Mirna Delivery to Inhibit Atherosclerosis in Apoe(-/-) Mice. ACS Nano 2015, 9, 8885-8897. (41) Fredman, G.; Kamaly, N.; Spolitu, S.; Milton, J.; Ghorpade, D.; Chiasson, R.; Kuriakose, G.; Perretti, M.; Farokhzad, O.; Tabas, I. Targeted Nanoparticles Containing the Proresolving Peptide Ac2-26 Protect against ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 32 of 43
Advanced Atherosclerosis in Hypercholesterolemic Mice. Sci. Transl. Med. 2015, 7, 275ra220. (42) Duivenvoorden, R.; Tang, J.; Cormode, D. P.; Mieszawska, A. J.; Izquierdo-Garcia, D.; Ozcan, C.; Otten, M. J.; Zaidi, N.; Lobatto, M. E.; van Rijs, S. M.; Priem, B.; Kuan, E. L.; Martel, C.; Hewing, B.; Sager, H.; Nahrendorf, M.; Randolph, G. J.; Stroes, E. S.; Fuster, V.; Fisher, E. A., et al. A Statin-Loaded Reconstituted High-Density Lipoprotein Nanoparticle Inhibits Atherosclerotic Plaque Inflammation. Nat. Commun. 2014, 5, 3065. (43) Leuschner, F.; Dutta, P.; Gorbatov, R.; Novobrantseva, T. I.; Donahoe, J. S.; Courties, G.; Lee, K. M.; Kim, J. I.; Markmann, J. F.; Marinelli, B.; Panizzi, P.; Lee, W. W.; Iwamoto, Y.; Milstein, S.; Epstein-Barash, H.; Cantley, W.; Wong, J.; Cortez-Retamozo, V.; Newton, A.; Love, K., et al. Therapeutic Sirna Silencing in Inflammatory Monocytes in Mice. Nat. Biotechnol. 2011, 29, 1005-1010. (44) Kamaly, N.; Fredman, G.; Fojas, J. J.; Subramanian, M.; Choi, W. I.; Zepeda, K.; Vilos, C.; Yu, M.; Gadde, S.; Wu, J.; Milton, J.; Carvalho Leitao, R.; Rosa Fernandes, L.; Hasan, M.; Gao, H.; Nguyen, V.; Harris, J.; Tabas, I.; Farokhzad, O. C. Targeted Interleukin-10 Nanotherapeutics Developed with a Microfluidic Chip Enhance Resolution of Inflammation in Advanced Atherosclerosis. ACS Nano 2016, 10, 5280-5292. (45) Lewis, D. R.; Petersen, L. K.; York, A. W.; Zablocki, K. R.; Joseph, L. B.; Kholodovych, V.; Prud'homme, R. K.; Uhrich, K. E.; Moghe, P. V. Sugar-Based Amphiphilic Nanoparticles Arrest Atherosclerosis In Vivo. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 2693-2698. (46) Sha, S.; Vong, L. B.; Chonpathompikunlert, P.; Yoshitomi, T.; Matsui, H.; Nagasaki, Y. Suppression of Nsaid-Induced Small Intestinal Inflammation by Orally Administered Redox Nanoparticles. Biomaterials 2013, 34, 8393-8400. (47) Eguchi, A.; Yoshitomi, T.; Lazic, M.; Johnson, C. D.; Vong, L. B.; Wree, A.; Povero, D.; Papouchado, B. G.; Nagasaki, Y.; Feldstein, A. E. Redox Nanoparticles as a Novel Treatment Approach for Inflammation and Fibrosis Associated with Nonalcoholic Steatohepatitis. Nanomedicine 2015, 10, 2697-2708. (48) Kannan, S.; Dai, H.; Navath, R. S.; Balakrishnan, B.; Jyoti, A.; Janisse, J.; Romero, R.; Kannan, R. M. Dendrimer-Based Postnatal Therapy for Neuroinflammation and Cerebral Palsy in a Rabbit Model. Sci. Transl. Med. 2012, 4, 130ra146. (49) Cho, B. R.; Ryu, D. R.; Lee, K. S.; Lee, D. K.; Bae, S.; Kang, D. G.; Ke, Q.; Singh, S. S.; Ha, K. S.; Kwon, Y. G.; Lee, D.; Kang, P. M.; Kim, Y. M. P-Hydroxybenzyl Alcohol-Containing Biodegradable Nanoparticle Improves Functional Blood Flow through Angiogenesis in a Mouse Model of Hindlimb Ischemia. Biomaterials 2015, 53, 679-687. (50) Zhang, Q.; Zhang, F.; Chen, Y.; Dou, Y.; Tao, H.; Zhang, D.; Wang, R.; Li, X.; Zhang, J. Structure-Property Correlations of Reactive Oxygen Species-Responsive and Hydrogen Peroxide-Eliminating Materials with Anti-Oxidant and Anti-Inflammatory Activities. Chem. Mater. 2017, 29, 8221-8238. (51) Wilcox, C. S. Effects of Tempol and Redox-Cycling Nitroxides in Models of Oxidative Stress. Pharmacol. Ther. 2010, 126, 119-145. (52) Okawa, M.; Kinjo, J.; Nohara, T.; Ono, M. Dpph (1,1-Diphenyl-2-Picrylhydrazyl) Radical Scavenging Activity of Flavonoids Obtained from Some Medicinal Plants. Biol. Pharm. Bull. 2001, 24, 1202-1205. (53) Guo, J.; Tao, H.; Dou, Y.; Li, L.; Xu, X.; Zhang, Q.; Cheng, J.; Han, S.; Huang, J.; Li, X.; Li, X.; Zhang, J. A Myeloperoxidase-Responsive and Biodegradable Luminescent Material for Real-Time Imaging of Inflammatory Diseases. Mater. Today 2017, 20, 493-500. (54) Immordino, M. L.; Dosio, F.; Cattel, L. Stealth Liposomes: Review of the Basic Science, Rationale, and Clinical Applications, Existing and Potential. Int. J. Nanomed. 2006, 1, 297-315. (55) Zhang, Q.; Tao, H.; Lin, Y.; Hu, Y.; An, H.; Zhang, D.; Feng, S.; Hu, H.; Wang, R.; Li, X.; Zhang, J. A Superoxide Dismutase/Catalase Mimetic Nanomedicine for Targeted Therapy of Inflammatory Bowel Disease. ACS Paragon Plus Environment
Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Biomaterials 2016, 105, 206-221. (56) Moore, K.; Sheedy, F.; Fisher, E. Macrophages in Atherosclerosis: A Dynamic Balance. Nat. Rev. Immunol. 2013, 13, 709-721. (57) Bennett, M. R.; Sinha, S.; Owens, G. K. Vascular Smooth Muscle Cells in Atherosclerosis. Cir. Res. 2016, 118, 692-702. (58) Cannon, G. J.; Swanson, J. A. The Macrophage Capacity for Phagocytosis. J. Cell Sci. 1992, 101, 907-913. (59) Sack, M. N.; Fyhrquist, F. Y.; Saijonmaa, O. J.; Fuster, V.; Kovacic, J. C. Basic Biology of Oxidative Stress and the Cardiovascular System: Part 1 of a 3-Part Series. J. Am. Coll. Cardiol. 2017, 70, 196-211. (60) Chatterjee, P. K.; Cuzzocrea, S.; Brown, P. A.; Zacharowski, K.; Stewart, K. N.; Mota-Filipe, H.; Thiemermann, C. Tempol, a Membrane-Permeable Radical Scavenger, Reduces Oxidant Stress-Mediated Renal Dysfunction and Injury in the Rat. Kidney Int. 2000, 58, 658-673. (61) Morrell, C. N. Reactive Oxygen Species: Finding the Right Balance. Circ. Res. 2008, 103, 571-572. (62) Tacconelli, S.; Capone, M. L.; Patrignani, P. Measurement of 8-Iso-Prostaglandin F2alpha in Biological Fluids as a Measure of Lipid Peroxidation. Methods Mol. Biol. 2010, 644, 165-178. (63) Kroese, L. J.; Scheffer, P. G. 8-Hydroxy-2'-Deoxyguanosine and Cardiovascular Disease: A Systematic Review. Curr. Atheroscler. Rep. 2014, 16, 452. (64) Nagy, L.; Tontonoz, P.; Alvarez, J. G. A.; Chen, H.; Evans, R. M. Oxidized Ldl Regulates Macrophage Gene Expression through Ligand Activation of Pparg. Cell 1998, 93, 229-240. (65) Chellan, B.; Reardon, C. A.; Getz, G. S.; Hofmann Bowman, M. A. Enzymatically Modified Ldl Promotes Foam Cell Formation in Smooth Muscle Cells Via Macropinocytosis and Enhances Receptor Mediated Uptake of Oxidized Ldl. Arterioscler. Thromb. Vasc. Biol. 2016, 36, 1101-1113. (66) Allahverdian, S.; Pannu, P. S.; Francis, G. A. Contribution of Monocyte-Derived Macrophages and Smooth Muscle Cells to Arterial Foam Cell Formation. Cardiovasc. Res. 2012, 95, 165-172. (67) Steinberg, D. The Ldl Modification Hypothesis of Atherogenesis: An Update. J. Lipid Res. 2009, 50, S376-381. (68) Nakashima, Y.; Plump, A. S.; Raines, E. W.; Breslow, J. L.; Ross, R. Apoe-Deficient Mice Develop Lesions of All Phases of Atherosclerosis Throughout the Arterial Tree. Arterioscler. Thromb. Vasc. Biol. 1994, 14, 133-140. (69) Kim, Y.; Lobatto, M. E.; Kawahara, T.; Lee Chung, B.; Mieszawska, A. J.; Sanchez-Gaytan, B. L.; Fay, F.; Senders, M. L.; Calcagno, C.; Becraft, J.; Tun Saung, M.; Gordon, R. E.; Stroes, E. S. G.; Ma, M.; Farokhzad, O. C.; Fayad, Z. A.; Mulder, W. J. M.; Langer, R. Probing Nanoparticle Translocation across the Permeable Endothelium in Experimental Atherosclerosis. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 1078-1083. (70) Soehnlein, O. Multiple Roles for Neutrophils in Atherosclerosis. Cir. Res. 2012, 110, 875-888. (71) Daugherty, A.; Zweifel, B. S.; Schonfeld, G. The Effects of Probucol on the Progression of Atherosclerosis in Mature Watanabe Heritable Hyperlipidaemic Rabbits. Br. J. Pharmacol. 1991, 103, 1013-1018. (72) Braun, A.; Zhang, S.; Miettinen, H. E.; Ebrahim, S.; Holm, T. M.; Vasile, E.; Post, M. J.; Yoerger, D. M.; Picard, M. H.; Krieger, J. L.; Andrews, N. C.; Simons, M.; Krieger, M. Probucol Prevents Early Coronary Heart Disease and Death in the High-Density Lipoprotein Receptor Sr-Bi/Apolipoprotein E Double Knockout Mouse. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7283-7288. (73) Walldius, G.; Erikson, U.; Olsson, A. G.; Bergstrand, L.; Hådell, K.; Johansson, J.; Kaijser, L.; Lassvik, C.; Mölgaard, J.; Nilsson, S.; Schäfer-Elinder, L.; Stenport, G.; Holme, I. The Effect of Probucol on Femoral Atherosclerosis: The Probucol Quantitative Regression Swedish Trial (Pqrst). Am. J. Cardiol. 1994, 74, ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 34 of 43
875-883. (74) Zimmer, S.; Grebe, A.; Bakke, S. S.; Bode, N.; Halvorsen, B.; Ulas, T.; Skjelland, M.; De Nardo, D.; Labzin, L. I.; Kerksiek, A.; Hempel, C.; Heneka, M. T.; Hawxhurst, V.; Fitzgerald, M. L.; Trebicka, J.; Bjorkhem, I.; Gustafsson, J. A.; Westerterp, M.; Tall, A. R.; Wright, S. D., et al. Cyclodextrin Promotes Atherosclerosis Regression Via Macrophage Reprogramming. Sci. Transl. Med. 2016, 8, 333ra350. (75) Razani, B.; Feng, C.; Coleman, T.; Emanuel, R.; Wen, H.; Hwang, S.; Ting, J. P.; Virgin, H. W.; Kastan, M. B.; Semenkovich, C. F. Autophagy Links Inflammasomes to Atherosclerotic Progression. Cell Metab. 2012, 15, 534-544. (76) Korytowski, W.; Wawak, K.; Pabisz, P.; Schmitt, J. C.; Chadwick, A. C.; Sahoo, D.; Girotti, A. W. Impairment of Macrophage Cholesterol Efflux by Cholesterol Hydroperoxide Trafficking: Implications for Atherogenesis under Oxidative Stress. Arterioscler. Thromb. Vasc. Biol. 2015, 35, 2104-2113. (77) Silvestre-Roig, C.; de Winther, M. P.; Weber, C.; Daemen, M. J.; Lutgens, E.; Soehnlein, O. Atherosclerotic Plaque Destabilization. Cir. Res. 2014, 114, 214-226. (78) Owusu-Ansah, E.; Yavari, A.; Banerjee, U. A Protocol for In Vivo Detection of Reactive Oxygen Species. Protocol Exchange 2008, doi:10.1038/nprot.2008.1023. (79) Swirski, F. K.; Libby, P.; Aikawa, E.; Alcaide, P.; Luscinskas, F. W.; Weissleder, R.; Pittet, M. J. Ly-6chi Monocytes Dominate Hypercholesterolemia-Associated Monocytosis and Give Rise to Macrophages in Atheromata. J. Clin. Invest. 2007, 117, 195-205. (80) Serbina, N. V.; Pamer, E. G. Monocyte Emigration from Bone Marrow During Bacterial Infection Requires Signals Mediated by Chemokine Receptor Ccr2. Nat. Immunol. 2006, 7, 311-317. (81) Galkina, E.; Ley, K. Immune and Inflammatory Mechanisms of Atherosclerosis. Ann. Rev. Immunol. 2009, 27, 165-197. (82) Wang, J.; Ma, A.; Zhao, M.; Zhu, H. Ampk Activation Reduces the Number of Atheromata Macrophages in Apoe Deficient Mice. Atherosclerosis 2017, 258, 97-107. (83) Zhang, D. L.; Wei, Y. L.; Chen, K.; Zhang, X. J.; Xu, X. Q.; Shi, Q.; Han, S. L.; Chen, X.; Gong, H.; Li, X. H.; Zhang, J. X. Biocompatible Reactive Oxygen Species (Ros)-Responsive Nanoparticles as Superior Drug Delivery Vehicles. Adv. Healthc. Mater. 2015, 4, 69-76. (84) Feliciano, C. P.; Tsuboi, K.; Suzuki, K.; Kimura, H.; Nagasaki, Y. Long-Term Bioavailability of Redox Nanoparticles Effectively Reduces Organ Dysfunctions and Death in Whole-Body Irradiated Mice. Biomaterials 2017, 129, 68-82. (85) Virdis, A.; Santini, F.; Colucci, R.; Duranti, E.; Salvetti, G.; Rugani, I.; Segnani, C.; Anselmino, M.; Bernardini, N.; Blandizzi, C.; Salvetti, A.; Pinchera, A.; Taddei, S. Vascular Generation of Tumor Necrosis Factor-Alpha Reduces Nitric Oxide Availability in Small Arteries from Visceral Fat of Obese Patients. J. Am. Coll. Cardiol. 2011, 58, 238-247. (86) Duewell, P.; Kono, H.; Rayner, K. J.; Sirois, C. M.; Vladimer, G.; Bauernfeind, F. G.; Abela, G. S.; Franchi, L.; Nunez, G.; Schnurr, M.; Espevik, T.; Lien, E.; Fitzgerald, K. A.; Rock, K. L.; Moore, K. J.; Wright, S. D.; Hornung, V.; Latz, E. Nlrp3 Inflammasomes Are Required for Atherogenesis and Activated by Cholesterol Crystals. Nature 2010, 464, 1357-1361.
ACS Paragon Plus Environment
Page 35 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 1. Schematic of engineering of a broad-spectrum ROS-scavenging nanoparticle and targeted therapy of atherosclerosis. (A) Chemical structure of a broad-spectrum ROS-eliminating material TPCD and development of a TPCD nanoparticle (TPCD NP). (B) Sketch of targeted treatment of atherosclerosis by eliminating ROS through i.v. administration of engineered TPCD NP.
ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 36 of 43
Figure 2. ROS-eliminating capability of TPCD and characterization of TPCD NP. (A-D) Dose-dependent elimination of H2O2 (A), DHHP radical (B), superoxide anion (C), and hypochlorite (D) by TPCD. (E-G) Representative TEM image (E), SEM image (F), and size distribution profile (G) of TPCD NP. (H) TEM image of TPCD NP after phosphotungstic acid-staining. (I-J) FT-IR (I) and 1H NMR (J) spectra of TPCD and TPCD NP. (K) In vitro hydrolysis kinetics of TPCD NP in PBS containing different concentrations of H2O2. Data in (A-D,K) are mean ± SE (n = 3).
ACS Paragon Plus Environment
Page 37 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 3. Cellular uptake of TPCD NP and its biological effects in RAW264.7 macrophages. (A) Confocal microscopy images of time-dependent cellular uptake of Cy5-labeled TPCD NP. After RAW264.7 cells were incubated with Cy5/TPCD NP for various periods of time, nuclei were stained with DAPI (blue), while late endosomes and lysosomes were stained with LysoTracker Green (Lyso, green). (B) Flow cytometric quantification of mean fluorescence intensity (MFI) in RAW264.7 cells after incubation with Cy5/TPCD NP for varied times. (C) Fluorescence images showing dose-dependent internalization of Cy5/TPCD NP after 2 h of incubation. (D) Quantification of MFI data. (E-F) Fluorescence images (E) and quantification by flow cytometry (F) showing intracellular ROS generation after treatment with different formulations and stimulation with LPS/IFN-γ. RAW264.7 cells were incubated with medium alone or various doses of Tpl or TPCD NP for 2 h, followed by stimulation with LPS/IFN-γ for 4 h. Cells unstimulated with LPS/IFN-γ served as the normal control. Then confocal microscopic and flow cytometric analyses were performed. (G-I) Typical inflammatory cytokines TNF-α (G), IL-1β (H), and MCP-1 (I) secreted by RAW264.7 cells. Cells were pre-incubated with medium or various doses of Tpl or TPCD NP for 2 h, followed by stimulation with LPS/IFN-γ for 24 h. The levels of cytokines were detected by ELISA. (J-K) Expression levels of 8-iso-PGF2α (J) and 8-OHdG (K). After pretreatment with Tpl or TPCD NP for 2 h, RAW264.7 cells were incubated with H2O2. Both 8-iso-PGF2α and 8-OHdG were detected by ELISA. (L) Apoptosis of RAW264.7 cells. After incubation with medium or various doses of Tpl or TPCD NP for 2 h, cells were exposed to H2O2. Flow cytometric analysis was performed after cells were stained using FITC Annexin V apoptosis detection kit with PI. Scale bars, 20 µm (A,C), 50 µm (E). Data are mean ± SE (B,D,F,L, n = 3; G-K, n = 4). ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 38 of 43
Figure 4. The effect of TPCD NP on cellular uptake of oxLDL and foam cell formation in macrophages and VSMCs. (A-B) Confocal fluorescence images (A) and quantified MFI data (B) show cellular internalization of DiI-labeled oxidized LDL (DiI-oxLDL) in RAW264.7 cells. After pre-incubation with fresh medium (the model group) or various doses of Tpl or TPCD NP for 2 h, cells were cultured with DiI-oxLDL for 4 h, followed by confocal microscopic observation and flow cytometric analysis. Cells in the normal control group were not incubated with DiI-oxLDL. For fluorescence observation, nuclei were stained with DAPI (blue), while the cell membrane was labeled with DiO (green). (C-D) Confocal images (C) and quantified MFI data (D) of DiI-oxLDL in MOVAS cells. Cells were pre-treated with Tpl or TPCD NP for 2 h, and then incubated with DiI-oxLDL for 3 h, followed by different analyses. (E) Optical microscopy images showing oxLDL-induced foam cell formation in macrophages and MOVAS cells. (F-G) The quantified contents of ORO in foam cells derived from RAW264.7 (F) and MOVAS (G) cells. Cells were pre-incubated with medium (the model group), Tpl, or TPCD NP for 2 h, and then cultured with 50 µg/mL oxLDL for 48 h to induce foam cell formation. The normal control group was not induced with oxLDL. Foam cells were stained with ORO for microscopy observation, while intracellular ORO was extracted with isopropanol for quantification. Scale bars, 20 µm. Data are mean ± SE (B,D, n = 3; F,G, n = 4).
ACS Paragon Plus Environment
Page 39 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 5. In vivo circulation and targeting capability of TPCD NP in mice after i.v. administration. (A-B) In vivo pharmacokinetic performance of Cy7.5-labeled TPCD NP after i.v. injection in mice. (A) Ex vivo images of whole blood collected at various time points after i.v. administration. (B) The quantified data of fluorescence intensities. The control group in (A) shows results of whole blood from mice without treatment with Cy7.5/TPCD NP. (C-D) Ex vivo fluorescence images (C) and quantitative data (D) illustrating accumulation of Cy7.5 fluorescent signals in the aorta. ApoE-/- mice fed with high-fat diet for 3 months were i.v. injected with Cy7.5/TPCD NP. At 3 or 12 h after administration, mice were euthanized and aortas were isolated for ex vivo imaging. Mice in the control group were treated with the same volume of saline. (E-F) Immunofluorescence analysis of the co-localization of Cy5/TPCD NP and CD68+ macrophages in cryosections (E) of the aortic root, aortic arch, and brachiocephalic artery or isolated cells (F). (G) Fluorescence images indicate the localization of Cy5/TPCD NP and Ly-6G+ neutrophils in cryosections of the aortic root, aortic arch, and brachiocephalic artery. For all immunofluorescence analyses, ApoE-/- mice fed with high-fat diet for 3 months were i.v. injected with Cy5/TPCD NP. At 4 h after administration, mice were euthanized and different aortic tissues were isolated for preparation of cryosections. In separate experiments, cells in the aorta were isolated and stained with CD68 antibody for fluorescence observation. (H-I) Quantification of the distribution of Cy5/TPCD NP in blood cells after i.v. injection in C57BL/6 mice. At 30 min after injection, the distribution of Cy5/TPCD NP in typical blood cells was detected by flow cytometry. Data are mean ± SE (B,D, n = 3; I, n = 6). ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 40 of 43
Figure 6. Therapeutic effects of i.v. delivered TPCD NP in ApoE-/- mice. (A) Schematic illustration of the treatment protocols. (B) Representative photographs of en face ORO-stained aortas from mice after treatment with different formulations. (C) Quantitative analysis of the lesion area in aortas. (D) ORO-stained cryosections of the aortic root, aortic arch, and brachiocephalic artery. (E-G) Quantitative analysis of the relative plaque area in sections of the aortic root (E), aortic arch (F), and brachiocephalic artery (G). ApoE-/- mice were fed a cholesterol-rich and high-fat diet for 3 months. After the first month, all mice received various treatments by i.v. injection once a week for additional 2 months. Mice in the saline group were treated with saline alone, while other groups were separately administered with Tpl (17.2 mg/kg), probucol (5.2 mg/kg), and TPCD NP at low (50 mg/kg) or high (100 mg/kg) doses. Scale bars, 200 µm. Data in (E-G) are mean ± SE (n = 4-5). ACS Paragon Plus Environment
Page 41 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 7. Histochemistry analyses of aortic root sections from ApoE-/- mice after different treatments. (A) Representative images of aortic root sections stained with H&E, antibody to CD68, antibody to MMP-9, Masson's trichrome, and antibody to α-SMA. (B-F) Quantitative analysis of the necrotic core area relative to plaque area (B), plaque macrophage area relative to total arterial wall area (C), plaque MMP-9 area relative to total arterial wall area (D), plaque collagen area relative to plaque area (E), and plaque VSMCs area relative to plaque area (F). ApoE-/- mice were fed a cholesterol-rich and high-fat diet for 3 months. After the first month, all mice received various treatments by i.v. injection once a week for additional 2 months. Mice in the saline group were treated with saline alone, while other groups were separately administered with Tpl (17.2 mg/kg), probucol (5.2 mg/kg), and TPCD NP at low (50 mg/kg) or high (100 mg/kg) doses. Data in (B-F) are mean ± SE (n = 4-5).
ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 42 of 43
Figure 8. Inhibition of oxidative stress and inflammation in ApoE-/- mice by TPCD NP treatment. (A-B) Fluorescence images (A) and quantitative analysis (B) of DHE-stained sections of brachiocephalic artery from normal (C57BL/6 mice) or ApoE-/- mice subjected to different treatments. (C-D) The serum levels of H2O2 (C) and oxLDL (D). (E-F)The levels of TNF-α (E) and IL-1β (F) in aortic tissues from ApoE-/- mice treated with different formulations. (G-H) The serum levels of TNF-α (G) and IL-1β (H). In these cases, ApoE-/- mice were fed a cholesterol-rich and high fat diet for 3 months. After the first month, all mice concomitantly received various treatments by i.v. injection once a week for additional 2 months. Mice in the saline group were treated with saline alone, while other groups were separately administered with Tpl (17.2 mg/kg), probucol (5.2 mg/kg), and TPCD NP at low (50 mg/kg) or high (100 mg/kg) doses. (I-J) The effect of TPCD NP treatment on inflammatory monocytes in the blood. (I) Representative dot-plots illustrate the gating strategy and flow cytometric profiles. (J-K) The quantitative data of Ly-6Chigh (J) and CCR2+Ly-6Chigh (K) monocytes. ApoE-/- mice fed with high-fat diet for 3 months were i.v. injected with TPCD NP at 100 mg/kg once every two days for 3 times. At 12 h after the last injection, the counts of Ly-6Chigh and CCR2+Ly-6Chigh monocytes were analyzed by flow cytometry. Data are mean ± SE (B-H, n = 4-5; J-K, n = 6).
ACS Paragon Plus Environment
Page 43 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Table of Contents Graphic (TOC)
ACS Paragon Plus Environment
