SR-A-Targeted Phase-Transition Nanoparticles for the Detection and

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Biological and Medical Applications of Materials and Interfaces

SR-A-Targeted Phase Transition Nanoparticles for the Detection and Treatment of Atherosclerotic Vulnerable Plaques Man Ye, Jun Zhou, Yixin Zhong, Jie Xu, Jingxin Hou, Xingyue Wang, Zhi-Gang Wang, and Dajing Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18190 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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ACS Applied Materials & Interfaces

SR-A-Targeted Phase Transition Nanoparticles for the Detection and Treatment of Atherosclerotic Vulnerable Plaques

Man Ye†&, Jun Zhou†&, Yixin Zhong†, Jie Xu†, Jingxin Hou†, Xingyue Wang‡, Zhigang Wang‡, Dajing Guo†*

†Department

of Radiology, the Second Affiliated Hospital of Chongqing

Medical University, No. 74 Linjiang Rd, Yuzhong District, 400010 Chongqing, China ‡Institute

of Ultrasound Imaging, Department of Ultrasound, the Second

Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Rd, Yuzhong District, 400010 Chongqing, China

Keywords: Atherosclerotic plaque; Perfluorohexane; Dextran sulfate; Phase transition; Low intensity focused ultrasound

Abstract Atherosclerosis is a major cause of sudden death and myocardial infarction, instigated by unstable plaques. Thus, the early detection of unstable plaques and corresponding treatment can improve the prognosis and reduce mortality. In this study, we describe a protocol for the preparation of nanoparticles (NPs) combined with the phase transitional material

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perfluorohexane (PFH) and with dextran sulfate (DS) targeting class A scavenger receptors (SR-A) for the diagnosis and treatment of atherosclerotic vulnerable plaques. The results showed that the Fe-PFHpoly (lactic-co-glycolic acid) (PLGA)/chitosan (CS)-DS NPs were fabricated successfully, with the ability to undergo phase transition by low intensity focused ultrasound (LIFU) irradiation to achieve ultrasound (US) imaging; a high carrier rate of Fe3O4 had a good negative enhancement effect on magnetic resonance imaging (MRI). The NPs had a high binding affinity for activated macrophages and could be endocytosed by the macrophages and notably induced apoptosis under LIFU irradiation by an acoustic droplet vaporization (ADV) effect in vitro. Furthermore, in an ex vivo atherosclerotic plaque model of apolipoprotein E (ApoE) knockout (KO) (apoE -/-) mice induced by high cholesterol, the NPs selectively accumulated at the sites of SR-A expressed on the activated macrophages of the aortic region. This result was also confirmed by MRI in vivo, where the NPs could be targeted to the aortic plaque and reduced the T2* signal. The LIFU-induced phase transition could lead to the apoptosis of macrophages on plaques in vivo. In summary, Fe-PFH-PLGA/CS-DS NPs may be applied as multimodal and multifunctional probes and are expected to enable the specific diagnosis and targeted therapy of vulnerable plaques.

1. Introduction


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Atherosclerosis is an important pathological basis for the occurrence and development of cardiovascular disease (CVD), a major disease and a leading cause of morbidity and mortality throughout the world.1-2 The atherosclerotic vulnerable plaque is prone to rupture suddenly, leading to fatal events, and the rupture of an atherosclerotic plaque is closely related to the composition of the plaque.3 Therefore, the noninvasive precise detection of the composition of atherosclerotic plaques and the evaluation of plaque vulnerability are of great clinical value in the treatment and prognostic evaluation of atherosclerosis patients. Macrophages are the most abundant cellular components in vulnerable plaques, accounting for more than 80 % of the composition.4 When the metabolism of low-density lipoprotein (LDL) is abnormal, the increased LDL in the blood will penetrate directly into the inner subcutaneous of the vascular wall through the loose gap between the endothelial cells. Then, the endothelial LDL oxidizes to produce mildly modified oxidized low density lipoprotein (oxLDL). OxLDL is phagocytosed by macrophages, causing macrophages to accumulate large amounts of cholesterol and become foam cells. Foam cells continue to accumulate, forming fatty striations in the arterial wall that eventually lead to the development of atherosclerosis.5 The characteristic early pathological changes of atherosclerosis are the activation of macrophages with the surface over-expression of class A scavenger receptors (SR-A).6



SR-A are only over-expressed in atherosclerosis-activated macrophages, not in normal vascular wall cells.7 Petersen et al. fabricated amphiphilic macromolecules that can competitively inhibit the uptake of oxLDL via SR-A in a sustained manner to alleviate atherosclerosis.8 In this study, SRA in activated macrophages were selected as targets to achieve semiquantitative evaluation at the cellular level of plaque vulnerability. Dextran sulfate (DS) is one of the ligands of SR-A. It is a polyanion compound of dextran carrying large amounts of negative charge and has good biocompatibility, biodegradability and safety.9-10 Some studies have used DS to target over-expressed macrophage SR-A in vulnerable plaques and have confirmed its good targeting ability.7, 11 In addition, DS has the potential to regress and stabilize the atherosclerotic plaques, which can prevent the further development and deterioration of atherosclerosis.12 Several studies have used DS as a ligand to prepare adsorbent materials to reduce the LDL content in patients' blood and improve their physical condition.12-13 The use of contrast agents by molecular targeting technology can accurately determine the composition and activity of plaques and has the potential to identify vulnerable and high-risk plaques. In addition, molecular imaging can provide information about the disease before the onset of clinical symptoms, while guiding and implementing treatment at the molecular level of the disease.14-15 Ultrasound (US) imaging, a mature


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application for vascular diseases often used for real-time imaging, can effectively diagnose and evaluate arterial wall lesions. However, detecting plaques in deep and small vascular structures and determining the composition of plaques with US is difficult. While magnetic resonance imaging (MRI) has the advantage of a high soft-tissue resolution, multiparameter and multi-directional imaging can be used for qualitative and quantitative analyses, even for deep and small vascular structures, to make up for this deficiency. Yao et al. developed a specific dual-modality fluorescent and MRI nanoprobe targeting activated macrophages for noninvasively visualizing vulnerable plaques.16 Multimodal contrast agents combined with two or more imaging modes are the research hotspot of modern molecular imaging,17-18 enabling the various imaging methods to complement each other and provide deeper information on tissues or diseases more intuitively and vividly. As a negative MRI agent, Fe3O4 have been widely used in the diagnosis and treatment of diseases in various systems and have long circulating properties in the bloodstream after intravenous administration, making them particularly suitable for the diagnosis of vascular diseases.19 In our previous studies, we used oleic acid-modified Fe3O4 to construct nanoparticles (NPs), and these NPs had good MR negative contrast enhancement for arterial thrombosis.20-21 Phase transition agents have been successfully used in US molecular imaging to enhance the imaging effect.



In addition, a series of biological effects, such as sonoporation and cavitation effects, are generated in the process of the phase transition, and have the potential to treat tumors and cardiovascular diseases.22-23 Zhu et al. successfully developed a novel NP combining low intensity focused ultrasound (LIFU) with an intracellular ‘explosion effect’ caused by the acoustic droplet vaporization (ADV) effect, realizing precise tumor therapy.22 In the process of ADV, a series of violent dynamic processes such as oscillation, expansion, contraction and even the collapse of tiny bubbles in the liquid under the action of sound waves are generated, thereby generating phenomena such as chemical reactions, luminescence and subharmonics. These processes may damage the ultrastructure of the cells and induce apoptosis to achieve a therapeutic effect.24 For vascular diseases, our research group also confirmed the therapeutic effect of the perfluorohexane (PFH) phase transition on thrombi for the first time.21 However, there are very few reports in the literature on multimodal and multifunctional molecular probes for the diagnosis and treatment of vulnerable plaques. Thus, we considered whether vulnerable plaques could also be treated with PFH phase transition. The main traditional methods for treating atherosclerotic plaques include lipid-lowering and antiplatelet drugs, which mainly make plaques stable and cannot significantly reverse plaques. The relatively slow curative effect requires long-term use, as well as endarterectomy and stent implantation, which are invasive methods.


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During the development of atherosclerosis, macrophage-derived foam cells are unable to effectively be removed and form the core of necrosis, leading to an instability of the atherosclerotic plaques. Thus, strategies targeting macrophages may reverse atherosclerotic plaque formation. If the PFH phase transition can achieve the ablation of vulnerable plaques by reducing the number of macrophages in the inflammatory response to delay the disease progression noninvasively and conveniently, the limitations of traditional treatments can be overcome. Therefore, a SR-A-targeted phase transition multimodal and multifunctional molecular probe was fabricated in this study. Poly (lacticco-glycolic acid) (PLGA) was used as a carrier to prepare NPs by a double emulsification solvent evaporation method. The oleic acid-modified Fe3O4 was embedded in the shell membrane of the NPs, PFH was encapsulated in the nuclei of the NPs, and DS was electrostatically adsorbed onto the NPs using chitosan (CS) without involving harsh synthesis steps. This process specifically targets the SR-A of macrophages, thus enabling the diagnosis of atherosclerotic plaques and evaluation of plaque vulnerability followed by phase transition inside macrophages to damage the macrophages at the cellular level (Figure 1). The results are expected to facilitate early diagnosis and timely interventions for vulnerable plaques in the absence of clinical symptoms and to prevent the further development and deterioration of atherosclerosis.



2. Materials and methods 2.1 Materials PLGA (lactide: glycolide = 75:25, with a molecular weight of 8,000 Da) was purchased from the Jinan Daigang Biological Material Company, Ltd. (Jinan, Shangdong, China). Iron oxide NPs (Fe3O4, 10 nm, 25 mg/mL) that had been surface-modified with oleic acid were provided by Ocean Nano Technology Co., Ltd. (Springdale, AR, USA). PFH was obtained from J&K Scientific, Ltd. (Beijing, China). CS with a molecular weight of 50,000 Da was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). DS with a molecular weight of 5,000 Da was provided by Aladdin Reagent Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate dextran sulfate sodium 10 (FITC-DS) was purchased from Seebio Biotechnology Co., Ltd. (Shanghai, China). Polyvinyl alcohol (PVA, with a molecular weight of 30,000-70,000 Da) and fluorescent dyes including 1,1 ′ -dioctadecyl-3,3,3 ′ ,3 ′ -tetramethylindocarbocyanine perchlorate (DiI) and 4 ′ ,6-diamidino-2-phenylindole (DAPI) were provided by Sigma-Aldrich Corporation (St Louis, MO, USA). Cell Counting Kit-8 and Live-dead viability/cytotoxicity assay kit for animal cells was purchased from Dojindo Laboratories (Mashiki-machi, Tabaru, Japan). Rabbit Anti-SRA antibody was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China). MAC-3 antibody was obtained


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from Proteintech Group, Inc. (Wuhan, Hubei, China). The corresponding PE-conjugated goat anti-rabbit IgG and donkey anti-rat FITC secondary antibodies were provided by Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). The TUNEL (TdT-mediated dUTP nick end labelling) apoptosis assay kit was purchased from Shanghai Roche Pharmaceutical Co., Ltd. (Shanghai, China). All other reagents used were of at least analytical grade. 2.2 Preparation of the NPs The double emulsification method was used to prepare the Fe-PFH-PLGA NPs. After 50 mg PLGA was added to 2 mL of dichloromethane (CH2Cl2), 100 μL of Fe3O4 was added until fully dissolved. Then, 200 μL of PFH was added as the inner aqueous phase. The first emulsion was processed for 3 min (5 s on and 5 s off) using an ultrasonic oscillation instrument (VCY500, YYSonics Co., Ltd., Shanghai, China) with a power of 130 W. The above emulsified solution was poured into 6 mL of a 4 % PVA solution as the outer aqueous phase and acoustically emulsified for 5 min (5 s on and 5 s off) to produce a brown solution. Next, 10 mL of a 2 % isopropanol solution was added and stirred continuously for 3 h until the surface solidification of the Fe-PFH-PLGA NPs and complete volatilization of the organic solvents. All the above-mentioned processes were conducted under ice bath conditions. Finally, the Fe-PFH-PLGA NPs were collected for



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further use by centrifugal washing with double-distilled water three times to remove the supernatant. To fabricate the Fe-PFH-PLGA/CS-DS NPs, we first prepared FePFH-PLGA/CS NPs. The same processes were followed as for fabricating the Fe-PFH-PLGA NPs except that 3 mL of a 4 % PVA solution and 3 mL of a 2 % CS solution (dissolved in 3 % acetic acid) were added together as the outer aqueous phase, instead of 6 mL of the 4 % PVA solution.25 The Fe-PFH-PLGA/CS NPs were re-dissolved to 5 mL after centrifugation. Then, 10 mg DS was added to the Fe-PFH-PLGA/CS dispersion under an ice rocker with vigorous stirring for 1 h to prepare the Fe-PFH-PLGA/CSDS NPs via electrostatic absorption.26-27 The Fe-PLGA/CS-DS NPs were prepared using the same procedure described above, adding doubledistilled water as the inner aqueous phase rather than PFH. For fluorescently labelled NPs, the various NPs were dyed by DiI when PLGA was dissolved in CH2Cl2, or the FITC-DS was substituted for DS according to the different purposes. Finally, the samples were lyophilized for further study and long-term storage. 2.3 Physical and chemical characterization of the NPs The zeta potential, mean particle size and polydispersity index (PDI) of FePFH-PLGA,

Fe-PFH-PLGA/CS,

Fe-PFH-PLGA/CS-DS,

and

Fe-

PLGA/CS-DS NPs were determined by a dynamic light scattering detector (DLS, Malvern Instruments, Malvern, UK). Their internal structures were


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analyzed using transmission electron microscopy (TEM, Hitachi 7500, Hitachi, Ltd., Tokyo, Japan). The NPs were dispersed in double-distilled water in an appropriate amount, and their distribution and morphological characterization were observed with an optical microscope (Leica DMi8, Leica Co. Ltd., Austria, Germany). To observe the vaporization process directly, 2 mL of 2 mg/mL Fe-PFH-PLGA/CS-DS NPs in a 3 % agarose gel model were irradiated using an LIFU instrument (Haifu Medical Technology Co., Ltd, Chongqing, China) at a power density of 1 to 4 W/cm2 for different durations (1 to 8 min). The samples were viewed using an optical microscope. The Fe concentration was analyzed using an atomic absorption spectrometer (Hitachi model Z-5000, Hitachi, Ltd., Tokyo, Japan).21 2.4 Confirmation of DS coating The fourier transform infrared spectroscopy (FTIR) spectra were obtained using a Spectrum GX series (Nicolet iS50, USA) equipped with an MIRTGS detector and KBr beam splitter. The FTIR spectra of CS, DS, and the Fe-PFH-PLGA/CS-DS NPs were obtained in the wave number range from 4,000 cm-1 to 500 cm-1 under a dry air purge at room temperature.26, 28

A KBr disc was used as a sample holder, and then its spectrum was

subtracted from each sample spectrum. To further confirm the connection of DS and NPs, Fe-PFHPLGA/CS-DS NPs dyed by DiI combined with the FITC-DS were



observed using a confocal laser scanning microscope (CLSM, Nikon A1, Japan). To measure and calculate the DS carrying rate using flow cytometry analysis (FCM, FACSVantage, Becton, Dickinson and Company, USA), the Fe-PFH-PLGA NPs were used as the control group, while the DiI and FITC-labelled Fe-PFH-PLGA/CS-DS NPs were used as the experimental groups. 2.5 Multimodal imaging properties of the NPs For US images, the Fe-PFH-PLGA/CS-DS NPs in conventional B mode and contrast-enhanced ultrasound mode (CEUS) were collected after LIFU irradiation at 4 W/cm2 for 8 min according to our above research. The saline, Fe-PLGA/CS-DS NPs and Fe-PFH-PLGA/CS NPs were used as control groups. Finally, the acoustic intensities of the US images were measured with DFY grey scale quantitative analysis software (Ultrasound Molecular Imaging Institute, Chongqing, China). To obtain the MR images, the Fe-PFH-PLGA/CS-DS NPs were mixed with a 1 % agar solution to create various concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL, which were equivalent to iron theoretical concentrations of 0, 0.096, 0.192, 0.288, 0.384, and 0.480 mM. Then, the samples were transferred into 5 mL eppendorf tubes and placed in a plastic container full of water to reduce the artifacts after the mixture was fully dissolved. Using a head coil on a 1.5 T MRI scanner (HDXT2012, GE Medical Systems, USA), T2*-weighted imaging was performed with the


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following parameters: gradient echo sequence; echo time (TE), 10.7 ms; repetition time (TR), 520 ms; field of view (FOV), 180 mm; flip angle (FA), 45°; and slice thickness, 3 mm. The transverse relaxation rates (R2*) of the Fe-PFH-PLGA/CS-DS NPs were calculated using the following parameters: 16 increasing TEs from 1.5 to 18.8 ms; TR, 35 ms; number of excitations (NEX), 1; matrix, 320 × 192; and slice thickness, 1.5 mm. The R2* values were measured using the research R2* software package on an ADW4.6 image workstation. The diagram of the relaxivity (r2) was drawn according to the relation between the Fe concentrations in the agar solutions and the R2* values. Finally, the r2 was determined by a linear fit. 2.6 Cell experiments 2.6.1 Cell culture RAW 264.7 cells were obtained from the Key Lab of Lipid and Glucose Metabolism of Chongqing Medical University. The cells were cultured in DMEM supplemented with 10 % fetal bovine serum and 1 % penicillin– streptomycin and added to a 75 cm2 cell culture flask with a humidified atmosphere containing 5 % carbon dioxide (CO2) at 37°C. The cells in a culture flask containing 6 mL of complete medium were activated with 100 ng/mL lipopolysaccharide (LPS) for 24 h,29 using the cells in the logarithmic growth phase for the experiments. All cell culture reagents were provided by Invitrogen (Carlsbad, CA, USA). 2.6.2 Targeting ability to cells



The targeting ability of the Fe-PFH-PLGA/CS-DS NPs towards the activated and unactivated RAW 264.7 cells was observed using both CLSM and FCM. The activated and unactivated cells were seeded into φ15 mm cell culture dishes with a density of 2.0 × 105 cells per dish for 24 h. After incubation, the original medium was replaced with medium containing the DiI-labelled Fe-PFH-PLGA/CS-DS or Fe-PFH-PLGA-/CS NPs. The concentration of NPs in all groups was 0.5 mg/mL. Then, the incubation was continued for 2 h. For CLSM, the cells in dishes were washed three times with PBS and then fixed with 4 % paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed again with PBS, and the cell nuclei were stained with DAPI for 10 min. The remaining DAPI was washed off with PBS, and the fixed cells were imaged using a CLSM with a 10× eyepiece and a 40× objective. For FCM, the activated and unactivated cells cocultured with the NPs were also washed three times with PBS, followed by digestion with trypsin. The cells were collected by centrifugation for 5 min at 1000 rpm and then briefly washed with PBS two additional times prior to the final suspension in 300 μL of PBS. Finally, the fluorescence intensity of the cellular uptake was detected by FCM. For the blocking experiment, the LPS-treated cells and free DS solution were allowed to attach overnight before incubation with medium containing the DiI-labelled Fe-PFH-PLGA/CS-DS NPs.


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For the time-point experiment, the RAW 264.7 cells were incubated with 100 ng/mL LPS for 24 h at 37°C, followed by incubation with DiIlabelled Fe-PFH-PLGA/CS-DS or Fe-PFH-PLGA-/CS NPs for 0, 0.5, 2, and 4 h. Both CLSM and FCM were used as described above. 2.6.3 Cell MRI 2 × 105 RAW 264.7 cells per well were seeded onto sterile 12-well plates activated with LPS in a humidified atmosphere containing 5 % CO2 at 37°C and incubated for 24 h. Then, the old medium was replaced with 500 µL of fresh complete medium containing different concentrations of the FePFH-PLGA/CS-DS and Fe-PFH-PLGA/CS NPs with iron concentrations of 0, 0.096, 0.192, 0.288, 0.384, and 0.480 mM. The cells were incubated for another 4 h and washed three times with PBS. Next, the cells were detached for 1 min, suspended in a 1 % agar solution, and transferred into 5 mL eppendorf tubes. MR T2*-weighted images were obtained using a 1.5 T MRI system with the same parameters as mentioned above. The central position of each tube was selected as the region of interest (ROI), and each ROI was defined as 100 pixels. The average signal intensity (SI) of each concentration was measured. The SI data were divided by the background noise to yield the signal-to-noise ratio (SNR): SNR = SI/noise. 2.6.4 Cytotoxicity test The cytotoxicity of the Fe-PFH-PLGA/CS-DS NPs in vitro was evaluated by using a Cell Counting Kit-8 (CCK8) test for activated and unactivated



RAW 264.7 cells. The activated and unactivated cells were seeded into 96well plates at a density of 1 × 104 cells per well and incubated for 24 h. Then, the old medium was replaced with 100 μL of medium containing the Fe-PFH-PLGA/CS-DS NPs at various concentrations from 0.05 to 2.5 mg/mL. Untreated cells in growth media were used as the control groups, and the wells without cells were used as blank groups. The cells were incubated for another 24 h and then washed three times with PBS. Subsequently, 10 μL of CCK8 solution was added and incubated for a further 2 h. When the brown color appeared in the wells, the absorbance of the solution in each well was measured on a microplate reader (Synergy HT, BioTek Instruments, Inc., USA) at 450 nm. The cell viability (%) relative to that of the control cells was calculated according to the instructions of the CCK8 assay. 2.6.5 ADV effect on cells To investigate the in vitro ADV effect of NPs on activated macrophages, LPS-activated macrophages were prepared using the same methods as in the above. The cells were cultured in φ15 mm cell culture dishes with a density of 2.0 × 105/dish for 24 h and then randomly divided into four groups: control group, LIFU group, non-PFH + LIFU group, and PFH + LIFU group. For the control group, the cells were treated with complete culture medium. For the LIFU group, cells were irradiated with LIFU under 4 W/cm2 for 3 min in the absence of NPs. For the non-PFH + LIFU


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group and PFH + LIFU group, the cells were treated with 0.5 mg/mL of Fe-PLGA/CS-DS or Fe-PFH-PLGA/CS-DS NPs that were suspended in serum-free culture medium. After 4 h incubation, the medium was changed with a fresh cell-culture medium and then irradiated with LIFU under 4 W/cm2 for 3 min. The cells were then further incubated for 12 h, washed twice and stained with calcein acetomethoxy (Calcein-AM, 10 mM in PBS) and propidium iodide (PI, 2 mM in PBS) for 15 min at 37°C under 5 % CO2 atmosphere. For CLSM imaging, cells were subjected to imaging analysis with confocal microscopy after replacing the staining solution with PBS within 1 h. Calcein-AM and PI were excited by 490 ± 10 nm and 545 nm lasers, respectively. 2.7 Animal experiments 2.7.1 Establishment of the animal model The animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chongqing Medical University. Normal age-matched wild-type C57BL/6 mice were provided by Chongqing Medical University as the control group, fed a normal diet (ND，10 % fat, 70 % carbohydrate, 20 % protein). To induce atherosclerotic plaque formation in the mouse aortic region, male apoE -/- mice of the C57BL/6J lineage, which were purchased from Beijing Hua Fukang Biotechnology Co., Ltd., were housed in a specific-pathogen-



free room and were fed a normal diet until they were 6 weeks old. Then, the apoE -/- mice were fed a western diet (WD, 40 % fat, 40 % carbohydrate, 20 % protein) containing high cholesterol levels (D12108Chigh-fat rodent diet with 1.25 % cholesterol, FBSH, Shanghai, China) for 8 to 12 weeks. To confirm the formation of atherosclerotic plaques, the mice were euthanized, and the aortas were isolated and cleaned in 1.5 mL eppendorf tubes. Oil Red O staining was performed on the aortas of the apoE -/- mice.30-31 2.7.2 SR-A expression in macrophages To detect the presence of SR-A in the macrophages, age-matched apoE -/mice and normal wild-type C57BL/6 mice (n=3/group) were euthanized and immediately perfused with PBS. The aortic arches were dissected, fixed with 4 % paraformaldehyde for 24 h, embedded in paraffin, and then used to generate H&E staining sections. For the arteries from the apoE -/mice, sections were collected from regions with atherosclerotic plaques to observe the characteristics of the atherosclerotic lesions. For the wild-type mice, sections were taken from the regions with a normal arterial wall as a control. Then, corresponding paraffin sections were stained by using standard and widely accepted immunostaining techniques for MAC-3 and CD204 (SR-A) to detect the presence of SR-A on the macrophages. To confirm the SR-A and macrophage co-localization, sections were coincubated with primary polyclonal rabbit CD204 and monoclonal mouse


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MAC-3 antibodies followed by the corresponding PE-conjugated goat anti-rabbit IgG (red) or donkey anti-rat FITC (green) secondary antibodies. The co-localization images were obtained using a fluorescence microscope (Olympus CKX41, Olympus, Tokyo, Japan). 2.7.3 Targeting performance ex vivo Freshly isolated aortic arches from apoE -/- mice (n=3) were embedded in Tissue-Tek OCT compound, followed by continuous cryosectioning (10 μm). The sections were subjected to Oil Red O staining to determine the locations of the atherosclerotic plaques. Afterwards, the next slices were fixed in 10 % phosphate buffered formalin for 20 min and then incubated with 0.5 mg/mL DiI-labelled Fe-PFH-PLGA/CS-DS or Fe-PFH-PLGA/CS NPs for 1 h. Next, the cell nucleus was stained with DAPI for 5 min. Finally, the samples were washed with PBS three times before observation under a fluorescence microscope. 2.7.4 Targeted treatment of plaques ex vivo The isolated aortic arches (n=3/group) were added to the agarose model with 30 mL of 5 mg/mL Fe-PLGA/CS-DS or Fe-PFH-PLGA/CS-DS NPs for 1 h, placed in double-distilled water, and irradiated by LIFU at an intensity of 4 W/cm2 for 10 min, 20 min, and 30 min, respectively. All aortic arches were fixed with 4 % paraformaldehyde for 24 h, embedded in paraffin and used to generate H&E staining sections to observe the results using an optical microscope.



2.7.5 Toxicity study of NPs in vivo The apoE -/- mice were studied for toxicity experiments in vivo. The mice were divided into two groups (n=3/group) after adaptation to the environment. Next, 0.16 mL of the 1 mg/mL of Fe-PFH-PLGA/CS or FePFH-PLGA/CS-DS NPs were injected via the tail vein. The overall health of the animal at regular intervals was observed to detect signs of irritation, pain, discomfort or inflammation. The organs of the heart, liver, spleen, lung, and kidney were collected on the 7th day after the NP injection and fixed with 4 % paraformaldehyde for 24 h to prepare H&E stained sections. 2.7.6 Effect of targeting and treatment of plaques in vivo MRI was performed on apoE -/- mice fed a high cholesterol diet for 8 to 12 weeks. The mice were randomly divided into two groups (n=6/group) and scanned before and 2 h after the injection of 0.16 mL of 1 mg/mL of the Fe-PFH-PLGA/CS or Fe-PFH-PLGA/CS-DS NPs via the tail vein. All MRI was performed on a 3.0 T MRI scanner (Achieva 3.0 T TX, Philips Healthcare, Best, the Netherlands) with a rat experiment coil (CG-MUC 18-H300-AP, Shanghai Chenguang Medical Technologies Co., Ltd., Shanghai, China). The axial T2*-weighted 3D gradient echo sequence was performed using the following parameters: TE, 6.9 ms; TR, 21 ms; FOV, 25 × 25 × 9 mm; voxel size, 0.3 × 0.3 × 0.4 mm; FA, 25°. The areas of the vascular wall showing a decrease in the T2* signal 2 h after the injection of NPs were measured. The area change rate was calculated using the


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following formula: area change rate (%) = area of the decrease of T2* signal / area of the vascular lumen × 100%. Then, the aortic arches of the mice were irradiated by LIFU at an intensity of 4 W/cm2 for 10 min. Subsequently, the mice were euthanized, and the aortic arches were isolated for further histological analysis. As previously described, the TUNEL assay was performed to detect apoptosis in atherosclerotic plaques.32 The macrophage apoptosis rate (%) = TUNEL positive cells / MAC-3 positive cells × 100%. 2.8 Statistical analysis Data were analyzed using GraphPad Prism v. 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). Continuous variables are presented as the mean ± the standard deviation (SD), and categorical variables are reported numerically and as percentages. Each experiment was repeated three times in duplicate unless stated otherwise. Statistical differences for multiple groups were determined using a one-way ANOVA, and individual groups were compared using independent sample t-tests. Statistical significance was defined at P < 0.05.

3. Results 3.1 Physical and chemical characterization of the NPs The results of the zeta potential, mean particle size and PDI of the Fe-PFHPLGA, Fe-PFH-PLGA/CS, Fe-PFH-PLGA/CS-DS, and Fe-PLGA/CS-DS



NPs were listed in Table 1 and showed among the zeta potentials of the four kinds of NPs, only Fe-PFH-PLGA/CS NPs were positively charged. With the addition of DS, the charge of the NPs became a negative value. Finally, the zeta potential of the Fe-PFH-PLGA/CS-DS NPs were -17.70 ± 0.35 mV. Besides, a mean particle size in the range of 258.60 to 374.63 nm, with a PDI < 0.21. The TEM images showed that the iron oxides were distributed in the nanospherical shell, and the transparent structure of CS was observed around the NPs that contained CS (Figure 2a). In addition, the diameter of the Fe-PLGA/CS-DS NPs were a little smaller than that of the other three NPs in which the core was PFH. The results were consistent with those of Table 1 that the Fe-PLGA/CS-DS NPs had a diameter of 258.60 ± 13.59 nm. Optical microscopy showed relatively homogeneous particle sizes in the Fe-PFH-PLGA/CS-DS NPs with spherical shape and good dispersions (Figure 2b, 1st line). After the LIFU sonication of the FePFH-PLGA/CS-DS NPs, the optical microscopic results showed that many nanodroplets enlarged to form microbubbles and multiplied as the power density increased and time elapsed. Furthermore, at the top of the model, white phase transition microbubbles were found upon gross observation. Representative images, including 3 min and 8 min timepoints at power densities of 1 W/cm2, 2 W/cm2, and 4 W/cm2, are presented in Figure 2b. The Fe3O4 carrying rates of the Fe-PFH-PLGA/CS and Fe-PFHPLGA/CS-DS NPs were calculated to be 69.85 ± 6.09 % and 53.79 ± 3.36


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%. 3.2 Confirmation of DS coating and measurement of the DS carrying rate The FTIR spectra obtained for CS, DS, and Fe-PFH-PLGA/CS-DS NPs are shown in Figure 3a. The DS spectrum showed an asymmetrical S=O stretching vibration band at a wave number 1,266 cm-1, and CS showed a very low IR absorption at 1,266 cm-1 but indicated characteristic N - H bending at 1,594 and 1,663 cm-1, respectively. The spectrum of the FePFH-PLGA/CS-DS NPs exhibited altered absorbance at the S=O and N– H groups. The asymmetrical S=O stretching vibration band at 1,266 cm-1 was split into 2 well-defined bands at 1,188 and 1,268 cm-1, and the N–H bending absorption band at 1,663 cm-1 was shifted to 1,648 cm-1. These changes indicated that the CS-DS coating of the Fe-PFH-PLGA/CS-DS NPs were formed by electrostatic absorption between the positively charged amine groups of CS with the negatively charged sulfate group of DS. Confocal laser scanning microscopy showed that FITC-DS had a high degree of coincidence with DiI-labelled PLGA shell (Figure 3b), which demonstrated that the DS labelled with FITC was successfully connected to the PLGA NPs. Compared with that of the control group, the shell wavelength of the DiI and FITC-labelled Fe-PFH-PLGA/CS-DS NPs were



changed due to the binding of the DS labelled with FITC. The FCM results revealed that the DS carrying rate was 94.46 %. 3.3 Multimodal imaging in vitro of NPs The US images of the different NPs were investigated after irradiation with LIFU at a power density of 4 W/cm2 for 8 min. The results showed that the phase transition microbubbles and US signals were not obtained in the saline and the Fe-PLGA/CS-DS NP group. However, in the Fe-PFHPLGA/CS and Fe-PFH-PLGA/CS-DS NP groups, the US signals were detected in both B mode and contrast mode, respectively (Figure 4a). As shown in Figure 4b, in B mode, the acoustic intensities of the Fe-PFHPLGA/CS and Fe-PFH-PLGA/CS-DS NP groups were 52.16 dB and 54.59 dB, respectively. However, the saline and Fe-PLGA/CS-DS NP group were both 0.91 dB. In contrast mode, the acoustic intensities of the FePFH-PLGA/CS and Fe-PFH-PLGA/CS-DS NP groups were 68.23 dB and 70.66 dB, while those of saline and Fe-PLGA/CS-DS NP groups were both as low as 0 dB. In sum, the acoustic intensities of the PFH-contained NPs were significantly higher than those of the non-PFH NPs (B mode and contrast mode: all P < 0.001). There was no significant difference between the acoustic intensities of the Fe-PFH-PLGA/CS and Fe-PFH-PLGA/CSDS NPs (B mode and contrast mode: both P > 0.05). In addition to being used as US contrast agents, the Fe-PFHPLGA/CS-DS NPs were able to reduce the T2* signal intensity. MR images


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showed that, with an increase in the Fe-PFH-PLGA/CS-DS NP concentration, the T2* signal intensity was reduced gradually. In addition, there was a strong linear correlation between the 1/T2* values and Fe concentration, with the r2 calculated to be 91.9472 mM-1·s-1 (Figure 4c). 3.4 Cell experiments 3.4.1 Targeting ability in vitro The targeting ability of the Fe-PFH-PLGA/CS-DS NPs labelled with DiI towards the activated and unactivated RAW264.7 cells was observed using CLSM (Figure 5a). In the activated cells, a great number of the Fe-PFHPLGA/CS-DS NPs were adhered to the cells, as observed through strong red fluorescence; nevertheless, there was weak fluorescence from the FePFH-PLGA/CS NPs, indicating very limited uptake. In addition, in the blocking experiment, the fluorescence was also weak from the DS solution + Fe-PFH-PLGA/CS-DS group. The FCM results showed that the normalized fluorescence of Fe-PFH-PLGA/CS-DS NPs were 67.76%, while the other groups were less than 4.65% (Figure 5c), illustrating that the Fe-PFH-PLGA/CS-DS NPs were more prone to be taken into the activated cells than other groups. Similarly, in the unactivated cells, weak fluorescence was observed between the Fe-PFH-PLGA/CS-DS and FePFH-PLGA/CS NPs in both the CLSM and FCM (Figure 5a and c). To further examine the binding efficiency, the activated cells were incubated with Fe-PFH-PLGA/CS-DS and Fe-PFH-PLGA/CS NPs for the time



different points. In the Fe-PFH-PLGA/CS-DS group, a few NPs were found to accumulate around the cells at 0.5 h, and some NPs appeared around the nucleus at 2 h. By 4 h, a large number of NPs were swallowed by the cells and gathered around the nucleus. In the same time point the fluorescence intensity of the Fe-PFH-PLGA/CS-DS NPs increased gradually as the Fe-PFH-PLGA/CS NPs became less obvious. For instance, the normalized fluorescence of the Fe-PFH-PLGA/CS-DS NPs were 89.46 %, while that of the Fe-PFH-PLGA/CS NPs were only 12.25 % at 4 h (Figure 5b and d). 3.4.2 MRI of macrophages In in vitro MR images, the activated macrophages incubated with the SRA-targeted NPs showed significantly lower T2* signals than cells incubated with the non-targeted NPs at the same iron concentration. In addition, as the concentration of iron increased, the SNR values of Fe-PFH-PLGA/CSDS NPs decreased more significantly than Fe-PFH-PLGA/CS NPs (Figure 4d). 3.4.3 Cytotoxicity test A CCK-8 viability assay was performed to assess the cytotoxicity of the Fe-PFH-PLGA/CS-DS NPs. No cytotoxicity was observed in the activated or unactivated RAW264.7 cells treated with the various concentrations of the Fe-PFH-PLGA/CS-DS NPs, and there was no significant difference in cell viability between the activated and unactivated groups (P > 0.05)


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(Figure 5e). 3.4.4 ADV effect on cells The in vitro ADV effect of NPs on activated RAW264.7 cells was evaluated using live/dead cell staining. Confocal laser scanning microscopy showed that the cells in the PFH + LIFU group exhibited bright red fluorescence, indicating late apoptotic or dead RAW264.7 cells. By contrast, the cells in the control group, the LIFU group and non-PFH + LIFU group showed intense green fluorescence, indicative of living cells. The maximum cell survival was observed in the control group as well as the LIFU group (Figure 6), illustrating that the use of LIFU irradiation alone had no effect on cell viability. 3.5 Animal experiments Gross vessels of the aortic arches were observed in macroscopic images, after the careful removal of any residual adventitial fat that may have adhered to the exterior of the adventitia (Figure 7a1). In addition, some lipid-rich plaques were obtained by Oil Red O staining compared with the control group in the isolated aortic arch (Figure 7a2 and a3). 3.5.1 SR-A expression in macrophages ex vivo The H&E staining of the aortic root in apoE -/- mice showed welldeveloped atherosclerotic plaques with the accumulation of lipid in the aortic root wall, while no atherosclerotic plaques were found in the normal wild-type mouse thoracic aorta (Figure 7b, 1st and 2nd column). The



macrophages in the aortic root lesions were assessed, and the results of immunohistochemistry showed that a large number of macrophages were present in the lesion area of the aortic root compared with the normal thoracic aorta (Figure 7b, 3rd column). The expression of SR-A in the atherosclerotic plaques was pronounced in the cross-sectional area of the aortic root; in contrast, no SR-A were expressed in the control thoracic aorta (Figure 7b, 4th column). As shown in Figure 7c, red-fluorescent PElabelled anti-CD204, an antibody that specifically targets SR-A, colocalized with green-fluorescent FITC-labelled anti-MAC-3 in the atherosclerotic plaques. 3.5.2 Targeting performance ex vivo Age-matched apoE -/- mice were sacrificed, and then their aortic arches were excised and continuously cryosectioned. Using Oil Red O staining, the locations of plaques in which the fat was stained to bright red were then visually verified. After incubating with the DiI-labelled Fe-PFH-PLGA/CS or Fe-PFH-PLGA/CS-DS NPs, the slices were washed and observed under a fluorescence microscope. There was a high amount of fluorescence for the Fe-PFH-PLGA/CS-DS NPs across almost the entire plaque area, whereas the Fe-PFH-PLGA/CS NP group exhibited a negligible fluorescence. Normal vessel walls without plaques could not be bound by the NPs in both groups (Figure 8). 3.5.3 Targeted treatment of plaques ex vivo


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After an incubation of the aortic arch with the Fe-PFH-PLGA/CS-DS NPs and irradiation by LIFU with 4 W/cm2 for 30 min, some holes of varying sizes appeared in the plaque slice, and the continuity of the fibre cap was interrupted. However, no identifiable holes were observed, and the fibre cap was continuous and relatively smooth in the plaque slices that had been incubated with the Fe-PFH-PLGA/CS-DS NPs and irradiation for 10 min and 20 min, or incubated with the Fe-PLGA/CS-DS NPs and irradiation for different time points (Figure 9). 3.5.4 Toxicity study of NPs in vivo ApoE -/- mice did not die during the 7-day observation period and were emotionally stable, with no inflammatory response. In addition, no abnormal cell morphology or necrosis was observed on H&E-stained sections of the heart, liver, spleen, lung or kidney (Figure 10). The Fe-PFHPLGA/CS or Fe-PFH-PLGA/CS-DS NPs had no significant toxicity and thus can be further applied. 3.5.5 Targeting and treatment of plaques in vivo Cross-sectional T2*-weighted images of the ascending aortas of apoE -/mice showed a bright circular lumen before the injection of NPs. Two hours after injection, decrease of T2* signal was observed in the wall of the ascending aortic plaques of apoE -/- mice in the Fe-PFH-PLGA/CS-DS group but not obviously observed in the Fe-PFH-PLGA/CS group. The signal reduction area was a mural crescent (Figure 11a, lower 2nd column).



The area change rate of Fe-PFH-PLGA/CS-DS group was as high as 30.80 %, and that of Fe-PFH-PLGA/CS group was 3.56 %, the differences in the area change rates between the two groups were statistically significant (P < 0.0001) (Figure 11b). Histological examination was performed after aortic arches were isolated. In both groups, brightfield showed no significant changes in plaques (Figure 11a, 3rd column). Compared with the Fe-PFH-PLGA/CS group, a significant number of apoptotic cells were observed in the FePFH-PLGA/CS-DS group, and the majority of apoptotic cells in the plaques of the Fe-PFH-PLGA/CS-DS group expressed the macrophage marker MAC-3 (Figure 11a, 4th column). The macrophage apoptosis rate of the Fe-PFH-PLGA/CS-DS group (20.46%) was significantly higher than that of the Fe-PFH-PLGA/CS group (3.05%) (P < 0.0001) (Figure 11c).

4. Discussion Considering the design of NPs, a too complicated preparation method accompanying unpredictable interactions between the components of NPs reduces the diagnostic and treatment effect of the NPs. Yi et al. fabricated an atorvastatin calcium (AT)-loaded DS-coated core−shell reconstituted high density lipoprotein (rHDL) in which the NPs were functionalized with DS via electrostatic interaction and could maintain their integrity and


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stability in cell culture medium.11 So, we chose to use the positive charge of the CS to connect the targeting agent DS with a large amount of negative charges by electrostatic adsorption instead of complex and timeconsuming organic chemical reactions. The prepared particles in the size range 258-374 nm were suitable for atherosclerotic imaging in the blood pool. The large particles are easily captured by the reticuloendothelial system (RES), which shortens the duration of the blood circulation and reduces their accumulation within the lesion, and the too small particles exhibited difficulties associated with phase transition.33-34 Transition in the zeta potential from positive to negative power altered the absorbance at the S=O and N–H groups of the Fe-PFH-PLGA/CS-DS NPs in the FTIR spectra, and the green fluorescence of NPs in confocal laser imaging demonstrated that DS was successfully attached to the nanoshell, thereby guaranteeing further targeting ability. From optical-microscope and TEM images, we could tell that the NPs were uniform in shape and moderate in volume. In PFH-free NPs (Fe-PLGA/CS-DS), the PLGA core was smaller than the others, and the iron oxide NPs were distributed in the nanospherical shell of the Fe-containing NPs, which makes it suitable for MRI; moreover, the ability to undergo a phase transition during LIFU irradiation makes PFH-containing NPs suitable for US imaging. In the multimodal imaging of NPs, it was verified that PFH-containing NPs became microbubbles after the phase transition, and US signals were



detected in both B mode and contrast mode. Simultaneously, in Fe-PFHPLGA/CS-DS NPs, the T2* signal intensity was reduced gradually with an increase the in Fe concentration, which was consistent with previous studies.21, 25 Our in vitro targeting performance showed that a large number of FePFH-PLGA/CS-DS NPs were endocytosed by activated macrophages 2 h after incubating with RAW 264.7 cells, which indicated the targetmediated cellular internalization of Fe-PFH-PLGA/CS-DS NPs, whereas in the Fe-PFH-PLGA/CS NP and the DS solution + Fe-PFH-PLGA/CSDS NP groups, a small amount of red fluorescence could be seen around the nucleus. In the MRI cell experiment, the SNR values decreased significantly with the increasing concentration of the Fe-PFH-PLGA/CSDS NP group, which indicated that more NPs were targeted to RAW264.7 cells and could achieve MR imaging. This targeting effect could resist the scouring effect of the blood flow to a certain extent and enable NP binding to macrophages for further imaging and treatment. It was concluded that the magnitude of the foreign body reaction caused by NPs followed by endocytosis was related to the state of macrophage polarization—in other words, whether it was activated.35 Therefore, in unactivated RAW 264.7 cells, the result was just the opposite: only a small number of NPs adhered to the unactivated cells in the Fe-PFH-PLGA/CS-DS and Fe-PFHPLGA/CS NP groups due to physical adsorption.


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The theoretical basis for targeting SR-A is that SR-A were highly expressed in macrophages in unstable atherosclerotic plaques in this study. Histopathological and biochemical results indicate that local macrophage infiltration is associated with the plaque characteristics and susceptibility to rupture.36 Therefore, SR-A are important molecular imaging targets for visualizing the plaque vulnerability. Immunohistochemical evidence showed the SR-A expression by macrophages localized to atherosclerotic vulnerable plaques formed in the aortic arch of high fat-fed apoE -/- mice. After incubation with the DiI-labelled NPs, red Fe-PFH-PLGA/CS-DS NPs were co-localized within macrophages in the same plaques, but no obvious red fluorescence was found in the Fe-PFH-PLGA/CS NPs. The present experiment validated this novel probe as being suitable for identifying and quantifying the cellular component of atherosclerotic lesions and the potential visualization of vulnerable plaques. In cell experiments, Fe-PFH-PLGA/CS-DS NPs were incubated with RAW 264.7 cells at 37°C in cell medium, and the phase transition of PFHcontaining NPs was not found to damage cells, suggesting that the phasechange material PFH had good stability and safety at 37°C. Compared with the low boiling point of perfluoropentane (PFP) and the high boiling points of perfluorooctyl bromide (PFOB) and perfluorodecalin (PFD), PFH with a boiling point of 56°C37 is more suitable for controllable phase transition in vivo.



A liquid fluorocarbon combined with the process of phase transition is capable of causing intracellular damage, which could be used for the treatment of tumors, thus achieving molecular imaging and therapy inside cells.22, 38-39 With the help of a targeting agent, the therapy of the tumor can be achieved at the cellular level. In vascular disease, the phase transition of PFH has rarely been applied for treatment purposes. Our research group attempted to apply it for the treatment of thrombi for the first time in a previous study;21 both a decrease in thrombus weight and a large amount of holes in the thrombi were observed after a LIFU-induced phase transition, demonstrating the thrombolytic effectiveness of the phase transition of PFH. However, would such a treatment be effective for another vascular disease, namely, atherosclerotic plaques? Considering the vulnerability of atherosclerotic plaques, the rupture and shedding of plaques from vessel walls and their entry into the bloodstream may result in stroke, heart attack and death.40 Thus, targeting for specific sites and a controlled ADV effect is vitally important when treating atherosclerotic plaques by LIFU-induced phase transition. It has been confirmed that Fe-PFH-PLGA/CS-DS NPs have good targeting ability for macrophages and may be endocytosed by activated macrophages to induce cell apoptosis and necrosis under LIFU irradiation by the ADV effect in cell experiments. Furthermore, the cells of the control, under simple LIFU irradiation and LIFU irradiation with non-PFH NPs,


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displayed almost full cell viability, suggesting that the use of LIFU irradiation alone or no ADV effect had no adverse effect on the activated macrophages. This ensures that it can induce apoptosis in only macrophages that endocytose PFH-containing NPs when used in vivo and has no effect on the surrounding normal tissues. In ex vivo experiments, the excessive time of the LIFU irradiation could cause the discontinuity of the fibre cap. Therefore, for the sake of safety, based on ex vivo experimental conditions, the irradiation time of LIFU were controlled relatively short in in vivo experiment. In our in vivo experiments, 2 h after the injection of the Fe-PFHPLGA/CS-DS NPs, decrease of T2* signal was observed in the wall of the ascending aortic plaques on apoE -/- mice, which was attributed to the ligand-receptor mediated targeting performance. By contrast, in the FePFH-PLGA/CS group, no identifiable signal reduction area was observed. After irradiation by LIFU at an intensity of 4 W/cm2 for 10 min, a significant number of apoptotic cells were observed in the Fe-PFHPLGA/CS-DS group. Importantly, the apoptotic cells co-localized with MAC-3 positive cells, demonstrating the macrophage avidity of the FePFH-PLGA/CS-DS NPs in vivo. In conclusion, Fe-PFH-PLGA/CS-DS NPs could bind to SR-A expressed on macrophages and be swallowed by macrophages; consequently, phase transition occurred inside the macrophages and made it possible to cause specific damage to and the



apoptosis of the macrophages. Thus, these NPs have great potential for stabilizing of the plaque by reducing the inflammation mediated by macrophages. This conjecture of cellular-level therapy was consistent with the research of Yi et al.,29 who synthesized a nanoagent that had excellent phototoxicity to the activated macrophages under laser irradiation and may be great for atherosclerosis-plaque stabilization. In addition, McCarthy et al. believed that macrophage ablation with a nanoagent has the effect of stabilizing atherosclerotic plaques.41 The findings in the present study provide a new perspective for the treatment of plaque because traditional interventional surgeries such as carotid endarterectomy were invasive methods, while the efficacy of traditional drugs is slow. The other two ways to treat atherosclerotic plaques are photodynamic therapy29 and sonodynamic therapy,32 which are limited to some superficial tissues due to insufficient light penetration or have certain phototoxic side effects. Our Fe-PFH-PLGA/CS-DS NPs can target atherosclerotic regions that express SR-A and could be endocytosed by the macrophages and notably induced apoptosis under LIFU irradiation by an ADV effect. The ADV effect might stabilize the atherosclerotic plaque by damaging the ultrastructure of the cells, causing apoptosis to ameliorate the inflammation, which are safe, highly efficient, and controllable, could overcome the deficiency of photodynamic therapy and sonodynamic therapy. Despite these encouraging results, our research still has some limitations.


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As a double-edged sword, the phase transition induced macrophage apoptosis under LIFU irradiation, but inappropriate experimental conditions that mainly include an inappropriate duration and power of LIFU may damage the plaque structure and potentially result in plaque destabilization, plaque rupture and subsequent thrombus formation. Suitable LIFU parameters and methods for assessing the integrity of plaque structure and its cellular content before and at different time points after LIFU irradiation and changes in related inflammatory factors in vivo require further study to evaluate the effect of the phase transition on plaque stabilization. What’s more, considering the safety of PFH application in the treatment of atherosclerotic vulnerable plaques, many aspects such as the biodistribution and the blood pharmacokinetics of the NPs will be further validated in our subsequent experiments.

5. Conclusion In this study, we successfully fabricated US and MRI multimodal molecular imaging NPs (Fe-PFH-PLGA/CS-DS) using simple and reproducible double emulsification method and electrostatic adsorption for the noninvasive assessment of SR-A on macrophages in atherosclerotic plaques. The Fe-PFH-PLGA/CS-DS NPs could selectively accumulate within the activated macrophages of the aortic plaques, causing apoptosis by LIFU irradiation in vitro. Furthermore, this had been confirmed in vivo.



The SR-A targeted NPs combined with multimodal molecular imaging may have the potential for providing important information for the characterization and treatment of atherosclerotic vulnerable plaques.

AUTHOR INFORMATION Corresponding author: *E-mail: [email protected] ORCID DajingGuo: 0000-0001-8655-6621

&These authors contributed equally to this work and should be considered co-first authors.

Acknowledgements The authors are grateful to Shike Wang for his assistance with the MRI technical support and American Journal Experts (AJE) for their assistance with language editing. This study was supported by the National Natural Science Foundation of China (Grants No. 81571663 and No. 81701650).

Disclosure statement We declare that we have no conflicts of interest.


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Table 1. Characteristics of different NPs (n = 3) Polydispersity index Sample

Zeta potential（mV）

Diameter（nm）（PDI）

Fe-PFH-PLGA

-18.63 ± 0.21

290.03 ± 2.75


0.152 ± 0.053


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Fe-PFH-PLGA/CS

14.03 ± 0.59

321.30 ± 2.86

0.207 ± 0.021

Fe-PFH-PLGA/CS-DS

-17.70 ± 0.35

374.63 ± 10.87

0.186 ± 0.045

Fe-PLGA/CS-DS

-20.50 ± 2.87

258.60 ± 13.59

0.053 ± 0.060

Figure 1. Schematic diagram of the preparation of the Fe-PFH-PLGA/CSDS NPs and the NPs targeting SR-A expressed on macrophage surfaces and endocytosed by macrophages, followed by cell damage due to phase transition under LIFU irradiation.


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Figure 2. Characterization of the NPs. TEM images of the Fe-PFH-PLGA, Fe-PFH-PLGA/CS, Fe-PFH-PLGA/CS-DS, and Fe-PLGA/CS-DS NPs (scale bar = 100 nm) (a). Optical microscopy images of the Fe-PFHPLGA/CS-DS NPs before and 3 min and 8 min after LIFU irradiation at power densities of 1 W/cm2, 2 W/cm2, and 4 W/cm2 (scale bar = 10 μm) (b).



Figure 3. Confirmation of DS coating. Fourier transform infrared spectroscopy spectra of CS, DS, and Fe-PFH-PLGA/CS-DS NPs (a). The DiI and FITC-labelled Fe-PFH-PLGA/CS-DS NPs were visualized by the colocalization of the DiI-labelled PLGA shell (red) and FITC-labelled DS (green) using confocal laser scanning microscopy (scale bar = 2 μm) (b).


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Figure 4. Multimodal imaging of NPs in vitro. US images of the B mode and contrast mode after LIFU irradiation for the Fe-PFH-PLGA/CS-DS NPs compared to the three control groups: saline, Fe-PLGA/CS-DS NPs, and Fe-PFH-PLGA/CS NPs (a). Acoustic intensities of the saline ， FePLGA/CS-DS, Fe-PFH-PLGA/CS, and Fe-PFH-PLGA/CS-DS NPs after LIFU irradiation (b) (***P < 0.001). The T2*-weighted MR images and the transverse relaxivity (r2) of the Fe-PFH-PLGA/CS-DS NPs at different Fe concentrations (c). The T2*-weighted MR images and SNR values of the Fe-PFH-PLGA/CS-DS NPs or Fe-PFH-PLGA/CS NPs after incubation with activated RAW 264.7 cells at different Fe concentrations (d). ACS Paragon Plus Environment


Figure 5. The targeting ability to cells and the cytotoxicity of NPs. The CLSM images (a) and FCM results (c) of RAW 264.7 cells with nucleus


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staining by DAPI incubated with DiI-labelled Fe-PFH-PLGA/CS-DS NPs or Fe-PFH-PLGA/CS NPs for 2 h after activation by LPS (+) or LPS (-). Activated cells were treated with DS solution + Fe-PFH-PLGA/CS-DS NPs (middle column). Time-point study (b, d) of activated RAW 264.7 cells under incubation with DiI-labelled Fe-PFH-PLGA/CS-DS NPs or FePFH-PLGA/CS NPs. (blue = nucleus, red = NP, scale bar = 10 μm, ****P < 0.0001). Cytotoxicity of the activated and unactivated RAW 264.7 cells incubated with Fe-PFH-PLGA/CS-DS NPs for 24 h in vitro (e).



Figure 6. ADV effect of NPs on activated macrophages. CLSM images of activated RAW264.7 cells stained with Calcein-AM and PI after incubation with 0.5 mg/mL of Fe-PLGA/CS-DS or Fe-PFH-PLGA/CS-DS NPs and LIFU irradiation under 4 W/cm2 for 3 min. (green = living cell, red = dead cell, scale bar = 50 μm).

Figure 7. Atherosclerotic plaque model in aorta and SR-A expression in macrophages. Gross aortic vessel isolated from age-matched mice (a1).


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Representative macroscopic image of lipid-rich plaques by Oil Red O staining (a2). Representative macroscopic image in the control group (a3). Representative images of the aortic root with atherosclerotic plaques and the thoracic aorta without atherosclerosis lesions (scale bar = 100 μm) (b). Images of colocalization between anti-CD204 stained SR-A and antiMAC-3 stained macrophages within a plaque. (blue = nucleus, green = anti-MAC-3, red = anti-CD204, scale bar = 20 μm) (c).

Figure 8. Targeting performance ex vivo. Frozen section image of plaque stained bright red by Oil Red O (a). The fluorescent images of the Fe-PFHPLGA/CS or Fe-PFH-PLGA/CS-DS NPs binding to slices of an atherosclerotic aortic arch from apoE -/- mice (blue = nucleus, red = NP, scale bar = 100 μm) (b). ACS Paragon Plus Environment


Figure 9. Ex vivo treatment of plaques. H&E staining of plaques after the incubation of the aortic arch with Fe-PLGA/CS-DS or Fe-PFH-PLGA/CSDS NPs and irradiation by LIFU with 4 W/cm2 for different time points (yellow dashed line = plaque area, blank arrows= irregular defect area, scale bar = 100 μm).


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Figure 10. Toxicity study in vivo. Pathological H&E-stained slices of the heart, liver, spleen, lung, or kidney in the Fe-PFH-PLGA/CS or Fe-PFHPLGA/CS-DS NP group (scale bar = 50 μm).

Figure 11. In vivo targeting and treatment of plaques. MR T2*-weighted images of before and 2 h after the injection of the Fe-PFH-PLGA/CS or Fe-PFH-PLGA/CS-DS NPs. Representative images after in vivo LIFU irradiation of corresponding brightfield and TUNEL staining for apoptosis (blue = nucleus, green = anti-MAC-3, red = apoptotic cell, scale bar = 50 μm) (a). The area change rate of the Fe-PFH-PLGA/CS and Fe-PFHPLGA/CS-DS groups (b) (****P < 0.0001). The quantification of



TUNEL-positive cells in the Fe-PFH-PLGA/CS and Fe-PFH-PLGA/CSDS groups (c) (****P < 0.0001).

Graphical Abstract


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SR-A-Targeted Phase-Transition Nanoparticles for the Detection and

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