Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis - PubMed (original) (raw)

. 2017 Jun 27;11(6):5785-5799.

doi: 10.1021/acsnano.7b01385. Epub 2017 May 15.

Max L Senders, Amr Alaarg 1 2, Carlos Pérez-Medina 1, Jun Tang 1 3, Yiming Zhao 1, Francois Fay 1, Jacqueline Deichmöller 4, Benjamin Born, Emilie Desclos, Nicole N van der Wel, Ron A Hoebe, Fortune Kohen, Elena Kartvelishvily, Michal Neeman, Thomas Reiner 3 5, Claudia Calcagno 1, Zahi A Fayad 1, Menno P J de Winther 6, Esther Lutgens 6, Willem J M Mulder 1, Ewelina Kluza

Affiliations

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis

Thijs J Beldman et al. ACS Nano. 2017.

Abstract

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Keywords: PET imaging; anti-inflammatory effects; antiatherogenic therapy; atherosclerosis; hyaluronan nanoparticles; inflammation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1

Figure 1

(A) Hyaluronan nanoparticles (HA-NPs) (upper panel) and HA-NPs after hyaluronidase (HYAL) treatment (lower panel) visualized by atomic force microscopy (AFM) (first panel), cryo-scanning electron microscopy (cryo-SEM) (second panel), environmental scanning electron microscopy (ESEM) (third panel), and direct stochastic optical reconstruction microscopy (dSTORM) (fourth panel). The scale bars shown in the lower images refer also to the upper images. White arrowheads in ESEM images indicate the location of nanoparticles. The image insets in the fourth panel show representative dSTORM images of a single nanoparticle at higher magnification. (B) Normalized distribution of nanoparticle diameter obtained from AFM (left panel) and dSTORM (right panel) data. In the left panel, the size distribution of HA-NPs and hydrolyzed HA-NPs (HA-NPs + HYAL) is shown in gray and red, respectively, and, in the right panel, in black and gray, respectively. (C) Amount of terminal _N_-acetlyglucosamine (NAcGlc) released from hyaluronan (HA) and HA-NPs during hydrolysis with HYAL, which is expressed as the percentage of total NAcGlc of substrate hyaluronan. (D) Transmitted light image of stained polyacrylamide gel showing the migration spots of different molecular weight hyaluronan, i.e., ∼75 kDa (75k), ∼400 kDa (400k), and ∼1.5 MDa (1.5M), nonlabeled hyaluronan nanoparticles (NPs), fluorescently labeled HA-NPs (NPsL), and their hydrolysis products. Symbol “+” indicates the hydrolyzed samples. Two black arrowheads point at the poorly migrating high-molecular-weight hyaluronan in HA-NP samples. (E) Wide-field (left panel) and two-color dSTORM (right panel) microscopy images of aortic endothelium of a mouse that received an injection of Cy5.5-HA-NPs 2 h before sacrifice. Endothelial cells were stained with CD31-Alexa Fluor 555. (F) Comparison of HA-NP morphology in a mouse aortic endothelium after the in vivo administration of Cy5.5-HA-NPs and on a coverglass after the in vitro seeding of Cy5.5-HA-NPs, assessed by dSTORM. Scale bars in lower images refer to those in the upper panel.

Figure 2

Figure 2

(A) Flow cytometry analysis of the cellular uptake of Cy5.5-labeled hyaluronan nanoparticles (HA-NPs) in different phenotypes of bone-marrow-derived macrophages. The macrophages are divided into three main phenotypic groups: naive (−) (white bars), interleukin 4 (IL-4)-stimulated (gray bars), and lipopolysaccharide (LPS) and interferon γ (INFγ)-stimulated (black bars). Moreover, three concentrations of oxidized low-density lipoprotein (oxLDL) were used for costimulation, i.e., 25, 50, and 100 μg/mL. The cellular uptake of HA-NPs is expressed as the median fluorescence intensity per cell (MFI). Bars represent mean MFI/condition ± SD (ns = 3). Symbol “*” indicates a significant difference and “#” indicates significantly higher MFI compared to all other conditions at p < 0.05. (B) Uptake efficacy of Cy7-HA-NPs in aortic, splenic, and bone marrow macrophages measured by flow cytometry. Black and gray bars represent the data obtained for mice fed with a high-fat diet for 6 (6w HFD) and 12 weeks (12w HFD), respectively. Symbol “*” indicates intergroup differences, whereas “#” and “&” indicate significantly higher MFI compared to all other macrophage populations within the 6w HFD and 12w HFD groups, respectively, and at p < 0.05. (C) Scatter plots showing the relation between the uptake efficacy of HA-NPs and free hyaluronan (HA)-binding (upper panel) and expression of CD44 receptor (lower panel). All data were obtained by flow cytometry and are expressed as the median fluorescence intensity per cell. The correlations were investigated for pooled aortic leukocyte populations, i.e, macrophages, Ly6chigh and Ly6clow monocytes, and neutrophils, in 6w HFD (left panel, black circles) and 12w HFD mice (right panel, gray squares). The black lines serve as guides for the eye. The correlation coefficient, R, was obtained from the nonparametric Spearman’s correlation test. (D) Representative confocal microcopy images of aortic lesions detected in 6w HFD (left image) and 12w HFD group (right image). The Cy5.5-HA-NPs are shown in red, CD68 staining of macrophages is shown in green, and cell nuclei are blue. (E) Selectivity of HA-NPs toward plaque-associated macrophages expressed as the percentage of HA-NP-positive area that colocalizes with CD68-positive macrophage area. Bars represent mean ± SD. Symbol “*” represents significant difference at p < 0.05.

Figure 3

Figure 3

(A) Autoradiography images of aortas excised from a wild-type mouse (WT, left) and two atherosclerotic mice that were on a high-fat diet for either 6 weeks (6w HFD, middle) or 12 weeks (12w HFD, right). Twenty-four hours before sacrifice, the mice received intravenous injection of 89Zr-HA-NPs. In the images, dark spots indicate higher radioactivity deposition. (B) Comparison between the radioactivity levels in healthy aortas (WT, white bar) and aortas with early (6w HFD, black bars) or advanced atherosclerosis (12w HFD, gray bars). The radioactivity was measured for the entire aorta by gamma counting 24 h after i.v. administration of 89Zr-HA-NPs, and it is expressed as the percentage of injected dose (%ID). (C) Blood clearance kinetics of 89Zr-HA-NPs determined by gamma counting in blood samples probed at different time points after NP injection. The data were obtained for the above-mentioned mouse groups and are presented as the mean ± SD of %ID per gram of blood (%ID/g) over time. (D) Coronal PET/MRI fusion images of an atherosclerotic rabbit, showing the organ radioactivity distribution at different time points after intravenous injection of 89Zr-HA-NPs. (E) Clearance kinetics of 89Zr-HA-NPs determined noninvasively in rabbits by measuring standardized uptake values (SUV) in the aortic blood. (F) Time-dependent biodistribution of 89Zr-HA-NPs determined by dynamic (20 min to 2 h) and static (12 and 24 h) PET imaging in the spleen (black squares), liver (gray circles), and kidney (gray triangles). (G) Left panel displays a representative PET/MRI fusion image of an atherosclerotic rabbit 12 h after the administration of 89Zr-HA-NPs. PET signal hot spot can be observed in the abdominal aorta (white arrowhead). In the right panel, the bar chart shows the difference between SUV of the aorta and skeletal muscle at 12 h postinjection. (H) Biodistribution of 89Zr-HA-NPs in different rabbit organs quantified ex vivo by gamma counting and presented as %ID/g of tissue. (I) Confocal microscopy images of the abdominal (upper left) and thoracic aorta (upper right) from a rabbit that was co-injected with both 89Zr-HA-NPs and Cy5.5-HA-NPs. The Cy5.5-HA-NPs are displayed in red, RAM-11 staining of macrophages is shown in green, and cell nuclei are blue. In the lower panel, higher magnification images of abdominal aorta show the engulfed HA-NPs by macrophages. In all bar charts, bars represent mean ± SD and symbol “*” indicates the significant difference at p < 0.05.

Figure 4

Figure 4

(A) Representative images of aortic roots from mice that received either PBS (control, left image panel), HA-NPs (middle image panel), or free HA (right image panel) during a 12-week high-fat feeding period. The sections were stained with hematoxylin and eosin (H&E), macrophage-specific antibody (MAC-3), or sirius red (collagen). Scale bar in the upper right image refers to all H&E-stained sections. Bar charts display the mean plaque area (top), percentage of plaque area containing macrophages (middle), and collagen (bottom) in the aforementioned treatment groups. (B) Flow cytometry analysis of aortic arches of the treated mice. Left panel shows representative cell scatter plots and histograms obtained for a control (upper panel) and HA-NPs-treated mouse (lower panel). The immune cells are defined as CD45-positive cells. Bar chart compares the total immune cell count in the control, HA-NPs-treated, and free HA-treated mice. In all bar charts, bars represent mean ± SD and symbol “*” indicates the significant difference at p < 0.05.

References

    1. Laurent T. C.Structure of Hyaluronic Acid. In Chemistry and Molecular Biology of the Intercellular Matrix; Balasz E. A., Ed.; Academic Press: London, 1970; Vol. 2, pp 703–732.
    1. Toole B. P. Hyaluronan: From Extracellular Glue to Pericellular Cue. Nat. Rev. Cancer 2004, 4, 528–539. 10.1038/nrc1391. - DOI - PubMed
    1. Petrey A. C.; de la Motte C. A.. Hyaluronan, a Crucial Regulator of Inflammation. Front. Immunol. 2014, 5, Article 101 10.3389/fimmu.2014.00101. - DOI - PMC - PubMed
    1. Toole B. P. Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities. Clin. Cancer Res. 2009, 15, 7462–7468. 10.1158/1078-0432.CCR-09-0479. - DOI - PMC - PubMed
    1. Zöller M. CD44: Can a Cancer-Initiating Cell Profit from an Abundantly Expressed Molecule?. Nat. Rev. Cancer 2011, 11, 254–267. 10.1038/nrc3023. - DOI - PubMed

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