Cyclodextrin polymer improves atherosclerosis therapy and reduces ototoxicity

Journal Pre-proof Cyclodextrin polymer improves atherosclerosis therapy and reduces ototoxicity

Heegon Kim, Junhee Han, Ji-Ho Park PII:

S0168-3659(19)30740-0

DOI:

https://doi.org/10.1016/j.jconrel.2019.12.021

Reference:

COREL 10064

To appear in:

Journal of Controlled Release

Received date:

8 August 2019

Revised date:

10 December 2019

Accepted date:

12 December 2019

Please cite this article as: H. Kim, J. Han and J.-H. Park, Cyclodextrin polymer improves atherosclerosis therapy and reduces ototoxicity, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.12.021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

Cyclodextrin polymer improves atherosclerosis therapy and reduces ototoxicity

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Heegon Kim, Junhee Han, and Ji-Ho Park*

Department of Bio and Brain Engineering and KAIST Institute for Health Science and

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Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141,

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Republic of Korea.

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*To whom correspondence should be addressed: [email protected]

Abstract

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Journal Pre-proof Recently, cyclodextrin (CD) has shown the potential for effective treatment of atherosclerotic plaques in mice by solubilizing plaque cholesterol. While promising as a new therapy for atherosclerosis, poor pharmacokinetics and ototoxicity of CD pose a therapeutic challenge. Thus far, however, there has been no attempts to overcome such limitations. Here, we showed that cyclodextrin polymer (CDP) with a diameter of ~ 10 nm exhibits outstanding pharmacokinetics and plaque targeting efficacy compared to a monomeric CD. Furthermore, we found out that

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CDP does not induce plasma membrane disruption as opposed to CD, which eliminated

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cytotoxicity and hemolytic activity of CD. In a mouse model of atherosclerosis, subcutaneous

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injections of beta-cyclodextrin polymer (βCDP) significantly inhibited plaque growth compared to monomeric hydroxypropyl-beta-cyclodextrin (HPβCD) at the same dose (1 g/kg). More

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importantly, βCDP did not induce significant ototoxicity at a high-dose (8 g/kg) where HPβCD

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reduced the outer hair cell content by 36 %. These findings suggest that the polymerization of

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CD can overcome major limitations of CD therapy for treatment of atherosclerosis.

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Keywords: Atherosclerosis, cyclodextrin polymer, plaque therapy, ototoxicity

Graphical Abstract

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Cyclodextrin polymer (CDP)

Plaque treatment

Renal clearance

Control

Glomerular filtration

Plaque targeting

CD

CDP

EPR effect Plaque

Ototoxicity

Plasma membrane

Plasma membrane disruption

Cholesterol extraction

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~ 10 nm

Control

1. Introduction

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CD

CDP

Journal Pre-proof High blood cholesterol concentration is a critical factor in progression of atherosclerosis. Cholesterol is not only a main component of atherosclerotic plaques, but also key inflammationtriggering source [1-3]. Clinically, the most successful approach to prevent and manage atherosclerosis has been to lower the low-density lipoprotein (LDL) level using statins [4]. However, even daily administration of maximal doses of statins over 2 years could not regress the atherosclerotic plaques [5], which has restricted improving the quality of life in patients with

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atherosclerosis. Considering the limited efficacy of statins on atherosclerotic plaque regression

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and the fact that cardiovascular disease still remains the major cause of death worldwide [6],

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such systemic therapy to lower the LDL cholesterol level is not effective enough and more direct and local therapy may be necessary.

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2-Hydroxypropyl-beta-cyclodextrin (HPβCD) is well known to bind and solubilize

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hydrophobic molecules such as cholesterol [7]. FDA approved HPβCD as a hydrophilic

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excipient [8] and also granted orphan drug designation to HPβCD for the treatment of NiemannPick Type C disease [9]. Recently, HPβCD has also shown great potential for the treatments of

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other cholesterol-related diseases such as atherosclerosis [10] and multiple sclerosis [11]. Despite

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such promising results, HPβCD possesses notable shortcomings that pose a therapeutic challenge for clinical use: HPβCD exhibits poor pharmacokinetics. HPβCD with a molecular weight less than 1500 Da and high hydrophilicity is rapidly eliminated from blood mainly via glomerular filtration in the kidney and thus exhibits a blood half-life shorter than 1 hour in mice, rats, and humans [12, 13]. The unfavorable pharmacokinetics requires high-doses of HPβCD to exert therapeutic effects and expose subjects to dose-limiting side effects such as hearing loss [14, 15]. Water-soluble low-molecular-weight drugs can be formulated in various ways to improve their pharmacokinetics and therapeutic efficacy. Drug formulations, particularly in the size range

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Journal Pre-proof of 10-100 nm, can avoid rapid glomerular filtration [16], and efficiently extravasate dysfunctional endothelium of inflamed tissues such as cancer [17] and atherosclerosis [18, 19], and remain long in the extracellular environments. With known therapeutic efficacy of CD for the treatment of atherosclerosis and its therapeutic challenges due to its poor pharmacokinetics, we hypothesized that the polymeric formulation of CD in the size range of 10-100 nm can improve CD-based therapy. Although CD has been engineered into various nano-sized

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formulations [20-23], their main role was to incorporate a drug of interest for drug delivery and

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they were not regarded as therapeutic agents themselves. Here, we show that the cyclodextrin

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polymer (CDP) prepared by covalent crosslinking of CD molecules improves pharmacokinetics

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and plaque targeting, and reduces plasma membrane disruption compared to CD, thus enabling

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effective and safe CD-based treatment of atherosclerosis (Scheme 1).

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Scheme 1. Cyclodextrin polymer for effective and safe treatment of atherosclerosis.

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2. Materials and methods Study design: The main objective of our study was to investigate whether cyclodextrin polymer exhibits enhanced pharmacokinetics and improved atherosclerosis therapy compared to free cyclodextrin. For this, we studied pharmacokinetics, biodistribution, plaque targeting, therapeutic efficacy, and ototoxicity in mice. Anti-atherogenic properties and cytotoxicity were studied in vitro. Specific details of the individual experiments are described below.

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Preparation and characterization of cyclodextrin and cyclodextrin polymer: All

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cyclodextrins were obtained from Cyclolab (Budapest, Hungary): 2-Hydroxypropyl-beta-

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cyclodextrin (HPβCD; degree of substitution, DS~4.5; Catalog Number: 128446-35-5), methyl-

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beta-cyclodextrin (MβCD; DS~12; Catalog Number: 128446-36-6), carboxymethyl-betacyclodextrin (CMβCD; DS~3.5; Catalog Number: CY-2006.0), carboxymethyl-beta-cyclodextrin

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polymer (CMβCDP, Catalog Number: CY-2010), beta-cyclodextrin polymer (βCDP; Catalong

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Number: CY-2009), and Rhodamine labeled βCD (Rho-βCD; CY-RL-2009). As indicated by the manufacturer, CMβCDP and βCDP are formulated by random crosslinking of the cyclodextrins

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with epichlorohydrin and have the approximate molecular weight of 150 kDa with estimated

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cyclodextrin content of 50-70 %. All cyclodextrins had purity higher than 95 %. For the conjugation of Cyanine 7 to CMβCD and CMβCDP (Cy7-CD and Cy7-CDP), carboxylates of CMβCD and CMβCDP were activated with equimolar N-ethyl-N -[3-dimethyl- aminopropyl] carbodiimide (EDC, Sigma Aldrich) and a 2-fold molar excess of N-hydroxy succinimide (NHS; Sigma Aldrich) for 30 minutes at room temperature. Then, Sulfo-Cyanine 7 amine (Lumiprobe; Catalog Number: 253C0) was added (Cy7:CD = 0.5:1 molar ratio), incubated for 2 hours at room temperature, and the unconjugated Cy7 was removed using size exclusion chromatography. The size of cyclodextrin polymers and cyclodextrins was measured using dynamic light scattering

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Journal Pre-proof (Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK). For Transmission electron microscopy, 5 µl of samples were deposited on Formvar carbon-coated EM grids (Ted Pella, Inc., Redding, CA) until they were completely dried. Transmission electron microscopic images were obtained using a JEM-2100F HRTEM operating at 200 kV (JEOL, Tokyo, Japan). Mice: For an atherosclerosis model, ten-week-old male ApoE–/– mice were purchased from Jackson Laboratory (Bar Harbor, ME). Five-to-seven-week old male wild-type C57/BL6

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mice were purchased from Koatech (Gyeonggi-do, South Korea). For euthanasia of mice, they

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were culled using carbon dioxide (CO2) inhalation. All animal procedures were approved by the

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Animal Care and Use committees at KAIST.

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Pharmacokinetics and biodistribution: For measurement of blood half-life, aliquots of 200 µl of Cy7-CD and Cy7-CDP (50 mg/kg CD) were injected into wild-type C57/BL6 mice via

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either intravenous or subcutaneous injection. Retro-orbital bleeding to collect blood was

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performed at 0.03, 1, 2, 6, 12, 24, 48, 72, and 96 hours post-injection. The fluorescence of blood samples was measured to obtain plasma concentration. Pharmacokinetics parameters were

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obtained by fitting the data using the non-compartmental model. For biodistribution study, the

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wild-type C57/BL6 mice were sacrificed at 24 hours and 72 hours post-injection for Cy7-CD and Cy7-CDP, respectively. Organs including liver, spleen, heart, lung, and kidney were harvested and the fluorescence was measured using an NIR fluorescent imaging system (LI-COR, Lincoln, NE). Plaque targeting: For plaque targeting study, ten-week-old male ApoE–/– mice were fed with Paigen’s high-fat diet (HFD, Catalog Number D12336; Research Diets, New Brunswick, NJ) containing 1.25% cholesterol, 16% fat, and 0.5% cholic acid for 12 weeks and then subcutaneously injected with Cy7-CD and Cy7-CDP (100 mg/kg CD) and sacrificed at 24 hours

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Journal Pre-proof and 72 hours post-injection, respectively. Organs including aorta, liver, spleen, heart, lung, kidney, and brain were harvested and the fluorescence was measured using an NIR fluorescent imaging system (LI-COR, Lincoln, NE). Preparation of cholesterol crystal (CC): Ethanol containing cholesterol (2 mg/ml; Avanti Polar Lipids) was prepared. Crystallization was induced by the addition of distilled water (30 % v/v) to the solution. The solution was dried completely under a vacuum, resuspended in PBS, and

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sonicated for size control. For preparation of fluorescent CC, we used 25-[N-[(7-nitro-2-1,3-

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benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (NBD cholesterol; Avanti Polar Lipids),

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which was dissolved with cholesterol in ethanol at a molar ratio of 1:10, to prepare NBD-labelled

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CC.

Extracellular cholesterol crystal binding and dissolution: For binding assay, NBD-

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labeled CC (50 μM in PBS) were incubated with Cy7-CD and Cy7-CDP (50 μM CD) for 5

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minutes at room temperature. Then, 100 μl of the solution was dropped onto a microscope slide, covered with a cover slip, and imaged under a 60x objective lens on a confocal microscope

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(Nikon, Tokyo, Japan). For dissolution assay, NBD-labeled CC (50 μM in PBS) were incubated

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with cyclodextrin polymers (βCDP and CMβCD) and free cyclodextrins (MβCD, HPβCD, and CMβCD) at different cyclodextrin concentrations (0, 0.1, 0.5, 1, 5, and 10 mM) overnight. The mixtures were then filtrated through 0.22-μm syringe filters and the fluorescence of NBDcholesterol in the filtrates was measured. Cell lines: Raw264.7 murine macrophage cells (ATCC TIB-71) were maintained in Dulbecco’s Modified Eagle’s Medium (Hyclone, South Logan, UT). The media were supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin

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Journal Pre-proof (Hyclone). All cells were cultured in tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 95% air and 5% carbon dioxide. Hemolysis assay: 200 μl of whole blood from wild-type C57/B6 mice were mixed with 200μl of CD solutions (1, 5, 7.5, 10, 15, 30, and 50 mM) for 1 hour at 37ºC in a shaking incubator. The mixture was centrifuged at 3,000g for 5 minutes and then the supernatant was used to measure observance at 540 nm.

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In vitro cell studies: To observe cellular localization of CC, CDP, and CD within Raw

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264.7 cells, the cells were seeded in 6-well plates for 24 hours at a density of 5×104 cells per

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well. For CC-laden Raw 264.7 cells, they were treated with NBD-CC (50 μM) for 24 hours.

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Then, the cells were treated with Cy7-CD and Cy7-CDP for 6 hours and observed under a 60x objective lens on a Nikon confocal microscope (Nikon, Tokyo, Japan). For an intracellular CC

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removal assay, Raw 264.7 cells were seeded in 48-well plates for 24 hours at a density of 2×104

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cells per well. The cells were then treated with NBD-CC (50 μM) for 24 hours. Cells were washed thoroughly and then treated with cyclodextrin polymers (βCDP and CMβCD) and free

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cyclodextrins (MβCD, HPβCD, and CMβCD) for 24 hours. The fluorescence of NBD-

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cholesterol inside cells was measured to quantify the effect of various cyclodextrins on the cholesterol removal. The cell culture media was aspirated and then 200 μl of DMSO was added to lyse cells and dissolve intracellular NBD-cholesterol, which was quantified by measuring its fluorescence (excitation: 480 nm/emission: 530 nm). For size exclusion chromatography, CCladen Raw 264.7 cells were treated with 5 mM of βCDP conjugated with Rhodamine via a thioureido group (Rho-βCD). At 6 hours post-incubation, the cells were washed thoroughly and incubated overnight. The supernatant was collected and 200 μl of the solution was used for size exclusion chromatography using Sephadex G-50 medium (GE healthcare) and the fractions were

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Journal Pre-proof examined by measuring the fluorescence of Rhodamine (excitation: 530 nm/emission: 570) and NBD (excitation; 480 nm/emission: 530 nm). Atherosclerosis therapy in vivo: ApoE–/– mice were fed the Paigen’s HFD for 12 weeks and the mice were injected subcutaneously with either PBS, HPβCD, or βCDP (1 g/kg CD) twice a week for an additional 4 weeks. During treatment, the diet was switched to normal chow diet (NCD). The mice were sacrificed at week 16 for analysis.

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Histological analysis of plaque: Hearts with ascending aortas were collected, fixed with

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3.7% formaldehyde, embedded in Tissue-Tek optimal cutting temperature (OCT) compound

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(Sakura Finetek), frozen in liquid nitrogen, cut into sections 10 μm thick, and then stored at –

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80°C until use. Frozen sections were serially collected when the three leaflets of aortic valve were recognized on unstained sections. For Oil-Red-O staining, the sections were washed with

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distilled water and rinsed with 60 % isopropanol (Sigma Aldrich). The sections were then stained

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with 0.3 % Oil-Red-O solution for 15 minutes. Subsequently, they were rinsed with isopropanol (60 %) and counterstained with hematoxylin (Sigma Aldrich) for 2 minutes. After final washing

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with distilled water, the sections were mounted in aqueous mounting medium. The aortic roots

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were imaged using 4x objective lens on light microscope (Nikon). For collagen staining, picrosirius red stain kit (Catalog Number ab150681; Abcam) was used in accordance with the instructions provide by the manufacturer and the samples were imaged under a 20x objective lens on a light microscope (Nikon). Photographs of the stained specimens were digitized for data analysis using Image J. Localization of βCDP within plaques: For localization study, Rho-βCDP was injected subcutaneously and mice were sacrificed at 72 hours post-injection. The aortic root sections were fixed with formaldehyde, embedded in Tissue-Tek optimal cutting temperature (OCT)

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Journal Pre-proof compound (Sakura Finetek, Alphen aan den Rijn, The Netherlands), cut into sections 10 μm thick. For filipin staining, the sections were washed with PBS and incubated with 50 μg/ml filipin (Sigma-Aldrich) for 2 hours at room temperature. They were then rinsed with PBS and mounted using aqueous mounting medium. For CD68 staining, the sections were first treated with blocking solution (1% BSA, 5% goat serum, and 0.02% Tween) for 60 minutes at room temperature. They were incubated with primary rat anti-mouse CD68 antibody (2 μg/ml in 10%

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normal goat serum, Catalog Number 137001; Biolegend, San Diego, CA) overnight at 4°C, and

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then incubated with secondary goat anti-rat IgG (4 μg/ml in 10% normal goat serum, Catalog

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Number 405416; Biolegend) for 60 minutes at room temperature. The sections were washed with

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PBS and counterstained with Hoechst 33342 (5 g/ml in PBS; Thermo Fisher Scientific) for 2 minutes at room temperature. After a final wash with PBS, the sections were mounted using

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aqueous mounting medium.

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Cholesterol efflux from plaque tissue: ApoE–/– mice were fed the Paigen’s HFD for 12 weeks and the mice were injected subcutaneously with either PBS, HPβCD, or βCDP (1 g/kg

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CD). Mouse atherosclerotic plaques in the brachiocephalic artery region were obtained from

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mice at 24 hours for PBS and HPβCD groups and 72 hours for βCDP group. The samples were cut into similar sizes and were cultured ex vivo in 200 μl Dulbecco’s modified Eagle’s medium (DMEM; Gibco) for 24 h. For the extraction of remaining cholesterol from the plaque samples, they were dried and then put into 100 μl of chlorofrom/methanol 2:1 (v:v) solvent for 2 hours at 37 ºC with gentle stirring. The plaque and culture media were analyzed for free cholesterol content using Amplex™ Red Cholesterol Assay Kit (Thermo Fisher; Catalog Number: A12216). Cholesterol efflux from plaque tissue into supernatants was displayed as % of total cholesterol per sample.

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Journal Pre-proof Ototoxicity: For the observation of ototoxicity, wild-type C57/B6 were injected subcutaneously with either PBS, HPβCD, or βCDP (8 g/kg CD) and sacrificed at 7 days postinjection for the extraction of cochleae. After fixation and decalcification, cochleae were dissected following the procedures described by Scott et al [24]. F-actin was staining using phalloidin- iFluor 488 (Catalog Number ab176753; Abcam) and nucleus was stained using Hoechst 33342 (Thermo Fisher Scientific). The samples were imaged under a 20x objective lens

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on a confocal microscope (Nikon).

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Blood tests and cholesterol analysis: Whole blood was collected using heparinized

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syringes right after the mice were sacrificed. Plasma lipids [total cholesterol, triglycerides, high-

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density lipoproteins (HDL), and LDL] were analyzed using a CX7 biochemical analyzer (Beckman Coulter, Brea, CA). Blood concentrations of alanine transaminase and aspartate

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transaminase were analyzed using an AU480 chemistry analyzer (Beckman Coulter).

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Data analysis and statistics: All data are presented as mean ± SEM. An unpaired, twotailed Student’s t-test was used for statistical analysis of two groups. Analysis of Variance

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(ANOVA) followed by Tukey's multiple comparisons test was used for comparison of three or

(Version 7.0).

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more groups. Data analysis and figures were prepared using the GraphPad Prism software

3. Results 3.1 Pharmacokinetics and biodistribution Firstly, we set out to investigate whether CDP exhibits improved pharmacokinetics and biodistribution compare to monomeric CD using a near-infrared (NIR) fluorescence imaging

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Journal Pre-proof system. Cy7 fluorescent dyes were conjugated to CD and CDP (Cy7-CD and Cy7-CDP) for NIR fluorescence imaging. For conjugation, we used CD and CDP that contain carboxymethyl groups, carboxymethyl-beta-cyclodextrin (CMβCD) and carboxymethyl-beta-cyclodextrin poylmer (CMβCDP), which were activated with EDC and then reacted with amine groups of Cy 7 (Supplementary Fig. 1). At a CD concentration of 5 mM, the hydrodynamic size of Cy7-CDP was measured to be 10.1 ± 0.6 nm in the dynamic light scattering (DLS) measurements while the

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size of Cy7-CD was undetectable (Figs. 1A and 1B). Transmission electron microscopy revealed

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that Cy7-CDP appeared as globular particles with a size similar to that measured in the DLS ( Fig.

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1C). When injected intravenously into mice, Cy7-CD was eliminated rapidly from the

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bloodstream with a half-life of 0.46 hours while Cy7-CDP extended the half-life 58-fold to 26.8 hours (Fig. 1E). Since we observed that they were eliminated from the bloodstream at different

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times after injection, biodistributions of Cy7-CD and Cy7βCDP were observed at 24 hour and 72

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hour postinjection, respectively. NIR fluorescence images of major organs revealed that Cy7CDP was cleared out mainly in the liver, which is the main organ of mononuclear phagocyte

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system [25, 26], while most of Cy7-CD was cleared in kidneys (Figs. 1E and 1F). These results

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show that the improved pharmacokinetics and biodistribution of CDP is mainly attributed to its increase in size by evading rapid renal clearance that particles smaller than ~ 8 nm should undergo after intravenous administration[16]. When subcutaneously injected, Cy7-CD in blood peaked at 2 hours and dropped rapidly while Cy7-CDP peaked at approximately 24 hours and decreased slowly over 100 hours (Fig. 1G). The peak of Cy7-CD was observed much faster than that of Cy7-CDP because the low molecular weight of monomeric CD allows its direct entrance into blood vessels after subcutaneous injection while CDP exceeds the size limit for the direct blood entrance (~ 20 kDa) and has to travel via peripheral lymph for systemic circulation [27].

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Journal Pre-proof The bioavailabilities of Cy7-CD and Cy7-CDP after subcutaneous injection were measured to be 97.2 ± 3.5% and 93 ± 1.7%, respectively. Due to their high bioavailabilities, both forms of CD exhibited the biodistribution similar to what was observed after intravenous injection (Figs. 1H and 1I). Therefore, we performed subcutaneous injection in the following experiments. Collectively, these results suggest that the polymeric formulation of CD can significantly

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improve pharmacokinetics and biodistribution profiles of CD.

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Figure 1. Pharmacokinetics and biodistribution of Cy7-CDP and Cy7-CD. (A) Hydrodynamic sizes of Cy7-CD and Cy7-CDP as measured by dynamic light scattering (n=4). (B) Distribution of hydrodynamic sizes of Cy7-CDP as measured by dynamic light scattering. (C) Representative transmission electron microscopic image of Cy7-CDP. The scale bar indicates 100 nm. (D) Blood half-life of Cy7-CD and Cy7-CDP after intravenous injection (n=3 per group). (E) Representative ex vivo fluorescence images of the dissected organs. Mice were intravenously injected with Cy7-CD and Cy7-CDP and sacrificed at 24 hours and 72 hours postinjection, respectively (n=3 per group). The scale bar indicates 1 cm. (F) Quantification of fluorescence in the dissected organs in (E) as measured by NIR fluorescence imaging system. (G) Blood half-life of Cy7-CD and Cy7-CDP after subcutaneous injection (n=3 per group). (H) Representative ex vivo fluorescence images of the dissected organs. Mice were subcutaneously injected with Cy7-CD and Cy7-CDP and sacrificed at 24 hours and 72 hours post-injection, respectively (n=3 per group). The scale bar indicates 1 cm. (I) Quantification of fluorescence in the dissected organs in (H) as measured by NIR fluorescence imaging system. Data are mean ± s.e.m.

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Journal Pre-proof 3.2 Plaque accumulation Having observed the improved pharmacokinetics and biodistribution of CDP compared to CD, we investigated whether CDP can effectively accumulate within atherosclerotic plaques. Ten-week-old ApoE-/- mice were fed with a high fat diet (HFD) for 12 weeks to induce atherosclerosis and then injected with either Cy7-CD or Cy7-CDP subcutaneously on the right flank. NIR fluorescence images of dissected aortic tree revealed that plaque accumulation of

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Cy7-CDP was 14.2-fold higher than that of Cy7-CD (Figs. 2A and 2B). The biodistributions of

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Cy7-CD and Cy7-CDP in the HFD-fed ApoE-/- mice were similar to those observed in the wild-

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type mice (Fig. 2C). These results suggest that the polymeric formulation of CD can

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preferentially accumulate within atherosclerotic plaques through dysfunctional endothelium and neovessels of the vasa vasorum [19], presumably due to its long blood half-life and relatively

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large size.

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Figure 2. Preferential accumulation of CDP in atherosclerotic plaques. (A) Representative ex vivo bright-field and fluorescence images of the dissected aorta. ApoE-/- mice were fed highfat-diet for 12 weeks and then subcutaneously injected with Cy7-CD and Cy7-CDP and sacrificed at 24 hours and 72 hours post-injection, respectively (n=3 per group). The scale bar indicates 1 mm. (B) Quantification of fluorescence in the dissected aorta in (A) as measured by NIR fluorescence imaging system. (C) Quantification of fluorescence in major organs as measured by NIR fluorescence imaging system. Data are mean ± s.e.m.

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Journal Pre-proof 3.3 Cholesterol crystal dissolution Having observed that CDP exhibits superior pharmacokinetics and plaque targeting efficacy compared to CD, we assessed whether CDP retain cholesterol-dissolving function comparable to CD in vitro. Firstly, to study whether CDP shows affinity to cholesterol, fluorescently-labeled CC were incubated with Cy7-CD or Cy7-CDP and then imaged with NIR fluorescence confocal microscopy. Fluorescence microscopic images revealed that Cy7-CDP

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bound effectively to CC, which was similar to what was observed with Cy7-CD (Fig. 3A). Then,

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we performed a CC dissolution assay using various types of CDP including CMβCDP and βCDP,

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and CD including methyl-beta-cyclodextrin (MβCD), CMβCD, and HPβCD because the type

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and number of functional groups of CD are directly associated with their ability to interact with and dissolve cholesterol [28] . We incubated fluorescently-labeled CC with each sample at

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different concentrations overnight and measured the fluorescence of dissolved cholesterol. βCDP

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exhibited the most effective cholesterol dissolution, followed by MβCD, CMβCDP, HPβCD, and CMβCD (Fig. 3B). Among CD, the cholesterol-solubilizing efficiency was in the order MβCD >

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HPβCD > CMβCD, which was consistent with a previous study [28]. Overall, these results

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indicate that the polymerization of CD into CDP with a diameter of ~ 10 nm does not hamper the ability of CDP to bind with and dissolve CC in solution.

3.4 Cytotoxicity and hemolytic activity It has been reported that there is correlation between cholesterol-solubilizing efficiency, cytotoxicity, and hemolytic activity of various types of CD: the better a CD solubilizes cholesterol, the higher cytotoxicity and hemolytic activity was observed [28-31]. Firstly, we examined the cytotoxic effects of various types of CDP and CD in vitro. Raw 264.7

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Journal Pre-proof macrophages were treated with each sample at different concentrations for 24 hours and the cell viability was examined by using a MTT assay. Interestingly, both types of CDP showed almost no cytotoxicity even at very high concentrations while all types of CD showed dose-dependent cytotoxicity with MβCD being the most cytotoxic, followed by HPβCD and CMβCD (Fig. 3C). Next, we performed a hemolysis assay and found that both types of CDP did not induce hemolysis while all types of CD exhibited dose-dependent hemolytic activities with MβCD

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being the most cytotoxic, followed by HPβCD and CMβCD (Fig. 3D). These results demonstrate

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that there is high correlation between cholesterol-solubilizing efficacy, hemolytic activity and

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cytotoxicity of various types of CD, as previously reported [28]. However, both types of CDP

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showed no such correlation. These results suggest that while both types of CDP can solubilize non-membrane bound cholesterol such as CC in solution, they cannot efficiently extract plasma

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membrane-bound cholesterol, presumably due to the polymeric structure that limits the close

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interaction of the cholesterol-binding cavity of CD with the membrane-embedded cholesterol [32, 33]. The differential affinity of CDP to cholesterol implies that CDP may have less detrimental

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effects on hair cells of the cochlea [34], which can greatly reduce the chance of hearing loss

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accompanied by CD therapy.

3.5 Intracellular cholesterol removal from macrophages Since CD can enter macrophages, interact with intracellular cholesterol, and induce cholesterol removal from macrophages [10, 35], we assessed whether CDP exhibits similar functions in vitro in a range of CD concentrations that does not affect cell viability. Through NIR fluorescence confocal microscopy, we observed that both Cy7-CD and Cy7-CDP were effectively internalized by macrophages and colocalized with intracellular CC (Fig. 3E, 3F and

20

Journal Pre-proof Supplementary Fig. 2). For the cholesterol removal assay, raw 264.7 macrophages were preloaded with fluorescently- labeled CC and then treated with various types of CDP and CD at different concentrations. The fluorescence of CC-laden macrophages was measured prior to and 24 hours after treatment to quantify the cholesterol removal. At a CD concentration of 5 mM, βCDP and MβCD exhibited comparable intracellular CC removal, decreasing the amount of intracellular cholesterol by approximately 50 % compared to the amount prior to treatment.

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CMβCDP and HPβCD were moderately effective above the concentration of 5 mM while

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CMβCD showed no significant effect over the entire concentration range. Among CD, the

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cholesterol removal efficiency was in the order MβCD > HPβCD > CMβCD, which was consistent with a previous study [35]. While βCDP and MβCD were the most potent agents for

re

intracellular CC removal, βCDP showed superior cholesterol removal at a CD concentration of

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10 mM where MβCD exhibited significant cytotoxicity (Fig. 3G). Furthermore, through size

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exclusion chromatography, we found that βCDP are exocytosed without significant degradation by forming complexes with cholesterol (Supplementary Fig. 3). Overall, these results suggest

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that βCDP can remove intracellular cholesterol from macrophages more effectively without

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cytotoxicity compared to various types of CD tested in the current study.

21

Journal Pre-proof

DIC

Cy7-βCD

Cholesterol ( % of total)

B

A Merged

CHOL

Cy7-βCDP

0 0.1 1 10 100 CD concentration (mM)

50 0

Merged

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Cy7-βCDP

DIC

of G

Intracellular cholesterol (%)

CHOL

0 0.1 1 10 CD concentration (mM)

Cy7-βCD

Cy7-βCDP

Merged

1 10 CD concentration (mM)

lP

Cy7-βCD

CMβCDP bCDP MbCD HPbCD CMβCD

ur

DIC

100

na

F

20

-p

50

CMβCDP bCDP MbCD HPbCD CMβCD

40

re

100

60

ro

150 Hemoylsis (%)

Viability (%)

150

80

E

D

C

CMβCDP bCDP MbCD HPbCD CMβCD

100

100

50

0 0.1

CMβCDP bCDP MbCD HPbCD CMβCD 1 10 CD concentration (mM)

Figure 3. Superior anti-atherogenic properties of CDP compared to CD in vitro. (A) Representative confocal microscopic images of Cy7-CD and Cy7-CDP binding to cholesterol crystals (CC). The scale bar indicates 50 μm. (B) Cholesterol dissolution of various CDP and CD (n=3). (C) Dose-dependent effects of various CDP and CD on cell viability (n=5). (D) Dosedependent effects of various CDP and CD on hemolysis (n=3). (E) Representative confocal microscopic images of macrophages treated with Cy7-CD and Cy7-CDP. The scale bar indicates 20 μm. (F) Representative confocal microscopic images CC-laden macrophages taking up Cy7CD and Cy7-CDP. The scale bar indicates 20 μm. (G) Dose-dependent effects of various CDP and CD on intracellular cholesterol removal in CC-laden macrophages (n=5). Data are mean ± s.e.m.

22

Journal Pre-proof 3.6 Atherosclerosis therapy Having observed the superior anti-atherogenic properties in vitro, we assessed whether βCDP can lead to effective atherosclerosis therapy in vivo. HPβCD was chosen as a representative CD for atherosclerosis therapy because it is a FDA-approved excipient used to solubilize various lipophilic drugs for therapeutic delivery in humans [36], and shown to be effective for the treatment of atherosclerosis [10]. Although MβCD was more effective in

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dissolving cholesterol in vitro than HPβCD, we excluded its use for in vivo atherosclerosis

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therapy because of its known side effects after parenteral administration [37, 38]. The

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hydrodynamic size of βCDP was measured to be 10.6 ± 0.7 nm in the DLS measurements while

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the size of HPβCD was undetectable (Supplementary Fig. 4). Ten-week-old ApoE-/- mice were fed with a HFD for 12 weeks to induce atherosclerosis and then treated twice a week for 4 weeks

lP

at a dose of 1 g CD/kg (Fig. 4A). En face aorta images and quantification of lesion areas after

na

Oil-Red-O staining revealed that βCDP exhibited significantly reduced atherosclerotic lesions in the aortic arch and thoracic aorta than HPβCD (Figs. 4B-D). In the histopathological analysis,

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βCDP also exhibited significantly reduced plaques in the aortic root than HPβCD ( Fig. 4E and

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4F). Considering that βCDP effectively colocalized with macrophages and cholesterol within plaques, we assumed that βCDP exerted therapeutic effect via removal of intracellular and extracellular cholesterol within plaques (Supplementary Fig. 5). Ex vivo culture of atherosclerotic plaques dissected from mice previously injected once with either HPβCD for 24 hours or βCDP for 72 hours at a dose of 1 g CD/kg showed that βCDP induced superior cholesterol efflux from plaque tissue (Supplementary Fig. 6). While HPβCD showed moderate cholesterol dissolution and cholesterol removal from macrophages in vitro, its cholesterol efflux from plaque tissue was hardly appreciated in vivo possibly due to its poor pharmacokinetics and

23

Journal Pre-proof plaque targeting. Importantly, the plaque stability, which was examined by the collagen content, was not affected by βCDP treatment (Fig. 4G and 4H), indicating that βCDP exerted antiatherogenic effects without increasing the vulnerability of the plaques. In addition, βCDP treatment did not affect plasma cholesterol and liver-associated enzyme levels (Fig. 4I and Suppleme ntary Fig. 7) and body weight (Fig. 4J). Taken together, these results suggest that βCDP can exert greater therapeutic effects for the treatment of atherosclerosis without significant

Jo

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na

lP

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ro

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systemic toxicity than HPβCD.

24

Journal Pre-proof A

Treatments HFD

0 Weeks

NCD

12

16 Sacrifice

C Control

Lesion area (%)

B

80 60

**

40 20

HPβCD

C on tr HP o l βC D βC DP

0

D

of

Lesion area (%)

l

D P βC βCD HP

100 NS

60 40 20 0 C

Jo

r HP o l βC D βC DP NS

600 400 200

J

0 C on tr HP o l βC D βC DP

10

80

0 C

20

C

10

Plasma cholesterol (mg/dL)

30

on tr H ol Pβ C D βC D P

H

40

tro on

20

I

*** **

0

*

30

Body weight (g)

βCDP

Collagen content (%)

HPβCD

50

40

on t

ro re lP na

Control

F

Plaque area (x104 mm2)

βCDP

HPβCD

ur

G

Control

-p

βCDP

E

50

50

NS

40 30 20 10 0 C

on

tro

l HP

D P βC βCD

Figure 4. Improved anti-atherosclerotic efficacy of βCDP compared to HPβCD in mice. (A) Schematic of experimental plan for atherosclerosis therapy. ApoE–/– mice were fed with a HFD for 12 weeks and then normal chow-diet (NCD) for 4 weeks. The mice were injected twice a week intravenously with either saline, HPβCD or βCDP at a dose of 1 g/kg cyclodextrin from week 12 post-HFD. At week 16, the mice were sacrificed for analysis (n=6 per group). (B) Representative en face aorta images after Oil-Red-O staining. The scale bar indicates 2 mm. (C) Quantification of the lesion area in the aortic arch in (B). (D) Quantification of the lesion area in the thoracic aorta in (B). (E) Representative histological images of the aortic root sections after Oil-Red-O staining. The scale bar indicates 500 μm. (F) Quantification of the plaque area in (E). (G) Representative histological images of the aortic root sections after picrosirius-red staining. The scale bar indicates 500 μm. (H) Quantification of the collagen content in (G). (I) Plasma cholesterol concentration after treatments. (J) Body weight after treatments. Data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey’s multiple comparisons test.

25

Journal Pre-proof 3.7 Ototoxicity CD therapies have accompanied the chance of hearing loss due to the detrimental effects of CD on outer hair cells (OHC) of the cochlea [34] although they have shown promising results for the treatments of various cholesterol-related diseases. This ototoxicity is caused by the disruption of plasma membranes via cholesterol extraction [34], and has been found not only in mice[15] but also in humans [9, 14]. Having observing that βCDP did not induce the disruption

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of plasma membranes in vitro, we investigated whether βCDP can reduce ototoxicity compared

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to HPβCD at a high-dose. Mice were given a single injection of PBS, HPβCD or βCDP at a dose

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of 8 g CD/kg that is known to cause ototoxicity in mice [15], and sacrificed for analysis one week after injection. While the mice treated with HPβCD exhibited significant loss of OHC in

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the mid-to-basal regions of the cochlea, the mice treated with βCDP showed no significant OHC

lP

loss (Fig. 5A and 5B). Furthermore, high-dose administrations of HPβCD and βCDP did not

na

affect plasma cholesterol and liver-associated enzyme levels (Supplementary Fig. 8). These findings have an important implication for clinical translation of CDP for the treatment of

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limiting side effects.

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atherosclerosis because significant therapeutic effects can be achieved without significant dose-

26

Journal Pre-proof

Control

B

βCDP

HPβCD

OHC(%)

A

Hoechst

150 100

***

50

Phalloidin

HP

C

o

β

l ro

nt

Merged

CD CDP β

0

Jo

ur

na

lP

re

-p

ro

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Figure 5. No observed ototoxicity of βCDP compared to HPβCD in mice. (A) Representative confocal microscopic images after high-dose treatment. (B) Quantification of outer hair cells in (A). Wild-type C57/B6 mice were injected subcutaneously with either saline, HPβCD, or βCDP (8 g/kg CD) and sacrificed at 7 days post-injection. (n=6 per group). The scale bar indicates 20μm. Data are mean ± s.e.m. ***P < 0.001 by one-way ANOVA and Tukey’s multiple comparisons test.

27

Journal Pre-proof

4. Discussion In this study, we demonstrate that CDP has great potential for the treatment of atherosclerosis by overcoming therapeutic challenges posed by CD therapy. In the previous study, CD has shown excellent efficacy in the treatment of atherosclerotic plaques in mice [10]. However, we did not observe significant anti-atherogenic effects of CD in our experiments

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because we injected half the dose used in the previous study and used the more advanced

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atherosclerosis model. Compared to CD, CDP exerted superior anti-atherogenic effects on more

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advanced atherosclerosis at the same dose and negligible plasma membrane disruption,

re

suggesting that CDP can provide a more effective and safe therapeutic option for the treatment of

lP

atherosclerosis.

CDP was prepared by covalent crosslinking of CD molecules. However, the therapeutic

na

function of CD to dissolve CC was maintained without requiring degradation into individual CD. We assume that CDP could retain its ability to interact with and dissolve CC because the

ur

hydrophobic cavity of CD required for interaction with cholesterol was not affected during the

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polymerization process [39]. Although the polymeric structure and dimension of CDP used in the current study did not affect its ability to dissolve CC in solution, they had a notable impact on extraction of cholesterol from plasma membrane [40, 41] considering that CDP did not induce cytotoxicity and hemolysis in vitro, and ototoxicity in vivo. It can be reasoned that the polymeric structure and dimension limits the accessibility of CD to the membrane-bound cholesterol [32, 33]. Therefore, we suggest that CDP that can avoid plasma membrane disruption can provide a safer therapeutic option than CD. Further optimizations in the types of substituents, degree of substitution, and size of CDP will help enhance the therapeutic outcome. Furthermore, while we

28

Journal Pre-proof assessed the therapeutic efficacy of CDP for the treatment of atherosclerosis, we believe that our findings that CDP can effectively dissolve cholesterol away without significant ototoxicity will have critical implications for the treatment of other cholesterol-related diseases such as Niemann-Pick Type C. One limitation of our study is that in vivo anti-atherogenic mechanisms of CDP have to be further defined. While we attributed the superior therapeutic efficacy of CDP to their

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enhanced pharmacokinetics in vivo and ability to dissolve cholesterol in vitro, reducing plaque

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size without hampering the stability of plaques would accompany complex biological processes

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besides simple dissolution of extra- and intracellular cholesterol within plaques. Considering the

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critical importance of inflammatory status in atherosclerosis, we believe that CDP played significant roles in the resolution of inflammation via cholesterol dissolution. However, the

lP

effects of CDP on various anti-inflammatory pathways such as efferocytosis [42, 43], monocyte

na

recruitment [44, 45], and smooth muscle cell proliferation [46] should be further studied. Furthermore, considering that CD was shown to induce liver X receptor (LXR) reprogramming

ur

of macrophages and have LXR-dependent cholesterol efflux in vivo [10], studying whether CDP

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exhibits similar mechanisms would help elucidate the therapy mechanism. Lastly, while we showed that CDP is exocytosed from macrophages without degradation in vitro, its degradation, metabolism, and excretion in vivo requires further demonstration for its clinical translation. Since CDP mainly accumulated in the liver and did not cause liver toxicity even at a very high dose (8 g/kg), we can assume that the liver would play a critical role for the clearance of CDP. Also, while CD was shown to increase cholesterol excretion into the feces and urine [10], CDP could exhibit different excretion profile compared to CD because of its relatively large size.

29

Journal Pre-proof Our results demonstrate that the polymeric formulation of CD enhances therapeutic efficacy and reduces ototoxicity compared to CD and support the feasibility of CD-based therapy for the treatment of atherosclerosis. Considering that CD is already being investigated in clinical trials [9], we expect that CDP can be developed for effective removal of atherosclerotic plaques

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in human patients in combination with conventional therapy for systemic cholesterol control.

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Acknowledgements

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This work was supported by the Basic Science Research Program through the National Research

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Foundation (NRF- 2017R1E1A1A01074847) funded by the Ministry of Science and ICT,

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Conflict of Interest

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Republic of Korea.

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The authors declare no competing financial interests.

References [1] A. Grebe, E. Latz, Cholesterol crystals and inflammation, Curr. Rheumatol. Rep. 15 (2013) 313. [2] P. Duewell, H. Kono, K.J. Rayner, C.M. Sirois, G. Vladimer, F.G. Bauernfeind, G.S. Abela, L. Franchi, G. Nuñez, M. Schnurr, T. Espevik, E. Lien, K.A. Fitzgerald, K.L. Rock, K.J. Moore, S.D. Wright, V. Hornung, E. Latz, NLRP3 inflammasomes are required for

30

Journal Pre-proof atherogenesis and activated by cholesterol crystals, Nature 464 (2010) 1357-1361. [3] E.O. Samstad, N. Niyonzima, S. Nymo, M.H. Aune, L. Ryan, S.S. Bakke, K.T. Lappegard, O.L. Brekke, J.D. Lambris, J.K. Damas, E. Latz, T.E. Mollnes, T. Espevik, Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release, J. Immunol. 192 (2014) 2837-2845. [4] G.N. Levine, J.F. Keaney, Jr., J.A. Vita, Cholesterol reduction in cardiovascular disease.

of

Clinical benefits and possible mechanisms, N. Engl. J. Med. 332 (1995) 512-521.

ro

[5] S.J. Nicholls, C.M. Ballantyne, P.J. Barter, M.J. Chapman, R.M. Erbel, P. Libby, J.S.

-p

Raichlen, K. Uno, M. Borgman, K. Wolski, S.E. Nissen, Effect of two intensive statin regimens on progression of coronary disease, N. Engl. J. Med. 365 (2011) 2078-2087.

re

[6] N.J. Pagidipati, T.A. Gaziano, Estimating deaths from cardiovascular disease: a review of

lP

global methodologies of mortality measurement, Circulation 127 (2013) 749-756.

na

[7] R.O. Williams, 3rd, V. Mahaguna, M. Sriwongjanya, Characterization of an inclusion

(1998) 355-360.

ur

complex of cholesterol and hydroxypropyl-beta-cyclodextrin, Eur. J. Pharm. Biopharm. 46

Jo

[8] R.G. Strickley, Solubilizing excipients in oral and injectable formulations, Pharm. Res. 21 (2004) 201-230.

[9] D.S. Ory, E.A. Ottinger, N.Y. Farhat, K.A. King, X. Jiang, L. Weissfeld, E. Berry-Kravis, C.D. Davidson, S. Bianconi, L.A. Keener, R. Rao, A. Soldatos, R. Sidhu, K.A. Walters, X. Xu, A. Thurm, B. Solomon, W.J. Pavan, B.N. Machielse, M. Kao, S.A. Silber, J.C. McKew, C.C. Brewer, C.H. Vite, S.U. Walkley, C.P. Austin, F.D. Porter, Intrathecal 2-hydroxypropylbeta-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial, Lancet 390 (2017) 1758-1768.

31

Journal Pre-proof [10] S. Zimmer, A. Grebe, S.S. Bakke, N. Bode, B. Halvorsen, T. Ulas, M. Skjelland, D. De Nardo, L.I. Labzin, A. Kerksiek, C. Hempel, M.T. Heneka, V. Hawxhurst, M.L. Fitzgerald, J. Trebicka, I. Bjorkhem, J.A. Gustafsson, M. Westerterp, A.R. Tall, S.D. Wright, T. Espevik, J.L. Schultze, G. Nickenig, D. Lutjohann, E. Latz, Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming, Sci. Transl. Med. 8 (2016) 333ra350. [11] L. Cantuti-Castelvetri, D. Fitzner, M. Bosch-Queralt, M.T. Weil, M. Su, P. Sen, T.

of

Ruhwedel, M. Mitkovski, G. Trendelenburg, D. Lutjohann, W. Mobius, M. Simons,

ro

Defective cholesterol clearance limits remyelination in the aged central nervous system,

-p

Science 359 (2018) 684-688.

[12] Y. Tanaka, Y. Yamada, Y. Ishitsuka, M. Matsuo, K. Shiraishi, K. Wada, Y. Uchio, Y. Kondo,

re

T. Takeo, N. Nakagata, T. Higashi, K. Motoyama, H. Arima, S. Mochinaga, K. Higaki, K.

lP

Ohno, T. Irie, Efficacy of 2-hydroxypropyl-beta-cyclodextrin in Niemann-Pick disease type

Bull. 38 (2015) 844-851.

na

C model mice and its pharmacokinetic analysis in a patient with the disease, Biol. Pharm.

ur

[13] E.A. Thackaberry, S. Kopytek, P. Sherratt, K. Trouba, B. McIntyre, Comprehensive

Jo

investigation of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-beta-cyclodextrin for use in general toxicology studies, Toxicol. Sci. 117 (2010) 485-492.

[14] S. Cronin, A. Lin, K. Thompson, M. Hoenerhoff, R.K. Duncan, Hearing loss and otopathology following systemic and intracerebroventricular delivery of 2-hydroxypropylbeta-cyclodextrin, J. Assoc. Res. Otolaryngol. 16 (2015) 599-611. [15] M.A. Crumling, L. Liu, P.V. Thomas, J. Benson, A. Kanicki, L. Kabara, K. Halsey, D. Dolan, R.K. Duncan, Hearing loss and hair cell death in mice given the cholesterol-

32

Journal Pre-proof chelating agent hydroxypropyl-beta-cyclodextrin, PLoS One 7 (2012) e53280. [16] M.J. Ernsting, M. Murakami, A. Roy, S.D. Li, Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles, J. Control. Release 172 (2013) 782-794. [17] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407 (2000) 249257.

of

[18] E. Falk, Pathogenesis of atherosclerosis, J. Am. Coll. Cardiol. 47 (2006) C7-12.

ro

[19] M.E. Lobatto, V. Fuster, Z.A. Fayad, W.J.M. Mulder, Perspectives and opportunities for

-p

nanomedicine in the management of atherosclerosis, Nat. Rev. Drug Discov. 10 (2011) 845852.

re

[20] S. Feng, Y. Hu, S. Peng, S. Han, H. Tao, Q. Zhang, X. Xu, J. Zhang, H. Hu, Nanoparticles

lP

responsive to the inflammatory microenvironment for targeted treatment of arterial

na

restenosis, Biomaterials 105 (2016) 167-184. [21] Y. Wang, L. Li, W. Zhao, Y. Dou, H. An, H. Tao, X. Xu, Y. Jia, S. Lu, J. Zhang, H. Hu,

ur

Targeted therapy of atherosclerosis by a broad-spectrum reactive oxygen species scavenging

Jo

nanoparticle with intrinsic anti-inflammatory activity, ACS Nano 12 (2018) 8943-8960. [22] C.B. Rodell, S.P. Arlauckas, M.F. Cuccarese, C.S. Garris, R. Li, M.S. Ahmed, R.H. Kohler, M.J. Pittet, R. Weissleder, TLR7/8-agonist- loaded nanoparticles promote the polarization of tumour-associated macrophages to enhance cancer immunotherapy, Nat. Biomed. Eng. 2 (2018) 578-588. [23] H. Shelley, R.J. Babu, Role of cyclodextrins in Nanoparticle-based drug delivery systems, J. Pharm. Sci. 107 (2018) 1741-1753. [24] S.C. Montgomery, B.C. Cox, Whole mount dissection and immunofluorescence of the adult

33

Journal Pre-proof mouse cochlea, J. Vis. Exp. 1 (2016). [25] D.A. Hume, The mononuclear phagocyte system, Curr. Opin. Immunol. 18 (2006) 49-53. [26] S.B. Yong, Y. Song, H.J. Kim, Q. Ul Ain, Y.H. Kim, Mononuclear phagocytes as a target, not a barrier, for drug delivery, J. Control. Release 259 (2017) 53-61. [27] G.B. Jones, D.S. Collins, M.W. Harrison, N.R. Thyagarajapuram, J.M. Wright, Subcutaneous drug delivery: An evolving enterprise, Sci. Transl. Med. 9 (2017).

of

[28] T. Kiss, F. Fenyvesi, I. Bacskay, J. Varadi, E. Fenyvesi, R. Ivanyi, L. Szente, A. Tosaki, M.

ro

Vecsernyes, Evaluation of the cytotoxicity of beta-cyclodextrin derivatives: evidence for the

-p

role of cholesterol extraction, Eur. J. Pharm. Sci. 40 (2010) 376-380. [29] D. Castagne, M. Fillet, L. Delattre, B. Evrard, B. Nusgens, G. Piel, Study of the cholesterol

re

extraction capacity of beta-cyclodextrin and its derivatives, relationships with their effects

lP

on endothelial cell viability and on membrane models, J. Incl. Phenom. Macrocycl. Chem.

na

63 (2009) 225-231.

[30] Y. Ohtani, T. Irie, K. Uekama, K. Fukunaga, J. Pitha, Differential effects of alpha-, beta- and

ur

gamma-cyclodextrins on human erythrocytes, Eur. J. Biochem. 186 (1989) 17-22.

Jo

[31] T. Irie, M. Otagiri, M. Sunada, K. Uekama, Y. Ohtani, Y. Yamada, Y. Sugiyama, Cyclodextrin-induced hemolysis and shape changes of human-erythrocytes in vitro, J. Pharmacobiodyn. 5 (1982) 741-744. [32] C.A. Lopez, A.H. de Vries, S.J. Marrink, Computational microscopy of cyclodextrin mediated cholesterol extraction from lipid model membranes, Sci. Rep. 3 (2013) 2071. [33] C.A. Lopez, A.H. de Vries, S.J. Marrink, Molecular mechanism of cyclodextrin mediated cholesterol extraction, PLoS Comput. Biol. 7 (2011) e1002020. [34] S. Takahashi, K. Homma, Y.J. Zhou, S. Nishimura, C.W. Duan, J. Chen, A. Ahmad, M.A.

34

Journal Pre-proof Cheatham, J. Zheng, Susceptibility of outer hair cells to cholesterol chelator 2hydroxypropyl-beta-cyclodextrine is prestin-dependent, Sci. Rep. 6 (2016). [35] V.M. Atger, M.D. Moya, G.W. Stoudt, W.V. Rodrigueza, M.C. Phillips, G.H. Rothblat, Cyclodextrins as catalysts for the removal of cholestero l from macrophage foam cells, J. Clin. Invest. 99 (1997) 773-780.

bioavailability, J. Control. Release 123 (2007) 78-99.

of

[36] R.L. Carrier, L.A. Miller, I. Ahmed, The utility of cyclodextrins for enhancing oral

ro

[37] V.J. Stella, Q.R. He, Cyclodextrins, Toxicol. Pathol. 36 (2008) 30-42.

-p

[38] M.E. Bellringer, T.G. Smith, R. Read, C. Gopinath, P. Olivier, Beta-cyclodextrin - 52-week toxicity studies in the rat and dog, Food. Chem. Toxicol. 33 (1995) 367-376.

re

[39] J. Nishijo, S. Moriyama, S. Shiota, Interactions of cholesterol with cyclodextrins in aqueous

lP

solution, Chem. Pharm. Bull. 51 (2003) 1253-1257.

na

[40] M.P. Besenicar, A. Bavdek, A. Kladnik, P. Macek, G. Anderluh, Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin - A surface plasmon

ur

resonance approach, BBA-Biomembranes 1778 (2008) 175-184.

Jo

[41] R. Zidovetzki, I. Levitan, Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies, BBA-Biomembranes 1768 (2007) 1311-1324.

[42] Y. Kojima, I.L. Weissman, N.J. Leeper, The Role of Efferocytosis in Atherosclerosis, Circulation 135 (2017) 476-489. [43] Y. Kojima, J.P. Volkmer, K. McKenna, M. Civelek, A.J. Lusis, C.L. Miller, D. Direnzo, V. Nanda, J. Ye, A.J. Connolly, E.E. Schadt, T. Quertermous, P. Betancur, L. Maegdefessel, L.P. Matic, U. Hedin, I.L. Weissman, N.J. Leeper, CD47-blocking antibodies restore

35

Journal Pre-proof phagocytosis and prevent atherosclerosis, Nature 536 (2016) 86-90. [44] F.K. Swirski, M. Nahrendorf, Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure, Science 339 (2013) 161-166. [45] M. Lameijer, T. Binderup, M.M.T. van Leent, M.L. Senders, F. Fay, J. Malkus, B.L. Sanchez-Gaytan, A.J.P. Teunissen, N. Karakatsanis, P. Robson, X.X. Zhou, Y.X. Ye, G. Wojtkiewicz, J. Tang, T.T.P. Seijkens, J. Kroon, E.S.G. Stroes, A. Kjaer, J. Ochando, T.

of

Reiner, C. Perez-Medina, C. Calcagno, E.A. Fisher, B. Zhang, R.E. Temel, F.K. Swirski, M.

ro

Nahrendorf, Z.A. Fayad, E. Lutgens, W.J.M. Mulder, R. Duivenvoorden, Efficacy and safety

-p

assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and nonhuman primates, Nat. Biomed. Eng. 2 (2018) 623-623.

lP

Jo

ur

na

BMB Rep. 47 (2014) 1-7.

re

[46] S. Lim, S. Park, Role of vascular smooth muscle cell in the inflammation of atherosclerosis,

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Journal Pre-proof Cyclodextrins have shown the potential for effective treatment of atherosclerotic plaques but exhibit ototoxicity at high doses.



Cyclodextrin polymer was prepared by cross -linking of cyclodextrins and has a diameter of ~10 nm.



Cyclodextrin polymer exhibited comparable efficacy to monomeric cyclodextrin in cholesterol dissolution and cholesterol efflux in vitro.



Cyclodextrin polymer showed enhanced pharmacokinetics, plaque targeting, and therapeutic efficacy compared to monomeric cyclodextrin.



At high doses, cyclodextrin polymer showed no significant effect on hair cells while monomeric cyclodextrin induced significant hair cell loss.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5