Multifunctional Dextran Sulfate-Coated Reconstituted High Density

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Multifunctional dextran sulfate-coated reconstituted high density lipoproteins target macrophages and promote beneficial antiatherosclerotic mechanisms Yi Zhao, Cuiping Jiang, Jianhua He, Qing Guo, Jing Lu, Yun Yang, Wenli Zhang, and Jianping Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00600 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 30, 2016

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Bioconjugate Chemistry

Multifunctional lipoproteins

dextran target

sulfate-coated macrophages

reconstituted and

high

promote

density

beneficial

antiatherosclerotic mechanisms Yi Zhao†, Cuiping Jiang†, Jianhua He†, Qing Guo†, Jing Lu†, Yun Yang†, Wenli Zhang*,†, Jianping Liu*,† †

Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009,

PR China

ABSTRACT An atorvastatin calcium (AT)-loaded dextran sulfate (DXS)-coated core-shell reconstituted high density lipoprotein (rHDL), termed as AT-DXS-LP-rHDL, was developed for targeted drug delivery to macrophages and suppression of inflammation via the high affinity of DXS with scavenge receptor class AI (SR-AI) as well as depletion of intracellular cholesterol by apolipoprotein A-I (apoA-I)-mediated cholesterol efflux. These core-shell nanoparticles comprising an AT-loaded negatively charged poly(lactide-co-glycolide) (PLGA) core and a cationic lipid bilayer shell, were prepared by nanoprecipitation method followed by thin film hydration and extrusion. The nanoparticles were further functionalized with apoA-I and DXS via sodium cholate mediation and electrostatic interaction, respectively. The core-shell structure and the surface coating of apoA-I and DXS were verified by the increased particle size, inverted zeta potential and reduced in vitro drug release rate. The TEM image further confirmed the entrapment of the PLGA nanoparticles in the aqueous interior of the liposomes. In vitro cell viability assay showed the biocompatibility of the AT-loaded nanocarriers. The cellular uptake study illustrated that the targeting efficacy to macrophages increased followed as PLGA nanoparticles (P-NP), core-shell nanoparticles (LP-NP), core-shell rHDL (LP-rHDL) and DXS-LP-rHDL. Moreover, cellular drug efficacy of AT-loaded nanoparticles in preventing macrophage-derived

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foam cells formation and inflammation such as intracellular lipid deposition, cholesterol esters content, DiI-oxLDL uptake, cholesterol efflux and secretion of TNF-α, IL-6, and IL-10 was much better than that of the drug-free nanoparticles, consistent with the results of cellular uptake study. Collectively, AT-DXS-LP-rHDL, as multifunctional carriers, could not only deliver more drug to macrophages, but also present antiatherogenic actions of the bio-functional nanocarriers through damping oxidized low density lipoproteins (oxLDL) uptake and promoting cholesterol efflux.

Graphical abstract

INTRODUCTION Cardiovascular disease underlies a leading cause of human morbidity and mortality worldwide and is caused in large part by atherosclerosis, which is a progressive disease characterized by chronic inflammation together with lipid accumulation in the arterial wall.1,2 Localized cholesterol and atherogenic inflammatory cycle are the primary challenges for current therapeutic strategies. The major antiatherosclerotic regimens aim to lower systemic levels of cholesterol via restricting liver-based synthetic pathways. Although lipid lowering therapy may have some impact on atherosclerosis prevention, its capabilities in inflammatory inhibition and vascular remodeling are limited. Monocytes-derived macrophages play a pivotal role in the pathogenesis of atherosclerosis through low density lipoproteins (LDL) uptake, which


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further triggers local sub-endothelial inflammation, arterial damage, endothelial cell dysfunction and plaque rupture.3 Therefore, direct drug delivery into the macrophages in atherosclerotic plaque may represent a promising avenue for therapeutic intervention. High density lipoprotein (HDL)-based nanoparticle is an appealing vector in plaque targeting due to its high affinity with scavenge receptor class BI (SR-BI), which is abundant in macrophages. HDL mainly with two different architectures, discoidal and spherical HDL, has been proposed to possess several antiatherogenic functions, including reverse cholesterol transport (RCT), anti-inflammatory, anti-oxidant and vascular-protective properties.4-7 The most predominant bio-function of HDL is RCT, a process that mainly relies on the remodeling behavior of discoidal HDL. In discoidal HDL, phospholipids form a bilayer interspersed with some free cholesterol and circumscribed by two apolipoprotein A-I (apoA-I) molecules. Spherical HDL has a core of hydrophobic lipids containing triglycerides (TG) and cholesterol esters (CE), which is covered by a monolayer of phospholipids with apoA-I embedded in. ApoA-I in discoidal HDL can engage cholesterol efflux receptors, mediating the efflux of free cholesterol to the surface of HDL. Under the action of lecithin cholesterol acyltransferase (LCAT), which is activated by apoA-I, free cholesterol is esterified to cholesteryl ester followed by being internalized within the bilayer, driving more cholesterol efflux. With continual cholesteryl ester moving to the core, discoidal HDL is converted to spherical HDL. In the last step of RCT, the cholesterol-loaded mature spherical HDL is taken up by liver for biliary excretion. Overall, apoA-I (interacting with cholesterol efflux receptors and activating LCAT) and lipid bilayer (receiving cholesterol) are essential for cholesterol efflux. Utilizing reconstituted high density lipoprotein (rHDL) could be an attractive avenue for harnessing the biology of HDL in a therapeutic context. A large number of clinical researches suggested that the infusion of rHDL could reduce lipid content, macrophage infiltration and inflammation, which are beneficial to plaque stabilization.8-10 Besides, rHDL displays plenty of other attractive attributes including biodegradability, biocompatibility, long



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circulation time in plasma. Recently, rHDL has been used as a delivery vehicle for membrane proteins, small interfering RNA (siRNA), anti-cancer drugs and cardiovascular drugs.11-14 Considering the biological functions of HDL in cardiovascular disease, we paid attention to the application of rHDL in anti-atherosclerotic drug delivery. In our previous studies, cardiovascular drug-loaded discoidal rHDL and spherical rHDL have been constructed to achieve the bio-functions and effective plaque targeting via SR-BI mediated endocytosis.15-17 Despite of the improved drug efficacy by using rHDL carrier, most of drug was inevitably retained by liver due to the over-expressed SR-BI in the hepatic cells.18 By exploiting discoidal rHDL, the unintended drug leakage during the remodeling behavior attenuated drug accumulation in the atherosclerotic lesions.19 As for spherical rHDL, although it was more stable and could better retain drug, its capability in RCT was limited due to a saturated core of cholesterol. Therefore, how to develop a novel rHDL nanosystem with improved targeting efficacy, biological functions and stable structure would pose a challenge to researchers. It has been reported that CD44, SR-BI and scavenge receptor class AI (SR-AI) are highly expressed on the surface of activated macrophages and foam cells.20,21 Among these receptors, SR-AI is a key molecule involved in the recognition of modified lipoproteins and accumulation of macrophages.22,23 Specifically, SR-AI-mediated increased oxidized low density lipoproteins (oxLDL) uptake and defective cholesterol efflux result in foam cells formation and pro-inflammatory cytokines secretion. Moreover, increased cell spreading and adhesion to extracellular matrix mediated by SR-AI would cause macrophages retention in the lesions. It was noted that a marked decrease in the progression of advanced necrotic lesions was achieved through the targeted deletion of SR-AI.24 Dextran sulfate (DXS), a polyanionic derivative of dextran, is a specific ligand for SR-AI, which can bind to the positive pocket of SR-AI residues via electrostatic interaction and competitively inhibit cellular internalization of oxLDL via SR-AI blockage.25,26 Pioneering studies have exploited


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DXS-coated iron oxide nanoparticles for atherosclerotic plaques imaging, which showed improved efficiency compared to non-targeted probes.27-29 Therefore, drug-loaded nanocarriers with DXS as a ligand may enhance plaque-targeted drug delivery, meanwhile retard foam cells formation and cytokines secretion through competitive blockage of SR-AI receptor. In the present study, an atorvastatin calcium (AT)-loaded DXS-coated core-shell rHDL

was

designed

and

fabricated

by

encapsulating

spherical

Poly

(lactide-co-glycolide) (PLGA) nanoparticles into liposomes, followed by incubation with apoA-I, then decoration with DXS on the surface. The modification of DXS could not only interfere with the recognition by liver, but also maintain the bio-function of apoA-I-mediated cholesterol efflux and simultaneously exert the atheroprotective roles of DXS. The stable core-shell structure could provide lipidic bilayer space for cholesterol efflux from foam cells and inhibit the drug leakage. In addition, the lipid bilayer structure resembling biological membrane is prone to fuse with cell membrane to deliver more drug intracellularly.30 The in vitro physicochemical properties were characterized in terms of particle size, zeta potential, encapsulation efficiency (EE), particle morphology,

drug release kinetics.

Furthermore, in vitro cell viability, cellular uptake behavior and the drug efficacies including intracellular lipid dispositions, CE content, DiI-oxLDL uptake, cholesterol efflux and TNF-α, IL-6, and IL-10 secretion of each preparation were comprehensively investigated. RESULTS AND DISCUSSION Preparation of the nanoparticles. The schematic diagram on preparation procedures was represented in Figure 1. AT-loaded PLGA nanoparticles (AT-P-NP) were fabricated by nanoprecipitation method. Thin-film hydration method followed by extrusion was adopted to encapsulate AT-P-NP into liposomes for the preparation of AT-loaded core-shell nanoparticles (AT-LP-NP). Poloxamer 188, a FDA-approved surfactant which could be applied in clinical intravenous therapy, was added into



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water phase to stabilize the AT-P-NP during precipitation. Owing to the lipophilicity, AT could easily be encapsulated into P-NP via hydrophobic interaction. The thin lipid film comprising PC, cholesterol and cationic DOTAP, was prepared by rotary evaporation, followed by thin film hydration and extrusion at 45 °C. The increase in thermal and mechanical energy allowed the lipid to disperse and self-assemble, forming a bilayer around the core. Afterwards, apoA-I was combined via sodium cholate mediation and DXS was decorated via electrostatic interaction to prepare AT-loaded core-shell rHDL (AT-LP-rHDL) and DXS-coated AT-LP-rHDL (AT-DXS-LP-rHDL), respectively. The DXS-coated core-shell nanoparticles were termed as AT-DXS-LP-NP.

Figure 1. Schematic diagram on preparation procedures of AT-DXS-LP-rHDL.

Characterization of the nanoparticles. In vitro characterization was carried out to examine the physicochemical properties of the nanoparticles. Figure 2 depicted the particle size, zeta potential and EE of the prepared nanoparticles. The hydrodynamic diameter of AT-P-NP was 97.8±1.0 nm, which was consistent with the particle size observed in TEM image. Meanwhile, AT-P-NP exhibited a negative surface charge of -23.1±1.6 mV owing to the terminal carboxyl acid of PLGA. In comparison, the increased particle size (130.8±1.1 nm) and the inverted zeta potential


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(31.7±1.4 mV) of AT-LP-NP indicated the fusion of cationic lipid shell on the surface of AT-P-NP. Furthermore, slight increment in particle size was observed after apoA-I conjugation (140.5±1.5 nm ) and DXS coating (156.5±0.5 nm). The decoration of apoA-I and DXS was also confirmed by the decreased zeta potential from 20.4±1.6 mV of AT-LP-rHDL to -38.48±0.6 mV of AT-DXS-LP-rHDL.

Figure 2. The particle size, zeta potential and encapsulation efficiency of different AT-loaded nanoparticles (n=3).

The EE of AT within AT-P-NP was only 72.1±0.8%, whereas approximately 87.7±1.2% of AT was encapsulated into AT-LP-NP. The increased EE could be attributed to the presence of lipid bilayer at the interface of AT-P-NP, which served as a molecular fence, retaining the drug within the nanoparticles. EE of AT in AT-LP-rHDL, AT-DXS-LP-NP, AT-DXS-LP-rHDL were all higher than 85%. The surface morphology of the nanoparticles was visualized by TEM as illustrated in Figure 3. The images showed that the nanoparticles were dispersed as individual particles with a well-defined spherical shape and homogeneous distribution. The TEM image of AT-LP-NP confirmed the core-shell structure with the entrapment of PLGA nanoparticles in the aqueous interior of liposomes.



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Figure 3. Size distribution and TEM micrographs of different AT-loaded nanoparticles.

The in vitro cumulative drug release profiles of AT-P-NP, AT-LP-NP, AT-LP-rHDL, AT-DXS-LP-NP, AT-DXS-LP-rHDL were shown in Figure 4. The release rate of AT from AT-P-NP was higher than that of other groups. For AT-P-NP, an initial drug burst of 62.3% within the first 6 h was observed, and virtually about 83.2% of AT was released within 72 h. Conversely, only 37.8% of AT was released from AT-LP-NP within 6 h, and nearly 66.5% of release was occurred after 72 h. It was worth noting that the coating of apoA-I and DXS further retarded the drug release. The AT-LP-rHDL, AT-DXS-LP-NP, AT-DXS-LP-rHDL released 58.4%, 44.3%, 38.3% of total AT after 72 h, respectively. It seems that the lipid bilayer, apoA-I and DXS played an important role in preventing the free diffusion of the drug out of the core and attenuating the hydrolysis rate of PLGA polymers, thereby resulting in the slower drug release rate.


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Figure 4. In vitro accumulative release (%) of AT from different AT-loaded nanoparticles in PBS (0.1 M, pH 7.4) for 72 h at 37 °C (n=3) (* p

Multifunctional Dextran Sulfate-Coated Reconstituted High Density

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