Faculty Bibliography

Introduction 2nd generation polymeric nanoparticles have shown significant advantages in drug delivery. They can be loaded with poorly water soluble drugs,1 their size can be judiciously controlled2 and their surface can be functionalized with a PEG coating and/or targeting ligands. Importantly, the polymeric core can be loaded with drugs and/or contrast agents for which the release rates can be controlled by the choice of polymer composition and molecular weight. High-density lipoprotein (HDL) is a natural nanoparticle that transports fats through the body, which has an inherent affinity for atherosclerotic plaques. HDL-like nanoparticles labeled with contrast agents have been shown suitable for molecular imaging as they effectively target atherosclerotic plaque. In the current study we developed a novel HDL-like hybrid nanoparticle using recently developed microfluidics technology.2 The nanoparticle is comprised of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core suitable for the delivery of drugs in a controlled manner. The versatility of the approach also allows the incorporation of functional lipids to render multifunctional nanoparticles with imaging, therapeutic and atherosclerosis targeting properties. Methods and Results Hybrid polymer-HDL nanoparticles with a PLGA core and a coating comprised of lipids and apolipoprotein A1 (PLGA-HDL) were synthesized using microfluidics. The synthetic approach consists of the rapid injection of the components in three different channels of a microfluidics chip. Amphiphilic phospholipids and PLGA were dissolved in a mixture of ethanol and acetonitrile. This solution was injected in the middle channel of the microfluidic chip and mixed with an aqueous apoprotein A1 (ApoA1) solution injected in the two outer channels. Inside the chip, controlled nanoprecipitation occurred through microvortices, resulting in the instantaneous and continuous production of hybrid PLGA-HDL nanoparticles with high reproducibility (!

High Density Lipoprotein (HDL) is a natural nanoparticle involved in the transport of cholesterol throughout the body. HDL has been shown to exhibit atheroprotective properties as it promotes cholesterol efflux from atherosclerotic plaque macrophages in the arterial wall. Various laboratories have focused on the reconstitution of HDL (rHDL) for a variety of reasons, ranging from a better understanding of the structural biology of apolipoproteins to the use of rHDL as an injectable therapeutic (1). A recent effort centers around the use of rHDL as a natural nanoparticle platform for the delivery of contrast agents such as gadolinium chelates, iron oxide or gold nanoparticles, and employing them as molecular imaging contrast agents (2). To date, multistep production protocols pose a limit on the synthesis of batch quantities and are sensitive to inter-batch variations. In order to scale up the production process and to judiciously control rHDL's composition we have developed a modular single-step approach based on recently introduced microfluidics technology (3) that enables the standardized mass production of such lipoprotein-based nanoparticles. Materials and methods Organic solutions containing phospholipids and imaging agents (QD, FeO-NP, Au-NP, DiO) were injected into a microfluidic chip alongside an aqueous solution containing ApoA1. Within the chip the controlled flow streams generate microvortices where fast mixing of the solutions leads to the instantaneous formation of HDL-like nanoparticles (Figure 1). HDL particles produced by this microfluidics method, which we refer to as muHDL, had similar physicochemical properties (size, morphology) to particles produced by conventional methods and natural HDL. Moreover cell based assays demonstrated that muHDL nanoparticles displayed a similar bioactivity profile to natural HDL. muHDL that encapsulated hydrophobic dies (DiO) or nanocrystals such as quantum dots (QD), gold (Au) or iron oxide (FeO) nanoparticles were characterized and evaluated in!

Introduction: Atherosclerosis is an inflammatory disease. Its major clinical manifestation, coronary artery disease, is the leading cause of death in the western world. The disease is caused by the rupture of macrophage-laden and highly-inflamed atherosclerotic plaques, which are prone to rupture and cause myocardial infarctions. Oral statin therapy is widely used to reduce blood cholesterol levels in patients with atherosclerosis, and it is believed to have modest anti-inflammatory effects. We previously developed a strategy that aims at amplifying these anti-inflammatory effects through statin delivery to plaque macrophages using a reconstituted high density lipoprotein (rHDL) nanoparticle as a delivery vehicle. This statin rHDL ([S]-rHDL) nanotherapy reduced plaque macrophages by 80% (Supplementary). In the current study, we set out a novel nanomedical treatment paradigm, based on the aforementioned [S]-rHDL nanotherapy, which aims at realizing rapid remodeling of advanced atherosclerotic plaques towards a favorable phenotype in Apolipoprotein E-/- (ApoE KO) mice. Design and Results: ApoE KO mice received 26 weeks of high-cholesterol diet to develop advanced atherosclerotic plaques. After the diet, the mice first received high dose [S]-rHDL for a week (60 mg/kg simvastatin, 4 intravenous injections /week), followed by either low dose [S]-rHDL (15 mg/kg simvastatin, 2 intravenous injections /week), oral simvastatin (15 mg/kg/day), or no treatment for another 8 weeks (A). We evaluated the efficacies of the therapies during the course of the 9-week treatment with a MATLAB-assisted histological assessment of aortic roots, and CD68 immunostaining and hematoxylin phloxine saffron stain (HPS)) were performed (B). In vivo magnetic resonance imaging (MRI) of abdominal aortas and blood tests were done as well. After one week high dose [S]-rHDL treatment, macrophage levels in aortic roots were reduced by 70% (P < 0.001). The low levels were maintained by a subsequent 8-week low dose [S]-rHDL (48 % lowe!

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

Cardiovascular disease represents the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD exhibit an atherogenic dyslipidemia that is characterized by an increased plasma concentration of triglycerides, reduced concentration of high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) particles that are smaller and more dense than normal. The pathogenesis of NAFLD-associated atherogenic dyslipidemia is multifaceted, but many aspects are attributable to manifestations of insulin resistance. Here the authors review the structure, function, and metabolism of lipoproteins, which are macromolecular particles of lipids and proteins that transport otherwise insoluble triglyceride and cholesterol molecules within the plasma. They provide a current explanation of the metabolic perturbations that are observed in the setting of insulin resistance. An improved understanding of the pathophysiology of atherogenic dyslipidemia would be expected to guide therapies aimed at reducing morbidity and mortality in patients with NAFLD.

BACKGROUND: Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall. METHODS AND RESULTS: A monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichment of apoA1 compared with normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed that only a minority (<3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.06390%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d>1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed approximately 80% lower cholesterol efflux activity and approximately 90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1. CONCLUSIONS: The function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired.

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.