Faculty Bibliography

One mechanism of the lipid-lowering effects of the fish oil n-3 fatty acids [e.g., docosahexaenoic acid (DHA)] in cell and animal models is induced hepatic apolipoprotein B100 (apoB) presecretory degradation. This degradation occurs post-endoplasmic reticulum, but whether DHA induces it before or after intracellular VLDL formation remains unanswered. We found in McA-RH7777 rat hepatic cells that DHA and oleic acid (OA) treatments allowed formation of pre-VLDL particles and their transport to the Golgi, but, in contrast to OA, with DHA pre-VLDL particles failed to quantitatively assemble into fully lipidated (mature) VLDL. This failure required lipid peroxidation and was accompanied by the formation of apoB aggregates (known to be degraded by autophagy). Preventing the exit of proteins from the Golgi blocked the aggregation of apoB but did not restore VLDL maturation, indicating that failure to fully lipidate apoB preceded its aggregation. ApoB autophagic degradation did not appear to require an intermediate step of cytosolic aggresome formation. Taken with other examples in the literature, the results of this study suggest that pre-VLDL particles that are competent to escape endoplasmic reticulum quality control mechanisms but fail to mature in the Golgi remain subject to quality control surveillance late in the secretory pathway.

It is well recognized that macrophages in many contexts in vitro and in vivo display a spectrum of inflammatory features and functional properties. A convenient system to group together different subsets of macrophages has been the M1 (inflammatory)/M2 (anti-inflammatory) classification. In addition to other sites of inflammation, it is now established that atherosclerotic plaques contain both M1 and M2 macrophages. We review results made possible by a number of recent mouse models of atherosclerotic regression that, taken with other literature, have shown the M1/M2 balance in plaques to be dynamic, with M1 predominating in disease progression and M2 in regression. The regulation of the macrophage phenotype in plaques and the functional consequences of the M1 and M2 states in atherosclerosis will also be discussed.

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

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 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 (!

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.