Detection of Macrophages via Paramagnetic Vesicles Incorporating Oxidatively Tailored Cholesterol Ester: An Approach for Atherosclerosis Imaging

Andrei Maiseyeu; Georgeta Mihai; Sashwati Roy; Nisharahmed Kherada; Orlando P Simonetti; Chandan K Sen; Qinghua Sun; Sampath Parthasarathy; Sanjay Rajagopalan

Disclosures

Nanomedicine. 2010;5(9):1341-1356. 

In This Article

Abstract and Introduction

Abstract

Aim: Macrophages play a key role in the initiation, progression and complications of atherosclerosis. In this article we describe the synthesis of biocompatible, paramagnetic, fluorescent phosphatidylserine vesicles containing cholesterol ester with a free carboxylic acid function and its use for targeted imaging of macrophages.
Methods & results: We synthesized anionic vesicles containing a combination of phosphatidylserine and a novel synthetic oxidized cholesterol ester derivative (cholesterol-9-carboxynonanoate [9-CCN]). In vitro studies to characterize particle size, MRI relaxation times and stability were performed. Vesicles containing 9-CCN demonstrated enhanced ability to bind human low-density lipoprotein and to be internalized by macrophages. Experiments in cultured macrophages with 9-CCN vesicles, alone and in the presence of low-density lipoprotein, indicated uptake of vesicles through scavenger receptor and integrin-dependent pathways. In vivo MRI using 9-CCN vesicles containing gadolinium in a rabbit model of atherosclerosis revealed protracted enhancement of 9-CCN vesicles and colocalization with arterial macrophages not seen with control vesicles. Pharmacokinetic experiments demonstrated prolonged plasma residence time of 9-CCN vesicles, perhaps due to its capacity to bind to low-density lipoprotein.
Conclusion: Vesicles containing 9-CCN demonstrate prolonged plasma and plaque retention in experimental atherosclerosis. Such a strategy may represent a simple yet clinically relevant approach for macrophage imaging.

Introduction

Imaging of macrophages in atherosclerotic plaque may allow derivation of prognostic information in addition to enabling targeted approaches to ablate or modify macrophage function. Prior studies have established the feasibility of imaging macrophages using contrast agents that incorporate antibodies,[1–3] lipoproteins[4–6] and synthetic peptides.[4,7] Combining affinity with biocompatibility, safety and ease of preparation is essential to any clinically applicable formulation. Recent studies suggest that uptake of apoptotic debris by macrophages is facilitated by specific 'eat-me' signals exteriorized on the plasma membrane surface of apoptotic cells.[8,9] Phosphatidylserine (PS) and oxidized lipids are pivotal cues in this process.[9,10] In atherosclerotic plaques, the genesis of foam cells is believed to relate to the engulfment of highly negatively charged oxidatively modified lipoproteins.[11,12] Cholesteryl or 7-ketocholesteryl esters of 9-oxononanoate represent a frequent type of oxidized lipid present both intracellularly within plaque macrophages and extracellularly in advanced atherosclerotic lesions.[13] We hypothesized that incorporation of PS and cholesteryl-9-carboxynonanoate (9-CCN), two highly anionic lipids, within a lipid-based delivery platform would allow recognition and subsequent uptake by macrophages present in atherosclerotic plaque. Accordingly, in this investigation we describe the synthesis of 9-CCN-containing anionic vesicles that additionally incorporate two different reporter molecules for their MRI and fluorescent detection in atherosclerotic lesions. In vitro and in vivo evaluation of these vesicles was carried out and compared with control PS vesicles without 9-CCN. Three vesicle formulations were studied: vesicles composed of PS and 9-CCN; vesicles containing PS (control); and vesicles containing PS and 9-CCN-OMe, a methyl ester of 9-CCN. These formulations were termed oxPL, PL and mePL, respectively (Figure 1). The latter has the carboxyl function blocked and, thus, would be expected to be less anionic. All vesicles were formulated with one of the aforementioned lipids in conjunction with paramagnetic gadolinium (Gd) lipid (for MRI detection), vehicle lipid (phosphatidylcholine) and fluorescent (rhodamine) lipid. Gd lipid and oxidatively modified cholesterol lipid (9-CCN) is easily synthesized using a one-step procedures in gram-scale quantities. Use of PS/phosphatidylcholine in approved clinical formulations[14,15] and ease of synthesis makes such a strategy attractive for scale-up and eventual human use.

Figure 1.

Schematic representation of synthesized vesicles. PL vesicles were generated from commercially available phospholipids and cholesterol. oxPL vesicles were synthesized with cholesteryl-9-carboxynanoate to mimic oxidized phospholipids while providing targeting to macrophages. mePL vesicles were formulated with cholesteryl-9-carboxynanoate methyl ester and served as nontargeted control. All formulations contained Gd lipids and rhodamine for MRI and fluorescence detection in cells and tissues.
Gd: Gadolinium; mePL: (3b)-cholest-5-en-3-yl methyl azelaate phospholipid vesicles; oxPL: Cholesteryl-9-carboxynonanoate phospholipid vesicles; PL: Phospholipid.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.

processing....