(794b) Investigation of Self-Assembled Peptide Amphiphile Micelles for Targeting Early Stage Atherosclerotic Plaques

Atherosclerosis, the leading cause of death in the United States, is characterized by the formation of plaques in the arterial wall, which progress through multiple stages.  If left untreated, these plaques can become unstable and rupture, leading to potentially fatal consequences.  Delivery of therapeutic drugs to these plaques using vehicles targeted directly to markers located on the plaque could allow for development of new therapies for atherosclerosis.  Our group has shown the ability to target fibrous cap formation, characteristic of late stage plaques, using self-assembled peptide amphiphile (PA) micelles [1].  Detection of plaques at an earlier stage, however, could lead to the potential for treatment prior to these plaques becoming unstable.  Here, we discuss micelles that are designed to bind to vascular cell adhesion molecule-1 (VCAM-1), an inflammatory marker of early stage atherosclerotic plaques.  PAs are formed by conjugating a hydrophobic, lipid tail to a hydrophilic peptide headgroup.  PAs under investigation in this study are composed of a peptide headgroup that recognizes and binds to VCAM-1, a 2,000 molecular weight polyethylene glycol (PEG) spacer, and a diC₁₈ hydrophobic tail to form a PA and resulting micelle termed DSPE-PEG-VCAM.

Dynamic light scattering (DLS) of DSPE-PEG-VCAM micelles shows the hydrodynamic diameter to be approximately 20 nm.  The large PEG spacer helps to confer a large headgroup and sphere formation is both predicted and observed using negative stain TEM.  The critical micelle concentration (CMC), or the concentration at which micelles begin to form, was found to be approximately 60 μM as determined by the DPH method [2].  Finally, circular dichroism shows that the peptide in solution as well as DSPE-PEG-VCAM, retain similar secondary structures, shown by fitting the resulting spectra (peptide: 33±1% beta sheet, 67±1% random coil; PA: 28±1% beta sheet, 72±1% random coil).

Human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (AEC) were shown to remain viable in the presence of DSPE-PEG-VCAM micelles. Furthermore, in vitro results have shown localization of DSPE-PEG-VCAM micelles to aortic endothelial cells using confocal microscopy with micelles fluorescently labeled with both fluorescein and Cy7. This may indicate binding between the micelle and the VCAM-1 marker and these results will be further discussed by examining the binding specificity of DSPE-PEG-VCAM micelles for AECs versus control micelles.  Specifically, the effects of micelle composition and PA formulation with the ability of the micelle to associate with AECs will be discussed.  Future work will determine where DSPE-PEG-VCAM PAs localize within AECs as well as assessing the ability to deliver a hydrophobic payload to both cells in an in vitro setting and atherosclerotic plaques in vivo.

[1] Peters, D, et al. Targeting Atherosclerosis by Using Modular, Multifunctional Micelles. PNAS. 2009; 106: 9815-9819.

[2] Chattopadhyay, A and London, E.  Fluorimetric Determination of Critical Micelle Concentration Avoiding Interference from Detergent Charge. Analytical Biochemistry. 1984; 139: 408-412.