UC Berkeley

The use of functionalized nanoparticles in biomedical applications has become increasingly more prevalent due to the promise of using these materials as drug delivery and molecular imaging agents. Targeting of specific markers of disease in vivo can be achieved using functionalization techniques, such as modification of nanoparticles with short peptides. Peptides exhibit definite advantages versus whole proteins and their small size leads to the ability to display tens to hundreds of targeting peptides per nanoparticle. In this work, peptide functionalization is utilized to target both model drug delivery and imaging nanoparticles to a site of interest displayed by atherosclerotic plaques. The biocompatibility and clearance of these particles is considered as well as the overall targeting ability. After first establishing the biocompatibility and safety of these materials, the ability to create targeted nanoparticles as well as modular, multifunctional materials through the combination of multiple targeting peptides was assessed.

Two strategies are employed in this work: the formation of self-assembled peptide amphiphile micelles as potential drug delivery agents and peptide-functionalized iron oxide particles for use as molecular imaging agents. The establishment of biocompatibility was first determined using a peptide amphiphile micelle, DSPE-PEG2000-CREKA, which specifically binds to fibrin, a marker of late stage atherosclerotic plaques. Both CREKA and PEG micelles were evaluated in vivo using a standard atherosclerosis mouse model, ApoE -/-. Atherosclerosis is characterized by the presentation of multiple markers of the disease and progresses from early to late stage through the growth and development of lipid-core plaques. Injection of small, spherical Cy7-labeled micelles allowed for in vivo near-infrared imaging. These studies showed that these micelles are cleared both through the liver and spleen, therefore involving the reticuloendothelial and renal system, respectively. Biocompatibility was observed through histological staining of excised tissues, determining the level of apoptosis in the liver and spleen, as well as testing for liver function. By varying the mole percent of Cy7 in the micelles, it was possible to determine an optimal regime for both whole body and ex vivo near-infrared imaging.

Utilizing the results of the biocompatibility and biodistribution of peptide amphiphile micelle study, it was found that 10 mole percent Cy7 was optimal for future in vivo studies. While late stage targeting of atherosclerotic plaques had previously been demonstrated, the ability to actively target earlier stages of plaque formation with a model drug delivery vehicle formed from peptide amphiphile micelles had yet to be observed. Therefore, the formation of early stage targeting was achieved through DSPE-PEG2000-VCAM micelles labeled with a near-infrared dye, Cy7. These micelles were designed to be small enough to extend their in vivo circulation time and also be spherical. The intended target, vascular cell adhesion molecule-1, or VCAM-1, is expressed by endothelial cells that line the developing plaque, making it a great target for intravenously injected particles. VCAM-1 targeting micelles were shown to accumulate in the cardiovascular system in early stage mice. Immunohistochemistry showed that VCAM-1 expression overlapped with Cy7 dye in the aortic tree, providing evidence for active targeting of early and mid-stage atherosclerotic plaques.

While active targeting was shown with VCAM-1 micelles, one of the central advantages to self-assembled micelles is the inherent ability to incorporate multifunctionality through mixing of different amphiphiles. To determine the ability to mix different peptide amphiphiles, micelles composed of DSPE-PEG2000-CREKA and DSPE-PEG2000-VCAM as well as diC16CREKA and diC16VCAM were analyzed. Both systems showed some degree of mixing via transmission electron microscopy and Förster resonance energy transfer and the region over which one population of mixed micelles were formed was established. Mixing was additionally determined using two amphiphiles that individually formed different geometry micelles, either spherical or cylindrical particles. The ability to incorporate otherwise cylindrical-forming amphiphiles into spherical micelles was demonstrated.

Each peptide amphiphile study focused on the formation of platform technologies for drug delivery. A strategy to form a targeted contrast agent was additionally developed via peptide functionalization of iron oxide particles. Using CREKA-functionalized contrast agents developed for magnetic particle imaging (MPI), the ability to bind to fibrin ex vivo was shown to be dependent on the amount of fibrin present, demonstrating the specific binding of these nanoparticles. The in vivo biodistribution additionally showed clearance through the liver, as is expected for iron oxide nanoparticles.

In each study presented, both the function of the peptide-functionalized nanoparticle system was established as well as the in vivo biodistribution. As the field of targeted delivery agents progresses, the design parameters as well as the establishment of biodistribution, safety, and targeting ability set forth in this work will be a guide for future studies using peptide-functionalized and targeted nanoparticles.