Poster Y17 - Characterization of Plaque-Targeted Peptide Amphiphile Nanoparticles for the Treatment of Atherosclerosis

Introduction: Atherosclerosis remains a significant cause of mortality and morbidity. Modern approaches to atherosclerotic plaque regression include endovascular plaque debulking, stenting, and endarterectomy procedures, all of which are invasive and subject to restenosis. Nanotherapeutic approaches to plaque targeting are a promising alternative to invasive surgical procedures. Peptide amphiphile (PA) molecules, which can self-assemble into supramolecular structures in an aqueous environment at physiologic conditions, are highly modifiable with targeting epitopes, cleavable linkers, and directly conjugated therapeutics. An attractive candidate for plaque targeting is vascular cell adhesion molecule 1 (VCAM-1) due to its high, inducible expression in early-stage atherosclerotic lesions and persistence of expression throughout disease progression. The peptide sequence VHPKQHRGG has been reported as a potential targeting moiety for VCAM-1 via in vivo phage display experiments. We hypothesize that incorporation of this sequence into a PA molecule will form supramolecular co-assemblies that target VCAM-1 expressed on the endothelial surface of atherosclerotic plaques in vivo.

Materials and

Methods: VCAM-1-targeted PA and backbone filler PA molecules were synthesized using Fmoc chemistry and purified using high-performance liquid chromatography (HPLC). PAs were co-assembled in various mole percent ratios (10-50%) of VCAM-1 to backbone PA by mixing in aqueous phase (2 mg/mL), followed by lyophilization and resuspension in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at 10 mg/mL. Co-assemblies were then sonicated for 10 minutes and all HFIP was removed using a Schlenk line. Next, co-assemblies were resuspended in normal saline (NS, 0.9% w/v NaCl), corrected to pH 7.35-7.45, and then lyophilized for long-term storage at -20°C. Co-assemblies were then characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). TEM grids were prepared by reconstituting PA co-assemblies at 1 mg/mL in NS and staining with 2% uranyl acetate. Images were acquired using an 80 kV, high-tension electron beam and analyzed in Fiji. For DLS and NTA, PA co-assemblies were reconstituted at 2 mg/mL in NS and analyzed for count rate, diameter, zeta potential, and polydispersity index (PDI). To increase nanoparticle monodispersity, measurements were repeated on the same samples after either 1-hour sonication or 0.22 um filtration. Finally, microscale thermophoresis (MST) was conducted using a recombinant VCAM-1 His-tagged protein, VHPKQHRGG peptide, and scrambled control (RQVGHGPHK) at a wide range of ligand concentrations (10^-7-10^-2 M) to determine the binding affinity of the VCAM-1-targeted peptide to its target in vitro.

Results, Conclusions, and Discussions: Two different PA molecules were synthesized: a VCAM-1-targeted PA (Figure 1A) with the VHPKQHRGG sequence and a backbone diluent PA (Figure 1B) without the VCAM-1-targeting epitope. Both PA molecules were >95% as measured by HPLC and showed expected molecular weights: VCAM-1-targeted PA 2132 g/mol (Figure 1C) and diluent PA 1249 g/mol (Figure 1D). VCAM-1-targeting PA (20 mole %) self-assembled into a relatively monodisperse population of nanoparticles with a mean diameter of 18.2 +/- 1.3 nm on TEM (Figure 1E). Using a fluorescent tag and recombinant VCAM-1 protein, an upward shift in fluorescence intensity was observed via MST upon VHPKQHRGG peptide addition (green line), indicative of stable binding complex formation (Figure 1F). Multiple concentrations of VHPKQHRGG peptide were complexed with a constant concentration of recombinant VCAM-1 to determine binding kinetics. Preliminary analysis from MST data indicates the dissociation constant, Kd, of the VHPKQHRGG peptide and VCAM-1 to be ~980 uM, providing strong supporting evidence for in vitro and in vivo binding kinetics (Figure 1G). VCAM-1-targeted nanoparticles increased in mean diameter with increasing mole % of VCAM-1-targeted PA (28.3 +/- 9.2 nm vs. 40.1 +/- 3.3 nm), as measured by NTA (Figure 1H). These results are corroborated by the size measurements acquired by TEM but may be slightly higher due to a higher concentration of nanoparticles required for DLS and NTA measurements to achieve a sufficient count rate. Initial PDI measurements of PA nanoparticles were extremely polydisperse (PDI > 0.8), so 1-hour sonication or 0.22 um filtration conditions were investigated for their potential to reduce PDI. Filtration (PDI 0.26 +/- 0.08), but not 1-hour sonication (PDI 0.94 +/- 0.12), was an effective method for reducing PDI, indicating a more monodisperse population of nanoparticles, possibly suitable for in vivo injection in future experiments (Figure 1I). In conclusion, these data show that VCAM-1-targeted PA molecules, with a backbone diluent PA, co-assemble at various molar ratios to form a monodisperse population of spherical nanoparticles that bind VCAM-1 in vitro. Future efforts will include reducing the zeta potential (data not shown) to a net negative charge to aid in hemocompatibility before in vivo plaque-targeting characterization.

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