Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms

Abstract

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Keywords: abdominal aortic aneurysm; matrix metalloproteinases; peptide amphiphile; self-assembly; targeted nanomaterials.

Figure 1.

Study design and AAA model characterization. (A) AAA study outline of key experimental time points. (B) Photomicrographs of aorta in situ before and 35 days after CaCl₂ exposure. Dashed lines indicate aortic margins. (C) Measurements of aortic diameter before and after CaCl₂ exposure. (D) Immunofluorescence imaging of suprarenal and infrarenal aorta stained for MMP-2. (E) Quantification of MMP-2 staining, *p<0.05. (F) Immunofluorescence imaging of suprarenal and infrarenal aorta stained for MT1-MMP. (G) Quantification of MT1-MMP staining, *p<0.05. (H) Photomicrographs of suprarenal and infrarenal aorta stained for collagen via Ver Hoeff Van Gieson (VVG) stain.

Figure 2.

Peptide amphiphile design and synthesis. (A) The chemical structure of peptide amphiphile (PA) molecules synthesized containing the derived targeting peptides. (B) Molecular graphics simulation of the structure of the PA molecule and (C) a PA nanofiber co-assembly. TAMRA = 5-carboxytetramethylrhodamine.

Figure 3.

Nanofiber formation screening. (A) Conventional transmission electron microscopy (TEM) images of PA co-assemblies consisting of 25 mole% to 95 mole% targeting PA. (B) TEM images of control and diluent PA co-assemblies. (C) Histogram of fiber length and mean fiber length for 25 mole% fragmented elastin PA, 95 mole% MT1-MMP PA, and 25 mole% MMP-2 PA co-assemblies.

Figure 4.

MMP-2 targeting PA nanofiber material characterization. (A) Small-angle X-ray scattering (SAXS) analysis of the 25 mole% MMP-2 targeting PA with polydisperse core-shell cylinder modeling overlaid. (B) Wide-angle X-ray scattering (WAXS) analysis of the 25 mole% MMP-2 targeting PA shows peaks typical of β-sheets. (C) Circular dichroism (CD) spectroscopy of the 25 mole% MMP-2 targeting PA shows maximum and minimum characteristic of β-sheets. MicroScale Thermophoresis analysis of MMP-2 targeting peptide shows binding kinetics to (D) rhMMP-2 and (E) BSA.

Figure 5.

AAA targeting by PAs. (A) Light sheet fluorescence microscopy (LSFM) images of rat aorta 2 hours post PA injection. White color represents tissue autofluorescence, and red to yellow represents the fluorescence of TAMRA-labeled PA. Fragmented elastin targeting PA is 25 mole%, MT1-MMP is 95 mole%, and MMP-2 Targeting PA is 25 mole%. (B) Plotted mean data of the ratio of PA volume to tissue volume for each AAA-targeting PA. *p= 5.61×10⁻⁸ vs. MMP-2 Scrambled PA.

Figure 6.

Targeting PA dosage optimization. (A) Nile Red Assay to determine critical aggregation concentration of 25 mole% MMP-2 targeting PA co-assemblies. (B) LSFM images of rat aorta 2 hours post PA injection of either 2.5 mg, 5 mg, or 10 mg of 25 mole% MMP-2 targeting PA. (C) Plotted mean data of the ratio of PA volume to tissue volume for each dose of PA.

Figure 7.

MMP-2 targeted PA localization duration. (A) LSFM images of rat aorta 2 hours, 24 hours, 48 hours, and 72 hours post injection of 5 mg 25 mole% MMP-2 targeting PA. (B) Plotted mean data of the ratio of PA volume to tissue volume for each time point.

Figure 8.

MMP-2 targeting PA predominantly localizes to the aorta. (A) Organ distribution of 25 mole% MMP-2 targeting PA in the kidney, liver, lung, spleen, and aorta of Sprague Dawley rats 2 hours post injection. White color represents autofluorescence and red to yellow represents TAMRA-labeled PA. (B) Plotted mean data of the ratio of PA to tissue in each organ.