K. Marulanda1, A. Mercel1, M. Gambarian1, D. C. Gillis1, K. Sun1, M. Karver2, N. D. Tsihlis1, S. E. McLean1, M. R. Kibbe1 1University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 2Northwestern University,Simpson Querrey Institute,Chicago, IL, USA
Introduction: Pulmonary hypertension (PH) is a highly morbid disease without an effective treatment. Our aim is to develop a systemically administered nanoparticle therapy that specifically targets the pulmonary vasculature. Angiotensin converting enzyme (ACE) is highly associated with PH pathogenesis, demonstrating increased expression in the diseased pulmonary vascular endothelium. To target ACE, self-assembled peptide amphiphile (PA) nanofibers are an ideal delivery vehicle, as they are readily modifiable, biocompatible, and can be re-dosed. We hypothesize that ACE-targeted PA nanofibers will localize to the pulmonary vasculature in a mouse model of chronic hypoxia.
Methods: Two ACE-targeted amino acid sequences, GNGSGYVSR (GNG) and RYDF, were covalently attached to a PA backbone. PAs were synthesized using solid phase peptide synthesis, then purified and characterized by high pressure liquid chromatography paired with mass spectrometry (HPLC-MS). The GNG- and RYDF-PA nanofibers were co-assembled using different ratios of backbone PA and fluorescently tagged PA. Conventional transmission electron microscopy (TEM) was used to assess nanofiber formation. Female and male C57BL/6J mice (8-10 weeks old) were exposed to chronic hypoxia (10% FiO2) for 3 weeks. Control mice were kept at room air (21% FiO2). To assess in vivo nanofiber localization, targeted nanofiber (10mg/kg) was administered to control and hypoxic mice via tail vein injection. Lungs were harvested after 30 minutes, and nanofiber fluorescence was quantified.
Results: HPLC-MS confirmed >95% purity of PAs, and TEM confirmed nanofiber formation for both ACE-targeted nanofibers. The mouse PH model was validated by observing pulmonary arterial muscularization per high power field on histology and elevated right ventricular systolic pressure with hemodynamic assessment. ACE immunostaining levels were 5-fold higher in the hypoxic versus control mouse lungs (3106 ± 287 vs. 644 ± 98 AU, n=3, p<0.0001), validating this protein as a useful target. After inducing PH in the mice, targeted nanofibers were injected systemically. Interestingly, the RYDF-PA nanofiber demonstrated extremely high (78-fold) binding affinity in hypoxic versus control lungs (390 ± 35 vs. 5 ± 2 AU, n=2-4/treatment group, p<0.0001) while the GNG-PA nanofiber demonstrated no difference in binding between the groups (15 ± 5 vs. 21 ± 5 AU, n=3-4, p=0.39). The pattern of binding of the RYDF-targeted nanofiber to the pulmonary vasculature had a similar distribution pattern as ACE immunoreactivity on fluorescent microscopy.
Conclusion: An ACE-targeted PA nanofiber was successfully designed and synthesized, and specifically localized to the pulmonary vasculature following intravascular administration in a mouse model of chronic hypoxia. Our findings lay the groundwork for incorporation of a therapeutic into the targeted nanoparticle to effectively mitigate pulmonary hypertension.