W. Jiang1,2, E. M. Bahnson1, M. R. Kibbe1 1Feinberg School Of Medicine – Northwestern University,Vascular Surgery,Chicago, IL, USA 2Northwestern University,McCormick School Of Engineering,Chicago, IL, USA
Introduction: We developed, characterized, and validated a collagen-binding peptide amphiphile (PA) nanofiber as a novel targeted delivery vehicle that specifically binds to and releases a therapeutic agent directly at the site of vascular injury and prevents neointimal hyperplasia following systemic administration. However, in order to translate this therapy to the clinical arena, the safety of this nanofiber as a drug delivery tool must be evaluated. Thus, the goal of this current project is to evaluate the safety of this systemically administered targeted nanofiber. We hypothesize that a systemically injected targeted nanofiber will be safe following in vivo administration.
Methods: Male Sprague-Dawley rats received a single dose of a fluorescently labeled collagen-binding PA (2.5mg, N=3). Liver, spleen, and kidney were harvested at different time points (24, 48, and 72h) for H&E staining and immunohistochemical evaluation for macrophage infiltration (ED1 antibody). Blood samples were collected for coagulation studies. C3a levels in plasma was assessed by ELISA. Functional complement activation was assessed by a hemolysis assay following incubation of human serum with PA (1 and 0.5mg/ml) for 1h. Platelet activation was evaluated by assessing adherence ex vivo after incubation of rat platelet rich plasma with PA (1 and 0.5mg/ml) for 30 min.
Result: Assessment of liver, spleen, and kidney histologically revealed the presence of fluorescence at 24, 48, and 72h, suggesting the presence of the targeted nanofiber. However, no change in organ architecture was observed in H&E stained slides and there was no difference in macrophage infiltration in the liver, kidney, and spleen of nanofiber-treated animals compared to control animals. The serum dose-response curves for the complement activation assay showed no difference between treated and untreated serum. At a 1:15 serum dilution, the percentage of residual complement for different concentrations of the PA (1 and 0.5mg/ml) was 99.8 +/- 7.7% and 96.4 +/- 8.8%, respectively vs. 100.7 +/- 9.9% for untreated serum, indicating no significant activation of complement. Similarly, C3a levels in rat plasma did not differ between control and PA-treated rats. Regarding the coagulation studies, the PT was increased to 22 +/- 4s at 24h (p=0.027) and 21 +/- 0.1s at 48h (p=0.044) after PA administration compared to 15 +/- 2s for control, but returned to normal at 72h (14 +/- 0.2s). PTT, fibrinogen and platelet count showed no statistically significant change after treatment. Finally, PA did not cause platelet activation ex vivo.
Conclusion: This data suggested that systemic administration of our novel targeted intravascular nanotherapy is safe, as organ morphology, inflammation, and complement and platelet aggravation were not impacted following administration in vivo. While there was a transient rise in the PTT, this returned to baseline within 3 days. Thus, no major safety concerns were found in our study.