Development of Brain Targeting Peptide Based MMP-9 Inhibiting Nanoparticles for the Treatment of Brain Diseases with Elevated MMP-9 Activity

Latent and active levels of cerebral matrix metalloproteinase 9 (MMP-9) are elevated in neurological diseases and brain injuries, contributing to neurological damage and poor clinical outcomes. This study aimed developing peptide-based nanoparticles with ability to cross the blood-brain-barrier (BBB) and inhibit MMP-9. Three amphiphilic peptides were synthesised containing brain-targeting ligands (HAIYPRH or CKAPETALC) conjugated with MMP-9 inhibiting peptide (CTTHWGFTLC) linked by glycine (spacer) at the N-terminus, and the peptide sequences were conjugated at the N- terminus to cholesterol. 19F-NMR assay was developed to measure MMP-9 inhibition. Cell toxicity was evaluated by the LDH assay, and dialysis studies were conducted with/without fetal bovine serum. An in vitro model was employed to evaluate the ability of nanoparticles crossing the BBB. The amphiphilic peptide (Cholesterol-GGGCTTHWGFTLCHAIYPRH) formed nanoparticles (average size of 202.8 nm) with ability to cross the BBB model. MMP-9 inhibiting nanoparticles were non-toxic to cells, and reduced MMP-9 activity from kobs of 4.5×10-6s-1 to complete inhibition. Dialysis studies showed that nanoparticles did not disassemble by extreme dilution (40 folds), but gradually hydrolysed by serum enzymes. In conclusion, the MMP-9 inhibiting nanoparticles reduced the activity of MMP-9, with acceptable serum stability, minimal cell toxicity and ability to cross the in vitro BBB model.