Functionalization of nanoparticles with targeting ligands

Functionalization of nanoparticles with vascular targeting ligands was accomplished via a sulfo-SMCC crosslinker. The nanoparticle composites, SPIO-NH₂ and NC-NH₂, had terminal amine groups that are readily available for conjugation with available thiol groups on the cysteine end of the targeting ligands. Here, P-selectin-targeting peptide CDAEWVDVS and fibrin-targeting peptide CREKA were used. Briefly, sulfo-SMCC contains two functional terminal groups on contralateral sides; an amine-reactive N-hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. First, sulfo-SMCC in a 2-molar excess to available amine groups on the SPIO-NH₂ solution and allowed to react for 30 minutes while shaking. Next, a 2:3 molar excess of sulfo-SMCC to targeting ligand was added and allowed to react for an additional 2 hours while shaking. The functionalized product was dialyzed against PBS using a 100,000 Da MW cut-off dialysis bag to remove excess peptide and crosslinker. For dual-ligand functionalization equal molar peptides were added with the overall molar excess to sulfo-SMCC still at a 2:3 molar ratio (2-moles sulfo-SMCC:1.5 moles fibrin-targeting peptide:1.5 moles P-selectin targeting peptide).

Bio-Rad DC protein assay was used to quantify the total number of conjugated peptides. Here, 200 μL of Bio-Rad dye solution (1 to 2 parts Bio-Rad dye and water) was added to an 800 μL solution of 10 mg/mL particles and vortexed. The absorbance of the sample was obtained at 595 nm after incubating the sample for 15 minutes. The absorbance value was compared to a standard curve, which was obtained by measuring the known absorbance of known concentrations of CREKA, P-selectin peptide or both.