Fluorstar optima fluorescent plate reader

Drug release was quantified by incubating the particles in dialysis membranes with a 10,000 Da MWCO (Thermo Fisher Scientific, Heysham, UK) at 37 °C with agitation. The release of the tobramycin was quantified by incubating 3 mg of particles in 1 mL of PBS in the donor compartment with 5 mL of PBS in the receiver compartment. At each time point the PBS was collected and replaced with fresh PBS release medium. Quantification of the tobramycin release was performed by diluting the release medium 1:1 with boric acid (0.4 M pH 9.7) prior to derivatisation with ortho-phthaldialdehyde (80 mg) in a solution containing 1 mL of 95% ethanol, 200 µL boric acid (0.4 M pH 9.7), 400 µL of β-mercaptoethanol and 200 µL diethyl ether. The fluorescent derivative was monitored by fluorescence at λ_ex/λ_em 360/460 nm, respectively using a BMG-LABTECH Fluorstar Optima fluorescent plate reader (BMG-LABTECH, Aylesbury, UK).

Anti-neutrophil elastase activity was monitored by incubation with the human neutrophil elastase (HNE) specific substrate methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide (Sigma, Aldrich, UK). Inhibitions of HNE (Elastin products, Owensville, MO, USA) were measured in the presence and absence of both blank and SLPI conjugated particles. The assay was carried out by incubating 10 µL (10 mg/mL particles) of blank and SLPI conjugated particles containing 100 µg/mL SLPI with 4 µL of HNE (100 µg/mL). HNE activity was measured by the cleavage of chromogenic methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide substrate by adding 50 µL of 0.2 mM substrate in 0.1 M HEPES buffer containing 0.5 M NaCl. Assays were conducted at 37 °C and the formation of the fluorescent product (p-nitroaniline) was measured continuously at 405 nm on a BMG-Labtech Fluorstar Optima fluorescent plate reader.