Inlab routine pro ph probe

UV-Vis absorption studies were performed using a Nicolet Evolution 300 UV-Vis spectrophotometer (Thermo Electron Corporation, Madison, WI, USA). Diffuse reflectance UV-Vis (DR UV-Vis) spectra were acquired using a Praying Mantis accessory (Harrick Scientific Products, Inc., Pleasantville, NY, USA) and a Spectralon^® baseline. Over the course of 12 weeks, pH values were obtained using a Mettler Toledo Seven Easy pH meter equipped with a Mettler Toledo InLab^® Routine Pro pH probe (Mettler Toledo, Columbus, OH, USA). Dilutions for pH tests were prepared using ultrapure water to make 11.1% w/v solutions at room temperature. Viscosity was analyzed using a Fungilab Viscolead Pro rotational viscometer (Fungilab, Barcelona, Spain). Viscosity measurements were obtained for S-nitrosated and non-nitrosated, hyaluronic acid-free samples using spindles L4 Fungilab at 60 revolutions per min (rpm) and L3 Fungilab at 12 rpm, respectively. The sample temperature was controlled with a water bath and maintained at 25 °C.

As described previously,^{22 (link), 23 (link)} hydrogen ion concentration of dissolved PLGA MPs was measured and converted to average pH of the intraparticle microenvironment, based on the total aqueous volume of hydrated MPs. Briefly, 10 mg of MPs were incubated in 1 mL of PBS (pH 7.4) on a roto-shaker at 37°C. At predetermined time points, the MP suspensions were centrifuged, and the supernatant was removed. The remaining MPs and associated aqueous microenvironment were then dissolved in 800 uL of acetonitrile (ACN) by vigorous vortexing. Tubes were centrifuged a second time to remove any undissolved PLGA, and 800 uL of this ACN+PBS+PLGA solution was added to 200 uL of deionized water (DIW) prior to pH measurements with an InLab Routine Pro pH probe (Mettler Toledo, Columbus, OH). To determine the pH of the MPs and aqueous microenvironment, we obtained a correlation between the pH of lactic acid monomers in PBS and lactic acid monomers in a mixture of PBS, ACN, and DIW (comparable to the dissolved PLGA MPs). Based on the measured pH values and total aqueous volume of the hydrated MPs, average intraparticle pH could be estimated. Supernatant pH was also measured.