Product recovery was by filtration and washing with 600 mL of distilled water. They were then oven-dried at 37 °C and stored in a dessicator until further analysis. For the NIMslurry formulation, 0.5 mL of the Nslurry was used instead of Ndried. HA-loaded microparticles were prepared for comparison

with the NIMs. These were prepared using a similar method to that used for the NIMs; however, in the absence of nanoparticles 0.015 g of HA was added directly to the 3 mL [o] phase of 1% w/w PLGA solution. Their average size was 113 ± 10 μm, with drug loading (see Section 2.4) of 3.43 ± 0.73%. Further studies to investigate how particle morphology and size could be manipulated were carried out with PLLA and PDLA (dissolved drug discovery in DCM). The PLA’s solutions were incorporated into the [o] phase with PLGA at a PLGA/PLA volume ratio of 1/2, all polymer solution at 1% w/w. Drug quantification was achieved using HPLC (Shimadzu HPLC system equipped with a SCL-10A system controller, LC-10AD pump, SIL-10AD auto injector, CTO-10A column

oven and SPD-10AV UV detector units) with a Sunfire™ column (C18 3.5 μm, 4.6 × 100 mm with a guard cartridge (4.6 × 20 mm) (Waters, UK). The chromatographic conditions were injection volume = 50 μL, flow rate = 1.0 mL min−1, mobile phase = 30/70 MeCN/NaOAc buffer (pH 2.65), and UV detection at λ = 248 nm. To determine drug loading, approximately 8–10 mg of drug-loaded particles was dissolved in 50 mL of MeCN. Prior to injection, 1 KU-57788 solubility dmso volume of the sample solution was mixed with 2 volumes of the mobile phase. Drug loading was defined as below: equation(1) %drug loading=[amount of drug/total dry particle mass]×100% In vitro drug release studies were carried out in a USP Type II dissolution apparatus. Approximately crotamiton 8–10 mg of drug-loaded particles was incubated in 1 L of citric acid buffer (pH 4, in which drug sink conditions could be readily maintained) at 37 °C and 150 rpm. Solution sampling was carried out at regular intervals. A 2 mL aliquot was collected at each sampling point and replaced

with an equal volume of fresh buffer. Drug concentration was determined using HPLC (as above). The particle size distributions of NIMs were measured using laser diffraction particle sizing (Mastersizer 2000, Malvern Instruments, UK) giving overall average from three independent formulations each measured at least three times (± standard error of the mean). Size analysis using photon correlation spectroscopy (High Performance Particle Sizer, Malvern Instruments, UK) showed the nanoparticles to be 513 ± 46 nm in z-average diameter. Fluorescent microscopy was carried out using an Axiolab (Carl Zeiss Ltd.) fluorescence microscope. Confocal imaging was done using a Carl Zeiss LSM 510 microscope equipped with an argon photon laser (laser power, 10–75%) with excitation wavelength, λ = 488 nm and LP 505 filter. Image viewing and processing were performed using LSM 510 software.