Spectramax m5 microplate spectrofluorometer

Eight batches of DOX-loaded PLGA microspheres were prepared as described above. To determine loading and loading efficiency, samples from each batch were tested. To determine loading, a sample of DOX-loaded microspheres (3–5 mg) post-lyophilization was dissolved in DMSO and the yield of DOX was calculated using a standard curve. Along with the standard curve samples, the test samples were measured for DOX at λ_ex485, λ_em570 using a SPECTRAmax M5 Microplate Spectrofluorometer (Molecular Devices, San Diego, CA). The yield of DOX was then divided by the weight of DOX-loaded microspheres (in the sample) to determine the loading per mg of microspheres. To determine loading efficiency, the known amount of total DOX-loaded PLGA particles from each batch post-lyophilization was multiplied by the calculated loading (as determined above) and then divided by the original 3 mg of DOX added to fabricate the particles. This value was then multiplied by 100 to achieve a percent value. The size (diameter) of the fabricated microspheres was calculated using scanning electron microscopic (SEM) images (Hitachi High-Technologies, Tokyo, Japan) and analysis using ImageJ software.

DOX-loaded microspheres ( ~3 mg) were added to amber microcentrifuge tubes containing 1 mL of PBS. After US application (as described in Section 2.3), the tubes were then placed in a shaker incubator set at 300 rpm and 37°C. Samples were collected after 1, 3, 6, 12, 24, 48, 72, 96, 168, 336, 672, 840, and 1008 h. Sampling involved centrifuging the microcentrifuge tubes at 180*g for 5 minutes from which 300 µl of supernatant was collected. Then 300 µl of fresh PBS was added back to the tubes and the microsphere pellets were resuspended. The collected samples were measured at λ_ex485, λ_em570 using a SPECTRAmax M5 Microplate Spectrofluorometer (Molecular Devices). These readings were then compared to a standard curve and normalized for photodegradation (data not shown) and weight to determine the amount of DOX released.

Release studies were performed with polymer and fluorophore combinations comprising either 4% (w/v) alginate (shell) or 1% (w/v) PLGA (core) with either fluorescein or rhodamine B. Alginate hydrogel was combined with rhodamine solution to yield a 0.80 mg/ml solution, while PLGA was solubilized and mixed with fluorescein or rhodamine B to yield a polymer-fluorophore mixture of 0.025 mg/ml and 0.80 mg/ml, respectively. Samples in one mL volumes of each desired polymer-fluorophore combinations were made, to which 3 mL of PBS was added and then placed in a shaker incubator set at 300 rpm and 37°C and were covered with aluminum foil. Samples were then collected after 1, 3, 6, 12, 24, 48, 72, 120, and 168 h. Sampling involved removing 300 μl out of each vial and replacing with 300 μl of PBS. The samples were measured for fluorescein (λ_ex494, λ_em521) and rhodamine B (λ_ex540, λ_em625) fluorescence using a SPECTRAmax M5 Microplate Spectrofluorometer (Molecular Devices). The readings were compared to a standard curve and photodegradation equations applied to normalize the results.