459651 Enhancing in Vitro Macrophage Drug Delivery Efficiency Via Co-Localization of Cells and Drug-Loaded Microcarriers in 3D Ultrasound Standing Wave Field
Materials and Methods: The calcein-AM-loaded PLGA microparticles (CAPMs) were fabricated by single oil-in-water emulsification in association with a solvent evaporation approach. The calcein-AM with expression of green fluorescence (GF) was served as the model drug. To investigate the efficacy of cellular uptake of CAPMs under USWF exposure, canis macrophages (DH82 cells) were exposed to 1-MHz USWF with output intensity of 0.5 W/cm2 for 0, 1, 3, 5, 10, and 15 min, respectively, following wash twice with PBS and incubation at 37°C for an additional 24 h. The drug delivery efficiency which was represented by the intensity of green fluorescence expressed (i.e., relative fluorescence units; RFUs) was detected using a fluorescent microscope and quantitatively analyzed through flow cytometry.
Results and Discussion: Based on the phase-contrast microscopic and SEM detections, the produced CAPMs remained intact spheroids without deformation after the fabrication process including centrifugation and vacuum-aid filtration. The mean size of the CAPMs was 2.68 ± 0.07 μm in which > 90% of the particles were in the range of 1 – 10 μm based on the Stable Light Scattering analysis. The mean surface charge of the CAPMs was -91.8 ± 2.82 mV, and the encapsulation efficiency of the calcein-AM was 76.8 ± 3.2%. Based on the delayed and prolonged GF expression in the CAPMs-treated DH82 macrophages, our data showed that the PLGA microspheres were able to protect the encapsulated calcein-AM molecules from enzymatic digestion in the phagocytic endolysosomal system and thus the effect of GF expression can be extended. This is particularly important for macrophage drug delivery because mostly the exogenous molecules are often quickly degraded by the phagocytic endolysosoms. Through the fluorescent microscopic and flow cytometric analyses, our results showed that both DH82 macrophages and CAPMs can be quickly brought to acoustic pressure nodes within 20 sec under USWF exposure, and were consequently aggregated throughout the time course. In this study, the efficacy of cellular uptake of CAPMs enhanced with increased USWF exposure time that a three-fold augmentation (P < 0.05) was obtained after 15 min of USWF exposure. We further demonstrated that the enhanced CAPM delivery efficiency was mainly contributed by the co-localization of cells and CAPMs due to USWF exposure, rather than from sonoporation..
Conclusions: In summary, the developed USWF-mediated microparticulate delivery approach provides a feasible means for macrophage drug delivery.
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