Spatiotemporal release of DZNep and gemcitabine using engineered nanoparticles reduced drug dose while potentiating chemosensitivity.

<p>A. Spatial distribution of DZNep and gemcitabine within NPs. Co-encapsulating double-emulsion formulations were created using PLGA-<i>b</i>-PEG-OH (<i>left</i>), DSPE-PEG-OH (<i>middle</i>), and PLGA-<i>b</i>-PEG-TPP (<i>right</i>). B and E. TEM illustrates the inner core and outer shell of all the double-emulsion NPs created. Insets show the NPs at higher magnification. <i>Bars</i>, SD. <i>n</i> = 3. C. Release kinetics indicates the similarity between DZNep and gemcitabine release using the PLGA-<i>b</i>-PEG-OH formulation. D. Gemcitabine (<i>top</i>) and DZNep (<i>bottom</i>) in individual PLGA-<i>b</i>-PEG-OH formulations distinctly increased cytotoxicity in MIA PaCa-2. Significance between nanoparticles and free formulation is shown. F. HPLC analyses demonstrate the rapid and sequential release of DZNep compared with gemcitabine in both DSPE (<i>top</i>) and TPP (<i>bottom</i>) formulations.G. Both engineered DSPE (<i>top</i>) and TPP (<i>bottom</i>) delayed-release NPs increased the cytotoxicity of MIA PaCa-2 even further compared with PLGA-<i>b</i>-PEG-OH NPs. Twenty-four hours after 5×10³ cells/well were seeded in a 96-well plate, cells were treated for 72 h. Cellular viabilities were measured using MTT assays. Cytotoxic IC₅₀ values are indicated. Significance between simultaneous and delayed NPs using the Student’s t test is shown. Bar graphs to the right indicate the relative levels of caspase-3 activity as measured by fluorescence intensity. Values were background-subtracted and are indicated as fold-change from simultaneous NPs to delayed NPs. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01. H. H3K27 trimethylation decreases with simultaneous and delayed-release NPs. MIA PaCa-2 was treated with empty, simultaneous, or delayed NPs as above and total cell lysates were analyzed for H3K27 trimethylation using Western Blotting. A clear reduction in H3K27 trimethylation was noticed in both simultaneous and delayed NPs, with DSPE (<i>left</i>) and TPP (<i>right</i>) delayed NPs producing a slightly greater reduction compared with simultaneous NPs.