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. 2013 Aug 6;8(8):e71196. doi: 10.1371/journal.pone.0071196

Figure 6. Spatiotemporal release of DZNep and gemcitabine using engineered nanoparticles reduced drug dose while potentiating chemosensitivity.

Figure 6

A. Spatial distribution of DZNep and gemcitabine within NPs. Co-encapsulating double-emulsion formulations were created using PLGA-b-PEG-OH (left), DSPE-PEG-OH (middle), and PLGA-b-PEG-TPP (right). 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. Bars, SD. n = 3. C. Release kinetics indicates the similarity between DZNep and gemcitabine release using the PLGA-b-PEG-OH formulation. D. Gemcitabine (top) and DZNep (bottom) in individual PLGA-b-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 (top) and TPP (bottom) formulations.G. Both engineered DSPE (top) and TPP (bottom) delayed-release NPs increased the cytotoxicity of MIA PaCa-2 even further compared with PLGA-b-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. Bars, SD. n = 3. *p<0.05, **p<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 (left) and TPP (right) delayed NPs producing a slightly greater reduction compared with simultaneous NPs.