Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention

Figures

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  Figure 1. Liposome characterization. (a) Schematic representation of free Ac-SDKP, a liposome (a lipid bilayer nanoparticle), and L-Ac-SDKP (Ac-SDKP inside the liposome). (b,c) Representative graphs of liposome size distribution and zeta potential, respectively, by nanoparticle tracking analysis. Comparative data for empty liposome (L-PBS) and L-Ac-SDKP for (d) size, (e) polydispersity, and (f) zeta potential. (g), Ac-SDKP concentration in the liposomal formulations (L-PBS vs. L-Ac-SDKP). Cryo-TEM images of L-Ac-SDKP (h) and empty liposome (L-PBS) (i) and a comparative data for size (j). Scale bars, 60 nm.

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  Figure 2. In vitro uptake of free Ac-SDKP and L-Ac-SDKP. RAW 263.2 macrophages (a,b) and primary mouse cardiac fibroblasts (c,d) were incubated with FITC-labeled Ac-SDKP (0.24 µg/mL) with or without liposomes (labeled with Rh-PE) for 4 h. Colocalization of the Ac-SDKP and liposomes was determined by the presence of FITC and Rh-PE positivity. Representative fluorescence images of FITC (green), Rh-PE (red), and DAPI (blue) and the composite demonstrating colocalization of the Ac-SDKP and liposomes within the cells with perinuclear accentuation within the macrophages (a) and nuclear localization within the fibroblasts (c). Scale bar, 50 μm. Representative flow cytometry results corresponding to Ac-SDKP (FITC, green) and L-Ac-SDKP (FITC + Rh-PE, yellow) uptake by macrophages (b) and fibroblasts (d). The gating was set to show the FITC+ Rh-PE− cell population in green, FITC− Rh-PE+ population in red, and FITC+ Rh-PE+ population in yellow; FITC− Rh-PE− (viable cell nuclei) cells are in blue. (e,f) show percent cell viability in raw macrophages and human cardiac fibroblasts with empty liposome (L-PBS), Ac-SDKP (0.24 ug/mL), and L-Ac-SDKP treatment (n = 7–8 in each treatment). (g) represents…

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  Figure 3. L-Ac-SDKP bioavailability after daily i.p. injection in mice with and without left chest radiation. Biodistribution of Ac-SDKP in the serum and heart was evaluated after four i.p. injections given daily one day after thoracic irradiation. (a), representative IVIS spectrum images (b), and biofluorescence quantification (c) of the tissues demonstrating fluorescence intensity in animals receiving L-Ac-SDKP with and without radiation normalized to that in PBS-treated (vehicle) controls. The coloring corresponds to the spectrum gradient bar for the radiant efficiency unit (p/s/cm2/sr). Scale bar, 5 mm.

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  Figure 4. Cardiac tissue and serum bioavailability after L-Ac-SDKP i.p. injection at intermittent dose in mice after 45 Gy chest radiation. Experimental timeline for data shown in panel (a). (b–e) Ac-SDKP levels in tissue homogenates (heart, left lungs, right lungs and serum) from irradiated mice treated with vehicle (PBS) or L-Ac-SDKP (n = 4) after three daily injections followed by a fourth dose 4 days later.

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  Figure 5. Effects of L-Ac-SDKP on cardiac function, macrophage infiltration, and fibrosis in mice after acute MI. C57BL/6J mice received Ac-SDKP or L-Ac-SDKP via i.p. injections. (a) Experimental timeline. (b) Survival curves of mice without MI, with MI, and with MI treated with L-Ac-SDKP or the free peptide. Echocardiography analyses of ejection fraction (EF) (c) and fractional shortening (FS) (d) in the L-Ac-SDKP- and Ac-SDKP-treated mice compared to sham and untreated control mice (n = 5–9). (e) Immunohistochemical analysis of LV sections showing infiltration of CD163+ macrophages in experimental mice (n = 5–9). Scale bar, 20 µm. (f) Interstitial collagen volume fractions and quantitative data of fibrosis in experimental mice (n = 5–8). Scale bar, 20 µm.

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  Figure 6. Effects of L-Ac-SDKP on fibroinflammatory gene changes 5 weeks after MI. Relative expression levels of genes predominantly involved in fibrotic (a–h) and inflammatory (i–q) processes in the myocardial tissues of mice without MI and those with MI untreated or treated with L-Ac-SDKP or Ac-SDKP (n = 5–9, L-Ac-SDKP MI vs. Ac-SDKP MI).