Comparative Study of the Pharmacological Properties and Biological Effects of Polygonum aviculare L. herba Extract-Entrapped Liposomes versus Quercetin-Entrapped Liposomes on Doxorubicin-Induced Toxicity on HUVECs

Figure 1. DLS analysis of the liposomal systems. Particle size distribution of the L1 (a), L2 (b), L3 (c), L4 (d), L5 (e) and L6 (f) formulations.

Figure 2. AFM images for the studied liposomes. The letters correspond to (A) L1, (B) L2, (C) L3, (D) L4, (E) L5 and (F) L6. The numbers correspond to (1) AFM image, (2) 3D image, (3) profile of the sample and (4) area selected for profiling.

Figure 3. The release of flavonoids from liposomes loaded with the PAH extract (L5 and L6) vs. liposomes loaded with quercetin (L3 and L4).

Figure 4. Cell viability of HUVECs treated with quercetin, PAH extract and different liposome formulations with quercetin and the PAH extract. HUVECs were exposed to quercetin and L3 and L4 formulations in concentrations ranging between 0.001-µg GAE/mL and 100-µg GAE/mL and to the PAH extract and L5 and L6 formulations in concentrations between 0.006-µg GAE/mL and 0.678-µg GAE/mL (data are presented as the mean of OD540 ± SD; n = 3 for each sample). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control, untreated cells.

Figure 5. Malondialdehyde levels in the cell lysates, and IL-6 secretion in the supernates of the HUVECs exposed to Doxo and quercetin, the PAH extract and different formulations of liposomes. (A) MDA increased significantly after Doxo administration and diminished in the cells treated with quercetin (p < 0.001), the PAH extract (p < 0.001), L3 and L4 with quercetin (p < 0.001) and L5 and L6 with the PAH extract (p < 0.001). (B) IL-6 secretion increased after Doxo exposure (p < 0.001) and diminished in the supernates of the cells treated with L3, L4 (p < 0.001), L5 and L6 (p < 0.05). The statistical significance of the differences between the treated and control groups was evaluated with one-way ANOVA, followed by Dunnett’s multiple range test. Data are presented as the mean and SD of triplicate samples. ### vs. control and * p < 0.05; *** p < 0.001 vs. Doxo-treated cells.

Figure 6. Caspases-3, -8 and -9 in the supernates of HUVECs exposed to Doxo and quercetin, the PAH extract and different liposomes formulations. Caspases-3 (A), -8 (B) and -9 (C) increased significantly after Doxo exposure and diminished both in cells treated with quercetin (p < 0.001) and L3 (p < 0.001). The PAH extract significantly reduced caspase-8 and caspase-9 (p < 0.001), while L5 and L6 decreased the caspase-3 and caspase-8 levels (p < 0.001). The statistical significance of the difference between the treated and control groups was evaluated with one-way ANOVA, followed by Dunnett’s multiple range test. Data are presented as the mean and SD of triplicate samples. ## p < 0.01; ### p < 0.001 vs. control and *** p < 0.001 vs. Doxo-treated cells.

Figure 7. NF-kB, pNF-kB, Nrf2 and γH2AX expressions in HUVEC lysates after exposure to Doxo and the treatments with quercetin; the PAH extract and the L3, L4, L5 and L6 formulations. Representative images of immunoblotting for NF-kB, pNF-kB, Nrf2, γH2AX and GAPDH in the HUVECs treated with quercetin, the PAH extract and liposomes loaded with quercetin and the PAH extract are shown in the upper panel (A), and the results of the statistical analysis for the ratio of Nrf2, γH2AX and GAPDH expressions are in the lower panels (C,D). For NF-kB and pNF-kB, the results were expressed as pNF-kB divided by the total NF-kB (B). The statistical significance of the differences between treated and control groups was evaluated with one-way ANOVA, followed by Dunnett’s multiple range test; # p < 0.05; ## p < 0.01 vs. control cells and * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Doxo-treated cells.