Evaluation of the Antitumor Effectiveness and Toxicity of pH-Sensitive Liposomes Coencapsulating Doxorubicin and Simvastatin in a Murine Breast Cancer Model

Figures

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  1. Temporal evolution of average diameter (a) and polydispersity index (b) of SpHL-D-S liposomes upon incubation at 37 °C and pH 7.4 with NaCl (blue), RPMI 1640 medium (red), or murine plasma (black) compared to SpHL-D-S control (without dilution). Data are presented as average ± standard deviation (n = 3). (c) Representative dynamic light scattering (DLS) measurements by the intensity of SpHL-D-S. Two peaks with size distributions at 20 and 137 nm were identified for SpHL-D-S incubated with plasma.

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  2. Cytotoxicity of different treatments with (a) free drugs (DOX and SIM) or (b) encapsulated in liposomes against 4T1 murine breast tumor cells. (c) IC50 values obtained for cell line 4T1 when exposed to different molar ratios between free DOX and SIM and coencapsulated in SpHL for 48 h. (d) Combination index values of DOX:SIM in free form and coencapsulated in long-circulating and pH-sensitive liposomes versus affected fraction. Three independent experiments were performed on different days and cell passages. Values are expressed as the average ± standard deviation (n = 3).

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  3. (a) Nuclear morphometric distribution cell nuclei of the 4T1 breast cell line exposed to 80 nM of treatment with free drugs and combined therapy. All groups were pretreated with 80 nM of free drug or combination for 48 h. N is normal; LR is large and regular; SR is small and regular; and I is irregular. (b) Percentage of cell migration of 4T1 cells after exposure to free drug treatment or combined therapy at the total concentration of 80 nM for 24 h. Data are expressed as mean ± standard deviation of three independent experiments. The symbols *, **, ***, and **** represent significant differences for p < 0.05, 0.01, 0.001, and 0.0001, respectively (ANOVA followed by Tukey’s multiple comparisons test).

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  4. Echocardiography parameter variation: (a) ejection fraction, (b) fractional area shortening, (c) left ventricular deformation, and (d) left atrial area. The parameters were measured in Swiss mice before and after repeated administration of SpHL, SpHL-S, SpHL-D, or SpHL-D-S, and the variation was obtained by ratio after/before × 100%. All data are represented as average ± standard deviation (n = 10). The symbols *, **, and *** represent significant differences for p < 0.05, 0.01, and 0.001, respectively (two-way ANOVA followed by Tukey’s multiple comparisons test).

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  5. Effect of different treatments on BALB/c mice bearing 4T1 tumor growth. (a) % variation tumor volume. (b) Tumor relative volume (TRV) analysis for each treatment. All data are represented as average ± standard deviation. The symbols *, **, and *** represent significant differences for p < 0.05, 0.01, and 0.001, respectively (two-way ANOVA followed by Tukey’s multiple comparisons test).

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  6. Photomicrographs of tumor tissue from mice in (a) control group, (b) free DOX, (c) SpHL-D, and (d) SpHL-D-S. Yellow arrows indicate the infiltration of tumor cells into muscle fibers of the tissue adjacent to the primary tumor. Hematoxylin–eosin staining, 40× magnification.

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  7. Representative histological sections of lungs and liver from female BALB/c mice carrying a 4T1 breast tumor treated with (a) control group, (b) free DOX, (c) SpHL-D, and (d) SpHL-D-S. (e) Incidence of metastasis in the lungs and liver after intravenous administration of different treatments in BALB/c mice subcutaneously transplanted with 4T1 breast cancer cells. Yellow arrows indicate metastatic foci. Hematoxylin–eosin staining, 20× magnification (lungs), 40× magnification (liver). Absence = no metastasis; focal = 1–3 metastasis foci; multiple = more than 4 metastasis foci.

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  8. (a) Percentage of variation in body weight of BALB/c mice subcutaneously transplanted with 4T1 breast cancer cells after different treatments for 8 days. Biochemical parameters (b) AST levels, (c) ALT levels, and (d) CK-MB levels evaluated in BALB/c mice bearing 4T1 tumor after different treatments. The symbols *, **, ***, and **** represent significant differences for p < 0.05, 0.01, 0.001, and 0.0001, respectively (two-way ANOVA followed by Tukey’s multiple comparisons test). Data are expressed as the mean ± standard deviation of the mean (n = 7).

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  9. Histological sections of hearts from female BALB/c mice carrying a 4T1 breast tumor treated with (a) the control group, (b) free DOX, (c) SpHL-D, and (d) SpHL-D-S. Yellow arrows indicate vacuolation of cardiomyocytes. Hematoxylin-eosin staining, 40× magnification.