Antitumor Efficacy, Pharmacokinetics, and Toxicity Studies of Novel Estrogen Receptors Targeted PEGylated Liposomes Encapsulating Paclitaxel and Cisplatin in SKOV-3 Tumor-Bearing Nude Mice, ICR Mice, and SD Rats

Figures

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  1. Schematic Illustration of ES-SSL-PTX/CDDP for Ovarian Cancer Therapy in Nude Mice

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  1. Cytotoxicity, combination index, and liposome characterization. (A) SKOV-3 cell viability was treated with a single drug or various ratios of combination PTX/CDDP for 48 h. Data were represented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. (B) IC50 values (μM) and CI values of various ratios of combination PTX/CDDP treated against SKOV-3 cells. Data were represented as mean ± SD, n = 3. *p < 0.05, compared with combination PTX/CDDP (1:2) group. (C) The morphology of ES-SSL-PTX/CDDP observed by TEM. Scale bar: 200 nm. Representative size distribution profiles were determined by DLS of (D) L-PTX/CDDP, (E) ES-L-PTX/CDDP, (F) SSL-PTX/CDDP, and (G) ES-SSL-PTX/CDDP. Release profiles of (H) PTX and (I) CDDP from different liposomal formulations (molar ratio of PTX to CDDP is 1:2) at 37 °C for 48 h. Data were represented as mean ± SD, n = 3. ***p < 0.001, compared with non-PEGylated liposome.

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  2. Stability of liposomes. (A) Changes in particle size of ES-SSL-PTX/CDDP in PBS, complete cell culture medium, and 10% FBS at 37 °C for 48 h. (B) Changes of PDI of ES-SSL-PTX/CDDP in PBS, complete cell culture medium, and 10% FBS at 37 °C for 48 h. (C) Changes in particle size and PDI of ES-SSL-PTX/CDDP at 4 °C for 28 days. (D) Changes in zeta potential of ES-SSL-PTX/CDDP at 4 °C for 28 days. (E) Leakage rate of PTX from different liposome formulations at 4 °C. (F) The leakage rate of CDDP from different liposome formulations at 4 °C. (G) The leakage rate of PTX from different liposome formulations at 25 °C. (H) The leakage rate of CDDP from different liposome formulations at 25 °C. Data were expressed as the mean ± SD, n = 3; *p < 0.05, ns = no significance.

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  3. in vitro cellular uptake of different liposomal formulations in SKOV-3 cells after (A) 1 h incubation, (B) 2 h incubation, (C) 3 h incubation, and (D) 4 h incubation observed by fluorescence microscope. Scale bar: 50 μm. (E) Mean fluorescence intensity quantified by ImageJ. Data were presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

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  4. Endocytosis mechanism of ES-SSL-PTX/CDDP. Fluorescence microscopy images captured under the fluorescence microscope (Ts2-FL, Nikon) after SKOV-3 cells were incubated with (A) ES-SSL-Rh B, (B) ES-SSL-Rh B plus Amiloride, (C) ES-SSL-Rh B plus Genistein, (D) ES-SSL-Rh B plus Sucrose, and (E) ES-SSL-Rh B plus ES. Scale bar, 50 μm. (F) Mean fluorescence intensity quantified by ImageJ. Data were presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

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  5. In vitro Cytotoxicity of PTX/CDDP and its liposomal formulations on SKOV-3 cells. Cell viability of SKOV-3 cells after incubation for (A) 24, (B) 48, and (C) 72 h incubation. Data were represented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. IC50 values were calculated after (D) 24, (E) 48, and (F) 72 h of incubation. Results were represented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the PTX/CDDP group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the L-PTX/CDDP group; && p < 0.01, compared with the ES-L-PTX/CDDP group; £ p < 0.05, compared with the SSL-PTX/CDDP group.

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  6. In vivo biodistribution of DiR, SSL-DiR, and ES-SSL-DiR. (A) IVIS images of SKOV-3 tumor bearing nu/nu mice at different time points after administration of DiR, SSL-DiR, and ES-SSL-DiR. Red circles represent the location of tumors, n = 3. (B) Histogram of DiR fluorescence intensity at tumor sites in vivo analyzed by ImageJ software. Data were represented as mean ± SD, n = 3. ***p < 0.001, compared with the DiR group; # p < 0.05, compared with the SSL-DiR group. (C) Ex vivo images of the heart, liver, spleen, lungs, kidneys, and tumor captured by the IVIS Spectrum, n = 3. (D) Quantitative analysis for the ex vivo tissues after 24 h was performed by ImageJ software. Data were represented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the DiR group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the SSL-DiR group.

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  7. In vivo antitumor efficacy of ES-SSL-PTX/CDDP. (A) Tumor volume changes of various groups. (B) The harvested tumors of various groups at the end of the antitumor experiment. (C) Tumor weights of various groups at the end of the antitumor experiment. (D) Tumor inhibition rate of various groups. (E) Images of H&E staining for tumor tissues. Scale bar, 100 μm. (F) Immunohistochemistry staining of Ki67 in tumor tissues. Scale bar, 100 μm. (G) Immunohistochemistry staining of PCNA in tumor tissues. Scale bar, 100 μm. (H) Quantitative Analysis of Ki67. (I) Quantitative analysis of PCNA. (J) Body weight changes of various groups. Data were shown as mean ± SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the Normal saline group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the PTX/CDDP group; & p < 0.05, && p < 0.01, &&& p < 0.001, compared with the L-PTX/CDDP group; £ p < 0.05, ££ p < 0.01, £££ p < 0.001, compared with the SSL-PTX/CDDP group.

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  8. In vivo pharmacokinetics and biodistribution study of PTX in different formulations. (A) Plasma concentration–time curve of PTX in ICR mice after intravenous administration of PTX/CDDP, L-PTX/CDDP, and ES-SSL-PTX/CDDP at equal doses of 9.0 mg/kg PTX and 6.0 mg/kg CDDP. The data were presented as mean ± SD, n = 3. Quantitative biodistribution data of PTX in different formulations at different time points in ICR mice, including (B) heart, (C) liver, (D) spleen, (E) lungs and (F) kidneys. Data were shown as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the PTX/CDDP group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the L-PTX/CDDP group.

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  9. In vivo pharmacokinetics and biodistribution study of CDDP in different formulations. (A) Plasma concentration–time curve of CDDP in ICR mice after intravenous administration of PTX/CDDP, L-PTX/CDDP, and ES-SSL-PTX/CDDP at equal doses of 9.0 mg/kg PTX and 6.0 mg/kg CDDP. The data were presented as mean ± SD, n = 3. Quantitative biodistribution data of CDDP in different formulations at different time points in ICR mice, including (B) heart, (C) liver, (D) spleen, (E) lungs and (F) kidneys. Data were shown as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the PTX/CDDP group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the L-PTX/CDDP group.

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  10. In vivo acute toxicity study. Survival curve and body weight change curve of healthy ICR mice after intravenous administration at different doses of (A) and (B), PTX/CDDP, (C) and (D), L-PTX/CDDP, and (E) and (F), ES-SSL-PTX/CDDP. The results were represented as mean ± SD, n = 10.

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  11. In vivo acute toxicity study. The results of hematological and blood biochemical tests of (A) White blood cell count (WBC), (B) Red blood cell (RBC), (C) Platelet (PLT), (D) Hemoglobin (HGB), (E) Blood urea nitrogen (BUN), and (F) Creatinine (Cre). Data were presented as mean ± SD, n = 10. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the Normal Saline group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the PTX/CDDP group; & p < 0.05, compared with the L-PTX/CDDP group; ns = no significance.