Targeting Ovarian Cancer Cells Overexpressing CD44 with Immunoliposomes Encapsulating Glycosylated Paclitaxel

Abstract

Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC₅₀) for gPTX range from 15⁻20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.

Keywords: CD44; glycosylated paclitaxel; liposome; modified paclitaxel; ovarian cancer; specific targeting.

Figure 1

SK-OV-3 cells are overexpressing CD44. (A) Western blot analysis of human ovary cancer derived cells probed with anti-hCD44 MAb and human beta-actin antibody. (B) Relative intensity of the bands of CD44 to beta-actin in Western blot was densitometrically analyzed by ImageJ. (C) Relative gene expression levels of CD44 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by RT-qPCR. The data presented as the mean ± SD from three independent experiments. The statistical significance in mean values of more than two groups was determined using one-way analysis of variance (ANOVA) and Dunnet multiple comparison test using CD44 expression of SK-OV-3 cells as control; (****) p < 0.001.

Figure 2

SK-OV-3 cells contain CD44⁺/CD24⁻ population as well as CD44⁺/CD24⁺ population. SK-OV-3, OVCAR-3, and OVK18 cells were analyzed by flow cytometry by staining for CD44 and CD24. The margins of CD24 and CD44 for each cell line were set up by non-stained cells as the negative control shown at the bottom of each analysis. Most of the population in SK-OV-3 cells were found CD44 positive.

Figure 3

SK-OV-3 cells and OVK18 cells sensitive to paclitaxel and glycosylated paclitaxel. (A) Paclitaxel (PTX) and glycosylated paclitaxel (gPTX) sensitivity graph, cytotoxicity of both drug was assessed on SK-OV-3 and OVK18 cells by MTT assay after 72h drug treatment. (B) IC₅₀ value of gPTX and PTX detemined by graph (A). The data presented as the mean ± SD from three independent experiment.

Figure 4

Immunoliposome enhanced cellular uptake in CD44 postive cells. (A,C) Confocal Microscopy image after 2 h treatment FAM-L and FAM-IL, Each scale bar shows 20 µm. (B,D) Flow cytometry analysis after 1 h and 3 h treatment FAM-L and FAM-IL. FAM-L and FAM-IL were evaluated for the cellular uptake in SK-OV-3 (A,B) and OVK18 (C,D), SSC-A is side scatter area. Data are representative of three replicates.

Figure 5

Transmission electron microscopy (TEM) images of liposome encapsulating gPTX. gPTX-IL showed unilamellar vesicles with diameter of approximately 100 nm similarly to gPTX-L. Each scale bar shows 100 nm.

Figure 6

gPTX-IL exhibited the lowest inhibition concentration (IC₅₀) in SK-OV-3 cells. In vitro cytotoxicity IC₅₀ of gPTX in different formulation after 24 h (A) and 72 h (B) of exposure to SK-OV-3 cells and OVK18 cells was evaluated. The data presented as the mean ± SD (n = 3). The statistical significance in mean values of more than two groups was determined using one-way analysis of variance (ANOVA) and Dunnet multiple comparison test using IC₅₀ value of gPTX-IL treatment as control, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; (****) p < 0.001; (NSD) no significant difference.

Figure 7

gPTX-IL suppressed tumor growth in the most effective manner in vivo. gPTX-IL (open circle), gPTX-L (open triangle), naked gPTX (open square), CEP-IL (immunoliposome of CEP solvent) (closed circle), Cremophor EL®, ethanol, and phosphate buffered saline (PBS) (CEP, closed square), or PBS (cross) was intravenously injected at day 0, 4, 8, 12, 16, and 20 (indicated by vertical arrows). (A) The effect of different formulations of gPTX on the volume of tumors. (B) Relative tumor volume at day 30. gPTX-IL was the most effective formulation to suppress the growth of tumor. The statistical significance in mean values of more than two groups was determined using one-way analysis of variance (ANOVA) and Dunnet multiple comparison test using relative tumor volume of gPTX-IL treatment as a control, (*) p < 0.05; (**) p < 0.01; (****) p < 0.001. (C) The tumors from the experiment (A) representing each group were displayed exhibiting the effect of each formulation of gPTX. Data are expressed as the mean with ± SD where n = 4.

Figure 8

gPTX-IL treatment showed no significance side effects. gPTX- IL (open circle with line), gPTX-L (open triangle), naked gPTX (open square), CEP-IL (closed circle), CEP (closed square), or PBS (cross) was intravenously injected at day 0, 4, 8, 12, 16, and 20 (indicated by vertical arrows). (A) Change of body weight of mice bearing tumors. (B) Relative body weight at day 30. The statistical significance in mean values of more than two groups was determined using one-way analysis of variance (ANOVA) and Dunnet multiple comparison test using relative body weight of PBS treatment as a control, NSD, no significant difference. (C) H & E staining of some vital organs of the drug treated animal groups, each scale bar of liver and kidney section shows 64 µm and spleen section shows 129 µm.