Bleomycin in octaarginine-modified fusogenic liposomes results in improved tumor growth inhibition

Abstract

Bleomycin (BLM) is an example of an anticancer drug that should be delivered into cytosol for its efficient therapeutic action. With this in mind, we developed octaarginine (R8)-modified fusogenic DOPE-liposomes (R8-DOPE-BLM). R8-modification dramatically increased (up to 50-fold) the cell-liposome interaction. R8-DOPE-liposomes were internalized via macropinocytosis and did not end up in the lysosomes. R8-DOPE-BLM led to a significantly stronger cell death and DNA damage in vitro relative to all controls. R8-DOPE-BLM demonstrated a prominent anticancer effect in the BALB/c mice bearing 4T1 tumors. Thus, R8-DOPE-BLM provided efficient intracellular delivery of BLM leading to strong tumor growth inhibition in vivo.

Fig. 1. Interaction of R8-Liposomes with HeLa cells by FACS

HeLa cells were treated with liposomes (50 μg/ml) for 1, 4 or 4+20 hrs in complete DMEM. The fluorescence of cells was measured with a FACS. (A) A typical histogram of the cells treated with R8-liposomes for 4 hrs: (i) untreated cells and the cells treated with (ii) P-DOPE-liposomes; (iii) R8-PC-liposomes; (iv) R8-DOPE-liposomes. (B) The mean fluorescence intensity of the liposome-treated cells. The data are a mean for 3 experiments ± SEM.

Fig. 2. Intracellular localization of R8-liposomes

HeLa cells were treated with liposomes (50 μg/ml) for 1, 4 or 4+20 hrs in complete DMEM. (A) The intracellular localization of liposomal Rh (red) evaluated by confocal microscopy. (B) Z-stacked images of a cell treated with R8-DOPE-liposomes for 4+20 hrs (1–8: from bottom to top, section intervals 0.7 μm). (C) Orthogonal projections reconstructed from Z-stacked images of the cells treated with R8-DOPE-liposomes for 4+20 hrs. The nuclei were visualized by staining with Hoechst 33342 (blue).

Fig. 3. Colocalization of the liposomal RhB with macropinocytotic (A) and lysosomal (B) markers

(A) HeLa cells were treated with either P-DOPE- or R8-DOPE-liposomes in DMEM complemented with FITC-Dextran_70kD (0.35 mg/ml) for 4 hrs. (B) HeLa cells treated with R8-PC- or R8-DOPE- liposomes in complete DMEM for 4+20 hrs were stained with Lamp-2 mAb. The nuclei were visualized by staining with Hoechst 33342 (blue). Colocalization (yellow) of liposomal RhB-PE (red) with the organelle’s markers (green) was evaluated by confocal microscopy.

Fig. 4. Intracellular distribution of the lipophilic RhB and hydrophilic FITC-Dextran4kD liposomal markers without (A) or in the presence (B) of chloroquine

HeLa cells were treated with FITC-Dextran_4kDa-loaded R8-DOPE-liposomes in a medium without (A) or in the presence of 50 μM chloroquine (B) for 4 hrs. The cells were washed and incubated in liposome/chloroquine-free medium for an additional 20 hrs. Colocalization (yellow) of the liposomal RhB-PE (red) and FITC-Dextran_4kD(green) was evaluated by confocal microscopy. (C) Mean values of Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOP) were calculated by ImageJ software for 6 images from two different experiments ± SEM.

Fig. 5. Effect of BLM-loaded R8-liposomes on cell viability

4T1 cells were treated with BLM-loaded liposomes or free BLM for 24 hrs in complete DMEM. The cell viability was evaluated by the Cell-Titer Blue assay. The data are presented as liposomal lipid (A) or encapsulated BLM concentrations (B). The mean values are for 7 experiments ± SEM.

Fig. 6

DNA damage by BLM-loaded R8-liposomes. 4T1 cells were treated with BLM-loaded liposomes (0.5 mg/ml) or free BLM (at concentration equal to BLM content in the liposomal formulations) for 24 hrs in complete DMEM. The cells were trypsinized, fixed with 4% paraformaldehyde, permeabilized with proteinase K and then subjected to TUNEL assay. The TUNEL-positive cells (FL-1 channel) were examined by FACS analysis. (A) A typical histogram of the cells treated with liposomes or free BLM and subjected to TUNEL assay: (i) untreated and cells treated with: (ii) R8-DOPE-liposomes; (iii) P-DOPE-BLM-liposomes; (iv) R8-DOPE-BLM-liposomes. (B) The percent of TUNEL-positive cells. The data are a mean for 5 experiments ± SEM.

Fig. 7. Anti-tumor and pro-apoptotic effect of R8-DOPE-BLM in vivo

The BALb/c mice bearing 4T1 breast cancer tumor were tail vein administrated with PBS, free BLM, R8-DOPE-liposomes, P-DOPE-BLM-liposomes or R8-DOPE-BLM-liposomes five times with two day interval. (A) Tumor volumes. (B) The tumor weights after removal at day 22. The data are the mean for 8–9 animals ± S.D. (C) The frozen tumor sections were subjected to TUNEL staining and examined by fluorescence microscopy. The upper line shows the sections stained with DAPI and the lower line indicates the TUNEL staining.