Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines - PubMed (original) (raw)

Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines

Tamer A Elbayoumi et al. Eur J Pharm Sci. 2007 Nov.

Abstract

Doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) were additionally modified with the nucleosome-specific monoclonal antibody 2C5 (mAb 2C5) recognizing a broad variety of tumor cells via the tumor cell surface-bound nucleosomes. These mAb 2C5-modified PEGylated liposomes demonstrated 3-8-fold increase in the in vitro binding and internalization by multiple cancer cell lines of diverse origins (murine LLC, 4T1, C26 and human BT-20, MCF-7, and PC3), as shown by flow cytometry (FACS) and epi and confocal microscopy. As a result, mAb 2C5-modified Doxil demonstrated significantly higher cytotoxicity towards various cancer cells, including those resistant to doxorubicin, than all control preparations. The specific internalization of the mAb 2C5-Doxil into cytosol, along with the nuclear localization of their drug load, inside the target cancer cells were mainly responsible the superior anticancer activity. The IC50 values of mAb 2C5-Doxil with various murine and human cancer cells were 5-8-fold lower than those of control doxorubicin-loaded liposomes, Doxil or Doxil modified with a nonspecific IgG.

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Figures

Figure 1

Figure 1

Flow cytometry analysis of the surface binding (A) and uptake (B) of mAb 2C5-targeted and control liposomes, labeled with FITC-PE, in murine breast carcinoma cells, 4T1 (I); murine colon carcinoma cells, C26 (II); and human prostate carcinoma cells, PC3 (III).

Figure 2

Figure 2

Fluorescence microscopy of 2 different carcinoma cell lines (murine C26, I; and human PC3, II), incubated for 1 hr at 37°C, with different Doxil™ formulations: mAb 2C5-mododified Doxil™ (A); IgG-modified Doxil™ (B); original Doxil™ (C). Row (1) represents doxorubicin distribution, images taken using Rh/TRITC filter; row (2) represents nuclei of C26 cells stained with DAPI stain; row (3) represents merged images.

Figure 3

Figure 3

Confocal fluorescence microscopy of C26 (A) and PC3 (B), cells incubated with mAb2C5-modified Doxil™ formulation for 1 and 2 hours respectively. Red color represents doxorubicin distribution, images taken using Rh/TRITC filter, row (1); Blue color represents nuclei of cells stained with DAPI stain, row (2); merged images with contrast bright light, row (3).

Figure 4

Figure 4

Time-dependent cell-associated doxorubicin fluorescence of cells incubated with various liposomal formulations at 37°C (lipid concentration is 0.25mg/ml), murine colon cancer (C26), panel A; human prostate carcinoma (PC3), panel B; (n=3)

Figure 5

Figure 5

In vitro cytotoxicity results using different cancer cell lines. Upper panels -cytoxic effect of different concentrations of IgG-Doxil™ (□) and mAb 2C5-Doxil™ (●) on cancer cells; lower panels – cytotoxicity of various preparations at the fixed concentration of IgG-Doxil™ and 2C5-Doxil™ (as 100 μg/ml free doxorubicin) and same concentration of 2C5-liposomes (as lipid). 4T1 murine cell line, I; murine C26 cell line, II; and human PC3 cell line, III.

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