Doxorubicin in TAT peptide-modified multifunctional immunoliposomes demonstrates increased activity against both drug-sensitive and drug-resistant ovarian cancer models

Abstract

Multidrug resistance (MDR) is a hallmark of cancer cells and a crucial factor in chemotherapy failure, cancer reappearance, and patient deterioration. We have previously described the physicochemical characteristics and the in vitro anticancer properties of a multifunctional doxorubicin-loaded liposomal formulation. Lipodox(®), a commercially available PEGylated liposomal doxorubicin, was made multifunctional by surface-decorating with a cell-penetrating peptide, TATp, conjugated to PEG 1000-PE, to enhance liposomal cell uptake. A pH-sensitive polymer, PEG 2000-Hz-PE, with a pH-sensitive hydrazone (Hz) bond to shield the peptide in the body and expose it only at the acidic tumor cell surface, was used as well. In addition, an anti-nucleosome monoclonal antibody 2C5 attached to a long-chain polymer to target nucleosomes overexpressed on the tumor cell surface was also present. Here, we report the in vitro cell uptake and cytotoxicity of the modified multifunctional immunoliposomes as well as the in vivo studies on tumor xenografts developed subcutaneously in nude mice with MDR and drug-sensitive human ovarian cancer cells (SKOV-3). Our results show the ability of multifunctional immunoliposomes to overcome MDR by enhancing cytotoxicity in drug-resistant cells, compared with non-modified liposomes. Furthermore, in comparison with the non-modified liposomes, upon intravenous injection of these multifunctional immunoliposomes into mice with tumor xenografts, a significant reduction in tumor growth and enhanced therapeutic efficacy of the drug in both drug-resistant and drug-sensitive mice was obtained. The use of "smart" multifunctional delivery systems may provide the basis for an effective strategy to develop, improve, and overcome MDR cancers in the future.

Keywords: 2C5 mAb; Lipodox; SKOV-3 cells; TAT peptide; doxil; doxorubicin; multidrug resistance; multifunctional immunoliposomes; pH-sensitive polymers.

Figure 1. Fluorescence microscopy (A) and flow cytometry (B and C) analysis of cells treated with multifunctional Rhodamine-labeled liposomes. Fluorescence microscopy (A) of Rhodamine-PE labeled (HSPC-Chol-PEG) plain liposomes, TATp liposomes, mAb 2C5 liposomes or multifunctional liposomes, pre-incubated at pH 5 or pH 7.4 with SKOV-3 drug-sensitive and -resistant cells. Flow cytometry analysis (B and C) showed an increase in the geometric mean fluorescence intensity of SKOV-3 drug-sensitive (B, upper panel) and -resistant (C, lower panel) cells incubated for 1 h with the various formulations. Multifunctional carriers were incubated with cells following their pre-exposure to normal/acidic conditions for 1 h. (*P ≤ 0.05, n = 3, mean ± SD).

Figure 2. In vitro cytotoxicity of plain Lipodox, TATp-modified Lipodox, mAb 2C5-modified Lipodox, and multifunctional (TATp-modified, pH-sensitive, immuno)-Lipodox, preincubated at pH 5.0 or pH 7.4 for 1 h at 37 °C. Upper panel shows the cytotoxicity of the different preparations over a range of concentrations of doxorubicin. The lower panel compares the viability of cells at a concentration of 12.5 μg/ml of doxorubicin. (*P ≤ 0.05, n = 3, mean ± SD).

Figure 3. Effect of i.v. administration of liposomal preparations on tumor growth in nude mice bearing SKOV-3 drug-sensitive (A) and drug-resistant (B) tumors shown as percentage change in tumor volume from initial, calculated as (V_n − V₀)/V₀ × 100, where V₀ is the tumor volume on day zero of drug administration and V_n is the volume on day “n” after drug administration has begun. Arrows indicate day of treatment with the formulations given after tumor size of 100 mm³ was reached. (n = 5, mean + SD). ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.05 calculated from day 10 to day 20, by ANOVA using the tukey post-hoc test. For SKOV-3-sensitive tumors,*P ≤ 0.05 is from day 15 to day 20 between multifunctional Lipodox and Lipodox-2C5. (C and D) describe the final tumor weights in nude mice bearing SKOV-3 sensitive and resistant tumors measured on day 20, following intravenous administration of liposomal preparations as described previously (n = 5, mean + S.D., *P < 0.05).

Figure 4. Biodistribution of doxorubicin (Dox) in mice bearing SKOV-3 drug-resistant tumors. The following liposomal formulations were used: multifunctional TATp-modified pH-sensitive immuno-Lipodox (multifunctional Lipodox), Non-pH sensitive multifunctional immuno-Lipodox (nonsense-Lipodox) and plain Lipodox. The data are a mean for 4 animals ± SEM. Significant differences: *P = 0.025 and ^#P = 0.008.

Figure 5. Detection of apoptotic activity by fluorescence microscopy in SKOV-3 drug-sensitive (right) and -resistant (left) tumor sections as shown by TUNEL staining. Left panel in each set shows DAPI staining while right panel shows TUNEL staining.