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

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.

Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity.

pH-sensitive PEGylated (with PEG-PE) long-circulating liposomes (HSPC:cholesterol and Doxil®), modified with cell-penetrating TAT peptide (TATp) moieties and cancer-specific mAb 2C5 were prepared. A degradable pH-sensitive hydrazone bond between a long shielding PEG chains and PE (PEG(2k)-Hz-PE) was introduced. TATp was conjugated with a short PEG(1k)-PE spacer and mAb 2C5 was attached to a long PEG chain (2C5-PEG(3.4k)-PE). The "shielding" effect of TATp by long PEG chains was investigated using three liposomal models. At normal pH, surface TATp moieties are "hidden" by the long PEG chains. Upon the exposure to lowered pH, this multifunctional carrier exposes TATp moieties after the degradation of the hydrazone bond and removal of the long PEG chains. Enhanced cellular uptake of the TATp-containing immunoliposomes was observed in vitro after pre-treatment at lowered pH (using flow cytometry and fluorescence microscopy techniques). The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, multifunctional immuno-Doxil® preparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. In conclusion, a multifunctional immunoliposomal nanocarrier containing a pH-sensitive PEG-PE component, TATp, and the cancer cell-specific mAb 2C5 promotes enhanced cytotoxicity and carrier internalization by cancer cells and demonstrates the potential for intracellular drug delivery after exposure to lowered pH environment, typical of solid tumors.

Cell-penetrating TAT peptide in drug delivery systems: proteolytic stability requirements.

The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a 'short' PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function.