2005) using a Hitachi U-1500 spectrophotometer, Hitachi Instruments (Schaumburg, IL). 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-to-8-fold lower than those of control doxorubicin-loaded liposomes, Doxil? or Doxil? modified Ac2-26 with a non-specific IgG. (Lee and Low 1995). Doxorubicin-loaded liposomes conjugated with folic acid were shown to be internalized upon their binding with folate receptors (Goren, Ac2-26 et al. 2000), suggesting the potential for such targeting and subsequent internalization strategy in the treatment of several MDR-tumors (Mamot, et al. 2003). This approach is additionally supported by the fact that endocytosis of the liposomal drugs is essential for bypassing multidrug resistant (MDR) efflux pumps, such as P-glycoprotein or Pgp, in drug-resistant tumor cells (Gabizon 2002, Reddy and Low 1998). Similar results have been obtained with doxorubicin-loaded long-circulating liposomes modified with RGD-peptide motif and capable of targeting the neovasculature of the angiogenic tumors (Xiong, et al. 2005). Using a small cell lung cancer cell line, it was shown that RGD-targeted liposomes were internalized much faster, delivered doxorubicin to the cell nuclei more efficiently, and were more cytotoxic compared to non-targeted liposomes (Moreira, et al. 2001). Doxorubicin-loaded liposomes modified with Fab fragments of anti-disialoganglioside antibodoes selectively and almost completely inhibited the metastatic growth of human neuroblastoma in nude mouse model (Pastorino, et al. 2003). From the list of targeting moieties, monoclonal antibodies and their fragments seem to have the highest potential in terms of specificity and variability (Torchilin 2000). Monoclonal antibodies have been obtained that can recognize specific antigens from the majority of known tumors, such as antibodies against ovarian cancer, prostate cancer or colorectal cancer (Agus, et al. 2000). Earlier, we have identified a family of natural antibodies with nucleosome-restricted specificity, which are capable of effective recognition and binding of a broad variety of live cancer cells (but not normal cells) via the nucleosomes originating from the apoptotically dying neighboring malignancy cells and attached to the surface of malignancy (but not normal) cells via characteristic nucleosome-binding sites (Iakoubov, et Ac2-26 al. 1995, Iakoubov and Torchilin 1998). In addition to their personal broad Ac2-26 anticancer potential (Chakilam 2004, Torchilin, et al. 2003), these antibodies and their representative, the monoclonal antibody 2C5 (mAb 2C5), becoming used in sub-therapeutic quantities, can serve as effective focusing on molecules for tumor-specific delivery of drug-loaded pharmaceutical nanocarriers (Torchilin, et al. 2003). To attach antibodies to Doxil? liposomes above the protecting coating of PEG, we have used earlier developed protocol of initial antibody changes with p-nitrophenyl-carbonyl-PEG-phosphatidyl ethanolamine (pNP-PEG-PE) conjugate (Torchilin, PGFL et al. 2001) with the subsequent incorporation of the revised antibody molecule into the membrane of PEGylated liposomes via the hydrophobic PE moiety. Earlier, we have acquired some encouraging initial data within the improved cytotoxicity of Doxil? revised by mAb 2C5 (Gupta, et al. 2005, Lukyanov, et al. 2004). Here, we present the results of our prolonged studies within the cytotoxicity of mAb 2C5-revised Doxil? towards a broad variety of tumor cell lines as well as within the mechanism of the internalization of mAb 2C5-PEG-liposomes by malignancy cells. 2. Materials and Methods 2.1. Materials Cholesterol (Chol), fully hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxy poly (ethylene glycol 2000)-1,2-distearoyl-release of doxorubicin from the different Doxil? formulations over a 48 hr period, was investigated in DMEM cell tradition medium with 10% FBS. One ml aliquots of liposomes at doxorubicin concentration of 0.5 mg/ml, diluted in the media, were sealed into dialysis tubes with the cutoff size of 12,000-to-14,000 Da. Then, the liposomes-loaded dialysis tubes were incubated in 50 ml of the press for 48 h at 37C, with continuous stirring at medium speed. At numerous time points, aliquots were withdrawn, and replaced with equal volume of the press. The doxorubicine concentrations were then measured at 485 nm (Xiong, et al. 2005) using a Hitachi U-1500 spectrophotometer, Hitachi Tools (Schaumburg, IL). The validity of the used protocol and the minimal influence of the medium components within the doxorubicin fluorescence have been shown earlier (Eliaz and Szoka 2001). 2.2.7. Cell cultures In order to investigate the broad tumor-specificity of the mAb 2C5-revised liposomes and their anticancer activity, several unrelated tumor cell lines have been used. Murine Lewis lung carcinoma (LLC), murine breast adenocarcinoma (4T1), murine colon cancer (C26), human being mammary adenocarcinoma (BT-20), human being prostate malignancy (Personal computer3), and estrogen receptor-sensitive human being breast carcinoma (MCF-7) were purchased from your American Type Tradition Collection (Manassas, VA). LLC, Personal computer3 and MCF-7 cells were managed in DMEM cell tradition medium supplemented with 10% FBS, 1 mM Na pyruvate, 50.