dioleoyl-phosphatidylethanolamine and octaarginine

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.

Topics: Animals; Apoptosis; Bleomycin; Cell Growth Processes; Disease Models, Animal; Female; HeLa Cells; Humans; Liposomes; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Oligopeptides; Phosphatidylethanolamines; Random Allocation; Xenograft Model Antitumor Assays

Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL or Lipodox) were surface functionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide was conjugated to the polyethylene glycol-dioleoyl phosphatidylethanolamine (PEG-DOPE) amphiphilic co-polymer. The resultant R8-PEG-PE conjugate was introduced into the lipid bilayer of liposomes at 2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modification did not alter the particle size distribution, as measured by Particle Size Analyzer and transmission electron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of the modified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular and intratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laser scanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes (Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes at higher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomes at tested time point of 1h. The significantly higher accumulation of loaded drug to its site of action for R8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 ± 7% for R8-Dox-L compared to 90.6 ± 2% for Dox-L at Dox dose of 50 μg/mL for 4h followed by 24h incubation) and enhanced suppression of tumor growth (348 ± 53 mm(3) for R8-Dox-L, compared to 504 ± 54 mm(3) for Dox-L treatment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeutic window of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.

Topics: Animals; Antineoplastic Agents; Cations; Cell Line, Tumor; Cell Separation; Doxorubicin; Drug Delivery Systems; Drug Design; Endosomes; Female; Flow Cytometry; Lipids; Liposomes; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Electron, Transmission; Neoplasm Transplantation; NIH 3T3 Cells; Oligopeptides; Phosphatidylethanolamines; Polyethylene Glycols