Long-circulating liposomes increase the duration of drug action by maintaining drug levels in the body and decrease the toxicity and other undesirable side effects of various drugs. They also demonstrate the ability to accumulate non-specifically in tissues with """"""""leaky"""""""" vasculature, such as infarcted areas and tumors, via the enhanced permeability and retention (EPR) effect. Long-circulating liposomes can also be made targeted by attaching specific ligands, such as monoclonal antibodies to their surface. In our earlier experiments, we have demonstrated the ability of long-circulating liposomes modified with appropriate antibodies to specifically deliver their load to ischemic areas and tumors in various models. Intracellular transport of different biologically active molecules is one of the key problems in drug delivery. It is of special importance in the delivery of DMA and various drug targeting intracellular sites, such as pro-apoptotic peptides. Multiple and only partially successful attempts have been made to deliver various drugs and drug carriers (liposomes) into the cell cytoplasm to enhance the efficiency of the therapy. Interestingly, it was recently demonstrated that certain proteins, such as trans-activating transcriptional activator (TAT) from HIV-1 and shorter peptides known as Cell Penetrating Peptides (CPPs), can enter cell cytoplasm and even target cell nuclei. CPPs have been successfully used for the intracellular delivery of a variety of loads via the energy-dependent macropinocytosis with subsequent escape from endosomes into the cytoplasm (large molecules and nanoparticulates) or via direct transduction involving electrostatic interactions and hydrogen bonding (small molecules). Currently, the next steps required in this area are specifically aimed to deliver DNA into various cells for desired transfection and deliver various pro-apoptotic drugs into cancer cells to treat tumors. In our recent experiments, we have been able to couple TAT peptide (TATp) to liposomes and achieve an efficient intracellular delivery of liposomes including DNA-loaded liposomes. We hypothesize that the modification of long-circulating (immuno)liposomes with CPPs can improve intracellular delivery of drugs and DNA aimed to protect or genetically modify ischemic cardiac cells; similar constructs can enhance the killing of cancer cells by intracellular delivery of pro-apoptotic peptides. In the present study we propose: (a) to prove that TATp-modified long-circulating (immuno)liposomes can efficiently deliver drugs, such as ATP, and DNA into ischemic cardiomyocytes in vitro, in a Langendorff isolated heart model, and in vivo; (b) to prepare long-circulating liposomes simultaneously bearing on their surface TATp and antibodies specifically recognizing ischemic cells (mAb 2G4) and capable of sequential delivery of such liposomes into the ischemic areas and then into the ischemic cells both in vitro and in vivo; and (c) to demonstrate the general applicability of this approach by preparing cancer cell-targeted long-circulating TATpliposomes loaded with pro-apoptotic peptide and demonstrate an increased level of cancer cell killing by such preparations in vitro and in vivo. This proposal is expected to develop a new generation of long-circulating liposomes additionally modified with TAT peptide and capable of the efficient intracellular delivery of various drugs and DNA into normal and ischemic cardiomyocytes and cancer cells in various experimental settings in vitro and in vivo. We also expect to develop early approaches for gene therapy of the cardiac muscle and obtain the preparations for enhanced killing of cancer cells via the intracellular delivery of pro-apoptotic peptides by means of TAT peptide-modified long-circulating (immuno)liposomes.
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