The current level of engineering pharmaceutical nanocarriers allows for drug delivery systems (DOS) demonstrating a combination of several desired properties. Ideally, """"""""smart"""""""" nanoparticular DOS should be able: (a) to accumulate in the required organ or tissue, and then (b) penetrate inside target cells delivering there its load (drug or DNA). Organ or tissue (tumor, infarct) accumulation could be achieved by the passive targeting via the enhanced permeability and retention (EPR) effect or by the antibody-mediated active targeting, while the intracellular delivery could be mediated by internalizable ligands or by cell-penetrating peptides (CPPs). For this, DOS should simultaneously carry on their surface various moieties capable of functioning in a certain orchestrated way, and be built in such a way that during the first phase of delivery, a cell-penetrating function is shielded by the function providing organ/tissue accumulation (sterically-protecting polymer or antibody). Inside the target, the protecting polymer or antibody attached to the surface of DOS via the stimuli- sensitive bond should rapidly detach under the action of local pathological conditions (abnormal pH or temperature) and expose the previously hidden function allowing for the delivery of the carrier and its cargo inside cells. So far, multiple and mainly unsuccessful attempts have been made to deliver various drug carriers (liposomes and micelles) directly into the cell cytoplasm bypassing the endocytic pathway, to protect drugs and DNA from the lysosomal degradation. CPPs (such as TAT peptide, TATp) can enter cell cytoplasm directly and have been successfully used for the intracellular delivery of small drug molecules, large molecules (enzymes, DNA) and nanoparticulates (quantum dots, iron oxide nanoparticles, liposomes). Currently, the next steps are required such as the development of CPP-based systems with controlled intracellular localization and the ability to deliver drug and DNA into drug resistant cancer cells. We were able to couple a large number of TATp molecules to a single liposome/micelle and achieved their efficient intracellular delivery; used TATp-liposomes/DNA complexes for an efficient and non-toxic cell transfection; and constructed multifunctional stimuli-sensitive pharmaceutical nanocarriers capable of switching their activity under acidic conditions. We have also in our possession monoclonal 2C5 antibody (mAb 2C5) recognizing a variety of tumors via tumor cell surface-bound nucleosomes (NS). We can also control the intracellular localization of nanocarriers. Our hypothesis is that the therapeutic activity of various Pharmaceuticals can be enhanced by loading them onto multifunctional stimuli-sensitive nanoparticular DOS allowing for prolonged circulation and target accumulation via the EPR effect and/or via specific antibody-mediated recognition, and subsequent fast CPP(TATp)-mediated internalization into target cells after the deprotection of previously hidden CPP function under the action of local stimuli. TATp-mediated delivery of DOS, such as liposomes and micelles, loaded with water-soluble and water-insoluble drugs and DNA, directly into the cell cytoplasm may be further influenced in terms of the intracellular trafficking, fate, and distribution by controlling DOS charge and composition. We propose: (a) to prepare and characterize liposome- and micelle-based multifunctional stimuli-sensitive DOS; (b) to develop DOS capable of drug/DNA delivery TO and INTO cancer cells using a combination of detachable PEG and/or cancer-specific monoclonal antibody and TATp; (c) to demonstrate the possibility of regulating the intracellular fate and distribution of TATp-liposomes/micelles by controlling their charge and composition; and (d) to show the efficiency of drug/DNA-loaded TATp-liposomes/micelles in controlling the growth of drug resistant cancer cells both in vitro and in vivo. ? ? ?
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