Emerging Technology and Best Practices Seminar Series

Nanotechnology in Medicine: From Diagnostics to Therapeutics

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NEW GENERATION LIPOSOMES AS MULTIFUNCTIONAL PHARMACEUTICAL NANOCARRIERS

Dr. Vladimir Torchilin

Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, USA

Pharmaceutical nanocarriers currently in use and under studies, such as liposomes, micelles, polymeric nanoparticles and many others, demonstrate a broad variety of attractive properties, such as longevity in the blood; specific targeting to disease sites; enhanced intracellular penetration; contrast properties; stimili-sensitivity, sensitivity towards magnetic field, etc. More rarely pharmaceutical nanocarriers are available combining several of the listed properties. Long-circulating immunoliposomes capable of prolonged residence in the blood and specific target recognition represent one of few examples of this kind. At the same time, the engineering of multifunctional pharmaceutical nanocarriers combining several useful properties in one particle can significantly enhance the efficacy of many therapeutic and diagnostic protocols.

Within the frame of this concept, it is important to develop multifunctional stimuli-responsive nanocarriers, i.e. nanocarriers that, depending on the particular requirements, can possess the combination of the following abilities: 1. circulate long; 2. target the site of the disease; 3. respond local stimuli characteristic of the pathological site; 4. provide an enhanced intracellular delivery of an entrapped drug. Additionally, these carriers can be provided with contrast moieties to follow their biodistribution and target accumulation. To be able to behave this way, drug carrier should simultaneously carry on its surface various moieties capable of functioning in a certain orchestrated order. Evidently, different modifiers can be combined on the surface of the same nanoparticular drug carrier providing it with a combination of useful properties (for example, longevity and targetability, targetability and stimuli-sensitivity, or targetability and contrast properties). We will consider here the current status and possible future directions in the emerging area of multifunctional nanocarriers using liposomes as an example and with primary attention on the combination of such properties as longevity, targetability, intracellular penetration and contrast loading.

Thus, the potential of long-circulating liposomes as drug carriers may be still further improved by attaching targeting ligands, including specific antibodies, to their surface. Using PEG-PE activated at its distal end with p-nitrophenylcarbonyl group, we attached various ligands (in particular, nucleosome-specific monoclonal 2C5 antibody with broad anti-cancer specificity) to the surface of drug-loaded liposomes. Protein attachment does not affect the size and stability of drug carriers, and proteins attached to the surface completely preserve their specific activity. Anticancer drug-loaded 2C5 antibody-bearing liposomes provide sharp increase in killing cancer cells in vitro and in vivo.

An important problem in drug delivery is that drugs and DNA delivered inside cells via the receptor-mediated endocytosis usually undergo a substantial degradation in cell lysosomes. Although certain drug carriers such as pH-sensitive liposomes, may provoke endosome destabilization and facilitated drug release into the cytoplasm, additional methods for the intracellular drug delivery are still at large. The coupling of HIV-1 TAT protein, or shorter TAT peptides and some other arginine-rich positively charged peptides to various molecules, including peptides and proteins (enzymes), or even to small colloidal particles dramatically facilitates their intracellular delivery. Even 200 nm liposomes can be successfully delivered into the cell cytoplasm if a sufficient number of TAT peptide molecules are attached to their surface. To apply such liposomes for intracellular delivery of DNA, TAT-liposomes have been prepared containing a sub-toxic amount of a positively charged lipid (less than 10% mol) and loaded with a plasmid encoding for the Green Fluorescent Protein (GFP). Their incubation with various cells resulted in an efficient transfection of all investigated cells (the green fluorescence of GFP appear in cell cytoplasm), while no or very little transfection was seen when cells were incubated with free plasmid or with TAT-free plasmid-loaded liposomes. The successful cell trancfection was also achieved in vivo, by administering TAT-liposome/DNA complexes directly into tumors in mice.

Multifunctional liposomes have been also engineered capable of both, specific drug delivery to target cells and then inside tumor cells. Such long-circulating, specifically targeted, and capable of cell penetration liposomes were built in such a way that during the first phase of delivery, cell-penetrating function is shielded by the function providing organ/tissue-specific delivery (sterically-protecting polymer or antibody). Upon accumulating in the target, protecting polymer or antibody attached to the surface of the liposome via the stimuli-sensitive bond should detach under the action of local pathological conditions (abnormal pH or temperature) and expose the previously hidden second function allowing for the subsequent delivery of the carrier and its cargo inside cells. TATp-bearing rhodamine-labeled liposomes modified with pH-cleavable or non-cleavable PEG-PE were injected intratumorarly to Lewis lung carcinoma (LLC)-bearing mice or infused into Langendorff’s isolated rat heart. Cryofixed tumor or normoxic and ischemic myocardial tissue sections were observed under the microscope for the rhodamine fluorescence. While TATp-liposomes coated with uncleavable PEG-PE did not demonstrate significant internalization by tumor cells (after 6 hours) or cardiomyocytes (after 0.5 hour), TATp-liposomes coated with cleavable PEG-HZ-PE showed a pronounced internalization (red fluorescence inside cells),

The combination of targeted delivery of drug-loaded nanocarriers and their subsequent delivery inside cancer cells might significantly improve the efficiency of therapy.