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In this study, transferrin (Tf)-modified poly(ethylene glycol)-phosphatidylethanolamine (mPEG-PE) micelles loaded with the poorly water-soluble drug, R547 (a potent and selective ATP-competitive cyclin-dependent kinase (CDK) inhibitor), were prepared and evaluated for their targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma cells, which overexpress transferrin receptors (TfR). At 10 mM lipid concentration, both Tf-modified and plain micelles solubilized 800 μg of R547. Tf-modified micelles showed enhanced interaction with A2780 ovarian carcinoma cells in vitro. The involvement of TfR in endocytosis of Tf-modified micelles was confirmed by colocalization studies of micelle-treated cells with the endosomal marker Tf-Alexa488. We confirmed endocytosis of micelles in an intact form with micelles loaded with a fluorescent dye and additionally labeled with fluorescent lipid. The in vitro cytotoxicity and in vivo tumor growth inhibition studies in A2780-tumor bearing mice confirmed the enhanced efficacy of Tf-modified R547-loaded micelles compared to free drug solution and to nonmodified micelles. The results of this study demonstrate the potential application of Tf-conjugated polymeric micelles in the treatment of tumors overexpressing TfR.

As part of our program to develop breast cancer specific therapeutic agents, we have synthesized a conjugate agent that is a conjugate of the steroidal anti-estrogen and the potent cytotoxin doxorubicin. In this effort, we employed a modular assembly approach to prepare a novel 11β-substituted steroidal anti-estrogen functionalized with an azido-tetraethylene glycol moiety, which could be coupled to a complementary doxorubicin benzoyl hydrazone functionalized with a propargyl tetraethylene glycol moiety. Huisgen [3 + 2] cycloaddition chemistry gave the final hybrid that was evaluated for selective uptake and cytotoxicity in ER(+)-MCF-7 and ER(-)-MDA-MB-231 breast cancer cell lines. The results demonstrated that the presence of the anti-estrogenic component in the hybrid compound was critical for selectivity and cytotoxicity in ER(+)-MCF-7 human breast cancer cells as the hybrid was ~70-fold more potent than doxorubicin in inhibition of cell proliferation and promoting cell death., (© 2012 American Chemical Society)

Nanotechnology is playing an increasing role in targeted drug delivery into pathological tissues. Drug-loaded pharmaceutical nanocarriers can be delivered into diseased sites by passive targeting (spontaneous accumulation of nanocarriers in the areas with affected vasculature) or by active targeting (via site-specific ligands attached to the surface of drug-loaded nanocarriers). Subsequent level of targeting requires cellular internalization of nanocarriers and their specific association with certain individual cell organelles. The control over intracellular distribution of pharmaceutical nanocarriers requires effective and noninvasive methods of their visualization inside cells. In an attempt to enhance cellular internalization of pharmaceutical nanocarriers and their association with mitochondria specifically, we have prepared three types of cationic liposomes and investigated their intracellular distribution. The analysis was performed using Raman microspectroscopy in order to provide morphological information as well as biochemical signatures of the sample. It was demonstrated that Raman microscopy allows evaluation of the extent of mitochondrial association depending on the liposome composition.

Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, whereafter successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy, and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal antibody 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound specifically with biotin-agarose. The study on the pH-dependence showed that almost 80% of the hydrazone bond was cleaved after rather brief preincubation of the immunoliposomes at pH 5 for 0.5 to 1 h. Fluorescence microscopy and flow cytometry analysis of cancer cells (HeLa and MCF-7) treated with cancer cell-specific targeting ligand mAb 2C5-bearing liposomes showed enhanced cellular binding. Studies at low pH clearly confirmed the easy cleavability of the targeting ligand from the liposomes resulting in significantly less or virtually no cellular association.

6-Ascorbate-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6-ascorbate-PEG-PE) was synthesized according to a two-step procedure: (1) activation of ascorbic acid with bromine, and (2) synthesis of 6-ascorbate-PEG-PE by reacting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly(ethylene glycol))-2000] with an excess of 6-Br-ascorbic acid. The 6-ascorbate-PEG-PE was recovered by precipitation in diethyl ether and purified by gel permeation chromatography. The analysis of the product by 1H NMR and UV-vis spectroscopy confirmed the identity of the conjugate. Liposomes and PEG-PE-based lipid-core micelles were prepared by thin film hydration technique incorporating 6-ascorbate-PEG-PE as targeting moiety. The targeting properties of the ascorbate-decorated nanosystems were tested by fluorescence-activated cell sorting (FACS) analysis and fluorescent microscopy on a panel of tumor cell lines preliminary selected for their ability to express the SVCT2 ascorbate transporter. Cell lines had been selected on the basis of the immunological properties assessed by FACS, which showed that two glioma cell lines, C6 and F98, and fibroblasts NIH/3T3 express plasma membrane-associated SVCT2 transporter for reduced ascorbic acid. Ascorbate-decorated pharmaceutical nanocarriers were endowed with selective targeting properties toward the SVCT2 transporter expressed in glioma cell models. This study shows that SVCT2 transporter for ascorbic acid expressed both in peculiar epithelial cells of the choroid plexus responsible for the filtering of vitamin C into the central nervous system (CNS) and, in some brain tumor cell lines, can be conceivably exploited as a potential target for delivery of drug-loaded pharmaceutical nanocarriers to the brain.