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Stable 30–50 nm polymeric polyethylene glycol–phosphatidylethanolamine (PEG–PE)-based micelles entrapping superparamagnetic iron oxide nanoparticles (SPION) have been prepared. At similar concentrations of SPION, the SPION-micelles had significantly better magnetic resonance imaging (MRI) T2 relaxation signal compared to ‘plain’ SPION. Freeze-fracture electron microscopy confirmed SPION entrapment in the lipid core of the PEG–PE micelles. To enhance the targeting capability of these micelles, their surface was modified with the cancer cell-specific anti-nucleosome monoclonal antibody 2C5 (mAb 2C5). Such mAb 2C5-SPION immunomicelles demonstrated specific binding with cancer cells in vitro and were able to bring more SPION to the cancer cells thus demonstrating the potential to be used as targeted MRI contrast agents for tumor imaging.

Surface architecture of pharmaceutical nanocarriers (using polymeric micelles as an example) and the length of the spacer group through which specific ligand is attached to the carrier surface determine the interaction of ligand-bearing nanocarrier with cells. We have prepared surface-modified polyethyleneglycol-phosphatidylethanolamine (PEG-PE) micelles containing TATp attached to PEG-PE with a PEG block longer or shorter (TATp-PEG(1000)-PE or TATp-PEG(3400)-PE) than the PEG block in the main micelle-forming material (PEG(750)-PE and/or PEG(2000)-PE). The length of the PEG spacer in TATp-PEG-PE should allow for a non-hindered interaction of TATp with the cell surface, but it should not be too long to allow for the conformational "folding in" of TATp moiety inside the PEG globule making it unable to interact with the cells. The "folding in" of the ligand attached to an unnecessary long PEG spacer was further supported by the fluorescence resonance energy transfer (FRET) study between fluorescently labeled lipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) inserted into the core of PEG(750)-PE micelles and micelle-incorporated rhodamine-labeled TATp-PEG-PE. Micelles containing rhodamine-labeled TATp-PEG-PE with the longest PEG spacer (3400 Da) demonstrated strongly enhanced quenching of NBD-PE fluorescence with rhodamine-TATp confirming the "folding in" of TATp moiety into PEG globule bringing it closer to the micelle core-incorporated NBD.

Long-circulating liposomes loaded with doxorubicin (Dox) were additionally modified with the prostate cell-specific monoclonal antibody 5D4 (mAb 5D4). The resultant Dox-loaded 5D4-immunoliposomes specifically recognized prostate cancer cell lines of several different types expressing the mAb 5D4 antigen, PSMA, and significantly enhanced cytotoxicity toward these cells compared with the non-targeted Dox-liposomes in vitro while no increased toxicity was observed toward non-prostate (lung) cancer cell line.

Dextran-polyethylene glycol (PEG) conjugates were synthesized by activating dextran hydroxy groups with carbonyldiimidazole, introducing amino groups by attaching ethylenediamine, and reacting amino groups with a succinimidyl-activated derivative of PEG. Conjugates with an average of 12 and 21 PEG (5 kDa) residues per single dextran (73 kDa) molecule were prepared. These conjugates have circulation half-lives of 5.3 h and 7.0 h, respectively, compared to 4.0 h for non-PEGylated dextran. The modification of dextran with PEG inhibits the uptake of polymer by the major organ of the reticuloendothelial system, the liver. Dextran-PEG conjugates may represent a convenient platform for long-circulating pharmaceutical preparations.

Two poorly soluble, potent anticancer drugs, paclitaxel and camptothecin, were successfully solubilized by mixed micelles of polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) and vitamin E. Drug containing micelles were additionally modified with anti-nucleosome monoclonal antibody 2C5 (mAb 2C5), which can specifically bring micelles to tumor cells in vitro. The optimized micelles had an average size of about 13-to-22 nm and the immuno-modification of micelles did not significantly change it. The solubilization of both drugs by the mixed micelles was more efficient than by micelles made of PEG-PE alone. Solubilization of camptothecin in micelles prevented also the hydrolysis of active lactone form of the drug to inactive carboxylate form. Drug loaded mixed micelles and mAb 2C5-immunomicelles demonstrated significantly higher in vitro cytotoxicity than free drug against various cancer cell lines.

Stable nanocolloids of insoluble drugs with very high drug content (up to 90% wt) can be easily and reproducibly prepared through the application of the layer-by-layer (LbL) technology, alternate adsorption of oppositely charged polyelectrolytes on the surface of drug nanoparticles produced by ultrasonication of larger drug crystals. Such polymeric coating prevents drug nanoparticle aggregation and creates a firm polymeric shell on their surface. Drug release rate from such nanocolloidal particles can be easily controlled by assembling multilayer shells with variable shell density and thickness. Various additional functions, such as specific targeting ligands, can be easily attached to the surface on nanocolloidal particles of poorly soluble drugs by using a polymer with free reactive groups for the "outer" coating. This may represent a novel approach to preparing convenient dosage forms of poorly soluble drugs.

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG(2000)-PE or PEG(5000)-PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 min) at lower pH values (pH 5.0-6.0), nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.

Mixed micelles prepared of poly(ethylene glycol)2000-phosphatidyl ethanolamine conjugate (PEG(2000)-PE) and d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) in 1:1 molar ratio have been investigated. Micelle formation was confirmed by NMR spectroscopy. CMC of the micelles was found to be 1.5 x 10(-5)M. Poorly soluble anti-cancer drug paclitaxel (PCL) was efficiently solubilized in 15 nm non-toxic PEG-PE/TPGS micelles. PCL entrapment was quite stable with only about 20% of the incorporated drug released from micelles after 48 h at 37 degrees C. In addition, PCL-containing PEG(2000)-PE/TPGS micelles were stable in vitro under various conditions modeling the physiological ones, in particular, at low pH values and in the presence of bile acids, which is especially important for their possible oral administration. Fluorescently labeled micelles demonstrated time-dependent internalization by human colon adenocarcinoma cell line, Caco-2. The internalization of PEG(2000)-PE/TPGS micelles loaded with P-glycoprotein (P-gp) substrate, rhodamine-123 (RH-123), opposite to the internalization of the free RH-123, was not influenced by the inhibition of the P-gp pump with verapamil hydrochloride, which assumes a P-gp-independent micelle internalization.