In order to develop targeted pharmaceutical carriers additionally capable of responding 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 7.5-8.0, 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-to-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 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.

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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.

Background Understanding hepatitis C virus (HCV) replication has been limited by access to serial samples of liver, the primary site of viral replication. Our understanding of how HCV replicates and develops drug resistant variants in the liver is limited. Methods We studied 15 patients chronically infected with genotype 1 HCV treated with telaprevir (TVR)/pegylated-interferon alfa/ribavirin. Hepatic fine needle aspiration was performed pretreatment and at hour 10, days 4 and 15, and week 8 after initiation of antiviral therapy. We measured viral kinetics, resistance patterns, TVR concentrations, and host transcription profiles. Results All patients completed all protocol defined procedures that were generally well tolerated. First phase HCV decline (baseline-treatment day 4) was significantly slower in liver than in plasma (slope plasma, −0.29; liver, −0.009 [p<0.001]) while second phase decline (post-treatment day 4 to 15) did not differ between the two body compartments (−0.11 and −0.15, respectively, p=0.1). TVR-resistant variants were first detected in the plasma, but not in the liver (where only wild-type virus was detected). Based upon NS3 sequence analysis, no compartmentalization of viral populations was observed between plasma and liver compartments. Gene expression profiling revealed strong tissue-specific expression signatures. Human intrahepatic TVR concentration, measured for the first time, was lower compared to plasma on a gram per milliliter basis. We found moderate heterogeneity between HCV RNA levels from different intrahepatic sites, indicating differences in hepatic microenvironments. Conclusion These data support an integrated model for HCV replication wherein the host hepatic milieu and innate immunity control the level of viral replication, and the early antiviral response observed in the plasma is predominantly driven by inhibition of hepatic high-level HCV replication sites.

Liposome loading with Gd via the membrane-incorporated polychelating amphiphilic polymers (PAPs) significantly increases the Gd content and relaxivity (T1 parameter) of PEGylated liposomes, which can be used as contrast agents for magnetic resonance imaging (MRI). Here, we demonstrate that such Gd-containing liposomes can be additionally modified with the monoclonal anticancer antibody 2C5 (mAb 2C5) possessing the nucleosome(NS)-restricted specificity via the PEG spacer. Liposome-bound antibody preserves its specific activity (ELISA) and such Gd-loaded PEGylated 2C5-immunoliposomes specifically recognize various cancer cells in vitro and target an increased amount of Gd to their surface compared to antibody-free Gd-liposomes or Gd-liposomes modified with tumor nonspecific antibody. Gd-loaded cancer cell-targeted immunoliposomes may represent promising agents for enhanced tumor MRI.

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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 surfacemodified 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 rhodamineTATp confirming the "folding in" of TATp moiety into PEG globule bringing it closer to the micelle core-incorporated NBD.