Reduction-sensitive, robust vesicles with a non-covalently modifiable surface as a multifunctional drug-delivery platform.

The design and synthesis of a novel reduction-sensitive, robust, and biocompatible vesicle (SSCB[6]VC) are reported, which is self-assembled from an amphiphilic cucurbit[6]uril (CB[6]) derivative that contains disulfide bonds between hexaethylene glycol units and a CB[6] core. The remarkable features of SSCB[6]VC include: 1) facile, non-destructive, non-covalent, and modular surface modification using exceptionally strong host-guest chemistry; 2) high structural stability; 3) facile internalization into targeted cells by receptor-mediated endocytosis, and 4) efficient triggered release of entrapped drugs in a reducing environment such as cytoplasm. Furthermore, a significantly increased cytotoxicity of the anticancer drug doxorubicin to cancer cells is demonstrated using doxorubicin-loaded SSCB[6]VC, the surface of which is decorated with functional moieties such as a folate-spermidine conjugate and fluorescein isothiocyanate-spermidine conjugate as targeting ligand and fluorescence imaging probe, respectively. SSCB[6]VC with such unique features can be used as a highly versatile multifunctional platform for targeted drug delivery, which may find useful applications in cancer therapy. This novel strategy based on supramolecular chemistry and the unique properties of CB[6] can be extended to design smart multifunctional materials for biomedical applications including gene delivery.

Regulation of CD40 reconstitution into a liposome using different ratios of solubilized LDAO to lipids.

The integral membrane protein CD40 was found on the surface of B lymphocytes that interact with CD40L on T cells during the immune response. The hydrophobic transmembrane domains of membrane proteins can be stabilized in detergent or in lipid bilayers such as liposomes. Membrane proteins can be incorporated into the liposome in a similar fashion to the way they are handled in vivo. In this study, a large amount of full-sequence CD40 was produced using a bacterial system that contained a Mistic construct. The CD40 was then reconstituted into liposomes by detergent-mediated reconstitution. All stages in the process of liposome disruption with various detergent ratios were easily observed by monitoring the optical density. The structure of the liposome and the reconstitution of CD40 were confirmed by cryo-TEM. The results of the present study show that the detergent ratio had an effect on the structure of the liposome and the amount of CD40 that was reconstituted into the liposome.

Polymeric nanoparticles for protein kinase activity.

Nanoparticles were prepared from poly-ion complexes, possessing both PEI-FITC-(PKA-specific substrate) (kemptide) and PAA-TRITC, which produce intermolecular FRET; the nanoparticles were dissociated by phosphorylation, presented a strong FITC intensity and can be applied to high-throughput screening for large chemical libraries, for drug discovery of kinase inhibitors.

Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.

Gadolinium-coordinated elastic nanogels for in vivo tumor targeting and imaging.

Coordination polymer gels have been recognized as promising hybrid nanoplatforms for imaging and therapeutic applications. Here we report functional metal-organic coordinated nanogels (GdNGs) for in vivo tumor imaging, whose non-crystalline and elastic nature allows for long blood circulation, as opposed to the rapid systemic clearance of common nanohybrids with rigid/crystalline frameworks. The deformable structure of GdNGs was constructed by random crosslinking of highly flexible polyethyleneimines (PEI) with gadolinium (Gd(3+)) coordination. The in vitro characterization revealed that GdNGs have elasticity with an apparent Young's modulus of 3.0 MPa as well as minimal cytotoxicity owing to the tight chelation of Gd(3+) ions. In contrast to common T1-enhancing gadolinium complexes, GdNGs showed the capability of enhancing negative T2 contrast (r2 = 82.6 mm(-1)s(-1)) due to the Gd(3+)-concentrated nanostructure. Systemic administration of fluorescently labeled GdNGs with core and overall hydrodynamic sizes of ~65 and ~160 nm manifested efficient targeting and dual-modality (magnetic resonance/fluorescence) imaging of tumor in a mouse model. The minimal filtration by the reticuloendothelial system (RES) suggests that the structural deformability helps the large colloids circulate in the blood stream for tumor accumulation. The unusual performance of a large Gd(3+)-complexed colloid (minimal RES sequestration and high T2 contrast enhancement) represents the versatile nature of nanoscopic organic-inorganic hybridization for biomedical applications.

In vivo NIRF imaging of tumor targetability of nanosized liposomes in tumor-bearing mice.

To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.

Multi-core vesicle nanoparticles based on vesicle fusion for delivery of chemotherapic drugs.

The Pluronic nanoparticles (NPs) composed of Pluronic (F-68) and liquid polyethylene glycol (PEG, molecular wt: 400) containing docetaxel (DTX) were stabilized with the vesicle fusion. When DTX-loaded Pluronic NPs were mixed with vesicles in the aqueous medium, DTX-loaded Pluronic NPs were incorporated into vesicles to form multi-core vesicle NPs. The morphology and size distribution of multi-core vesicle NPs were observed using FE-SEM, cryo-TEM and a particle size analyzer. To apply multi-core vesicle NPs as a delivery system for DTX, a model anti-cancer drug, the release pattern of DTX was observed and the tumor growth was monitored by injecting the DTX-loaded multi-core vesicle NPs into the tail veins of tumor-bearing mice. We also evaluated the time-dependent excretion profile, in vivo biodistribution, circulation time, and tumor targeting capability of multi-core vesicle NPs using a non-invasive live animal imaging technology.

Paclitaxel-loaded Pluronic nanoparticles formed by a temperature-induced phase transition for cancer therapy.

We prepared nanoparticles by a temperature-induced phase transition in a mixture of Pluronic F-68 and liquid PEG (polyethylene glycol, molecular weight: 400) containing paclitaxel (PTX) with a fast, simple, continuous and solvent-free process. The liquid PEG is used as solubilizer of PTX and the polymer for the encapsulation of PTX is composed of Pluronic F-68. At the phase transition temperature, the polymer mixture was changed to the liquid phase, and stirring the liquid 0 °C to form Pluronic nanoparticles. The morphology and size distribution of the prepared Pluronic nanoparticles were observed using FE-SEM and TEM, and a particle size analyzer and cryo-TEM were used to observe the shape of paclitaxel-loaded Pluronic nanoparticles in an aqueous state. To apply Pluronic nanoparticles as a delivery system for cancer therapy, the release pattern of PTX, a model anti-cancer drug, was observed and the tumor growth was monitored by injecting the PTX-loaded Pluronic nanoparticles into the tail veins of tumor-bearing mice. We also evaluated the time-dependent excretion profile, in vivo biodistribution, circulation time, and tumor targeting ability of PTX-loaded Pluronic nanoparticles using non-invasive live animal imaging technology. In the early stage within 7h of release, the loaded PTX was rapidly released and the sustained release was observed for up to 48 h. In vivo studies, PTX-loaded Pluronic nanoparticles were observed with higher anti-tumor efficacy compared with PTX formulated in Cremophor EL.

Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump.

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.

Depletion of nucleophosmin via transglutaminase 2 cross-linking increases drug resistance in cancer cells.

It has been suggested that nucleophosmin has an anti-apoptotic function via Bax binding. We found that nucleophosmin is a substrate of transglutaminase 2 (TGase 2) in cancer cells. Increased expression of TGase 2 expression is highly associated with drug resistance, and polymerization of nucleophosmin by TGase 2 also can be correlated with the drug resistance of cancer cells. In the present study, an accumulation of nucleophosmin in cytosol was detected when doxorubicin was treated to cancer cells, and it was found, moreover, that an increase of cytosolic nucleophosmin can result in drug-induced apoptosis. Nucleophosmin was polymerized by TGase 2, and the polymerization was inhibited with the TGase 2 inhibitor, cystamine, in vitro. The nucleophosmin level in the cytosolic cell fraction was reduced when TGase 2 was expressed, and the reduced nucleophosmin level was rescued by cystamine treatment. Moreover, nucleophosmin cross-linked by TGase 2 was eradicated in MCF7 cells via the ubiquitin-proteasomal pathway. In parallel with this nucleophosmin-level restoration, the pro-apoptotic Bax protein level was increased. Therefore, depletion of cytosolic nucleophosmin by TGase 2 can decrease Bax protein stability and lead to anti-apoptosis. Drug-resistant cancer cells became sensitive to doxorubicin treatment when nucleophosmin was expressed in cytosol. Taking these results together, it can be concluded that TGase 2 inhibits accumulation of cytosolic nucleophosmin through polymerization, which results in drug resistance in cancer cells.