Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80\u201390 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy

The cuttlefish Sepia officinalis (Sepiidae, Cephalopoda) constructs cuttlebone from a liquid-crystal precursor

Cuttlebone, the sophisticated buoyancy device of cuttlefish, is made of extensive superposed chambers that have a complex internal arrangement of calcified pillars and organic membranes. It has not been clear how this structure is assembled. We find that the membranes result from a myriad of minor membranes initially filling the whole chamber, made of nanofibres evenly oriented within each membrane and slightly rotated with respect to those of adjacent membranes, producing a helical arrangement. We propose that the organism secretes a chitin-protein complex, which self-organizes layer-by-layer as a cholesteric liquid crystal, whereas the pillars are made by viscous fingering. The liquid crystallization mechanism permits us to homologize the elements of the cuttlebone with those of other coleoids and with the nacreous septa and the shells of nautiloids. These results challenge our view of this ultra-light natural material possessing desirable mechanical, structural and biological properties, suggesting that two self-organizing physical principles suffice to understand its formation.Spanish Ministerio de Ciencia e Innovacion [CGL2010-20748-CO2-01, CGL2013-48247-P, FIS2013-48444-C2-2-P]; Andalusian Consejeria de Innovacion Ciencia y Tecnologia [RNM6433]; (Sepiatech, PROMAR program) of the Portuguese Ministerio da Agricultura e do Mar, Portugal [31.03.05.FEP.002]; Junta de Andalucia [RNM363]; FP7 COST Action of the European Community. [TD0903]info:eu-repo/semantics/publishedVersio

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo, Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.AJOM was financed by a PhD Grant of the FPI program from the Spanish Ministerio de Ciencia e Innovación; TCB's PhD Grant belonged to the FPU Program of the same Ministry. AJOM and AGC were supported by Projects CGL2010-20748-C02-01 and CGL2013-48247-P of the mentioned Ministry, and RNM6433 of the Consejería de Economía, Innovación y Ciencia of the Junta de Andalucía. The European COST Action TD0903 contributed via two Short Term Scientific Missions to AJOM in FM's lab in Dijon

PH and Salt Effects on Surface Activity and Self-Assembly of Copolymers Containing a Weak Polybase

Copolymers with well-defined architectures, controlled molecular weights, and narrow molar mass dispersities (A) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The resultant polymers contain different combinations of the pH-responsive monomer 2-(diethylaminoethyl) methacrylate (DEAEMA), the hydrophobic comonomer butyl methacrylate (BMA), and a neutral hydrophilic stabilizing monomer polyethylene glycol monomethyl ether methacrylate (designated O950). Surface tension and cryo-TEM measurements of native and heavy-atom stained samples were used to characterize the pH and salt responsiveness of the different polymers as a function of their composition. These studies indicate that while the polymers predominately self-assemble to form spherical micelles, a narrow size distribution is observed in aqueous solutions of poly(O950)-b-(BMA) and poly(O950)-b-(DEAEMA-co-BMA), whereas a broad size distribution characterizes the assemblies of poly(O950)-b-(DEAEMA) and poly(DEAEMA-co-BMA). In the latter case, micelles having diameters around 15-25 nm are found along with smaller aggregates (about 10 nm) mostly arranged in elongated necklace-like structures. The pH and salt-responsiveness of the DEAEMA residue, as indicated by the surface activity of the copolymers, was found to depend on the nature of the additional components: covalently linked hydrophobic groups (BMA) moderated the pH response of the copolymer as compared to nonionic and hydrophilic groups as in poly(O950)-b-(DEAEMA). These results suggest that mutual interactions among the building blocks of self-assembling copolymers should be taken into account when designing responsive copolymers

Formation of tabular single-domain magnetite induced by Geobacter metallireducens GS-15

Distinct morphological characteristics of magnetite formed intracellularly by magnetic bacteria (magnetosome) are invoked as compelling evidence for biological activity on Earth and possibly on Mars. Crystals of magnetite produced extracellularly by a variety of bacteria including Geobacter metallireducens GS-15, thermophilic bacteria, and psychrotolerant bacteria are, however, traditionally not thought to have nearly as distinct morphologies. The size and shape of extracellular magnetite depend on the culture conditions and type of bacteria. Under typical CO(2)-rich culture conditions, GS-15 is known to produce superparamagnetic magnetite (crystal diameters of approximately <30 nm). In the current study, we were able to produce a unique form of tabular, single-domain magnetite under nontraditional (low-CO(2)) culture conditions. This magnetite has a distinct crystal habit and magnetic properties. This magnetite could be used as a biosignature to recognize ancient biological activities in terrestrial and extraterrestrial environments and also may be a major carrier of the magnetization in natural sediments

Nanoporous Structure and Medium-Range Order in Synthetic Amorphous Calcium Carbonate

We adopt a reverse Monte Carlo refinement approach, using experimental X-ray total scattering data, to develop a structure model for synthetic, hydrated amorphous calcium carbonate (ACC). The ACC is revealed to consist of a porous calcium-rich framework that supports interconnected channels containing water and carbonate molecules. The existence of a previously unrecognized nanometer-scale channel network suggests mechanisms of how additives can be accommodated within the structure and provide temporary stabilization, as well as influence the crystallization process. Moreover, while lacking long-range order, the calcium-rich framework in the ACC contains similar Ca packing density to that present in calcite, aragonite, and vaterite, yielding clues of how the amorphous material converts into the different crystalline forms. Our results provide a new starting point for advancing our understanding of biomineralization as well as the development of biomimetic approaches to next-generation materials synthesis. © 2010 American Chemical Society