Mucoadhesive and mucopenetrating chitosan nanoparticles for glycopeptide antibiotic administration.

The use of nanoparticles (NPs) represents a useful strategy for peptide antibiotic delivery to mucosal membranes by either prolonging drug residence time at the target site (mucoadhesive NPs) or by enhancing diffusion across mucus layer to reach the underlying epithelium (mucopenetrating NPs). The purpose of this study was to design chitosan (CH) NPs and to evaluate their employment as mucoadhesive and/or mucopenetrating systems for vancomycin (VM) delivery. NPs were prepared by ionic gelation of CH with sodium carboxymethylcellulose (CMC), sodium alginate (ALG), sodium tripolyphosphate (TPP) or phytic acid (PA) and characterized in terms of size, zeta-potential, morphology, drug encapsulation efficiency, mucoadhesion and mucopenetrating ability. Moreover, in vitro tests were conducted to evaluate VM release and the antibacterial activity against Staphylococcus aureus and Bacillus subtilis. NPs showed sizes ranged from 150 nm to 350 nm with good polydispersity index and positive zeta-potential. The selection of the suitable crosslinker allowed to modulate the mucoadhesive/mucopenetrating properties: CH/TPP NPs showed the best mucoadhesive ability, while CH/PA and CH/CMC NPs were characterized by an improved diffusion across the mucus layer. Further, NPs allowed a fast and complete release of VM, maintaining the antibacterial activity against the tested bacteria species.

Neutral wetting brush layers for block copolymer thin films using homopolymer blends processed at high temperatures.

Binary homopolymer blends of two hydroxyl-terminated polystyrene (PS-OH) and polymethylmethacrylate (PMMA-OH) homopolymers (Mn âˆ¼ 16000 g mol(-1)) were grafted on SiO2 substrates by high-temperature (T > 150 °C), short-time (t < 600 s) thermal treatments. The resulting brush layer was tested to screen preferential interactions of the SiO2 substrate with the different symmetric and asymmetric PS-b-PMMA block copolymers deposited on top of the grafted molecules. By properly adjusting the blend composition and the processing parameters, an efficient surface neutralization path was identified, enabling the formation, in the block copolymer film, of homogeneous textures of lamellae or cylinders perpendicularly oriented with respect to the substrate. A critical interplay between the phase segregation of the homopolymer blends and their grafting process on the SiO2 was observed. In fact, the polar SiO2 is preferential for the PMMA-rich phase that forms a homogeneous layer on the substrate, while the PS-rich phase is located at the polymer-air interface. During the thermal treatment, phase segregation and grafting proceed simultaneously. Complete wetting of the PS rich phase on the PMMA rich phase leads to the formation of a PS/PMMA bilayer. In this case, the progressive diffusion of PS chains toward the polymer-SiO2 interface during the thermal treatment allows tuning of the brush layer composition.

A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

Protein unfolding and refolding under force: methodologies for nanomechanics.

An increasing number of inter- and intramolecular interactions can nowadays be probed using single-molecule manipulation techniques. Protein unfolding and refolding is the most representative--though complex--of these interactions. Herein, we review the main modes of performing a force unfolding experiment: the velocity clamp and the new force clamp mode. We also compare some of the physical aspects behind the two most frequently used single-molecule manipulation instrumentations: optical tweezers and atomic force microscopes.

DNA detection by integrable electronics.

This paper presents a new electronic methodology to detect DNA hybridization for rapid identification of diseases, as well as food and environmental monitoring on a genetic base. The proposed solution exploits a new (electrical) capacitive measurement circuit, not requiring any prior labeling of the DNA (as it is often the case with the commonly employed optical detection). The sensitivity, the reliability, and the reproducibility of this device have been evaluated by experiments performed with a (non-integrated) prototype implementation, easily integrable in IC and/or micro-fabricated lab-on-a-chip.

Complex associates of plasmid DNA and a novel class of block copolymers with PEG and cationic segments as new vectors for gene delivery.

Cationic block copolymers, consisting of a poly(ethylene glycol) block and a block deriving from the poly(dimethylamino)ethyl methacrylate were prepared via a two-step procedure, based on the use of macroinitiators. By appropriately changing the experimental conditions and reacting the poly(dimethylamino)ethyl methacrylate block with iodo- or bromo-alkyl derivatives, a variety of ionic block copolymers with tuned physicochemical properties were prepared. These block copolymers are able to spontaneously self-assemble with plasmid DNA to produce oriented and shielded vectors, with physicochemical properties appropriate for in vivo applications. In addition, the formation of a complex between the cationic block copolymer and the plasmid DNA results in a nuclease resistance increase due to the stable nature of the complex.

Mapping the intrinsic curvature and flexibility along the DNA chain.

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a "natural" DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chain's intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

Scanning force microscopy study on a single-stranded DNA: the genome of parvovirus B19.

The genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.

Polynucleotide:Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA.

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.

Visualization and analysis of chromatin by scanning force microscopy.

The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone.

Deposition of supercoiled DNA on mica for scanning force microscopy imaging.

The deposition of DNA molecules on mica is driven and controlled by the ionic densities around DNA and close to the surface of the substrate. Dramatic improvements in the efficiency and reproducibility of DNA depositions were due to the introduction of divalent cations in the deposition solutions. The ionic distributions on DNA and on mica determine the mobility of adsorbed DNA molecules, thus letting them assume thermodynamically equilibrated conformations, or alternatively trapping them in non-equilibrated conformations upon adsorption. With these prerequisites, mica does not seem like an inert substrate for DNA deposition for microscopy, and its properties greatly affect the efficiency of DNA deposition and the appearance of the molecules on the substrate. In our laboratory, we have some preliminary evidence that mica could also participate in DNA damage, most likely through its heavy metal impurities.