Santina Carnazza

Surface free energy and cell attachment onto ion-beam irradiated polymer surfaces

Abstract The paper reports evidence of the different cytocompatibility towards ion irradiated polymer surfaces. In particular, we studied the cell attachment, adhesion and spreading of normal human dermal fibroblast cells onto poly(hydroxymethylsiloxane) and poly(ethyleneterephthalate) surfaces modified by 50 keV Ar+ beams. The cell response is discussed in connection with the radiation-induced changes of the polymers surface chemical structure and related surface free energy, investigated by means of X-ray photoelectron spectroscopy and static contact angle measurements. The biological response is interpreted in terms of the different modification trends of the Surface Free Energy components, and its relationship to the protein adsorption processes from culture medium or to the direct cell-surface interaction in a protein-free saline solution. The results point towards a critical role of the electron-donor character of the surfaces, which seems able to trigger the optimal cell response to the employed polymeric surfaces.

Biosynthesis and structural characterization of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas aeruginosa from fatty acids.

In this study, we investigated the ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize poly(3-hydroxyalkanoates) (PHAs) from saturated fatty acids with an even number of carbon atoms, from eight to 22, and from oleic acid. In a non-limiting medium, all carbon sources but docosanoic acid supported cell growth and PHA production, with eicosanoic acid giving the highest yield. In magnesium-limiting conditions, higher yields were obtained from sources with up to 16 carbon atoms. Composition was estimated by gas chromatography of methanolyzed samples and (13)C nuclear magnetic resonance. The 3-hydroxyalkanoate units extended from hexanoate to tetradecanoate or tetradecenoate, with octanoate and decanoate as the predominant components. Weight average molecular weights ranged from 78,000 to 316,000. Fast atom bombardment mass spectrometry of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.

Phage-AgNPs complex as SERS probe for U937 cell identification.

The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy.

Protein adsorption and fibroblast adhesion on irradiated polysiloxane surfaces.

A very peculiar case of differential cell response towards polysiloxane surfaces of very similar composition is investigated. Poly(hydroxymethylsiloxane) (PHMS) surfaces treated either by O2-plasma or 6 keV Ar+-beams have been used to test the adhesion, proliferation and spreading of human fibroblasts. The surface chemical structure and nanomorphology were investigated by means of X-ray photoelectron spectroscopy (XPS), surface free energy measurements and atomic force microscopy (AFM). In spite of the close compositional and morphological similarity of the modified surfaces, the viability of the adhered cells, evaluated by means of optical microscopy and epifluorescence microscopy, was found to be very different in the two cases. The study of the features of the adsorbed protein adlayer on the two types of surfaces was performed by XPS and AFM and indicated that the overall cell behavior is connected to a quite different protein aggregation process, occurring respectively on the plasma- and Ar+-modified polysiloxane surfaces. It is suggested that the specific biological response of the modified surfaces is determined by the chemical structure at the nanometric level.

Recombinant phage probes for Listeria monocytogenes

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10 4 cells ml -1 . In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

Self-organization of yeast cells on modified polymer surfaces after dewetting: new perspectives in cellular patterning

In recent years, biological micro-electro-mechanical systems (commonly referred to as BioMEMS) have found widespread use, becoming increasingly prevalent in diagnostics and therapeutics. Cell-based sensors are nowadays gaining increasing attention, due to cellular built-in natural selectivity and physiologically relevant response to biologically active chemicals. On the other hand, surrogate microbial systems, including yeast models, have become a useful alternative to animal and mammalian cell systems for high-throughput screening for the identification of new pharmacological agents. A main obstacle in biosensor device fabrication is the need for localized geometric confinement of cells, without losing cell viability and sensing capability. Here we illustrate a new approach for cellular patterning using dewetting processes to control cell adhesion and spatial confinement on modified surfaces. By the control of simple system parameters, a rich variety of morphologies, ranging through hexagonal arrays, polygonal networks, bicontinuous structures, and elongated fingers, can be obtained.

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy

Bacterial resistance to antibiotics has made it necessary to resort to using antibacterial drugs that have considerable toxicities. Here, we show that conjugation of vancomycin-loaded nanoparticles with the cyclic 9-amino-acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of the nanoparticles in S. aureus-infected tissues and reduces the systemic dose needed, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing CARG in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissues may help combat difficult-to-treat infections.Nanoparticles carrying an antibiotic and conjugated with a peptide identified via phage display that binds specifically to Staphylococcus aureus effectively suppress staphylococcal infections in vivo.

Spatial Patterns of Microbial Retention on Polymer Surfaces

Microbial adhesion and retention on surfaces are complex phenomena, critical to the formation and development of biofilms. Recently, the focus of research has been more and more on the importance of retention of bacteria under fluctuating high shear forces in biofilm formation. The aim of the present work was to carry out a comparative study of the retention process of different bacterial and yeast species using: (1) a range of surfaces with different surface free energy properties and (2) a number of different bacterial cell physiological states. It was found for the first time that once a threshold cell number is retained on the surface, microbial retention patterns are formed following a power law, i.e., not stochastic. Our results demonstrated that the overall spatial patterns of microbial retention observed for the different substrates are similar for the all investigated cell types and that the drastic modification of the surface free energy does not affect this spatial organization. On the other hand, the microbial retention patterns appear to be significantly affected by the physiological state of the cells. Finally, the experimental retention patterns have been well simulated by a general agent-based model, confirming that the typical fractal distribution of retained cells is the result of a self-organization process.

Self-organization and emergent models in bacterial adhesion on engineered polymer surfaces

Bacterial adhesion on surfaces is a very important complex phenomenon, since it is a determinant for the overall biofilm formation; the presence of biofilm can be both beneficial (i.e. to degrade hazardous substances in the soil or in a bioreactor) or detrimental (as on food, ship hulls and biomaterials implants or in the oral cavity). This work starts from a series of experiments carried out on different engineered surfaces on which bacteria adhere in such a way a pattern emerges. In this paper a model based on self-organization and thus ruled by local laws among the elements of the complex system is introduced. It is shown how this model accounts for generation of the patterns in bacterial adhesion.

Tween 20 and Its Major Free Fatty Acids as Carbon Substrates for the Production of Polyhydroxyalkanoates in Pseudomonas aeruginosa ATCC 27853

The ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize polyhydroxyalkanoates (PHAs) using Tween 20 as the sole carbon source was investigated. Tween 20 could support cell growth and PHA production. The polymer produced from Tween 20 was compared with those produced from its major free fatty acids components: lauric (C12), myristic (C14), and palmitic (C16) acids. Gas-chromatographic analysis of methanolyzed samples and 13C-Nuclear Magnetic Resonance (NMR) showed that the PHAs obtained are composed of even carbon atoms 3-hydroxyalkanoates ranging from C6 to C14, with C8 and C10 as the predominant components. The nature of the carbon sources used had little influence on the composition, but was found to be important in determining the average molecular weight, shorter chain fatty acids yielding higher molecular weight products. Fast Atom Bombardment-Mass Spectrometry (FAB-MS) of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.