Salvador Borrós

Polymer Electrolyte Fuel Cells Based on Phosphoric Acid-Impregnated Poly(2,5-benzimidazole) Membranes

b Institut Quimic de Sarria `, Universitat Ramon Llull, E-08017 Barcelona, Spain Preparation and characterization of membranes of poly~2,5-benzimidazole !~ ABPBI !~ as thin as 20 mm! for polymer electrolyte membrane fuel cells are reported. These membranes were prepared by solution casting and then impregnated in phosphoric acid baths. Their characterization included thermogravimetric analyses, conductivity measurements, Fourier transform infrared spec- troscopy, X-ray diffraction, and scanning electron microscopy. These membranes have high thermal stability and good proton conductivity at temperatures up to 200°C (6.23 10 22

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-β-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-β-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-β-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.

Functionalized self-assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I – in the traditional culture sandwich technique – by a defined extracellular matrix analogue, the self‐assembling peptide hydrogel RAD16‐I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), α1‐microglobulin, α1‐macroglobulin, retinol binding protein, fibronectin, α1‐inhibitor III and biotin‐dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues – carrying the 67 kD laminin receptor ligand – enhanced the gene expression of albumin, HNF4‐α, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses.

Protection of Sensors for Biological Applications by Photoinitiated Chemical Vapor Deposition of Hydrogel Thin Films

We report photoinitiated chemical vapor deposition (piCVD), a gentle synthetic method for the preparation of ultrathin films (approximately 100 nm) of the hydrogel poly(hydroxyethyl methacrylate) (pHEMA). piCVD occurs near room temperature and requires only mild vacuum conditions. The deposited films swell rapidly and reversibly in buffer solution, and the swelling properties can be controlled via the deposition conditions. Analysis of the swelling data indicates that the mesh size of the hydrogel creates a selectively permeable coating. The mesh is large enough to allow small molecule analytes to permeate the film but small enough to prevent the transport of large biomolecules such as proteins. X-ray photoelectron spectroscopy (XPS) shows that the films decrease nonspecific adhesion of the protein albumin by nearly 8-fold over bare silicon. A dry process, piCVD is suitable for coating particles with diameters as small as 5 microm. The absence of solvents and plasmas in piCVD allows films to be directly synthesized on optode sensors without degradation of sensitivity or response time.

Interstitial fluid flow intensity modulates endothelial sprouting in restricted Src-activated cell clusters during capillary morphogenesis.

Development of tissues in vitro with dimensions larger than 150 to 200 microm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0-50 microm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 microm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis

Early Tissue Patterning Recreated by Mouse Embryonic Fibroblasts in a Three-Dimensional Environment

Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction,proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression ofSox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development,this model could lead to new ways to consider tissue engineering and regenerative medicine.

The effect of self-assembling peptide nanofiber scaffolds on mouse embryonic fibroblast implantation and proliferation.

Development of new materials for tissue engineering can be facilitated by the capacity to efficiently monitor in vivo the survival, proliferation and differentiation behaviour of cells implanted in different target tissues. We present here the application of a previously developed platform that allows to monitor in real time the survival and proliferative behaviour of implanted cells in two anatomical sites: subcutaneous and intramuscular. Basically, the system is based on the use of a non-invasive bioluminescence imaging (BLI) technique to detect luciferase expressing C57BL/6 cells, mouse embryonic fibroblasts, seeded in two sets of scaffolds: 1, a RAD16-I self-assembling peptide nanofiber matrix and 2, a composite consisted of the same RAD16-I nanofibers contained into a microporous biorubber scaffold. Interestingly, our results indicated considerable differences in the behaviour of implanted cells in each scaffold type. We observed that the self-assembling peptide scaffold alone foster cell survival and promotes cell proliferation where the composite scaffold not. Since self-assembling peptide scaffolds presents value stiffness proximal to the implanted tissues it is suggestive to think that harder materials will provide a physical constriction for cells to proliferate as well as mechanical discontinuity. We therefore propose that it is important to close match the implantation environment with the cell/material constructs in order to obtain the best response of the cells, illustrating the convenience of this strategy for the development of new tissue engineering platforms.

Nanoparticle diffusion within intestinal mucus: Three-dimensional response analysis dissecting the impact of particle surface charge, size and heterogeneity across polyelectrolyte, pegylated and viral particles.

Multiple particle tracking (MPT) methodology was used to dissect the impact of nanoparticle surface charge and size upon particle diffusion through freshly harvested porcine jejunum mucus. The mucus was characterised rheologically and by atomic force microscopy. To vary nanoparticle surface charge we used a series of self-assembly polyelectrolyte particles composed of varying ratios of the negatively charged polyacrylic acid polymer and the positively charged chitosan polymer. This series included a neutral or near-neutral particle to correspond to highly charged but near-neutral viral particles that appear to effectively permeate mucus. In order to negate the confounding issue of self-aggregation of such neutral synthetic particles a sonication step effectively reduced particle size (to less than 340 nm) for a sufficient period to conduct the tracking experiments. Across the polyelectrolyte particles a broad and meaningful relationship was observed between particle diffusion in mucus (×1000 difference between slowest and fastest particle types), particle size (104-373 nm) and particle surface charge (-29 mV to +19.5 mV), where the beneficial characteristic promoting diffusion was a neutral or near-neutral charge. The diffusion of the neutral polyelectrolyte particle (0.02887 cm S(-1)×10(-9)) compared favourably with that of a highly diffusive PEGylated-PLGA particle (0.03182 cm(2) S(-1)×10(-9)), despite the size of the latter (54 nm diameter) accommodating a reduced steric hindrance with the mucin network. Heterogeneity of particle diffusion within a given particle type revealed the most diffusive 10% sub-population for the neutral polyelectrolyte formulation (5.809 cm(2) S(-1)×10(-9)) to be faster than that of the most diffusive 10% sub-populations obtained either for the PEGylated-PLGA particle (4.061 cm(2) S(-1)×10(-9)) or for a capsid adenovirus particle (1.922 cm(2) S(-1)×10(-9)). While this study has used a simple self-assembly polyelectrolyte system it has substantiated the pursuance of other polymer synthesis approaches (such as living free-radical polymerisation) to deliver stable, size-controlled nanoparticles possessing a uniform high density charge distribution and yielding a net neutral surface potential. Such particles will provide an additional strategy to that of PEGylated systems where the interactions of mucosally delivered nanoparticles with the mucus barrier are to be minimised.

Immobilization of biomolecules to plasma polymerized pentafluorophenyl methacrylate.

Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Förch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

Oligopeptide-terminated poly(β-amino ester)s for highly efficient gene delivery and intracellular localization

The main limitation of gene therapy towards clinics is the lack of robust, safe and efficient gene delivery vectors. This paper describes new polycations for gene delivery based on poly(β-amino ester)s (pBAE) containing terminal oligopeptides. The authors developed oligopeptide-modified pBAE-pDNA nanoparticles that achieve better cellular viability and higher transfection efficacy than other end-modified pBAE and commercial transfection agents. Gene expression in highly permissive cell lines was remarkably high, but transfection efficiency in less-permissive cell lines was highly dependent on oligopeptide composition and nanoparticle formulation. Moreover, the use of selected oligopeptides in the pBAE formulation led to preferential intracellular localization of the particles. Particle analysis of highly efficient pBAE formulations revealed different particle sizes and charge features, which indicates chemical pseudotyping of the particle surface, related to the oligopeptide chemical nature. In conclusion, chemical modification at the termini of pBAE with amine-rich oligopeptides is a powerful strategy for developing delivery systems for future gene therapy applications.