Luis J. del Valle

Cellular Adhesion, Proliferation and Viability on Conducting Polymer Substrates

This work reports a comprehensive study about cell adhesion and proliferation on the surface of different electroactive substrates formed by pi-conjugated polymers. Biological assays were performed considering four different cellular lines: two epithelial and two fibroblasts. On the other hand, the electroactivity of the three conducting systems was determined in physiological conditions. Results indicate that the three substrates behave as a cellular matrix, even though compatibility with cells is larger for PPy and the 3-layered system. Furthermore, the three polymeric systems are electro-compatible with the cellular monolayers.

Calcium-induced decrease of the thermal stability and chaperone activity of α-crystallin

Abstract α-Crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. α-Crystallin aggregation is involved in lens opacification, and high [Ca2+] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca2+ on the thermal stability of α-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca2+-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca2+] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca2+ concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca2+ decreases both secondary and tertiary–quaternary structure stability of α-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca2+ alters the structural stability of α-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, α-crystallin aggregation facilitated by Ca2+ would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.

Bioactive and electroactive response of flexible polythiophene:polyester nanomembranes for tissue engineering

Properties of free-standing nanomembranes prepared by blending poly(3-thiophene methyl acetate) and poly(tetramethylene succinate), a soluble polythiophene derivative and a biodegradable polyester, respectively, have been examined. The outstanding flexibility and robustness of the nanomembranes floating in ethanol have been demonstrated through aspiration in pipette/release/shape recovery cycles, which were repeated without cracking the film. The blend retains the electrochemical properties (i.e. oxidation and reduction processes) of the individual conducting polymer in both physiological and organic environments. Hydrolytic and enzymatic degradation assays show that the degradation of the polyester domains produces the detachment of the conducting polymer domains. The cellular viability, which has been studied using four different cellular lines, is significantly higher for the blend than for the polyester, indicating that the former material is a potential bioactive platform for tissue engineering. Finally, the electrobioactivity of the individual materials and the blend coated with cellular monolayers shows some dependence on the cellular line.

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn2+ on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn2+ concentrations (0–50 μmZn2+). The thermal bleaching process is accelerated in the presence of Zn2+ with k rate constants, at 55 °C, of 0.028 ± 0.002 min−1 (0 μmZn2+) and 0.056 ± 0.003 min−1 (50 μm Zn2+), corresponding tot 1 2 values of 24.4 ± 1.6 min and 11.8 ± 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E a increase at 50 μm Zn2+ for the process, with ΔG ‡ decrease and increase in ΔH ‡ and ΔS ‡ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn2+ in normal and pathological retinal function.

Biodegradable free-standing nanomembranes of conducting polymer:polyester blends as bioactive platforms for tissue engineering

The present study reports the fabrication of free-standing nanomembranes with semiconducting and biodegradable properties. Nanomembranes have been prepared by spin-coating mixtures of a semiconducting polythiophene derivative, poly(3-thiophene methyl acetate), and a biodegradable polyester, poly(tetramethylene succinate). Both the roughness and thickness of the nanomembranes, which ranged from 3 to 20 nm and from 20 to 80 nm, respectively, were precisely controlled through the spin-coater speed and the solvent evaporation properties. Nanomembranes made of conducting polymer/polyester blends, which are able to retain the properties of the individual polymers, are stable in air and in ethanol solution for more than one year, facilitating their manipulation. Enzymatic degradation essays indicated that the ultra-thin films are biodegradable due to the presence of the aliphatic polyester. Interestingly, adhesion and proliferation assays with epithelial cells revealed that the behavior of the blend as cellular matrix is superior to that of the two individual polymers, validating the use of the nanomembranes as bioactive substrates for tissue regeneration.

Polylactide nanofibers loaded with vitamin B6 and polyphenols as bioactive platform for tissue engineering

AbstractElectrospun polylactide nanofibers loaded with different antioxidants (i.e. vitamin B6 in pyridoxine and pyridoxal form, p-coumaric acid and caffeic acid) are prepared from N,N-dimethylformamide/dimethylsulfoxide solutions. Morphology, structure and crystallinity of the nanofibers are evaluated by transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction and differential scanning calorimetry (DSC) techniques. Fibers are highly amorphous but able to crystallize easily due to the high molecular orientation induced by the electrospinning process. The drug molecules are incorporated into the polymeric matrix or formed isolated crystals. A fast release of loaded drug occurs within the first 8 h in hydrophobic medium; but, a slow and sustained release during several days occurs in a hydrophilic medium. Cell attachment on the loaded scaffolds was unaffected by the incorporation of the antioxidants. In contrast, cell proliferation increases with high antioxidative activity against free radicals responsible for cell damage. These new electrospun scaffolds provide high protection of cells against oxidative stress and resulting in innovative 3D fibrous platforms for tissue growth and proliferation.

An electroactive and biologically responsive hybrid conjugate based on chemical similarity

Synthetic amino acids have become very important tools for the design of new materials. In this work, an electroactive polymer–amino acid hybrid material has been synthesized by conjugating poly(3,4-ethylenedioxythiophene) (PEDOT), a well known conducting polymer, with a synthetic amino acid bearing 3,4-ethylenedioxythiophene, which has been explicitly designed and prepared for such a purpose. Nanometric films have been electrochemically generated using a two-step procedure to evaluate the properties and potential applications of the resulting hybrid material. The successful incorporation of the amino acid as end-capping of the PEDOT chains has been proved by FTIR, energy dispersive X-ray and X-ray photoelectron spectroscopies. The fabrication of the hybrid material using an engineered tissue has allowed us to preserve not only morphological and structural characteristics of the conducting polymer but also, and most importantly, to preserve the electrical conductivity, electroactivity, electrochemical stability and specific capacitance. Finally, the behavior of the hybrid material as a cellular matrix has been compared with that of PEDOT using cellular adhesion and proliferation assays. Results obtained in this work represent the success of a new strategy for the preparation of peptide-conducting polymer hybrid materials, which is currently being improved upon by transforming the 3,4-ethylenedioxythiophene-containing amino acid into a cell adhesive peptide.

Effect of dodecyl maltoside detergent on rhodopsin stability and function

Detergent-solubilized bovine rhodopsin produces mixed detergent/lipid/protein micelles. The effect of dodecyl maltoside detergent on the thermal stability of dark-state rhodopsin, and upon formation of the different intermediates after rhodopsin photobleaching (metarhodopsin II and metarhodopsin III), and upon transducin activation has been studied. No significant effect is observed for the thermal stability of dark-state rhodopsin in the range of detergent concentrations studied, but a decrease in the stability of metarhodopsin II and an increase in metarhodopsin III formation is observed with decreasing detergent concentrations. The transducin activation process is also affected by the presence of detergent indicating that this process is dependent on the lipid micro-environment and membrane fluidity, and this stresses the importance of the native lipid environment in rhodopsin normal function.

Polybiguanide (PHMB) loaded in PLA scaffolds displaying high hydrophobic, biocompatibility and antibacterial properties

Polyhexamethylenebiguanide hydrochloride (PHMB), a low molecular weight polymer related to chlorohexidine (CHX), is a well-known antibacterial agent. In this study, polylactide (PLA) nanofibers loaded with PHMB were produced by electrospinning to obtain 3D biodegradable scaffolds with antibacterial properties. PLA fibers loaded with CHX were used as control. The electrospun fibers were studied and analyzed by SEM, FTIR, DSC and contact angle measurements. PHMB and CHX release from loaded scaffolds was evaluated, as well as their antibacterial activity and biocompatibility. The results showed that the nanofibers became smoother and their diameter smaller with increasing the amount of loaded PHMB. This feature led to an increase of both surface roughness and hydrophobicity of the scaffold. PHMB release was highly dependent on the hydrophilicity of the medium and differed from that determined for CHX. Lastly, PHMB-loaded PLA scaffolds showed antibacterial properties since they inhibited adhesion and bacterial growth, and exhibited biocompatible characteristics for the adhesion and proliferation of both fibroblast and epithelial cell lines.

Micro-molding with ultrasonic vibration energy: New method to disperse nanoclays in polymer matrices

Ultrasound technology was proved as an efficient processing technique to obtain micro-molded specimens of polylactide (PLA) and polybutylene succinate (PBS), which were selected as examples of biodegradable polyesters widely employed in commodity and specialty applications. Operational parameters such as amplitude, molding force and processing time were successfully optimized to prepare samples with a decrease in the number average molecular weight lower than 6%. Ultrasonic waves also seemed an ideal energy source to provide effective disaggregation of clay silicate layers, and therefore exfoliated nanocomposites. X-ray diffraction patterns of nanocomposites prepared by direct micro-molding of PLA or PBS powder mixtures with natural montmorillonite or different organo-modified clays showed the disappearance of the 001 silicate reflection for specimens having up to 6 wt.% clay content. All electron micrographs revealed relatively homogeneous dispersion and sheet nanostructures oriented in the direction of the melt flow. Incorporation of clay particles during processing had practically no influence on PLA characteristics but enhanced PBS degradation when an organo-modifier was employed. This was in agreement with thermal stability data deduced from thermogravimetric analysis. Cold crystallization experiments directly performed on micro-molded PLA specimens pointed to a complex influence of clay particles reflected by the increase or decrease of the overall non-isothermal crystallization rate when compared to the neat polymer. In all cases, the addition of clay led to a clear decrease in the Avrami exponent.