Surviving mass extinctions through biomineralized DNA

Even in the worst of conditions, such as those which occurred during mass extinction events, life on Earth never totally stopped. Aggressive chemical and physical attacks able to sterilize or poison living organisms occurred repeatedly. Surprisingly, DNA was not degraded, denatured or modified to the point of losing the capability of transferring the genetic information to the next generations. After the events of mass extinction life was able to survive and thrive. DNA was passed on despite being an extremely fragile biomolecule. The potential implications of hydroxyapatite protection of DNA are discussed in this Concept article including how DNA acts as a template for hydroxyapatite (HAp) formation, how cell death can trigger biomineralization, and how DNA can be successfully released from HAp when the conditions are favorable for life.Peer ReviewedPostprint (published version

Sustainable synthesis of amino acids by catalytic fixation of molecular dinitrogen and carbon dioxide

The industrial process of nitrogen fixation is complex and results in a huge economic and environmental impact. It requires a catalyst and high temperature and pressure to induce the rupture of the strong N–N bond and subsequent hydrogenation. On the other hand, carbon dioxide removal from the atmosphere has become a priority objective due to the high amount of global carbon dioxide emissions (i.e. 36 200 million tons in 2015). In this work, we fix nitrogen from N2 and carbon from CO2 and CH4 to obtain both glycine and alanine (D/L racemic mixture), the two simplest amino acids. The synthesis, catalyzed by polarized hydroxyapatite under UV light irradiation and conducted in an inert reaction chamber, starts from a simple gas mixture containing N2, CO2, CH4 and H2O and uses mild reaction conditions. At atmospheric pressure and 95 °C, the glycine and alanine molar yields with respect to CH4 or CO2 are about 1.9% and 1.6%, respectively, but they grow to 3.4% and 2.4%, when the pressure increases to 6 bar and the temperature is maintained at 95 °C. Besides, the minimum temperature required for the successful production of detectable amounts of amino acids is 75 °C. Accordingly, an artificial photosynthetic process has been developed by using an electrophotocatalyst based on hydroxyapatite thermally and electrically stimulated and coated with zirconyl chloride and a phosphonate. The synthesis of amino acids by direct fixation of nitrogen and carbon from gas mixtures opens new avenues regarding the nitrogen fixation for industrial purposes and the recycling of carbon dioxide.Peer ReviewedPostprint (published version

Effects of hydroxyapatite (0001) Ca2+/Mg2+ substitution on adsorbed D-ribose ring puckering

Advanced Molecular Dynamics (MD) simulation protocols have been used to assess the ring puckering of cyclic D-ribose when the sugar is adsorbed on the most stable (0001) facet of calcium hydroxyapatite (HAp). In addition, sugar¿mineral interactions, which are crucial for transfection processes and prebiotic chemistry, have been studied for systems in which the Ca2+ ions of the above mentioned HAp facet were totally or partially replaced by Mg2+. The latter replacement is spatially and quantitatively limited and has been found to cause important alterations in the conformational behavior of D-ribose that are similar to those suffered in hairpin RNA from A to B helical structures. Accordingly, replacement of Ca2+ by Mg2+ has a dramatic effect on the functionality of the nucleic acid. These changes have been related to both the substitution site on the surface and the amount of ions. Our results show that when replacement by Mg2+ occurs in OH--coordinated Ca2+ ions, Mg2+¿D-ribose interactions are strong enough to prevent the interactions between the hydroxyl groups of the sugar and the remaining Ca2+ ions.Peer ReviewedPostprint (published version

Incorporation of biguanide compounds into poly(GL)-b-poly(GL-co-TMC-co-CL)-b-poly(GL) monofilament surgical sutures

A new biodegradable coating was developed for bioabsorbable monofilament sutures. Specifically, a random copolymer having 35 wt-% and 65 wt-% of lactide and trimethylene carbonate units showed appropriate flexibility, stickiness and degradation rate, as well as capability to produce a complete and uniform coating. Monofilament sutures of polyglycolide-b-poly(glycolide-co-trimethylene carbonate-co-e-caprolactone)-b-polyglycolide were loaded with chlorhexidine (CHX) and poly(hexamethylene biguanide) (PHMB) to explore the possibility to achieve antimicrobial activity without adverse cytotoxic effects. To this end, two processes based on single drug adsorption onto the suture surface and incorporation into the coating copolymer were used and subsequently evaluated. Although the second process could be considered more complex, clear benefits were observed in terms of drug loading efficiency, antimicrobial effect and even lack of cytotoxicity. In general, drugs could be loaded in an amount leading to a clear bacteriostatic effect for both Gram-negative and Gram-positive bacteria without causing significant cytotoxicity. Release profiles of PHMB and CHX were clearly different. Specifically, adsorption of the drug onto the fiber surface which prevented complete release was detected for PHMB. This polymer had advantages derived from its high molecular size, which hindered penetration into cells, thus resulting in lower cytotoxicity. Furthermore, bacterial growth kinetics measurements and bacterial adhesion assays showed greater effectiveness of this polymer.Peer ReviewedPostprint (author's final draft

Polypropylene mesh for hernia repair with controllable cell adhesion/de-adhesion properties

Herein, a versatile bilayersystem, composed by a polypropylene(PP)mesh and a covalently bonded poly(N-isopropylacrylamide) (PNIPAAm) hydrogel, is reported. The cell adhesion mechanism was successfully modulated by controlling the architecture of the hydrogel in terms of duration of PNIPAAm graftingtime, crosslinker content, and temperature of material exposure in PBS solutions (belowandabove the LCST of PNIPAAm). The best in vitroresults with fibroblast (COS-1) and epithelial (MCF-7) cells was obtained with a mesh modified with porous iPP-g-PNIPAAm bilayer system, prepared via PNIPAAm grafting for 2 h at the lowest N,N'-methylene bis(acrylamide) (MBA)concentration (1 mM). Under these conditions, the detachment of the fibroblast-like cells was 50% lower than that of the control, after 7 days of cell incubation, which represents a high de-adhesionof cellsin a short period. Moreover, the whole system showed an excellent stability in dry or wet media, proving that the thermosensitive hydrogel was well adhered to the polymer surface, after PP fibreactivation by cold plasma. This study opens new insights on the development of anti-adherent meshes for abdominal hernia repairs.Peer ReviewedPublished versio

Electrically polarized hydroxyapatite: influence of the polarization process on the microstructure and properties

Semipermanently polarized hydroxyapatite, named SP/HAp(w), is obtained by applying a constant dc electric field of 1–10 kV/cm at 300–850 °C to the samples previously sintered in water vapor, while permanently polarized hydroxyapatite, PP/HAp(a), is produced by applying a dc electric field of 3 kV/cm at 1000 °C to the samples sintered in air. SP/HAp(w) has been used for biomedical applications, while PP/HAp(a) has been proved to be a valuable catalyst for N2 and CO2 fixation. In this work, structural differences between SP/HAp(w) and PP/HAp(a) have been ascertained using Raman microscopy, wide-angle X-ray diffraction, scanning electronic microscopy, high-resolution transmission electron microscopy, and grazing incidence X-ray diffraction. Results prove the existence of crystal distortion in the form of amorphous calcium phosphate and ß-tricalcium phosphate (ß-TCP) phases close to the surface because of the atmosphere used in the sintering process. The existence of an amorphous layer in the surface and the phase transition through ß-TCP of SP/HAp(w) are the structural factors responsible for the differences with respect to PP/HAp(a). Moreover, a superstructure has been identified in PP/HAp(a) samples, which could be another structural factor associated with enhanced conductivity, permanent polarization, and catalytic activity of this materialPeer ReviewedPostprint (author's final draft

Scaffolds with tunable properties constituted by electrospun nanofibers of polyglycolide and poly(e-caprolactone)

Electrospun scaffolds constituted by different mixtures of two biodegradable polyesters are prepared. Specifically, materials with well differentiated properties can be derived from the blending of hydrophilic polyglycolide (PGA) and hydrophobic poly(e-caprolactone) (PCL), which are also two of the most applied polymers for biomedical uses. Electrospinning conditions are selected in order to get homogeneous and continuous fibers with diameters in the nano/micrometric range. These conditions are also applied to load the different scaffolds with curcumin (CUR) and polyhexamethylene biguanide (PHMB) as hydrophobic and hydrophilic bactericide compounds, respectively. Physicochemical characterization of both unloaded and loaded scaffolds is performed and involved Fourier transform infrared and 1H NMR spectroscopies, morphological observations by scanning electron microscopy, study of thermal properties through calorimetry and thermogravimetric analysis, and evaluation of surface characteristics through contact angle measurements. Release behavior of the loaded scaffolds is evaluated in two different media. Results point out a well differentiated behavior where the delivery of CUR and even PHMB are highly dependent on the PGA/PCL ratio, the capability of the medium to swell the polymer matrix, and the diffusion of the selected solvent into the electrospun fibers. All samples show a bactericide effect in both hydrophilic cell culture and hydrophobic agar media.Peer ReviewedPostprint (author's final draft

Spherulitic morphologies of the triblock Poly(GL)-b-poly(GL-co-TMC-co-CL)-b-poly(GL) copolymer: Isothermal and non-isothermal crystallization studies

Crystallization of a biodegradable segmented copolymer constituted by polyglycolide hard segments and a middle soft segment constituted by a random disposition of glycolyl, e-caproyl and trimethylene carbonyl units has been studied by means of optical microscopy, atomic force microscopy and time resolved X-ray diffraction techniques. This Poly(GL)-b-poly(GL-co-TMC-co-CL)-b-poly(GL) copolymer is widely employed as surgical suture and has similar characteristics than previously studied copolymers having a middle soft segment constituted by only two monomers (i.e. glycolide and trimethylene carbonate). FTIR and NMR spectroscopies demonstrated that the middle segment had an amorphous character and a random microstructure as consequence of transesterification reactions that took place during synthesis. Nevertheless, polyglycolide segments were able to crystallize giving rise to peculiar positive birefringent spherulites with a morphology, which depends on crystallization temperature (i.e. flat-on and edge-on crystals) as verified by AFM and electron diffraction patterns. Complete bell shaped curves that defined the temperature dependence of the crystal growth rate could be experimentally obtained from both, isothermal and non-isothermal crystallizations. Data from both analyses were in close agreement and pointed out a secondary nucleation constant (2.42–2.88 × 105 K2) which was clearly higher than that determined for the related system with two components. Lamellar morphologic parameters were similar for samples crystallized from the melt state and after the reordering process that took place on heating. Comparing to the bicomponent system, significant differences were again observed highlighting the influence of the soft segment on the crystallization behaviorPeer ReviewedPostprint (author's final draft

Tunable drug loading and reinforcement of polycaprolactone films by means of electrospun nanofibers of glycolide segmented copolymers

Electrospinning of a segmented copolymer having polyglycolide hard segments is successfully performed from 1,1,1,3,3,3-hexafluoroisopropanol solutions. During the process, a bactericidal agent, i.e., chlorhexidine (CHX), is effectively loaded, which results in nanofibers with a smaller diameter because of the change in solution conductivity. New fabrics based on molding of alternate layers of poly(e-caprolactone) (PCL) films and the electrospun scaffolds of the segmented copolymer are prepared and characterized. The thermal molding process renders a PCL matrix homogeneously reinforced with nanofibers that compensate for the loss of mechanical properties caused by incorporation of CHX. Release of CHX is evaluated in different media. Results vary depending on the layer where the drug is incorporated. Thus, systems with an immediate bacteriostatic effect, as well as systems with a potential long term antimicrobial effect, are obtained. Growth inhibition and adhesion assays demonstrate the fast bactericidal effect of samples with CHX loaded in its outer layers.Peer ReviewedPostprint (author's final draft

Dissolving hydroxyolite: a DNA molecule into its hydroxyapatite mold

In spite of the clinical importance of hydroxyapatite (HAp), the mechanism that controls its dissolution in acidic environments remains unclear. Knowledge of such a process is highly desirable to provide better understanding of different pathologies, as for example osteoporosis, and of the HAp potential as vehicle for gene delivery to replace damaged DNA. In this work, the mechanism of dissolution in acid conditions of HAp nanoparticles encapsulating double-stranded DNA has been investigated at the atomistic level using computer simulations. For this purpose, four consecutive (multi-step) molecular dynamics simulations, involving different temperatures and proton transfer processes, have been carried out. Results are consistent with a polynuclear decalcification mechanism in which proton transfer processes, from the surface to the internal regions of the particle, play a crucial role. In addition, the DNA remains protected by the mineral mold and transferred proton from both temperature and chemicals. These results, which indicate that biomineralization imparts very effective protection to DNA, also have important implications in other biomedical fields, as for example in the design of artificial bones or in the fight against osteoporosis by promoting the fixation of Ca2+ ions.Peer ReviewedPostprint (published version