A Comparison of Three-Layer and Single-Layer Small Vascular Grafts Manufactured via the Roto-Evaporation Method.

This study used the roto-evaporation technique to engineer a 6 mm three-layer polyurethane vascular graft (TVG) that mimics the architecture of human coronary artery native vessels. Two segmented polyurethanes were synthesized using lysine (SPUUK) and ascorbic acid (SPUAA), and the resulting materials were used to create the intima and adventitia layers, respectively. In contrast, the media layer of the TVG was composed of a commercially available polyurethane, Pearlbond 703 EXP. For comparison purposes, single-layer vascular grafts (SVGs) from individual polyurethanes and a polyurethane blend (MVG) were made and tested similarly and evaluated according to the ISO 7198 standard. The TVG exhibited the highest circumferential tensile strength and longitudinal forces compared to single-layer vascular grafts of lower thicknesses made from the same polyurethanes. The TVG also showed higher suture and burst strength values than native vessels. The TVG withstood up to 2087 ± 139 mmHg and exhibited a compliance of 0.15 ± 0.1%/100 mmHg, while SPUUK SVGs showed a compliance of 5.21 ± 1.29%/100 mmHg, akin to coronary arteries but superior to the saphenous vein. An indirect cytocompatibility test using the MDA-MB-231 cell line showed 90 to 100% viability for all polyurethanes, surpassing the minimum 70% threshold needed for biomaterials deemed cytocompatibility. Despite the non-cytotoxic nature of the polyurethane extracts when grown directly on the surface, they displayed poor fibroblast adhesion, except for SPUUK. All vascular grafts showed hemolysis values under the permissible limit of 5% and longer coagulation times.

Low-Density Lipoproteins Increase Proliferation, Invasion, and Chemoresistance via an Exosome Autocrine Mechanism in MDA-MB-231 Chemoresistant Cells.

Dyslipidemias involving high concentrations of low-density lipoproteins (LDLs) increase the risk of developing triple-negative breast cancer (TNBC), wherein cholesterol metabolism and protein translation initiation mechanisms have been linked with chemoresistance. Doxorubicin (Dox) treatment, a member of the anthracycline family, represents a typical therapeutic strategy; however, chemoresistance remains a significant challenge. Exosomes (Exs) secreted by tumoral cells have been implicated in cell communication pathways and chemoresistance mechanisms; the content of exosomes is an outcome of cellular cholesterol metabolism. We previously induced Dox resistance in TNBC cell models, characterizing a variant denominated as variant B cells. Our results suggest that LDL internalization in parental and chemoresistant variant B cells is associated with increased cell proliferation, migration, invasion, and spheroid growth. We identified the role of eIF4F translation initiation factor and the down-regulation of tumor suppressor gene PDCD4, an inhibitor of eIF4A, in chemoresistant variant B cells. In addition, the exomes secreted by variant B cells were characterized by the protein content, electronic microscopy, and cell internalization assays. Critically, exosomes purified from LDL-treated variant B cell promoted cell proliferation, migration, and an increment in lactate concentration. Our results suggest that an autocrine phenomenon induced by exosomes in chemoresistant cells may induce modifications on signaling mechanisms of the p53/Mdm2 axis and activation of p70 ribosomal protein kinase S6. Moreover, the specific down-regulated profile of chaperones Hsp90 and Hsp70 secretion inside the exosomes of the chemoresistant variant could be associated with this phenomenon. Therefore, autocrine activation mediated by exosomes and the effect of LDL internalization may influence changes in exosome chaperone content and modulate proliferative signaling pathways, increasing the aggressiveness of MDA-MB-231 chemoresistant cells.

Synthesis of Aza-BODIPYs, Their Differential Binding for Cu(II), and Results of Bioimaging as Fluorescent Dyes of Langerhans ß-Cells.

Cellular labeling through the use of dyes is of great interest to the biomedical sciences for the characterization of the location and distribution of biomolecules and also for the tracking of the course of biological processes in both health and illness. This paper reports the synthesis, characterization, and subsequent evaluation as metal sensors and cell staining probes of four aza-BODIPY compounds [herein referred to as 7(a-d)]. Compounds 7(b-d) were found to display an outstanding selectivity for Cu(II) because their emission band at 720 nm was progressively quenched by this metal, presenting fluorescence quenching between 75 and 95%. On the other hand, cell imaging studies with pancreatic ß-cells proved that aza-BODIPYs 7a and 7b showed selectivity for the cytoplasm, while 7c and 7d were selective for the cell membrane. Moreover, aza-BODIPY 7b allowed to characterize in a clear way a lipotoxic condition mediated by saturated fatty acids, a critical phenomenon on ß-cell damage associated with diabetes mellitus type II. Taken together, the presented results highlight the obtained aza-BODIPY compounds as selective sensing/staining probes with the potential to be used in the biomedical field.

Optimizing the Efficiency of a Cytocompatible Carbon-Dots-Based FRET Platform and Its Application as a Riboflavin Sensor in Beverages.

In this work, the Förster resonance energy transfer (FRET) between carbon dots (CDs) as energy donors and riboflavin (RF) as an energy acceptor was optimized and the main parameters that characterize the FRET process were determined. The results were successfully applied in the development of an ultrasensitive ratiometric fluorescent sensor for the selective and sensitive determination of RF in different beverages. Water-soluble CDs with a high quantum yield (54%) were synthesized by a facile and direct microwave-assisted technique. The CDs were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Zeta potential, and UV-visible and molecular fluorescence spectroscopy. The study of the FRET process at two donor concentrations showed that the energy transfer efficiency decreases as the donor concentration increases, confirming its dependence on the acceptor:donor ratio in nanoparticle-based systems. The results show the importance of optimizing the FRET process conditions to improve the corresponding output signal. The variation in the ratiometric signal with the concentration of RF showed linearity in a concentration range of 0 to 11 µM with R2 = 0.9973 and a detection limit of 0.025 µM. The developed nanosensor showed good selectivity over other possible types of interference. The sensor was then applied for the determination of RF in beverage samples using the standard addition method with recoveries between 96% and 106%. Preliminary cytocompatibility tests carried out with breast cancer cells (MDA-MB-231) revealed the nanosensor to be cytocompatible in its working concentration regime, even after long incubation times with cells. Altogether, the developed RF determination method was found to be fast, low-cost, highly sensitive, and selective and can be extended to other samples of interest in the biological and food sectors. Moreover, thanks to its long-lasting cytocompatibility, the developed platform can also be envisaged for other applications of biological interest, such as intracellular sensing and staining for live cell microscopy.

Lipid Modulation in the Formation of ß-Sheet Structures. Implications for De Novo Design of Human Islet Amyloid Polypeptide and the Impact on ß-Cell Homeostasis.

Human islet amyloid polypeptide (hIAPP) corresponds to a 37-residue hormone present in insulin granules that maintains a high propensity to form ß-sheet structures during co-secretion with insulin. Previously, employing a biomimetic approach, we proposed a panel of optimized IAPP sequences with only one residue substitution that shows the capability to reduce amyloidogenesis. Taking into account that specific membrane lipids have been considered as a key factor in the induction of cytotoxicity, in this study, following the same design strategy, we characterize the effect of a series of lipids upon several polypeptide domains that show the highest aggregation propensity. The characterization of the C-native segment of hIAPP (residues F23-Y37), together with novel variants F23R and I26A allowed us to demonstrate an effect upon the formation of ß-sheet structures. Our results suggest that zwitterionic phospholipids promote adsorption of the C-native segments at the lipid-interface and ß-sheet formation with the exception of the F23R variant. Moreover, the presence of cholesterol did not modify this behavior, and the ß-sheet structural transitions were not registered when the N-terminal domain of hIAPP (K1-S20) was characterized. Considering that insulin granules are enriched in phosphatidylserine (PS), the property of lipid vesicles containing negatively charged lipids was also evaluated. We found that these types of lipids promote ß-sheet conformational transitions in both the C-native segment and the new variants. Furthermore, these PS/peptides arrangements are internalized in Langerhans islet ß-cells, localized in the endoplasmic reticulum, and trigger critical pathways such as unfolded protein response (UPR), affecting insulin secretion. Since this phenomenon was associated with the presence of cytotoxicity on Langerhans islet ß-cells, it can be concluded that the anionic lipid environment and degree of solvation are critical conditions for the stability of segments with the propensity to form ß-sheet structures, a situation that will eventually affect the structural characteristics and stability of IAPP within insulin granules, thus modifying the insulin secretion.

Preparation of Polymeric Films of PVDMA-PEI Functionalized with Fatty Acids for Studying the Adherence and Proliferation of Langerhans ß-Cells.

This study reports the synthesis of thin polymeric films by the layer-by-layer deposition and covalent cross-linking of polyvinyl dimethylazlactone and polyethylene imine, which were functionalized with lauric (12-C), myristic (14-C), and palmitic (16-C) saturated fatty acids, whose high levels in the bloodstream are correlated with insulin resistance and the potential development of type 2 diabetes mellitus. Aiming to assess the effect of the fatty acids on the adhesion and proliferation of Langerhans ß-cells, all prepared films (35 and 35.5 bilayers with and without functionalization with the fatty acids) were characterized in terms of their physical, chemical, and biological properties by a battery of experimental techniques including 1H and 13C NMR, mass spectrometry, attenuated total reflectance-Fourier transform infrared spectroscopy, field emission scanning electron microscopy, atomic force microscopy, cell staining, and confocal laser scanning microscopy among others. In general, the developed films were found to be nanometric, transparent, resistant against manipulation, chemically reactive, and highly cytocompatible. On the other hand, in what the effect of the fatty acids is concerned, palmitic acid was found to impair the proliferation of the cultured ß-cells, contrary to its homologues which did not alter this biological process. In our opinion, the multidisciplinary study presented here might be of interest for the research community working on the development of cytocompatible 2D model substrates for the safe and reproducible characterization of cell responses.

Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity.

Gemini surfactants (GS) have been highlighted as attractive gene carriers for a few years now; however, key aspects of the role of the GS chemical structure on the DNA-GS complexation and subsequent biological activity remain to be determined. Aiming to elucidate the effects of the GS spacer hydrophobicity, this work was focused on the biophysical characterization of the self-assembly, DNA complexation, cytocompatibility, and DNA transfection of a series of bis-quaternary ammonium GS with fixed side alkyl chains of 14 carbons and varying head-to-head alkyl chain spacers of 4, 6, and 14 carbons (referred to as GS4, GS6, and GS14, respectively). The characterization was carried out by a battery of experimental techniques including UV-vis and fluorescence sprectroscopies, ζ potential, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and flow cytometry, among others. Overall, the spectroscopic results showed that the self-assembly of the GS was favored with the spacer hydrophobicity since lower values of critical micelle concentration (CMC) were observed for samples with longer spacer chains. On the other hand, the ITC results revealed that the DNA-GS complexation was driven by an initial electrostatic attraction between DNA and GS monomers/micelles followed by complementary hydrophobic interactions which strengthen the DNA-GS binding, the latter being more pronounced for GS with longer spacers. Finally, the biological tests demonstrated that while GS with moderate hydrophobicity (GS4 and GS6) yielded outstanding levels of cytocompatibility and DNA transfection over a range of concentrations, the most hydrophobic sample (GS14) proved to be cytotoxic upon administration to cultured HeLa cells (p < 0.05). In our opinion, the fundamental information here presented might be pivotal not only for understanding the DNA-GS complexation mechanism, but also for developing efficient GS-based carriers for gene therapy.

Photocrosslinked Alginate-Methacrylate Hydrogels with Modulable Mechanical Properties: Effect of the Molecular Conformation and Electron Density of the Methacrylate Reactive Group.

Hydrogels for load-bearing biomedical applications, such as soft tissue replacement, are required to be tough and biocompatible. In this sense, alginate-methacrylate hydrogels (H-ALGMx) are well known to present modulable levels of elasticity depending on the methacrylation degree; however, little is known about the role of additional structural parameters. In this work, we present an experimental-computational approach aimed to evaluate the effect of the molecular conformation and electron density of distinct methacrylate groups on the mechanical properties of photocrosslinked H-ALGMx hydrogels. Three alginate-methacrylate precursor macromers (ALGMx) were synthesized: alginate-glycidyl methacrylate (ALGM1), alginate-2-aminoethyl methacrylate (ALGM2), and alginate-methacrylic anhydride (ALGM3). The macromers were studied by Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H-NMR), and density functional theory method (DFT) calculations to assess their molecular/electronic configurations. In parallel, they were also employed to produce H-ALGMx hydrogels, which were characterized by compressive tests. The obtained results demonstrated that tougher hydrogels were produced from ALGMx macromers presenting the C=C reactive bond with an outward orientation relative to the polymer chain and showing free rotation, which favored in conjunction the covalent crosslinking. In addition, although playing a secondary role, it was also found that the presence of acid hydrogen atoms in the methacrylate unit enables the formation of supramolecular hydrogen bonds, thereby reinforcing the mechanical properties of the H-ALGMx hydrogels. By contrast, impaired mechanical properties resulted from macromer conditions in which the C=C bond adopted an inward orientation to the polymer chain accompanied by a torsional impediment.

Fatty Acid and Lipopolysaccharide Effect on Beta Cells Proteostasis and its Impact on Insulin Secretion.

Metabolic overload by saturated fatty acids (SFA), which comprises ß-cell function, and impaired glucose-stimulated insulin secretion are frequently observed in patients suffering from obesity and type 2 diabetes mellitus. The increase of intracellular Ca2+ triggers insulin granule release, therefore several mechanisms regulate Ca2+ efflux within the ß-cells, among others, the plasma membrane Ca2+-ATPase (PMCA). In this work, we describe that lipotoxicity mediated mainly by the saturated palmitic acid (PA) (16C) is associated with loss of protein homeostasis (proteostasis) and potentially cell viability, a phenomenon that was induced to a lesser extent by stearic (18C), myristic (14C) and lauric (12C) acids. PA was localized on endoplasmic reticulum, activating arms of the unfolded protein response (UPR), as also promoted by lipopolysaccharides (LPS)-endotoxins. In particular, our findings demonstrate an alteration in PMCA1/4 expression caused by PA and LPS which trigger the UPR, affecting not only insulin release and contributing to ß-cell mass reduction, but also increasing reactive nitrogen species. Nonetheless, stearic acid (SA) did not show these effects. Remarkably, the proteolytic degradation of PMCA1/4 prompted by PA and LPS was avoided by the action of monounsaturated fatty acids such as oleic and palmitoleic acid. Oleic acid recovered cell viability after treatment with PA/LPS and, more interestingly, relieved endoplasmic reticulum (ER) stress. While palmitoleic acid improved the insulin release, this fatty acid seems to have more relevant effects upon the expression of regulatory pumps of intracellular Ca2+. Therefore, chain length and unsaturation of fatty acids are determinant cues in proteostasis of ß-cells and, consequently, on the regulation of calcium and insulin secretion.

NIR-Emitting Alloyed CdTeSe QDs and Organic Dye Assemblies: A Nontoxic, Stable, and Efficient FRET System.

In the present work, we synthesize Near Infrared (NIR)-emitting alloyed mercaptopropionic acid (MPA)-capped CdTeSe quantum dots (QDs) in a single-step one-hour process, without the use of an inert atmosphere or any pyrophoric ligands. The quantum dots are water soluble, non-toxic, and highly photostable and have high quantum yields (QYs) up to 84%. The alloyed MPA-capped CdTeSe QDs exhibit a red-shifted emission, whose color can be tuned between visible and NIR regions (608-750 nm) by controlling the Te:Se molar ratio in the precursor mixtures and/or changing the time reaction. The MPA-capped QDs were characterized by UV-visible absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), and zeta potential measurements. Photostability studies were performed by irradiating the QDs with a high-power xenon lamp. The ternary MPA-CdTeSe QDs showed greater photostability than the corresponding binary MPA-CdTe QDs. We report the Förster resonance energy transfer (FRET) from the MPA-capped CdTeSe QDs as energy donors and Cyanine5 NHS-ester (Cy5) dye as an energy acceptor with efficiency (E) up to 95%. The distance between the QDs and dye (r), the Förster distance (R0), and the binding constant (K) are reported. Additionally, cytocompatibility and cell internalization experiments conducted on human cancer cells (HeLa) cells revealed that alloyed MPA-capped CdTeSe QDs are more cytocompatible than MPA-capped CdTe QDs and are capable of ordering homogeneously all over the cytoplasm, which allows their use as potential safe, green donors for biological FRET applications.

Biocompatible hollow polymeric particles produced by a mild solvent- and template free strategy.

Macroscopic hollow polymeric particles are attractive materials for various applications such as surgery, food industry, agriculture, etc. However, protocols reporting their synthesis have hitherto made use of organic solvents and/or sacrificial templates, compromising the encapsulation of different bioactive compounds and the process yield. Here, millimeter-size, hollow polymeric particles were synthesized, for the first time, in a solvent- and template free manner onto superhydrophobic surfaces (SHS). The particles were produced upon assembly and double superficial crosslinking of liquid droplets of DNA and methacrylamide chitosan aqueous solutions (CH:MA), leading to liquid-core particles with a hardened hydrogel shell. The particles displayed appealing physical and biological properties. The millimeter-size hydrogel shell, resulting from the double ionic/covalent crosslinking of CH:MA, endowed the hollow particles with softness to the touch and an outstanding structural stability against manipulation by hand and with forceps. Meanwhile, the liquid DNA core guaranteed a biocompatible cell encapsulation followed by a superior release and proliferation of viable cells, as compared to solid CH:MA particles prepared as a blank. Particles with these characteristics show promise for surgical protocols practiced in Tissue Engineering and Regenerative Medicine, where manipulable and biocompatible synthetic implants are often needed to supply living cells and other sensitive bioactive compounds.

Biocompatible hollow polymeric particles produced by a mild solvent- and template free strategy.

Macroscopic hollow polymeric particles are attractive materials for various applications such as surgery, food industry, agriculture, etc. However, protocols reporting their synthesis have hitherto made use of organic solvents and/or sacrificial templates, compromising the encapsulation of different bioactive compounds and the process yield. Here, millimeter-size, hollow polymeric particles were synthesized, for the first time, in a solvent- and template free manner onto superhydrophobic surfaces (SHS). The particles were produced upon assembly and double superficial crosslinking of liquid droplets of DNA and methacrylamide chitosan aqueous solutions (CH:MA), leading to liquid-core particles with a hardened hydrogel shell. The particles displayed appealing physical and biological properties. The millimeter-size hydrogel shell, resulting from the double ionic/covalent crosslinking of CH:MA, endowed the hollow particles with softness to the touch and an outstanding structural stability against manipulation by hand and with forceps. Meanwhile, the liquid DNA core guaranteed a biocompatible cell encapsulation followed by a superior release and proliferation of viable cells, as compared to solid CH:MA particles prepared as a blank. Particles with these characteristics show promise for surgical protocols practiced in Tissue Engineering and Regenerative Medicine, where manipulable and biocompatible synthetic implants are often needed to supply living cells and other sensitive bioactive compounds.

Polysaccharide-Based Nanobiomaterials as Controlled Release Systems for Tissue Engineering Applications.

Polysaccharides belong to a special class of biopolymers that has been used in different areas of research and technology for some years now. They present distinctive features attractive for the biomedical field. Among others, as extracted from natural sources, these materials are usually biocompatible and possess a significant ability to absorb water. Moreover, they can be conveniently modified by chemical means so as to display improved biological and physicochemical properties. The last but not the least, they are abundant in the natural Extracellular Matrix (ECM) and have a tremendous affinity for different endogenous macromolecules. Accordingly, these particular materials constitute outstanding candidates for a variety of biomimetic approaches entailing the entrapment/stabilization of bioactive molecules (e.g. growth factors, siRNA, and DNA) that could be delivered and have an effect on relevant cellular mechanisms, such as gene expression and cell viability, -proliferation, and -differentiation. This review will explore the current status of nano-scale drug delivery devices based on polysaccharides that could be used in tissue engineering and regenerative medicine (TERM). Aiming to contextualize the topics here discussed, especially for non-experts in the field, section 1 (Introduction) will present a brief overview of TERM and the principal polysaccharides herein employed. In order to get a broader perspective on both issues, this section will include a brief description of non-nanometric systems with relevant characteristics for TERM, such as injectable microparticles and macroscopic hydrogels, just to cite a few. Section 2 will illustrate the contributions of nanotechnology to the development of TERM, in particular to the development of biomimetic systems capable of replicating the natural, endogenous ECMs. Next, sections 3 to 6 will describe representative systems in the nanometric scale presenting 0D (nanoparticles), 1D (nanorods and nanowires), 2D (thin coatings/films or multilayered systems), and 3D (woven nanofibrillar mats and meshes) configurations, respectively. Special attention will be paid on how nanometric constructs with these configurations can be used as model systems in TERM to understand and/or manipulate biological functions at the cellular level. Finally, section 7 will provide an outlook on future perspectives in the field. Overall, the review is intended to constitute a critical source of information relative to the current status of polysaccharide- based biomaterials for TERM, in particular those at the nanometric scale.

Enhanced cell affinity of chitosan membranes mediated by superficial cross-linking: a straightforward method attainable by standard laboratory procedures.

It is well accepted that the surface modification of biomaterials can improve their biocompatibility. In this context, techniques like ion etching, plasma-mediated chemical functionalization, electrospinning, and contact microprinting have successfully been employed to promote the cell adhesion and proliferation of chitosan (CH) substrates. However, they prove to be time-consuming, highly dependent on environmental conditions, and/or limited to the use of expensive materials and sophisticated instruments not accessible to standard laboratories, hindering to a high extent their straightforward application. Filling this gap, this paper proposes the superficial cross-linking of CH as a much simpler and accessible means to modify its superficial properties in order to enhance its cellular affinity. CH membranes were prepared by solvent casting followed by a cross-linking step mediated by the chemical vapor deposition (CVD) of glutaraldehyde (GA). The membranes were characterized against non- and solution cross-linked membranes in terms of their mechanical/surface properties and biological performance. Among others, the CVD membranes proved (i) to be more mechanically stable against cell culture and sterilization than membranes cross-linked in solution and (ii) to prompt the adherence and sustained proliferation of healthy cells to levels even superior to commercial tissue culture plates (TCPs). Accordingly, the CVD cross-linking approach was demonstrated to be a simple and cost-effective alternative to the aforementioned conventional methods. Interestingly, this concept can also be applied to other biomaterials as long as GA (or other volatile components alike) can be employed as a cross-linker, making possible the cross-linking reaction at mild experimental conditions, neither requiring sophisticated lab implements nor using any potentially harmful procedure.