Inês C. Gonçalves

Graphene-based materials biocompatibility: A review

Graphene-based materials (GBMs) have broad potential applications in biomedical engineering and biotechnology. However, existing studies regarding biological effects of GBMs often present contradictory or inconclusive results. This work presents a review of published data in order to provide a critical overview of the state of the art. Firstly, the distinct physical-chemical nature of the GBMs available is clarified, as well as the production methods involved. The review then discusses the available in vitro (with bacterial and mammalian cells) and in vivo studies concerning evaluation of GBMs biocompatibility, as well as existing hemocompatibility studies. The biocompatibility issues concerning composite materials that incorporate GBMs are addressed in a separate section, since encapsulation in a polymer matrix modifies biological interactions. The most pertinent questions that should be addressed in future works are also emphasized.

Biocompatibility of poly(lactic acid) with incorporated graphene-based materials

The incorporation of graphene-based materials has been shown to improve mechanical properties of poly(lactic acid) (PLA). In this work, PLA films and composite PLA films incorporating two graphene-based materials - graphene oxide (GO) and graphene nanoplatelets (GNP) - were prepared and characterized regarding not only biocompatibility, but also surface topography, chemistry and wettability. The presence of both fillers changed the films surface topography, increasing the roughness, and modified the wettability - the polar component of surface free energy increased 59% with GO and decreased 56% with GNP. Mouse embryo fibroblasts incubated with both fillers exceeded the IC(50) in both cases with a concentration of 10 μg mL(-1). No variations in cell proliferation at the surface of the composite films were observed, except for those containing GO after 24 h incubation, which presented higher cell proliferation than pristine PLA films. Platelet adhesion to PLA and PLA/GNP films was lower in the presence of plasma proteins than when no proteins were present. Furthermore, incorporation of GNP into PLA reduced platelet activation in the presence of plasma proteins. The results indicated that low concentrations of GO and GNP may be incorporated safely in PLA to improve aspects relevant for biomedical applications, such as mechanical properties.

Modulation of stability and mucoadhesive properties of chitosan microspheres for therapeutic gastric application

Chitosan microspheres have been explored for pharmaceutical applications, namely as a drug delivery systems for Helicobacter pylori gastric infection treatment, due to their mucoadhesive capacity. In this study, a different application of chitosan microspheres is proposed aiming the creation of an H. pylori-binding system where, after oral administration, microspheres will capture and remove these bacteria from infected patients, taking advantage of their muco/bacterial adhesive process. However, mucoadhesion is influenced by the degree of crosslinking necessary to avoid microspheres dissolution in the acidic gastric environment. During this work, the effect of genipin crosslinking on the stability, size, charge and mucoadhesive properties of chitosan microspheres under acidic pH was studied. Chitosan microspheres with ∼170 μm were produced by ionotropic gelation and subsequently covalently crosslinked with genipin in different degrees. The crosslinking reaction was followed by infrared spectroscopy and time-lapse fluorescence microscopy, since we have demonstrated that the fluorescence intensity of chitosan microspheres increases with genipin chemical bonding to chitosan. Results showed that both the zeta potential and the swelling capacity of chitosan microspheres decrease with increasing crosslinking. When immersed in simulated gastric fluid (SGF) with pepsin for 7 days, chitosan microspheres crosslinked with 10mM of genipin for 1h did not dissolve and doubled their size to approximately 345 μm. Furthermore, they maintained their in vitro mucoadhesion to soluble gastric mucins at both pH tested (3.6 and 6.5) and presented an in vivo retention time of around 2h in the stomach of C57BL/6 mice.

Effect of gastric environment on Helicobacter pylori adhesion to a mucoadhesive polymer.

Helicobacter pylori infection has been associated with several gastric diseases. This bacterium colonizes the gastric mucosa of half of the worlds population, and available treatments are unsuccessful in practically one in every five patients. Mucoadhesive polymers, such as chitosan, are being investigated as gastric drug delivery systems. However, since chitosan is also known for its antimicrobial properties, this work aims to evaluate H. pylori interactions with chitosan under simulated gastric environments, namely using various pHs (2.6, 4 and 6), pepsin and urea. To enable the visualization of adherent bacteria, ultrathin chitosan films were produced by spin-coating on gold/glass surfaces, cross-linked with genipin and characterized by Fourier transform infrared reflection absorption spectroscopy, ellipsometry and electrokinetic analysis. Films with homogeneous thickness of 11.7±0.6 nm were produced, and were stable and protonated at all the pHs used. Furthermore, they adsorbed pepsin in all these pHs, in contrast to urea, of which a small adsorption was only observed at pH 6. H. pylori binding to chitosan was higher at pH2.6 although most of adherent bacteria were dead. The presence of pepsin decreased bacterial adhesion, but increased its viability while in a more stressed morphology (coccoid form). The presence of urea did not affect the amount, morphology or viability of chitosan-adherent bacteria. In suspension, the decrease in pH changed H. pylori zeta potential from negative to positive. Moreover, bacteria were only culturable when incubated in pH 6 with and without urea (without pepsin). This work demonstrates that chitosan has the capacity to bind and kill H. pylori in a range of pHs independently of urea. This opens new perspectives for the application of chitosan-based materials to the elimination of H. pylori gastric colonization, though pepsin might appear to be an obstacle.

Effect of surface chemistry on bacterial adhesion, viability, and morphology

Helicobacter pylori (H. pylori) is one of the most common infectious agents in the world and it is thought to colonize the gastric mucosa of about half of the worlds population causing several gastric diseases. In this work, the effect of surface chemistry on H. pylori nonspecific adhesion, viability, and morphology was evaluated using three H. pylori strains with different adhesins expression profile. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to obtain surfaces exposing different functional groups: OH, CH3, and ethylene glycol (EG4). Bacterial adhesion onto the surfaces reached a plateau at 2 h. There was a correlation between adhesion and the exposed surface group, with bacterial cells adhering preferentially to CH3-SAMs while EG4-SAMs prevented H. pylori adhesion during the entire adhesion test (24 h). Surfaces that presented the EG4 group were also the only ones that significantly reduced the viability of adhered bacteria. Surface chemistry also influenced the morphology of adhered bacteria. The H. pylori rod shape observed in the control (Tissue Culture Polyethylene-TCPE) was only retrieved on CH3-SAMs. This work demonstrates that surface chemistry, namely specific functional groups on the material, influence the nonspecific adsorption of H. pylori. Moreover, the features of the bacterial strain and the surface chemistry can alter the adhesion kinetics, as well as the morphology and viability of attached bacteria.

Protein adsorption and clotting time of pHEMA hydrogels modified with C18 ligands to adsorb albumin selectively and reversibly

This work intended to create a nanostructured biomaterial that would bind albumin in a selective and reversible way in order to inhibit the adsorption of other blood proteins and therefore minimize activation of coagulation. Different levels of C18 ligand have been immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA). We hypothesize that samples with intermediate amounts of C18 ligand would allow albumin to recognize them and bind through its hydrophobic pockets specific for long chain fatty acids. Surface characterization has confirmed increasing amounts of C18 ligand on pHEMA as the percentage of C18 in solution increases, with maximum coverage achieved in 10% C18 samples. Adsorption from pure albumin solution revealed a small decrease in albumin adsorption from pHEMA to 1% C18 and 2.5% C18 samples, but on surfaces with 5% or higher C18 the amount of adsorbed albumin increased as the percentage of C18 increased. Competitive adsorption studies in the presence of both albumin and fibrinogen, and in the presence of all plasma proteins showed that 1% C18 and 2.5% C18 were the only surfaces selective for albumin, and that the presence of all plasma proteins may even potentiate albumin adsorption. Reversibility studies demonstrated that both 2.5% C18 and 5% C18 samples exchange (125)I-albumin selectively in the presence of both unlabeled albumin and plasma, but 2.5% C18 samples presented higher exchangeability rates (58%). Clotting times using recalcified plasma revealed that samples with none or small amounts of C18 (pHEMA to 5% C18) did not shorten the clotting time compared to the negative control (polystyrene), indicating low activation of the intrinsic coagulation cascade.

Selective protein adsorption modulates platelet adhesion and activation to oligo(ethylene glycol)-terminated self-assembled monolayers with C18 ligands

This study focuses on the selective binding of albumin to a nanostructured surfaces to inhibit other blood proteins from adsorbing thereby reducing platelet adhesion and activation. Tetra (ethylene-glycol)-terminated self-assembled monolayers (EG4 SAMs) with different percentages of C18 ligands on the surface were characterized by contact angle measurements, X-ray photoelectron microscopy, infrared reflection-absorption spectroscopy, and ellipsometry. A specific surface (2.5% C18 SAM) was found to be selective for human serum albumin (HSA) in the presence of both albumin and fibrinogen (HFG). The importance of this concentration of C18 ligands was stressed in reversibility studies since that surface exchanged almost all the preadsorbed HSA by HSA in solution, but not by HFG. The effect of protein adsorption in the subsequent adhesion and activation of platelets was studied by pre-immersing the surfaces in albumin and plasma before contact with platelets. Scanning electron microscopy and glutaraldehyde induced fluorescence technique images showed that as surfaces got more hydrophobic due to the immobilization of C18 ligands, the number of adherent platelets increased and their morphology changed from round to fully spread. Pre-immersion in HSA led to an 80% decrease in platelet adhesion and reduction of activation. Pre-immersion in 1% plasma was only relevant in 2.5% C18 SAMs since this was the only surface that demonstrated less adhesion of platelets comparing with buffer pre-immersion. However, they still adsorb more platelets then when HSA was preadsorbed. This was confirmed in competition studies between HSA and plasma that suggested that other plasma proteins were also adsorbing to this surface.

Bacterial-binding chitosan microspheres for gastric infection treatment and prevention

Helicobacter pylori (H. pylori) colonizes the gastric mucosa of over 50% of the world population, causing several pathologies, such as gastric ulcers and gastric cancer. Since current antibiotic treatments are inefficient in 20% of cases alternative therapies are needed. This work reports the ability of chitosan microspheres to adhere to H. pylori and prevent/remove H. pylori colonization. Adhesion of H. pylori strains with different functional adhesins (BabA and/or SabA) to chitosan microspheres (diameter 167 ± 27 μm) occurs at both pH 2.6 and 6.0, but is higher at pH 6.0. Bacterial adhesion to a gastric cell line expressing sialylated carbohydrates (SabA receptors) was performed at the same pH values using H. pylori strains with and without SabA. At both pH values addition of microspheres to gastric cells before and after pre-incubation with H. pylori decreased bacterial adhesion to cells. Furthermore, the chitosan microspheres were non-cytotoxic. These findings reveal the potential of chitosan microspheres as an alternative or complementary treatment for H. pylori gastric eradication or prevention of H. pylori colonization.

Platelet and leukocyte adhesion to albumin binding self-assembled monolayers

This study reports the use of tetraethylene glycol-terminated self-assembled monolayers (EG4 SAMs) as a background non-fouling surface to study the effect of an 18 carbon ligand (C18) on albumin selective and reversible adsorption and subsequent platelet and leukocyte adhesion. Surface characterization techniques revealed an efficient immobilization of different levels of C18 ligand on EG4 SAMs and an increase of surface thickness and hydrophobicity with the increase of C18 ligands. Albumin adsorption increased as the percentage of C18 ligands on the surface increased, but only 2.5%C18 SAMs adsorbed albumin in a selective and reversible way. Adherent platelets also increased with the amount of immobilized C18. Pre-immersion of samples in albumin before contact with platelets demonstrated an 80% decrease in platelet adhesion. Pre-immersion in plasma was only relevant for 2.5%C18 SAMs since this was the only surface to have less platelet adhesion compared to buffer pre-immersion. EG4 SAMs adhered negligible amounts of leukocytes, but surfaces with C18 ligands have some adherent leukocytes. Except for 10%C18 SAMs, which increased leukocyte adhesion after albumin pre-adhesion, protein pre-immersion did not influence leukocyte adhesion. It has been shown that a surface with a specific surface concentration of albumin-binding ligands (2.5%C18 SAMs) can recruit albumin selectively and reversibly and minimize the adhesion of platelets, despite still adhering some leukocytes.

Poly(lactic acid) Composites Containing Carbon-Based Nanomaterials: A Review

Poly(lactic acid) (PLA) is a green alternative to petrochemical commodity plastics, used in packaging, agricultural products, disposable materials, textiles, and automotive composites. It is also approved by regulatory authorities for several biomedical applications. However, for some uses it is required that some of its properties be improved, namely in terms of thermo-mechanical and electrical performance. The incorporation of nanofillers is a common approach to attain this goal. The outstanding properties of carbon-based nanomaterials (CBN) have caused a surge in research works dealing with PLA/CBN composites. The available information is compiled and reviewed, focusing on PLA/CNT (carbon nanotubes) and PLA/GBM (graphene-based materials) composites. The production methods, and the effects of CBN loading on PLA properties, namely mechanical, thermal, electrical, and biological, are discussed.