Luciana Magalhães Rebêlo

Diverse deformation properties of polymeric nanocapsules and lipid-core nanocapsules

The deformation properties of submicrometric drug carriers can influence their tissue-penetration ability and thus the drug targeting. The aim of this study was to determine whether the oily core composition (raw oils or a dispersion of oils and solid lipid) surrounded by a polymeric wall [poly(e-caprolactone), (PCL)] can affect the deformation properties of nanocapsules (NCs) or lipid-core nanocapsules (LNCs). Formulations were prepared as aqueous suspensions using a polymer and either a mixture of caprylic/capric triglyceride (CCT) and octyl methoxycinnamate (OMC) or a mixture of CCT, OMC and sorbitan monostearate (SM) as core components, respectively. Formulations had mean diameters close to 200 nm presenting monomodal distributions. A polysorbate 80 coating rendered ζ-potential values close to zero, acting as a steric stabilizer. Atomic force microscopy (AFM) showed, through force curves analysis, that the cantilever deflection was more pronounced for the LNCs than for the NCs. The same force applied to NC produced an indentation around twice that observed for the LNCs. The Youngs modulus (E) values were 0.537 MPa (LNC) and 0.364 MPa (NC) considering conical geometry while E = 0.17 MPa (NC) and E = 0.241 (LNC) for spherical geometry. These data confirm that the LNCs are stiffer than the NCs. The rigidity of both the polymer wall and lipid core is higher for LNCs. In conclusion, LNCs presented distinct mechanical properties compared to the conventional polymeric NCs.

Thickness-corrected model for nanoindentation of thin films with conical indenters

Nanoindentation of soft materials is a growing research field, demanding sophisticated models to extract accurate information from these materials. In this work we investigate the nanoindentation of thin soft films by sharp conical indenters using Finite Elements Modeling. Based on the work of Dimitriadis et al. [Biophys. J. 82, 2798 (2002)], we propose that load-displacement (F × δ) curves for conical indenters can be described by F = FHertz(δ)g[χ(δ,h)], where FHertz(δ) is the regular Hertz model, and g[χ(δ,h)] is a correction function that includes finite thickness effects. To test the applicability of the model, we analyze the elastic modulus of fibroblast cells as measured by Atomic Force Microscopy. The elastic modulus obtained with Hertz model is overestimated by 50% (when compared to our thickness-corrected model) in the thickest parts of the cell (3.67 μm), and by approximately 128% in the lamellipodia region (0.45 μm), illustrating the importance of the sample thickness for the evaluation of the mechanical properties.

In vitro evaluation of anti-calcification and anti-coagulation on sulfonated chitosan and carrageenan surfaces

In recent years, great effort has been devoted to the development of biomaterials that come into contact with blood. The surfaces of these materials need to be of suitable mechanical strength, and present anti-thrombogenic and anti-calcification properties. Chitosan is a natural polymer that has attracted attention due to its potential to act as a biomaterial. However, chitosan contains amino groups in its structure that may promote thrombogenesis and calcification. A strategy to reduce these properties constitutes the introduction of sulfonate groups (R-SO3-) in the chitosan chain. Another interesting biopolymer with similar characteristics to those of heparin is carrageenan, which has sulfate groups in its structure. As such, we evaluated “in vitro” calcification and thrombogenic processes on surfaces of pristine and sulfonated chitosan and on polyelectrolyte complexes (PEC) of chitosan and carrageenan. Results indicate that PEC demonstrate significant reductions in calcification and thrombogenic potential, probably due to the presence of sulfonate groups in both the carrageenan and treated chitosan.

Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: Insights into the mechanism of action.

CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.

Micromorphology and microrheology of modified bitumen by atomic force microscopy

The rheological study of bitumen is essential to improve the performance of pavements. Bitumen properties are traditionally determined by means of dynamic shear rheometer measurements. In this work, we aim to demonstrate that atomic force microscopy (AFM) can be a powerful complementary tool to the characterisation of bitumen. In particular, we compare the mechanical properties of pure and modified bitumen. Two different anti-oxidant additives were used: PPA (poly-phosphoric acid) and LCC (cashew nut oil) at concentrations of 1% and 2%, respectively. We will show that the AFM technique is able to distinguish the effects of both additives in the morphological and micromechanical properties of bitumen films.

Glutamine and Alanyl-Glutamine Increase RhoA Expression and Reduce Clostridium difficile Toxin-A-Induced Intestinal Epithelial Cell Damage

Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.

Experimental Diabetes Alters the Morphology and Nano-Structure of the Achilles Tendon

Although of several studies that associate chronic hyperglycemia with tendinopathy, the connection between morphometric changes as witnessed by magnetic resonance (MR) images, nanostructural changes, and inflammatory markers have not yet been fully established. Therefore, the present study has as a hypothesis that the Achilles tendons of rats with diabetes mellitus (DM) exhibit structural changes. The animals were randomly divided into two experimental groups: Control Group (n = 06) injected with a vehicle (sodium citrate buffer solution) and Diabetic Group (n = 06) consisting of rats submitted to intraperitoneal administration of streptozotocin. MR was performed 24 days after the induction of diabetes and images were used for morphometry using ImageJ software. Morphology of the collagen fibers within tendons was examined using Atomic Force microscopy (AFM). An increase in the dimension of the coronal plane area was observed in the diabetic group (8.583 ± 0.646 mm2/100g) when compared to the control group (4.823 ± 0.267 mm2/100g) resulting in a significant difference (p = 0.003) upon evaluating the Achilles tendons. Similarly, our analysis found an increase in the size of the transverse section area in the diabetic group (1.328 ± 0.103 mm2/100g) in comparison to the control group (0.940 ± 0.01 mm2/100g) p = 0.021. The tendons of the diabetic group showed great irregularity in fiber bundles, including modified grain direction and jagged junctions and deformities in the form of collagen fibrils bulges. Despite the morphological changes observed in the Achilles tendon of diabetic animals, IL1 and TNF-α did not change. Our results suggest that DM promotes changes to the Achilles tendon with important structural modifications as seen by MR and AFM, excluding major inflammatory changes.