Diana Soares da Costa

Controlling Cancer Cell Fate Using Localized Biocatalytic Self-Assembly of an Aromatic Carbohydrate Amphiphile

We report on a simple carbohydrate amphiphile able to self-assemble into nanofibers upon enzymatic dephosphorylation. The self-assembly can be triggered by alkaline phosphatase (ALP) in solution or in situ by the ALP produced by osteosarcoma cell line, SaOs2. In the latter case, assembly and localized gelation occurs mainly on the cell surface. The gelation of the pericellular environment induces a reduction of the SaOs2 metabolic activity at an initial stage (≤7 h) that results in cell death at longer exposure periods (≥24 h). We show that this effect depends on the phosphatase concentration, and thus, it is cell-selective with prechondrocytes ATDC5 (that express ∼15-20 times lower ALP activity compared to SaOs2) not being affected at concentrations ≤1 mM. These results demonstrate that simple carbohydrate derivatives can be used in an antiosteosarcoma strategy with limited impact on the surrounding healthy cells/tissues.

Gel network photodisruption: a new strategy for the codelivery of plasmid DNA and drugs.

In the last 5 years, we have gained further insight on the physical/chemical field of DNA gels. Our expertise on the gel swelling behavior, compaction of DNA by cationic entities, as lipids and surfactants, as well as on the assembly structures of these complexes allow us for the development of novel systems to be used in a variety of biomedical applications. In our previous reports, the physicochemical characterization has been well-established, and now one can evolve to the challenge of using DNA-based carriers in the biological area. Moreover, a new plasmid DNA (pDNA) hydrogel that is porous, is able to swell in the presence of additives, is biocompatible and, thus, is suitable to be used therapeutically was prepared. Here, the dual release of pDNA and solutes with pharmaceutical interest was the main challenge, and thus, we report on the photodisruption of plasmid DNA (pDNA) gels cross-linked with ethylene glycol diglycidyl ether (EGDE) as a strategy for this simultaneous release. The disruption over time, after the irradiation of the gel with ultraviolet light (400 nm), was characterized through the cumulative plasmid DNA release, the evolution in dry weight, the extent of swelling, and also the variations in the gel mesh size. The controlled release of different molecular weight solutes from plasmid DNA gels was investigated, and the influence of both the hydrogel degradation and cross-linker density on the release kinetics were addressed. While the release of lysozyme follows a Fickian process, the release of bovine serum albumin (BSA) and fluoresceinisothiocyanato-dextran (FITC-dextran) is characteristic of a Super Case II release phenomena. In addition, the size of the three solutes partially influences the release behavior; polymer chain mobility and the degree of swelling also play a role. To gain a fundamental understanding of drug release profile from pDNA matrices, in vitro release studies were evaluated using several anti-inflammatory drugs. The quantification of the release mechanism indicates a Super Case II release profile, which can be related with the gel swelling degree. A correlation between the drug release trend and the drug hydrophobicity can be found, with more hydrophobic drugs showing a slower release rate. In brief, this new pDNA gel system is biocompatible, is degradable upon light irradiation, and allows for the controlled and sustained release of plasmid DNA and incorporated solutes. This codelivery of pDNA and drugs would find relevant clinical uses due to the possibility of gene and nongene therapy combination in order to improve the therapeutic efficiency.

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary.Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.

Interactions between exogenous FGF-2 and sulfonic groups: in situ characterization and impact on the morphology of human adipose-derived stem cells.

FGF-2 is often used as a supplement to stem cells culture medium aiming at preserving their self-renewal capacity and plasticity through the passages. However, little is known on the influence of the underlying substrate in these interactions. In this study, we have used mixed self-assembled monolayers with different ratios of -SO3H and -OH tail groups to investigate the influence of substrate properties (e.g., charge) on the FGF-2 adsorption and activity. QCM-D data demonstrated that, in the presence of -OH groups, the quantity of the adsorbed FGF-2 is proportional to the percentage of surface -SO3H groups. The bioactivity of the adsorbed FGF-2 follows the same tendency as demonstrated by its interactions with anti-FGF-2. Surprisingly, the adlayer of FGF-2 formed on the surface containing only SO3H-tailed SAMs was similar to the surface with 25% of -SO3H groups, demonstrating that FGF-2 adsorption is not solely driven by electrostatic interactions. We related these results with changes in the morphology of adipose-derived stem cells (ASCs) cultured on the same surfaces.

Functional biopolymer-based matrices for modulation of chronic wound enzyme activities

Collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan (CS) sponges loaded with epigallocatechin gallate (EGCG), catechin (CAT) and gallic acid (GA) were developed and evaluated as active chronic wound dressings. Their physico-mechanical properties, biostability, biocompatibility and ability to inhibit in vitro myeloperoxidase (MPO) and collagenase--major enzymes related with the persistent inflammation in chronic wounds--were investigated as a function of the biopolymer composition and the polyphenolic compound used. The results demonstrated that the molecular weight of HA influences significantly the bulk properties of the obtained materials: higher elastic modulus, swelling ability and biostability against collagenase were measured when HA with higher molecular weights (830 and 2000 kDa) were added to the collagen matrices. The addition of CS and the polyphenols increased further the biostability of the sponges. Preliminary in vitro tests with fibroblasts revealed that the cells were able to adhere to all sponges. Cell viability was not affected significantly by the addition of the polyphenols; however, the presence of CS or high molecular weight HA in the sponge composition was associated with lower cellular viability. Finally, all specimens containing polyphenols efficiently inhibited the MPO activity. The highest inhibition capacity was observed for EGCG (IC₅₀=15±1μM) and it was coupled to the highest extent of binding to the biopolymers (>80%) and optimal release profile from the sponges that allowed for prolonged (up to 3-5 days) effects.

Plasmid DNA Microgels for a Therapeutical Strategy Combining the Delivery of Genes and Anticancer Drugs

Plasmid DNA (pDNA) is encapsulated into biocompatible microgels by an inverse microemulsion polymerization method. Plasmid DNA and doxorubicin are successfully released from pDNA microgels and their release profiles are characterized by appropriate release models. The co-delivery of genes and drugs from the microgels is evaluated as an enhancer of clinical treatment. Moreover, the release of the encapsulated pDNA is capable of transfection in vitro resulting in the expression of p53 protein. As a whole, a novel pDNA-based system is described that may find biomedical uses, especially in the cancer treatment through the combined action of chemotherapy and gene delivery approach.

Sulfation of Glycosaminoglycans and Its Implications in Human Health and Disorders

Sulfation is a dynamic and complex posttranslational modification process. It can occur at various positions within the glycosaminoglycan (GAG) backbone and modulates extracellular signals such as cell-cell and cell-matrix interactions; different sulfation patterns have been identified for the same organs and cells during their development. Because of their high specificity in relation to function, GAG sulfation patterns are referred to as the sulfation code. This review explores the role of GAG sulfation in different biological processes at the cell, tissue, and organism levels. We address the connection between the sulfation patterns of GAGs and several physiological processes and discuss the misregulation of GAG sulfation and its involvement in several genetic and metabolic disorders. Finally, we present the therapeutic potential of GAGs and their synthetic mimics in the biomedical field.

Sulfonic groups induce formation of filopodia in mesenchymal stem cells

Glycosaminoglycans (GAGs) are an integral part of the extracellular matrix and glycocalix, i.e. the closest cellular environment. They are abundant in –OH groups and their bioactivity is also associated with the presence of negatively charged –SO3H functionalities. Therefore, we have investigated and discussed the influence of these functional units on mesenchymal stem cells (MSCs) behaviour using single component and mixed self-assembled monolayers of alkanethiols with –SO3H and –OH end groups. In the absence of serum, MSCs attachment, spreading, cytoskeleton organisation and motility were significantly influenced by the surface chemistry. We found that the sulfonic groups induce star-like cell shape with very intense actin staining and a high density net of filopodia that enlarge from the base of lamellipodia structures. Moreover, this response is concentration dependent and is apparent only for very short culture time in the presence of serum.

Dissecting cardosin B trafficking pathways in heterologous systems.

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins’ high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.

GAGs-thiolated chitosan assemblies for chronic wounds treatment: control of enzyme activity and cell attachment

Multilayered polyelectrolyte coatings comprising thiolated chitosan (TC) and glycosaminoglycans (GAGs), chondroitin sulphate and hyaluronic acid, were built using a layer-by-layer approach. The surface activity of these coatings for binding and inhibition of enzymes related to chronic inflammation, such as collagenase and myeloperoxidase, was assessed. The build-up of five bi-layers of TC/GAGs onto gold surfaces was monitored in situ by QCM-D. All experimental groups showed exponential growth of the coatings controlled by the degree of chitosan thiolation and the molecular weight of the GAGs. The degree of chitosan modification was also the key parameter influencing the enzyme activity: increasing the thiols content led to more efficient myeloperoxidase inhibition and was inversely proportional to the adsorption of collagenase. Enhanced fibroblast attachment and proliferation were observed when the multilayered polyelectrolyte constructs terminated with GAGs. The possibility to control either the activity of major wound enzymes by the thiolation degree of the coating or the cell adhesion and proliferation by proper selection of the ultimate layer makes these materials potentially useful in chronic wounds treatment and dermal tissue regeneration.