Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischemia, inflammation, and infection costing $7.5 billion/year in the US alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization, and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA microparticles that provides a sustained release of bioactive insulin for >20days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring, healing. Using a heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04mg insulin/cm2, every three days for 9 days, have faster closure, faster rate of disintegration of dead tissue, and decreased oxidative stress.In addition, in insulin-treated wounds the pattern of neutrophil inflammatory response suggests faster clearing of the burn dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibers organized more like a basket weave (normal skin) than aligned and crosslinked (scar tissue). In summary , application of ASD-containing insulin-loaded PLGA particles on burns every three days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Assessment of a protease inhibitor peptide for anti-ageing

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.

Poly(vinylidene fluoride-trifluoroethylene)/NAY zeolite hybrid membranes as a drug release platform applied to ibuprofen release

Poly(vinylidene fluoride-trifluoroethylene)/NaY zeolite composite membranes were prepared by solvent casting and evaluated as a suitable drug release platform through the evaluation of loading and release of ibuprofen. The membranes were characterized at the morphological, structural and mechanical levels. The 1H-NMR spectra indicate that only the membranes with 16 and 32 % of NaY were useful for IBU encapsulation and the drug release was followed by UV-Vis spectroscopy. The release profile is independent of the zeolite content and can be described by the Korsmeyer-Peppas model. The membrane with 32 % zeolite content releases more than double IBU amount when compared with the membrane with 16 % showing that zeolite content allows tailoring membrane drug release content for specific applications. The drug release platform developed in this work is suitable for other drugs and applications.

Photolabile protection for amino acids: studies on the release from novel benzoquinolone cages

The synthesis of a novel fused nitrogen heterocycle, benzoquinolone, for evaluation as a photocleavable protecting group is described for the first time, by coupling to model amino acids (alanine, phenylalanine and glutamic acid). Conversion of the phenylalanine ester conjugate to the thionated derivative was accomplished by reaction with Lawesson’s reagent. Photocleavage studies of the carbonyl and thiocarbonyl benzoquinolone conjugates in various solvents and at different wavelengths (300, 350 and 419 nm) showed that the most interesting result was obtained at 419 nm for the thioconjugate, revealing that the presence of the thiocarbonyl group clearly improved the photolysis rates, giving practicable irradiations times for the release of the amino acids (less than 1 minute).

Aminobenzocoumarinylmehtyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2Hnaphtho[1,2-b] pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. As well as the photolysis, the photophysics of the compounds were characterised by both steady state and time-resolved methods.

Pró-fármacos fotoativáveis do péptido AAPV: síntese e avaliação da libertação controlada por luz

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)

Biomimetic supramolecular designs for the controlled release of growth factors in bone regeneration

The extracellular matrix (ECM) of tissues is an assembly of insoluble macromolecules that specifically interact with soluble bioactive molecules and regulate their distribution and availability to cells. Recapitulating this ability has been an important target in controlled growth factor delivery strategies for tissue regeneration and requires the design of multifunctional carriers. This review describes the integration of supramolecular interactions on the design of delivery strategies that encompass self-assembling and engineered affinity components to construct advanced biomimetic carriers for growth factor delivery. Several glycan- and peptide-based self-assemblies reported in the literature are highlighted and commented upon. These examples demonstrate how molecular design and chemistry are successfully employed to create versatile multifunctional molecules which self-assemble/disassemble in a precisely predicted manner, thus controlling compartmentalization, transport and delivery. Finally, we discuss whether recent advances in the design and preparation of supramolecular delivery systems have been sufficient to drive real translation towards a clinical impact. 

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Development and characterization of lipid-based nanosystems for controlled release of bioactive compounds

Tese de Doutoramento em Engenharia Química e Biológica.

MET is required for the recruitment of anti-tumoural neutrophils

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-a or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Encapsulation and controlled release of bioactive compounds in lactoferrin-glycomacropeptide nanohydrogels : curcumin and caffeine as model compounds

Curcumin and caffeine (used as lipophilic and hydrophilic model compounds, respectively) were successfully encapsulated in lactoferrin-glycomacropeptide (Lf-GMP) nanohydrogels by thermal gelation showing high encapsulation efficiencies (>90 %). FTIR spectroscopy confirmed the encapsulation of bioactive compounds in Lf-GMP nanohydrogels and revealed that according to the encapsulated compound different interactions occur with the nanohydrogel matrix. The successful encapsulation of bioactive compounds in Lf-GMP nanohydrogels was also confirmed by fluorescence measurements and confocal laser scanning microscopy. TEM images showed that loaded nanohydrogels maintain their spherical shape with sizes of 112 and 126 nm for curcumin and caffeine encapsulated in Lf-GMP nanohydrogels, respectively; in both cases a polydispersity of 0.2 was obtained. The release mechanisms of bioactive compounds through Lf-GMP nanohydrogels were evaluated at pH 2 and pH 7, by fitting the Linear Superimposition Model to the experimental data. The bioactive compounds release was found to be pH-dependent: at pH 2, relaxation is the governing phenomenon for curcumin and caffeine compounds and at pH 7 Ficks diffusion is the main mechanism of caffeine release while curcumin was not released through Lf-GMP nanohydrogels.

Growth of sulfate reducing bacteria on the methanogenic inhibitor BES

The metabolism of methanogenic archaea is inhibited by 2-bromoethanesulfonate (BES). Methane production is blocked because BES is an analog of methyl-coenzyme M and competes with this key molecule in the last step of methanogenesis. For this reason, BES is commonly used in several studies to avoid growth of acetoclastic and hydrogenotrophic methanogens [1]. Despite its effectiveness as methanogenic inhibitor, BES was found to alter microbial communities’ structure, to inhibit the metabolism of non-methanogenic microorganisms and to stimulate homoacetogenic metabolism [2,3]. Even though sulfonates have been reported as electron acceptors for sulfate- and sulfite-reducing bacteria (SRB), only one study described the reduction of BES by complex microbial communities [4]. In this work, a sulfate-reducing bacterium belonging to Desulfovibrio genus (98 % identity at the 16S rRNA gene level with Desulfovibrio aminophilus) was isolated from anaerobic sludge after several successive transfers in anaerobic medium containing BES as sole substrate. Sulfate was not supplemented to the anaerobic growth medium. This microorganism was able to grow under the following conditions: on BES plus H2/CO2 in bicarbonate buffered medium; on BES without H2/CO2 in bicarbonate buffered medium; and on BES in phosphate buffered medium. The main products of BES utilization were sulfide and acetate, the former was produced by the reduction of sulfur from the sulfonate moiety of BES and the latter likely originated from the carbon backbone of the BES molecule. BES was found, in this study, to represent not only an alternative electron acceptor but also to serve as electron donor, and sole carbon and energy source, supporting growth of a Desulfovibrio sp. obtained in pure culture. This is the first study that reports growth of SRB with BES as electron donor and electron acceptor, showing that the methanogenic inhibitor is a substrate for anaerobic growth.

Development of a poly(L-Lactic acid) based drug release system

Dissertação de mestrado em Biofísica e Bionanossistemas

Development of nanohydrogels as a vehicle for the release of bioactive compounds in food matrices

PhD in Chemical and Biological Engineering