Nuno Azoia | Universidade do Minho (original) (raw)

Papers by Nuno Azoia

Current Pharmaceutical Design, 2016

Nanomedicine, the application of nanotechnology to medicine, is being increasingly used to improv... more Nanomedicine, the application of nanotechnology to medicine, is being increasingly used to improve and exploit the advantages of efficient drug delivery. Different nanodevices have been developed in recent years, among them protein-based nanoparticles which have gained considerable interest. Albumin is a versatile protein carrier with several characteristics that make it an ideal candidate for drug delivery, such as its availability, its biocompatibility, its biodegradability, and its lack of toxicity and immunogenicity. This review embodies an overview of different methods available for production of albumin-based nanoparticles, with focus on high-energy emulsification methods. A comparison between production by using sonication, which involves acoustic cavitation, and the high pressure homogenization method, where occurs hydrodynamic cavitation, is presented. Taking into account important properties of nanoparticles required for intravenous administration, the use of poloxamers, tri-block copolymer surfactants is discussed as it improves blood circulation time and bioavailability of nanoparticles. Thus, nanoparticles can be engineered to provide adequate features to therapeutic applications, in which can be included surface functionalization with targeting agents. Different albumin-based formulations and their therapeutic applications are presented in this review, with emphasis on applications in cancer therapy, where albumin-based strategies are promising for targeted drug delivery in innovative clinical strategies.

Skin permeation of large hydrophilic molecules remains a challenge. The barrier function of mamma... more Skin permeation of large hydrophilic molecules remains a challenge. The barrier function of mammalian skin is mainly attributed to the stratum corneum (SC), the outer protective layer, consisted by flat dead cells filled with keratin and surrounded by lipid bilayers. Within the SC, the lipid bilayers are the major responsible for the skin impermeability to relatively large compounds (molecular weight over 500 Da).

67 4,05 1,76 2,33 3,42 J 2ax-3ax = 11,4 J 3ax-3ea = 12,9 J 3eq-4ax = 5,4 J 2ax-3eq = 3,3 J 3ax-4a... more 67 4,05 1,76 2,33 3,42 J 2ax-3ax = 11,4 J 3ax-3ea = 12,9 J 3eq-4ax = 5,4 J 2ax-3eq = 3,3 J 3ax-4ax = 10,8

Protein and peptide letters, Jan 7, 2015

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloprote... more 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.

Biomacromolecules, 2015

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and i... more Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

In this text is presented a summary of the synthetic strategies used to build up either D-or L-py... more In this text is presented a summary of the synthetic strategies used to build up either D-or L-pyranose skeleton of sugar framework structures under stereoselective methodologies. Polihydroxylated piperidines were generated from simple starting material like amino-acids, tartaric acid or glyceraldehyde derivatives or even from achiral reagents. Introduction of chiral centres in achiral materials intermediates are generally achieved by catalytic methods, using different classes of ligands. In other cases diastereoselective methods and chemoenzymatic synthesis have been developed.

Colloids and Surfaces B: Biointerfaces, 2015

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri... more Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

Biochemical Engineering Journal, 2015

The keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin f... more The keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin features such as its assembling in the mentioned structures, its interaction with some compounds or mechanical properties is of great interest in the fight against some diseases or in the development and optimization of cosmetic products.

RSC Adv., 2015

ABSTRACT The study of the interaction between hair filaments and formulations or peptides is of u... more ABSTRACT The study of the interaction between hair filaments and formulations or peptides is of utmost importance in fields like cosmetic research. Keratin intermediate filaments structure is not fully described, limiting the molecular dynamics (MD) studies in this field although its high potential to improve the area. We developed a computational model of a truncated protofibril, simulated its behavior in alcoholic based formulations and with one peptide. The simulations showed a strong interaction between the benzyl alcohol molecules of the formulations and the model, leading to the disorganization of the keratin chains, which regress with the removal of the alcohol molecules. This behavior can explain the increase of peptide uptake in hair shafts evidenced in fluorescence microscopy pictures. The model developed is valid to computationally reproduce the interaction between hair and alcoholic formulations and provide a robust base for new MD studies about hair properties. It is shown that the MD simulations can improve hair cosmetic research, improving the uptake of a compound of interest.

Encapsulation in the form of micro and nanocapsules is an attractive route for controlling the de... more Encapsulation in the form of micro and nanocapsules is an attractive route for controlling the delivery and release of active proteins and peptides. Many approaches exist to probe the morphology of such capsules as well as their mechanisms of formation. By contrast, the release of proteins from such components in a complex biological environment has been challenging to probe directly. In this paper we show that the spectral differences between green fluorescent protein (GFP) in capsules and in its free form can be used to monitor in situ the release of the protein from the confinement of capsules. These findings represent a new route towards engineering the spectral characteristics of GFP through physical rather than chemical means. We demonstrate the use of GFP protein capsules for monitoring in real time the release of protein in live cells by exposing rat L6 myotubes to protein capsules. The GFP spheres with a blue fluorescent signal dissociate inside the L6 myotubes to individual GFP molecules with a change in fluorescent signal from blue to green. These sensitive spectral characteristics enabled us to resolve the dissociation of capsules inside the cells in both time and space. We discuss the implications of our results for quantifying parameters crucial for the delivery of proteins in biological environments.

Int J Cosmet Sci., Jun 2013

Human hair has an important and undeniable relevance in society due to its important role in visu... more Human hair has an important and undeniable relevance in society due to its important role in visual appearance and social communication. Hair is mainly composed of structural proteins, mainly keratin and keratin associated proteins and lipids. Herein, we report a comprehensive study of the content and distribution of the lipids among ethnic hair, African, Asian and Caucasian hair. More interestingly, we also report the study of the interaction between those two main components of hair, specifically, the influence of the hair internal lipids in the structure of the hair keratin. This was achieved by the use of a complete set of analytical tools, such as thin layer chromatography-flame ionization detector, X-ray analysis, molecular dynamics simulation and confocal microscopy. The experimental results indicated different amounts of lipids on ethnic hair compositions and higher percentage of hair internal lipids in African hair. In this type of hair, the axial diffraction of keratin was not observed in X-ray analysis, but after hair lipids removal, the keratin returned to its typical packing arrangement. In molecular dynamic simulation, lipids were shown to intercalate dimers of keratin, changing its structure. From those results, we assume that keratin structure may be influenced by higher concentration of lipids in African hair.

Engineering in Life Sciences, 2015

ABSTRACT Silk fibroin (SF) is a natural biopolymer that has been extensively studied in various a... more ABSTRACT Silk fibroin (SF) is a natural biopolymer that has been extensively studied in various applications due to its impressive mechanical properties and biocompatibility. Recently, SF-based particles have been proposed as controlled drug delivery systems. A new and efficient method to prepare SF microemulsions (SF-MEs) was developed by oil-in-water emulsions using high-pressure homogenization to promote emulsification. During SF-ME production, the secondary structure of SF changed to a more stable conformation (from random coil to β-sheets), thus allowing the formation of small and stable (140.7 ± 1.9 nm; polydispersity index, 0.25) SF microparticles (SF-MPs). The efficiency of SF-MP formation was 60%. Orange IV was used as a model compound for incorporation and release studies, although its incorporation into the SF-MEs significantly improved particle size and size distribution over at least 4 wk compared to traditional stabilizers (e.g., poloxamer 407, transcutol, Tween 80, and SDS). This should be a call of attention when using dyes as model compounds since they can influence particle properties and lead to misinterpretation of the results. Orange IV showed an incorporation efficiency of 91% and a controlled release over time. Stable SF-MP formulations, further enhanced by orange IV incorporation, provide an innovative method with potential application in pharmaceutical development due to its associated high biocompatibility and release profile.

Molecular Pharmaceutics, 2014

A novel transdermal hyaluronic acid (HA) conjugated with bovine serum albumin (BSA) was developed... more A novel transdermal hyaluronic acid (HA) conjugated with bovine serum albumin (BSA) was developed in the form of solid-in-oil (S/O) nanodispersion (129.7 nm mean diameter). Ex vivo skin penetration analysis by fluorescence and confocal observation of histological skin sections revealed the ability of BSA/HA nanodispersions to cross the stratum corneum and penetrate into the dermis. Furthermore, no significant toxicity was found in fibroblast and keratinocyte cells in vitro. These results proved the potential of the developed nanodispersion for transdermal delivery of hyaluronic acid constituting a high value to biopharmaceutical and cosmetics industries.

Molecular Pharmaceutics, 2012

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Journal of Biotechnology, 2012

In this work it is describe for the first time, the use of an esterase with null activity (Tfu 08... more In this work it is describe for the first time, the use of an esterase with null activity (Tfu 0883 bacterial cutinase from Thermobifida fusca) on the removal of fat from the surface of a cotton substrate. Similar levels of fat removal were found for both null and wild-type proteins, despite that only wild type protein yielded fatty acids. Our results show that molecular recognition of esterase plays a major role on the removal of fatty soils, allowing important guidelines for the design of detergent enzymes. Furthermore, the advantage of using null esterase enzymes lies in the avoidance of the rancid smell of short chained fatty acids, typical after esterase treatment.

Colloids and Surfaces B: Biointerfaces, 2013

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are generally defined as small... more Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are generally defined as small cationic peptides with the ability to interact with lipidic membranes, in a process driven by electrostatic and hydrophobic processes. The interaction with CPPs is known to lead to its translocation across the membrane, while with AMPs lead to membrane damage.

Applied Microbiology and Biotechnology, 2014

Needs from medical and cosmetic areas have led to the design of novel nanosized emulsions and sol... more Needs from medical and cosmetic areas have led to the design of novel nanosized emulsions and solid-in-oil dispersions of proteins. Here, we describe the production of those emulsions and dispersions using high-energy methodologies such as high-pressure homogenization or ultrasound. Recent work has resulted in new mechanistic insights related to the formation of protein emulsions and dispersions. The production method and composition of these formulations can determine major parameters such as size, stability, and functionality, and therefore their final application. Aqueous nanoemulsions of proteins can be used for drug delivery, while solid-in-oil dispersions are often used in transdermal applications.