Nuno G. Azoia

Insights on the mechanism of formation of protein microspheres in a biphasic system

Microspheres of bovine serum albumin (BSA) and silk fibroin are produced by applying ultrasound in a biphasic system consisting of an aqueous protein solution and an organic solvent. The protein microspheres are dispersed in an aqueous media where the protein remains at the interface covering the organic solvent. This only occurs when high shear forces are applied that induce changes to force the protein to the interface. Fourier transform infrared results indicate a large increase in the content of the β-sheet during the formation of silk fibroin microspheres. Molecular dynamics simulations show a clear adaption on the 3D structure of BSA when stabilized at the interface, without major changes in secondary structure. Further studies demonstrate that high water content, oil solvents, and larger peptides with separated and clear hydrophobic and hydrophilic areas lead to more stable and smaller spheres. This is the first time that these results are presented. We also present herein the rationale to produce tailored protein microspheres with a controlled size, controlled charge, and increased stability.

Diastereoselective synthesis of aziridines from (1R)-10-(N,N-dialkylsulfamoyl)isobornyl 2H-azirine-3-carboxylates

Reaction of the chiral azirine 1a with nucleophiles and dienes is described. Thiols, heteroaromatic nitrogen compounds and phenylmagnesium bromide add to the azirine 1a to give functionalised aziridines 2/3a–h. X-Ray crystal structures for the products 3e and 3g have been obtained. Diastereodifferentiation of the two faces of the azirine 1a is observed in most cases, but only thiophenol gives a single diastereomer (2a). Most mixtures of diastereomers were separated by dry flash chromatography. Benzylamine produced a dimer of the original azirine 1a, compound 9. Representative conjugated dienes (cyclopentadiene, furan and open chain dienes) were added to the chiral azirine 1a. Only poor selectivities were observed. The selectivity was not significantly enhanced in the cycloaddition of cyclopentadiene to the more bulky azirine 1b.

Keratins and lipids in ethnic hair

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.

Peptide Anchor for Folate-Targeted Liposomal Delivery

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 vitro and computational studies of transdermal perfusion of nanoformulations containing a large molecular weight protein

Transdermal perfusion of a large protein is reported for the first time, using a nanoemulsion of bovine serum albumin (66kDa) of 160nm prepared by a solid-in-oil (S/O) process. Molecular dynamics simulations confirmed skin permeation by these formulations, with integration of the protein into the lipid bilayers. These results demonstrate the real possibility of delivering large proteins transdermally for a range of medical and cosmetic applications.

Albumin-Based Nanodevices as Drug Carriers

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.

Size controlled protein nanoemulsions for active targeting of folate receptor positive cells

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.

Design of novel BSA/hyaluronic acid nanodispersions for transdermal pharma purposes.

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.

Tailoring elastase inhibition with synthetic peptides

Chronic wounds are the result of excessive amounts of tissue destructive proteases such as human neutrophil elastase (HNE). The high levels of this enzyme found on those types of wounds inactivate the endogenous inhibitor barrier thus, the search for new HNE inhibitors is required. This work presents two new HNE inhibitor peptides, which were synthesized based on the reactive-site loop of the Bowman-Birk inhibitor protein. The results obtained indicated that these new peptides are competitive inhibitors for HNE and, the inhibitory activity can be modulated by modifications introduced at the N- and C-terminal of the peptides. Furthermore, these peptides were also able to inhibit elastase from a human wound exudate while showing no cytotoxicity against human skin fibroblasts in vitro, greatly supporting their potential application in chronic wound treatment.

PEGylation greatly enhances laccase polymerase activity

Laccase catalyzes the oxidation and polymerization of phenolic compounds in the presence of oxygen. Herein, we report for the first time that a previous PEGylation of laccase enhances the polymerase activity 3‐fold compared with the reaction of the native enzyme, as confirmed by UV/Vis spectroscopy. The polymerization of catechol increased only 1.5‐fold if poly(ethylene glycol) (PEG) was added to the medium reaction. Molecular‐dynamics simulations suggest the formation of a miscible complex of polycatechol and PEG, which is responsible to push the reaction forward. In a negative control experiment set, all catalysts were entrapped inside polyacrylamide gels and here the native laccase showed a relatively higher activity. These results suggest that the mobility of PEG is a key feature for the enhancement of the reaction.