Artur Ribeiro

Biofunctional design of elastin-like polymers for advanced applications in nanobiotechnology

Elastin-like recombinant protein polymers are a new family of polymers which are captivating the attention of a broad audience ranging from nanotechnologists to biomaterials and more basic scientists. This is due to the extraordinary confluence of different properties shown by this kind of material that are not found together in other polymer systems. Elastin-like polymers are extraordinarily biocompatible, acutely smart and show uncommon self-assembling capabilities. Additionally, they are highly versatile, since these properties can be tuned and expanded in many different ways by substituting the amino acids of the dominating repeating peptide or by inserting, in the polymer architecture, (bio)functional domains extracted from other natural proteins or de novo designs. Recently, the potential shown by elastin-like polymers has, in addition, been boosted and amplified by the use of recombinant DNA technologies. By this means, complex molecular designs and extreme control over the amino-acid sequence can be attained. Nowadays, the degree of complexity and control shown by the elastin-like protein polymers is well beyond the reach of even the most advanced polymer chemistry technologies. This will open new possibilities in obtaining synthetic advanced bio- and nanomaterials. This review explores the present development of elastin-like protein polymers, with a particular emphasis for biomedical uses, along with some future directions that this field will likely explore in the near future.

Influence of the Amino-Acid Sequence on the Inverse Temperature Transition of Elastin-Like Polymers

This work explores the dependence of the inverse temperature transition of elastin-like polymers (ELPs) on the amino-acid sequence, i.e., the amino-acid arrangement along the macromolecule and the resulting linear distribution of the physical properties (mainly polarity) derived from it. The hypothesis of this work is that, in addition to mean polarity and molecular mass, the given amino-acid sequence, or its equivalent--the way in which polarity is arranged along the molecule--is also relevant for determining the transition temperature and the latent heat of that transition. To test this hypothesis, a set of linear and di- and triblock ELP copolymers were designed and produced as recombinant proteins. The absolute sequence control provided by recombinant technologies allows the effect of the amino-acid arrangement to be isolated while keeping the molecular mass or mean polarity under strict control. The selected block copolymers were made of two different ELPs: one exhibiting temperature and pH responsiveness, and one exhibiting temperature responsiveness only. By changing the arrangement and length of the blocks while keeping other parameters, such as the molecular mass or mean polarity, constant, we were able to show that the sequence plays a key role in the smart behavior of ELPs.

Temperature-triggered self-assembly of elastin-like block co-recombinamers:the controlled formation of micelles and vesicles in an aqueous medium.

The possibility of obtaining different self-assembled nanostructures in reversible systems based on elastin-like block corecombinamers is explored in this work. The results obtained show how an evolution from a more common micellar structure to a hollow vesicle can be attained simply by changing the block arrangements and lengths, even when other molecular properties, such as molecular weight or mean polarity, remain essentially unchanged. This work sheds light on the possibility of obtaining hollow nano-objects, based on elastin-like recombinamers, which can assemble and disassemble in response to a change in their surroundings. This kind of system can be an example of how high precision in the genetic production of synthetic macro-molecules can be used, on an exclusive basis, to control the shape and size of their derived nano-objects.

Nanobiotechnological approach to engineered biomaterial design: the example of elastin-like polymers

Today, the development of advanced biomaterials is still lacking an appropriate tailored engineering approach. Most of the biomaterials currently used have their origin in materials developed for other technological applications. This lack of adequate biomaterial design is probably due to the peculiar environment where those materials must operate. On the one hand, this environment is dominated by the immune rejection system. On the other hand, the functionality of natural biomolecules is based on complex topological physical-chemical function distributions at the nanometer level. This review presents arguments concerning the role of biotechnology and nanotechnology in the future development of new advanced biomaterials and the potential of these biomaterials as a way to achieve highly biofunctional and truly biocompatible biomaterials for hot areas, such as regenerative medicine and controlled release. Recombinant protein-polymers will be presented as an example of candidates for this new paradigm in biomaterial design and production.

Therapeutic l-asparaginase: upstream, downstream and beyond

Abstract l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

Ultrasound intensification suppresses the need of methanol excess during the biodiesel production with Lipozyme TL-IM

The synthesis of biodiesel from sunflower oil and methanol based on transesterification using the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM) has been investigated under silent conditions and under an ultrasound field. Ultrasound assisted process led to reduced processing time and requirement of lower enzyme dosage. We found for the first time that oil to methanol ratio of 1:3 was favored for the ultrasound assisted enzymatic process which is lower than that observed for the case of conventional stirring based approach (ratio of 1.4). Our results indicate that intensification provided by ultrasound suppresses the need of the excess of the methanol reactant during the enzymatic biodiesel production. Ultrasound assisted enzymatic biodiesel production is therefore a faster and a cleaner processes.

Exploiting the Sequence of Naturally Occurring Elastin: Construction, Production and Characterization of a Recombinant Thermoplastic Protein-Based Polymer

Genetic engineering was used to produce an elastin-like polymer (ELP) with precise amino acid composition, sequence and length, resulting in the absolute control of MW and stereochemistry. A synthetic monomer DNA sequence encoding for (VPAVG)20, was used to build a library of concatemer genes with precise control on sequence and size. The higher molecular weight polymer with 220 repeats of VPAVG was biologically produced in Escherichia coli and purified by hot and cold centrifugation cycles, based on the reversible inverse temperature transition property of ELPs. The use of low cost carbon sources like lactose and glycerol for bacteria cells culture media was explored using Central Composite Design approach allowing optimization of fermentation conditions. Due to its self-assembling behaviour near 33 °C stable spherical microparticles with a size ~ 1µm were obtained, redissolving when a strong undercooling is achieved. The polymer produced showed hysteresis behaviour with thermal absorbing/releasing components depending on the salt concentration of the polymer solution.

Ultrasound enhances lipase-catalyzed synthesis of poly (ethylene glutarate)

The present work explores the best conditions for the enzymatic synthesis of poly (ethylene glutarate) for the first time. The start-up materials are the liquids; diethyl glutarate and ethylene glycol diacetate, without the need of addition of extra solvent. The reactions are catalyzed by lipase B from Candida antarctica immobilized on glycidyl methacrylate-ter-divinylbenzene-ter-ethylene glycol dimethacrylate at 40°C during 18h in water bath with mechanical stirring or 1h in ultrasonic bath followed by 6h in vacuum in both the cases for evaporation of ethyl acetate. The application of ultrasound significantly intensified the polyesterification reaction with reduction of the processing time from 24h to 7h. The same degree of polymerization was obtained for the same enzyme loading in less time of reaction when using the ultrasound treatment. The degree of polymerization for long-term polyesterification was improved approximately 8-fold due to the presence of sonication during the reaction. The highest degree of polymerization achieved was 31, with a monomer conversion of 96.77%. The ultrasound treatment demonstrated to be an effective green approach to intensify the polyesterification reaction with enhanced initial kinetics and high degree of polymerization.

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.

Hybrid Nanotopographical Surfaces Obtained by Biomimetic Mineralization of Statherin-Inspired Elastin-Like Recombinamers

Modification of surfaces mimicking unique chemical and physical features of mineralized tissues is of major interest for obtaining biomaterials for replacing and regenerating biological tissues. Here, human salivary statherin-inspired genetically engineered recombinamers (ELRs, HSS) on biomedical surfaces regulates mineralization to form an amorphous-calcium-phosphate (ACP) layer that reproduces the original substrate nanotopography. The HSS-ELRs carry a statherin-derived peptide with high affinity to tooth enamel. They are tethered to nanorough surfaces and mineralized using an enzyme-directed process. A homogeneous layer of ACP-minerals forms on HSS-coated surfaces retaining the original nanotopography of the substrate. In contrast, biomineralization of control surfaces results in uncontrolled growth of minerals. This suggest the statherin-inspired ELRs have ability to induce and control growth of the minerals on the biofunctional surfaces. Likely, the HSS-ELR coating have similar bioactivity to that of statherin in human saliva. The hybrid nanorough surfaces improve adhesion and differentiation of preosteoblasts and show potential for dental and orthopedic implants integration. This method enables the combination and tailoring of nanotopographical and biochemical cues to design functionalized surfaces to investigate and potentially direct the stem cell fate.