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. H. S. Azevedo acknowledges the financial support of the European Union under the Marie Curie Career Integration Grant SuprHApolymers (PCIG14-GA-2013-631871). I. Pashkuleva is thankful to the Portuguese foundation for science and technology (IF/00032/2013) and to the European Union (REGPOT-2012-2013-1-316331)

Encapsulation of alpha-amylase into starch-based biomaterials : an enzymatic approach to tailor their degradation rate

This paper reports the effect of a-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable a-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28 days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with a-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that a-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of a-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated a-amylase into the matrix.This work was partially supported by Portuguese Foundation for Science and Technology (FCT) through funds from the POCTI and/or FEDER Programmes. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Effects of temperature on the cellulose binding ability of cellulase enzymes

The effects of high temperatures on catalytic activity and binding abilities of crude Trichoderma reesei cellulases in solution and adsorbed to a cotton fabric were studied. Above optimum temperature of 50 degrees C, catalytic activities were severely diminished but the binding behaviour was not found to be adversely affected. In order to verify possible applications of cellulases adsorbed to cotton fabrics as anchors for textile finishing purposes, we also checked the binding abilities after ironing. Previous ironing of cellulase adsorbed fabrics increased dyeability with an acid dye, but dye fastness was poor. Desorption of cellulases from cotton fabrics increased from pH 5 to pH 10. Dry ironing of fabrics resulted in less desorption, whereas wet ironing inhibited desorption at pH 5 and only 11% of protein were desorbed at pH 10. Ironing of the fabrics diminished enzyme activity of desorbed cellulases. Wet ironing resulted in complete denaturation of the proteins and no cellulolytic activity was found. The presence of water during thermal treatment of cellulases was found to be essential for complete denaturation and unfolding of the proteins. Dry heat only resulted in partial denaturation. Fluorescence measurements of cellulases adsorbed to cotton fabrics showed after ironing a significant shift in tryptophan fluorescence to higher wavelengths. This indicates unfolding and denaturation of the enzymes and revelation of more hydrophobic amino acids to the surface, which enables increased hydrophobic interactions with the fabric. (C) 1999 Elsevier Science B.V. All rights reserved

Possibilities for recycling cellulases after use in celllase processing - part I: effects of end-product inhibition, thermal and mechanical deactivation, and cellulase depletion by adsorption

Preliminary recycling experiments with cellulase enzymes after cotton treatments at 50°C showed that activity remaining in the treatment liquors was reduced by about 80% after five recycling steps. The potential problems of end-product inhibition, thermal and mechanical deactivation, and the loss of some components of the cellulase complex by preferential and or irreversible adsorption to cotton substrates were studied. End-product inhibition studies showed that the build-up of cellobiose and glucose would be expected to cause no more than 40% activity loss after five textile treatment cycles. Thermal and mechanical treatments of cellulases suggested that the enzymes start to be deactivated at 60°C and agitation levels similar to those used in textile processing did not cause significant enzyme deactivation. Analysis of cellulase solutions, by fast protein liquid chromatography, before and after adsorption on cotton fabrics, suggested that the cellobiohydrolase II (Cel6A) content of the cellulase complex was reduced, relative to the other components, by preferential adsorption. This would lead to a marked reduction in activity after several treatment cycles and top-up with pure cellobiohydrolase II would be necessary unless this component is easily recoverable from the treated fabric

Possibilities for recycling cellulases after use in cotton processing - part II: Separation of cellulases from reaction products and released dyestuffs by ultrafiltration

The adsorption and activity of a total cellulase (Trichoderma reesei) was measured and compared on undyed and dyed cotton fabrics. Recovery of enzymes from the reaction mixture and by desorption from the cotton substrate was evaluated. About 80% of the initial protein could be recovered. The removal of released products (soluble reducing sugars and dyes) from the treatment liquor and subsequent concentration of cellulase proteins was performed using an ultrafiltration membrane. Strong protein-dye interactions made it impossible to separate efficiently the dyes from the enzymecontaining treatment liquors. The use of surfactants did not enhance cellulase desorption from cotton fabric. Although anionic surfactants have a deactivating effect on cellulases, this effect seems to be reversible, since after ultrafiltration the cellulase activity was similar to that of enzymes desorbed with buffer only. Humicola insolens cellulases were shown to be much more sensitive to anionic surfactant than T. reesei cellulases. The use of cellulases that bind reversibly to cellulose is suggested for achieving more efficient cellulase recycling and for reducing backstaining by dye-cellulase complexes

Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV(humicola insolens) and CenA (cellulomonas fimi)

The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation. Similar activity measurements were also made by using the core domains of these cellulases. Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied. The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption. EGV showed low percentage of adsorption under both levels of agitation. It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA). (C) 2000 Elsevier Science Inc. All rights reserved

Desorption of cellulases from cotton powder

Cotton fabrics were treated with three different Trichoderma reesei cellulase preparations (total crude – TC, endoglucanase enriched – EG-rich, cellobiohydrolase enriched – CBH-rich) using mechanical agitation to produce cotton powder. Desorption of cellulase enzymes from the cotton powder was then performed by washing with buffer. After 3 washings most of the protein was desorbed from the cotton powder and the amount of sugars released in the latter washings was negligible. TC and CBH-rich preparations produced a finer cellulose powder than EGs. The desorption process caused a decrease in degree of polymerisation (DP) specially for the cotton treated with EGs and a marked increase in polydispersity (Pd ) for all preparations