RESUMO
ß-Cyclodextrin (ß-CD)-grafted dextrans with spacer arms of different length were employed to evaluate the impact of supramolecular interactions on invertase activity. The modified dextrans were used as single additives or combined with trehalose in freeze-dried formulations containing invertase. Enzyme activity conservation was analyzed after freeze-drying and thermal treatment. The change of glass transition temperature (Tg ) was also evaluated and related to effective interactions. Outstanding differences on enzyme stability were mainly related to the effect of the spacer arm length on polymer-enzyme interactions, since both the degree of substitution and the molecular weight were similar for the two polymers. This change of effective interactions was also manifested in the pronounced reduction of Tg values, and were related to the chemical modification of the backbone during oxidation, and to the attachment of the ß-CD units with spacer arms of different length on dextran.
Assuntos
Dextranos/química , Liofilização , beta-Ciclodextrinas/química , beta-Frutofuranosidase/química , Estabilidade Enzimática , Vidro/química , Peso Molecular , Polímeros/química , Temperatura de Transição , Trealose/químicaRESUMO
Polyethylene glycol (PEG)-based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze-thawing, freeze-drying, and thermal treatment. A novel functional dendrimer (DGo -CD) based on the known PEG's ability as cryo-protector and ß-CD as supramolecular stabilizing agent is presented. During freeze-thawing, PEG and ß-CD failed to prevent catalase denaturation, while dendrimers, and especially DGo -CD, offered the better protection to the enzyme. During freeze-drying, trehalose was the best protective additive but DGo -CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or ß-CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze-dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze-dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with ß-CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer-enzyme supramolecular interactions (host-guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix.
Assuntos
Catalase/química , Dendrímeros/química , Polietilenoglicóis/química , beta-Ciclodextrinas/química , Análise de Variância , Estabilidade Enzimática/efeitos dos fármacos , Excipientes/química , Excipientes/farmacologia , Liofilização , Hidrodinâmica , Tamanho da Partícula , Temperatura , TrealoseRESUMO
Several alternatives to the conventional alginate beads formulation were studied for encapsulation of invertase. Pectin was added to the alginate/enzyme solution while trehalose and ß-cyclodextrin were added to the calcium gelation media. The effect of composition changes, freezing, drying methods (freeze, vacuum, or air drying), and thermal treatment were evaluated on invertase stability and its release kinetics from beads. The enzyme release mechanism from wet beads depended on pH. The addition of trehalose, pectin, and ß-cyclodextrin modified the bead structure, leading in some cases to a release mechanism that included the relaxation of the polymer chains, besides Fickian diffusion. Enzyme release from vacuum-dried beads was much faster than from freeze-dried beads, probably due to their higher pore size. The inclusion of ß-cyclodextrin and especially of pectin prevented enzyme activity losses during bead generation, and trehalose addition was fundamental for achieving adequate invertase protection during freezing, drying, and thermal treatment. Present results showed that several alternatives such as drying method, composition, as well as pH of the relese medium can be managed to control enzyme release.
Assuntos
Alginatos/química , Preparações de Ação Retardada/química , Portadores de Fármacos/química , beta-Ciclodextrinas/química , beta-Frutofuranosidase/metabolismo , Varredura Diferencial de Calorimetria , Preparações de Ação Retardada/metabolismo , Dessecação , Portadores de Fármacos/metabolismo , Estabilidade Enzimática , Liofilização , Géis , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Pectinas/química , Solubilidade , Trealose/química , beta-Frutofuranosidase/químicaRESUMO
Structure/function relationships of different biopolymers (alginate, dextran, or beta-cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer beta-cyclodextrin-branched alginate (beta-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. Beta-CD-A (with or without trehalose), beta-cyclodextrin (beta-CD), or dextran with trehalose were the best protective agents during thermal treatment, while beta-CD and alginate showed a negative effect on invertase activity preservation. The beta-CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. Beta-CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns.