Application of self-assembled ultrathin film coatings to stabilize enzyme encapsulation and activity in alginate microspheres.

摘要

Alginate-based hydrogels have been used for the encapsulation of a variety of materials, including enzymes, proteins, and cells for a wide range of applications from drug delivery to biosensors and bioreactors. However, due to the high porosity of the matrix, it has been increasingly difficult to retain macromolecules inside the alginate matrix, leading to loss in functionality over time. In an effort to improve the stability for long-term biosensor use, this work investigated layer-by-layer self-assembly as a potential technique to provide a diffusion barrier to an encapsulated macromolecule. Alginate microspheres (∼2--50mum radius) were fabricated using an emulsification technique, and were ionically crosslinked with calcium ions and used for the encapsulation of macromolecules including dextran and the enzyme glucose oxidase. Stepwise-assembled polyelectrolyte nanofilm coatings of different composition and thickness were then formed on the microspheres, and the loss of enzyme was monitored over one week. The total loss of encapsulated material was reduced to less than 15% with the application of a single {lcub}PAH/PAA{rcub} coating, in comparison to ∼50% loss observed with uncoated and {lcub}PDDA/PSS{rcub}-coated microspheres. The activity of the encapsulated enzyme was also tested over twelve weeks, and it was found that {lcub}PAH/PSS{rcub}-coated spheres retained more than 84% of their initial activity after twelve weeks, whereas uncoated and {lcub}PDDA/PSS{rcub}-coated microspheres retained less than 20% of initial activity after twelve weeks. The activity was further stabilized when a chemical conjugation technique with water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) immobilized the enzyme in the alginate matrix. More than 90% activity retention was observed for all cases. Finally, novel biocompatible coating materials were used to coat the alginate microspheres to slow release and improve biocompatibility. The enzyme was crosslinked to the alginate matrix using EDC-NHSS conjugation techniques. The experimental results showed that the application of single layer thin films to the alginate microspheres was effective in reducing loss of the encapsulated enzyme from the microspheres with more than 80% enzyme retention reported for monolayer coatings and more than 95% enzyme retention for multilayer coatings. The encapsulated enzyme was also found to be highly active (>95% retention of activity) inside the uncoated microspheres and coated microspheres, and the activity results were compared over a period of three months. In vitro cytotoxicity tests were completed using 3T3 cells to determine the "optimum" coating material towards biosensor use. Of the coatings tested, chondroitin sulfate, humic acid, PEG bis(amine), and chitosan coatings were found to be suitable for in vivo testing. These results demonstrate that the simple application of ultrathin film coatings to functional micro-systems can be useful in prolonging the usability of the encapsulated material in those templates, thus leading to improved stability and increased longevity for biosensors and bioreactors.