(116bg) Stability of Hen Egg White Lysozyme Crystals in Polymeric Media

Protein encapsulation and crystallization are under much investigation due to their wide variety of applications such as use in biosensors and drug delivery. Encapsulation in hydrogels can provide protein crystals a suitable environment to maintain their biological stability; however, this process involves the placement of crystals in organic/monomeric media. Therefore, Hen Egg White Lysozyme (HEWL) crystals were studied in diverse monomeric based solutions to find the ideal stability conditions, proceeded by the actual encapsulation of the crystals within polymeric membranes using the found conditions, to then determine whether the crystals can be released from said membranes or if the crystals can maintain stable and active within the membranes of a long lapse of time.

HEWL was crystallized using both hanging drop and batch crystallization method. Afterwards, crystal stability was assessed as a function of time and composition of the media. Crystal stability on each individual component was assessed including ethanol and monomers. Crystals showed a higher stability in solutions of 30 % EtOH. This factor was used in the following stability tests in which the crystals' stability was studied in pre-polymeric solutions of anionic monomer methacrylic acid (MAA), neutral monomer 2-hydroxyethyl methacrylate (HEMA), and cationic monomer 2-(dimethylaminoethyl)methacrylate (DMAEM). The tests were completed by varying either the cross-linker ratio or the dilution factor and finding longer stability times for the crystals in these solutions. The crystals were more stable in MAA than in HEMA and DMAEM. These results demonstrate that the stability of the crystals is highly linked to the interaction of the charges from the crystals' surface and the charges of the solution in question, hence the charge of HEWL is positive, providing a better environment for interaction with the negative charge of MAA In addition to this, ideal morphologies for the polymerization solutions were found for MAA, HEMA and DMAEM. The actual polymerization of the crystals was studied, and crystals were successfully encapsulated in MAA membranes. The crystals have showed a high stability when encapsulated in the MAA membranes, surviving in a high pH environment while encapsulated for much longer than a month, which proves its potential to be used in a biosensor. The release of the protein from crystal dissociation is currently being evaluated for a possible drug delivery application. Thus, the encapsulation and stabilization of the crystals has been successful, however more research must be completed to find the ideal membrane that permits the crystals to be released in pH controlled environments, to broaden the range of applications for this project.