(446d) Polyanhydride Microspheres Encapsulating Lipocalin 2 Expedite Cell Migration

Lipocalin 2 (Lcn2) is an acute phase protein thought to play an important role in the wound healing process through its ability to promote epithelial cell migration. The structure of Lcn2 forms a pocket for binding siderophores, which is important for its function. Thus, a denatured protein would no longer be biologically active, making stabilization of the protein upon treatment a primary concern. This work describes the encapsulation of Lcn2 into polyanhydride microspheres and release of the functional protein. Polyanhydrides are biodegradable polymers with potential in drug delivery applications because of their ability to degrade into non-toxic, non-mutagenic biocompatible degradation products and to stabilize and release protein in a controlled manner. The polymers of interest in this work are based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG). When copolymerized together they result in an amphiphilic copolymer capable of stabilizing both globular and recombinant proteins and of releasing protein over a period of time, ranging from days to months. The copolymers 20:80 CPTEG:CPH and 50:50 CPTEG:CPH were chosen as candidates for encapsulating Lcn2 and studying their effect on in vitro cell migration of wounded epithelial (HCT 116) cells. The 24 h in vitro cell migration assay was carried out by making unique scalpel marks on the wound line which were used for imaging at t = 0 h and again at t = 24 h. This allowed for accurate image alignment and subsequent quantification of the cell migration distance. Initial studies investigated the effect of blank 20:80 CPTEG:CPH and blank 50:50 CPTEG:CPH microspheres on cell migration which were proven to be no different than the negative control group (serum-free medium). However, when the cells were treated with Lcn2-loaded 20:80 CPTEG:CPH and Lcn2-loaded 50:50 CPTEG:CPH microspheres for 24 h a significant increase in cell migration was observed over both the negative control and the soluble Lcn2 control. In fact, the cells were treated with twice the dose of the soluble Lcn2 as what was encapsulated into the microspheres. An in vitro release study of Lcn2 from both microsphere chemistries demonstrated that by 24 h only ~25% of the total amount of encapsulated protein is released. This indicates the importance of a delivery system capable of controlling the release rate for treatments involving fragile therapeutic proteins with short half-lives. These findings add to the large body of evidence supporting the use of polyanhydrides for applications in drug and protein delivery.