Lipocalin-2-loaded amphiphilic polyanhydride microparticles accelerate cell migration.

This work demonstrates that amphiphilic polyanhydride microparticles based on co-polymers of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,6-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) provide stabilizing environments for proteins. A cryogenic atomization method was used to fabricate protein-loaded polyanhydride microparticles. These microparticles were tested for their ability to provide controlled delivery of lipocalin 2 (Lcn2) and to maintain its structure and function. Lcn2 is an acute-phase protein suspected to play a role in cell migration and tissue repair. The in vitro release kinetics of Lcn2 from the microparticles were a function of the chemistry of the polymer carrier. The biological activity of Lcn2 released from polyanhydride microparticles was investigated by its ability to stimulate migration of human colon epithelial cells (HCT116). Lcn2 released from 50:50 and 20:80 CPTEG/CPH microparticles maintained its biological activity as demonstrated by the increased rate of cell migration. In addition, the Lcn2-loaded 50:50 and 20:80 CPTEG/CPH microparticles promoted cell migration over that of the Lcn2 administered alone. This was interpreted as the ability of the amphiphilic microparticles to stabilize the encapsulated protein and release it in a controlled manner over a period of time. This work demonstrates the potential for therapeutic use of amphiphilic polyanhydride microparticles as protein/drug carriers.

Amphiphilic polyanhydrides for protein stabilization and release.

The overall goal of this research is to design novel amphiphilic biodegradable systems based on polyanhydrides for the stabilization and sustained release of peptides and proteins. Accordingly, copolymers of the anhydrides, 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), which are monomer-containing oligomeric ethylene glycol moieties, have been synthesized. Microspheres of different CPTEG:CPH compositions have been fabricated by two non-aqueous methods: solid/oil/oil double emulsion and cryogenic atomization. The ability of this amphiphilic polymeric system to stabilize model proteins (i.e., lysozyme and ovalbumin) was investigated. The structure of both the encapsulated as well as the released protein was monitored using gel electrophoresis, circular dichroism, and fluorescence spectroscopy. It was found that the CPTEG:CPH system preserves the structural hierarchy of the encapsulated proteins. Activity studies of the released protein indicate the CPTEG:CPH system retains the biological activity of the released protein. These results are promising for future in vivo studies, which involve the design of novel biodegradable polyanhydride carriers for the stabilization and sustained release of therapeutic peptides and proteins.

Protein stability in the presence of polymer degradation products: consequences for controlled release formulations.

When encapsulating proteins in polymer microspheres for sustained drug delivery there are three stages during which the stability of the protein must be maintained: (1) the fabrication of the microspheres, (2) the storage of the microspheres, and (3) the release of the encapsulated protein. This study focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of tetanus toxoid, ovalbumin (Ova), and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride (sebacic acid and 1,6-bis(p-carboxyphenoxy)hexane) monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. Enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of tetanus toxoid and Ova and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. Tetanus toxoid was found to be the most stable in the presence of anhydride monomers, while Ova was most stable in the presence of sebacic acid, and lysozyme was stable when incubated with all of the monomers studied.

The role of microsphere fabrication methods on the stability and release kinetics of ovalbumin encapsulated in polyanhydride microspheres.

The effect of microsphere fabrication methods on the stability and release kinetics of ovalbumin encapsulated in polyanhydride microspheres was investigated. The polyanhydrides used were poly(sebacic anhydride) (poly(SA)) and a 20:80 random copolymer of poly[1,6-bis(p-carboxyphenoxy)hexane] (poly(CPH)) and poly(SA). Microspheres were fabricated using three double emulsion methods (water/oil/water, water/oil/oil and solid/oil/oil) and cryogenic atomization. The encapsulation efficiency was highest for cryogenic atomization and lowest when the w/o/w technique was used. Microspheres fabricated by the s/o/o method had the largest initial burst of released protein. All the methods resulted in zero-order release of the protein after the burst. The release of ovalbumin from poly(SA) and 20:80 (CPH:SA) microspheres lasted approximately 3 and approximately 6 weeks, respectively. For all fabrication methods the primary structure of released ovalbumin was conserved as determined by gel electrophoresis. The secondary structure of ovalbumin encapsulated in 20:80 (CPH:SA) w/o/w microspheres was not conserved.

Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres.

In order to determine the efficacy of using polyanhydrides as a carrier for therapeutic proteins, the model protein bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was encapsulated in microspheres of poly sebacic anhydride (poly(SA)), and random copolymers of poly(SA) and poly(1,6-bis-p-carboxyphenoxy)hexane (poly(CPH)). The microspheres were fabricated via the double emulsion (water/oil/water) technique and were characterized using scanning electron microscopy, gel permeation chromatography, confocal microscopy, and a Coulter counter. The effect of protein loading, protein distribution, and change in polymer composition was examined in an in vitro release study. The secondary structure of the encapsulated BSA-FITC was determined with Fourier transform infrared spectroscopy. The primary structure of the released protein was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Poly(SA) and 20:80 (CPH:SA) microspheres were found to conserve the primary structure of the released protein and the secondary structure of the encapsulated protein, and showed a sustained delivery for approximately 15 and 30 days, respectively. As the CPH content in the copolymer increased, the secondary structure of FITC-BSA was not conserved, as indicated by the steep decrease in the alpha-helix content.