title: Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab.
abstract: A light-activated polycaprolactone dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA) based gel network was developed to sustain the release of stable, active bevacizumab (an anti-VEGF antibody used to treat choroidal neovascularization) and used to assess sustained ex vivo delivery in rabbit eyes and in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space. PCM was synthesized from polycaprolactone diol (PCD) and evaluated using NMR spectroscopy. PCM was used to cross-link HEMA in the presence of 365 nm UV light and 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator. Bevacizumab was entrapped in the gel using three different cross-linking durations of 3, 7, and 10 min. In vitro release of bevacizumab in PBS pH 7.4 at 37 °C during a 4 month study was quantified using a VEGF-binding based ELISA. The stability of released bevacizumab was monitored by size exclusion chromatography (SEC) and circular dichroism. Alexa Fluor 488 dye conjugated bevacizumab mixed with polymers was injected suprachoroidally in rabbit eyes to study the effect of different cross-linking durations on the spread of the dye conjugated bevacizumab. In vivo delivery was assessed in Sprague-Dawley (SD) rats by injecting Alexa Fluor 488 dye conjugated bevacizumab mixed with polymers followed by cross-linking for 10 min. Spread in the rabbit eyes and in vivo delivery in rat eyes was monitored noninvasively using a fundus camera and Fluorotron Master. The formation of PCM was confirmed by the disappearance of hydroxyl peak in NMR spectra. A cross-linking duration of 10 min resulted in a burst release of 21% of bevacizumab. Other cross-linking durations had ≥62% burst release. Bevacizumab release from 10 min cross-linked gel was sustained for ∼4 months. Release samples contained ≥96.1% of bevacizumab in the monomeric form as observed in SEC chromatograms. Circular dichroism confirmed that secondary β-sheet structure of bevacizumab was maintained after release from the gel. As the cross-linking duration was increased to 10 min, the gel/antibody was better confined at the injection site in excised rabbit eye suprachoroidal space. Delivery of Alexa Fluor 488 dye conjugated bevacizumab was sustained for at least 60 days in the suprachoroidal space of SD rats. PCM and HEMA gel sustained bevacizumab release for 4 months and maintained the stability and VEGF-binding activity of bevacizumab. Therefore, light-activated PCM and HEMA gel is suitable for in situ gel formation and sustained protein delivery in the suprachoroidal space.
authors.affiliation: Nanomedicine and Drug Delivery Laboratory, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States.
authors.forename: Puneet
authors.initials: P
authors.lastname: Tyagi
authors.forename: Matthew
authors.initials: M
authors.lastname: Barros
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Stansbury
authors.forename: Uday B
authors.initials: UB
authors.lastname: Kompella
mesh_terms: D000098:Acetophenones / Q000737:chemistry; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage* / Q000737:chemistry*; D000068258:Bevacizumab; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011091:Polyesters / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000098:Acetophenones; D061067:Antibodies, Monoclonal, Humanized; D011091:Polyesters; D011108:Polymers; C452198:2,2-dimethoxy-2-phenylacetophenone; C016240:polycaprolactone; D000068258:Bevacizumab