title: Use of glycol chitosan modified by 5beta-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis.
abstract: Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol chitosan-5beta-cholanic acid conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission electron microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% released after 48 h. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age E13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology.
authors.affiliation: Department of Pediatrics, Division of Pediatric Endocrinology, University of North Carolina at Chapel Hill, Mason Farm Road, Campus Box: 7039, Chapel Hill, NC 27599-7039, USA.
authors.forename: Tieshi
authors.initials: T
authors.lastname: Li
authors.forename: Lara
authors.initials: L
authors.lastname: Longobardi
authors.forename: Froilan
authors.initials: F
authors.lastname: Granero-Molto
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Myers
authors.forename: Yun
authors.initials: Y
authors.lastname: Yan
authors.forename: Anna
authors.initials: A
authors.lastname: Spagnoli
mesh_terms: D000818:Animals; D002417:Cattle; D048271:Chitosan; D002793:Cholic Acids; D004622:Embryo, Mammalian; D006614:Hindlimb; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles; D011506:Proteins / Q000494:pharmacology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology*
chemical_list: D002793:Cholic Acids; D011506:Proteins; C118638:glycol-chitosan; D012710:Serum Albumin, Bovine; D048271:Chitosan; C007036:cholanic acid