TY - JOUR
T1 - Use of glycol chitosan modified by 5β-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis
AU - Li, Tieshi
AU - Longobardi, Lara
AU - Granero-Molto, Froilan
AU - Myers, Timothy J.
AU - Yan, Yun
AU - Spagnoli, Anna
N1 - Funding Information:
This work was supported by an Arthritis Foundation Investigator Award and NIH-R01 to A. Spagnoli. We acknowledge Dr. Joseph De Simone, Professor of Chemistry, University of North Carolina at Chapel Hill, for his assistance with the DLS studies.
PY - 2010/5
Y1 - 2010/5
N2 - 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-5β-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.
AB - 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-5β-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.
KW - Developmental biology
KW - Embryonic mouse limb implant
KW - Hydrophobically modified glycol chitosan
KW - Protein delivery system
KW - Skeletogenesis
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U2 - 10.1016/j.jconrel.2010.01.021
DO - 10.1016/j.jconrel.2010.01.021
M3 - Article
C2 - 20116406
AN - SCOPUS:77953809953
VL - 144
SP - 101
EP - 108
JO - Journal of Controlled Release
JF - Journal of Controlled Release
SN - 0168-3659
IS - 1
ER -