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.
J Control Release. 2010 May 21;144(1):101-8. doi: 10.1016/j.jconrel.2010.01.021. Epub 2010 Jan 28.
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.
鼠胚肢培养在研究骨骼发生中具有不可估量的作用。物质传递是发育生物学中一个不发达的领域，主要依赖于 Affi-Gel-Blue-琼脂糖珠。然而，关于琼脂糖珠负载和释放效率的信息缺乏以及单个珠粒植入的困难是其显著的局限性。我们优化了使用乙二醇壳聚糖-5β-胆酸缀合物（HGC）作为新型蛋白质传递系统在鼠胚肢中的应用。为此，我们将 HGC 与重组 Noggin 或牛血清白蛋白（BSA）负载。通过透射电子显微镜和动态光散射确定负载蛋白的 HGC 的大小、形态和稳定性。HGC-BSA 和 HGC-Noggin 的负载效率为 80-90%。时程研究表明，Noggin 和 BSA 在 48 小时后释放了 80-90%。我们开发了几种技术，从胚胎龄 E13.5 到 E15.5 将负载蛋白的 HGC 植入鼠胚关节中，包括分配纳升的微注射系统。HGC 不干扰骨骼发生。使用 CBR-3BA 染色，我们在植入组织中检测到 HGC 纳米颗粒。此外，在 HGC-BSA 和 HGC-Noggin 注射区域证明了 BSA 和 Noggin 的持续释放。HGC 释放的 Noggin 在体外间充质肢微体和体外肢培养物中具有生物活性，可阻断 BMP 信号。结果表明，HGC 是发育生物学中一种有价值的蛋白质传递系统。
