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.

Figure 1

HGC-Noggin preparation. (A) Schematic representation of the strategy used to obtain purified HGC-Noggin preparation. (B) Noggin WIB of fractions obtained during the loading-purification process. Starting solution (F1), after loading-evaporation (F2), unloaded “free” fraction (F3) and loaded fraction (F4). To evaluate the concentration of Noggin in the fractions, an internal standard curve using serial dilutions of recombinant Noggin was used.

Figure 2

Size distribution and morphology of (A) HGC nanoparticles, (B) HGC-BSA nanoparticles and (C) HGC-Noggin(BSA) nanoparticles, determined by DLS and TEM analyses.

Figure 3

Time-course analysis of Noggin release from HGC. (A) Refreshing dialyzing solution was obtained at the indicated time points. (B) Time-course of HGC-Noggin release by Noggin WIB analysis. a, HGC-Noggin, before release (expected 20ng); b, Released Noggin after 16 hours, S1; c, Released Noggin after 24 hours, S1+S2; d, Released Noggin after 40 hours, S1+S2+S3; e, Released Noggin after 48 hours, S1+S2+S3+S4; f, Released Noggin after 64 hours, S1+S2+S3+S4+S5; g, Released Noggin after 72 hours, S1+S2+S3+S4+S5+S6; h, Remaining Noggin; i-m, Noggin Standard curve: 1ng, 2ng, 5ng, 10ng, 20ng. (C) Release profile of Noggin from HGC-Noggin nanoparticles; three distinct data sets were obtained from respective WIB analyses that were averaged and represented as mean±standard deviation.

Figure 4

Noggin loaded and released from HGC maintains its biological activity. (A) HGC-Noggin maintains the ability to form dimers as determined by Noggin WIB under non-reducing conditions. (S0): native Noggin; (S4): Noggin loaded to HGC. (B) Noggin released from HGC inhibits Smad-1,5,8 phosphorylation. HGC-Noggin, HGC-BSA, HGC alone were incubated for 48 hours in SF medium that was collected, dialyzed and used to treat limb mesenchyme micromasses for 20 minutes. a, control medium; b, medium from HGC alone; c, medium from HGC-BSA; d, medium from HGC-Noggin; e, 200 ng of native Noggin

Figure 5

HGC-BSA nanoparticles can be implanted into developing autopods using a microinjection unit. Dissected autopods from wild type C57BL/6 mice at either E13.5 (A-C) or E15.5 (D-L) were imaged before injection (non-injected, left panels) and then subjected to Methylene Blue-HGC-BSA implantation at one site (middle panels) followed by a second site (right panels). Red circles indicate the position where the pipette was inserted. Black arrows indicate where the Methylene Blue-HGC-BSA implants were delivered. Picture was taken immediately after delivery. See text for more details.

Figure 6

Implanted HGC-BSA can be visualized with CBR-3BA staining and IHC for BSA. CBR-3BA staining (A-C) was performed on frozen sections from wild type C57BL/6 mice at E15.5 implanted either with HGC-BSA (A) or HGC (B) or left non-injected (C). IHC for BSA was performed on adjacent frozen sections (D-F). 100X magnifications are depicted. Pictures of unsectioned limbs as well as 20X magnifications including the region from where the 100X magnifications were obtained can be found in Figure S4

Figure 7

Implanted HGC-BSA showed sustained BSA release. BSA IHC was performed on frozen sections from wild type C57BL/6 mice at E15.5 implanted either with HGC-BSA (A-C), or native BSA (D-F) and cultured for 1 and 2 days. 100X magnifications are depicted.

Figure 8

HGC-Noggin is released and inhibits Smad-1,5,8 phophorylation after implantation into embryonic limbs and ex-vivo culturing. Noggin IHC and pSmad1,5,8 IHC were performed on adjacent paraffin sections obtained from E14.5 autopods implanted either with HGC-BSA (A) or HGC-Noggin (B). 20X and 100X magnifications are depicted. Red arrows indicate Noggin IHC and black arrows indicate inhibited Smad-1,5,8 phosphorylation in the same region.