Controlled release of NELL-1 protein from chitosan/hydroxyapatite-modified TCP particles

Abstract

NEL-like molecule-1 (NELL-1) is a novel osteogenic protein that showing high specificity to osteochondral cells. It was widely used in bone regeneration research by loading onto carriers such as tricalcium phosphate (TCP) particles. However, there has been little research on protein controlled release from this material and its potential application. In this study, TCP was first modified with a hydroxyapatite coating followed by a chitosan coating to prepare chitosan/hydroxyapatite-coated TCP particles (Chi/HA-TCP). The preparation was characterized by SEM, EDX, FTIR, XRD, FM and Zeta potential measurements. The NELL-1 loaded Chi/HA-TCP particles and the release kinetics were investigated in vitro. It was observed that the Chi/HA-TCP particles prepared with the 0.3% (wt/wt) chitosan solution were able to successfully control the release of NELL-1 and maintain a slow, steady release for up to 28 days. Furthermore, more than 78% of the loaded protein's bioactivity was preserved in Chi/HA-TCP particles over the period of the investigation, which was significantly higher than that of the protein released from hydroxyapatite coated TCP (HA-TCP) particles. Collectively, this study suggests that the osteogenic protein NELL-1 showed a sustained release pattern after being encapsulated into the modified Chi/HA-TCP particles, and the NELL-1 integrated composite of Chi/HA-TCP showed a potential to function as a protein delivery carrier and as an improved bone matrix for use in bone regeneration research.

Keywords: Bioactivity; Chitosan; Hydroxyapatite; NELL-1; Release kinetics; TCP.

Fig.1

Schematic graph of Chi/HA-TCP preparation and the mechanism of protein or chitosan interacting with HA-TCP particles. TCP particles were first coated with hydroxyapatite by incubating in SBF solutions, and then coated with chitosan to prepare Chi/HA-TCP particles. The NELL-1 protein could be loaded onto HA-TCP particles by lyophilization. Mannitol was included in the protein formulation to preserve protein conformation and to prevent its denaturation and incomplete release caused by electrostatic interactions between the protein and particle surfaces.

Fig.2

SEM images of β-TCP particles (A,B), HA-TCP particles (C,D), and Chi/HA-TCP particles (E,F) at different magnifications. The corresponding EDX analysis of the particles is presented in the top right corner of the figures (B,D,F). (G) Particle size histogram of the sieved β-TCP particles. (H) Thickness of the hydroxyapatite layer was 4.67±0.88 μm. The flake-like apatite and pores between the flakes were clearly visible (D). After chitosan coating, chitosan film was filled in the pores of the apatite layer (F). The left scale bar is 100 μm, and the right one is 30 μm.

Fig.3

ATR-FTIR spectra of HA-TCP, Chitosan and Chi/HA-TCP particles. The characteristic peaks of chitosan were labeled as: intermolecular hydrogen bond stretching (v_OH, v_NH) centered at 3446 cm⁻¹, Amide I band corresponding to v_C=O vibration in chitosan’s acetyl groups at 1650 cm⁻¹, Amide III band at 1332 cm⁻¹ due to combination of NH deformation and v_CN stretching. These peaks were also visible in the Chi/HA-TCP spectrum, confirming the presence of chitosan on the particles.

Fig. 4

XRD pattern of HA-TCP, Chitosan and Chi/HA-TCP particles. After being coated with chitosan, the crystallization of the HA-TCP did not change.

Fig.5

FM image of Chi/HA-TCP particles using FITC-chitosan at 0.3% (wt/wt) concentration. Separately scattered particles were observed when using 0.3% (wt/wt) chitosan. Scale bar: 200 μm.

Fig.6

Zeta potential values of the particles with different coatings. The measured zeta potential values for TCP, HA-TCP, and Chi/HA-TCP (0.3%) were −18±2 mV, −28±2 mV, and +22±1 mV, respectively.

Fig.7

The effect of different excipients on the NELL-1 release from HA-TCP particles in PBS medium. The data were fitted with linear regression model.

Fig.8

Comparison of the release profiles for unmodified HA-TCP and Chi/HA-TCP particles (0.1, 0.3, 0.6% Chitosan) in release medium containing 10% FBS and 1% Pent/Strep. HA-TCP particles showed a fast release and 70% of NELL-1 was released on the very first day, but Chi/HA-TCP particles had a controlled and sustained protein release profiles.

Fig.9

The bioactivity of released NELL-1 from ATCP and Chi/HA-TCP particles. Bioactivity was measured by testing its ability to increase ALP expression in mouse calvarial osteoblast cells. Chi/HA-TCP particles without NELL-1 were used as a negative control. The bioactivity of NELL-1 released from Chi/HA-TCP was consistently higher than that of NELL-1 released from HA-TCP at all time points.