In situ fabrication of biphasic-induced magnetic nano-hydroxyapatite chitosan-based composite microcapsules for bone regeneration

摘要

Introduction: This research aims to in situ fabricate and investigate the biphasic-induced endogenous magnetic microspheres (BIEMM) as a promising environmental stimuli-responsive delivery vehicle to dispose the problem of drug burst effect. Icariin (ICA), a plant-derived flavonol glycoside, was combined to study drug release properties of BIEMM, which is ascribed to its outstanding function of promoting osteoblasts adhesion and proliferation. The ICA loaded magnetic chitosanano hydroxyapatite (CSHA/MNP) microcapsules were prepared by the high voltage static microcapsule forming device. Fe3O4 magnetic nanoparticles (MNP) and nHA were in situ crystallized simultaneously by one - step process and BIEMM were characterized. It was governed by the formation mechanism that the amino groups (-NH3) of CS could be used as binding sites for the synthesis of both Fe3O4 MNP and nHA. Therefore, the novel ICA loaded biphasic-induced endogenous CSHA/MNP microcapsules are expected to find application in bone regeneration. Materials and Methods: Briefly, moderate CS powder was dissolved in 53 ml of 1 % acetic acid solution stirred for 1 h to form a homogeneous CS solution. The alcohol solution of ICA was added and stirred for 1.5 h magnetically at room temperature, and then added FeCl3·6H2O and FeCl2·4H2O to form 0.15mol·L-1 and 0.075mol·L-1 solution, according the mole ratio of Fe3+/ Fe2+=2, stirred for 45 min, respectively. Similarly, followed by adding Ca(NO3)2·4H2O and K2HPO4 solution according the ratio of Ca/P=1.67, stirred for 3 h, to prepare the BIEMM precursor solution. By the high voltage electrostatic micro capsule forming device, precursor solution was drop-wise added in a 250 ml beaker containing 4 % NaOH aqueous solution (as coagulation bath). The mixtures were thoroughly mixed by occasional shaking and incubated for 12 h at 37 oC to form microsphere. After several cycles of centrifugation at 8,000 rpm for 5 min and washing with deionized water, the media gradually became a neutral and the microspheres were lyophilized for further use. Results and Discussion: Fig. 1 In situ fabrication of BIMCM. (A) Stereoscopic microscope images, (B) SEM images, (C) Cumulative release curves of ICA, (D) Magnetic hysteresis curves of (1) CS/MNP, (2) CSHA/MNP and (3) BIMCM. (E) Digital photographs of the dispersion of BIMCM in the separation with an external field (1) and absence of external magnetic field (2). BIMCM with equiaxed and homogeneous morphology were obtained in Fig. 1 A. In addition, BIMCM showed relatively porous and rough surface with no obvious collapse structure, as displayed on Fig. 1B. We investigated the morphology of automated in situ dick Fe3O4 MNP and nHA particles on the surface structure, which may be closely related to the deposition of Fe3O4 MNP and nHA particles into the BIMCM, as well as the bareness of ICA on the surface of microcapsules. In Fig. 1E, BIMCM could be easily attached to the sidewall from water solution within eternal magnetic field. The successful introduction of the in situ Fe3O4 MNP provided an alternative strategy for ICA sustained release in Fig. 1C. Therefore, the microcapsules possessed magnet-induced sensitivity, indicated the BIMCM could meet the requirement of magnetic drug targeting. Fig. 2 Wright-Giemsa dying assay (A1A2) and AO/EB fluorescent staining (A1B2) for proliferation of osteoblasts cultured with BIMCM for 3 and 5 d, respectively. Wright-Giemsa dying assay (A1A2) and AO/EB fluorescent staining (B1B2) of osteoblasts cultured with BIMCM for 3 and 5 d respectively were depicted, as shown in Fig. 2. Our in vitro toxicity text results demonstrated that BIMCM had a good biocompatibility and could be very conducive to promoting osteoblast proliferation. Conclusion: It can be speculated that the successful introduction of in situ Fe3O4 MNP into bone repair provided a robust platform as a function of the properties of magnetic sensitivity and sustained release. As such, the innovative biphasic-induced ICA loaded CSHA/MNP microcapsules were expected to find potential application in drug delivery system and bone repair in the future.