Development of liposome nanoparticles for delivering recombinant human secretory leukocyte protease inhibitor (rhSLPI) for enhancing osteoblast proliferation, adhesion, and differentiation

Abstract

The global number of elderly people has been increasing, and the world can now be considered an ageing society. One of the most concerning health problems in the elderly is a musculoskeletal disease, in which fractures and osteoporosis are the most common problems. The deteriorating bone quality in the elderly is a limitation for treatment and patient recovery. Several drugs and biomolecules have been studied for enhance osteoblast properties and differentiation. The secretory leukocyte protease inhibitor (SLPI), which is a serine protease inhibitory protein, has been reported to enhance osteoblast cell adhesion, proliferation, and differentiation. However, the application of SLPI in real clinical settings is limited due to its short half-life in circulation and the fact that it can be destroyed by circulating protease enzymes. Therefore, the application of nanoparticle encapsulation might beneficial for SLPI delivery. This study aims to fabricate liposome nanoparticles encapsulating recombinant human SLPI, or rhSLPI (rhSLPI-LNPs) for augmenting the half-life and enhancing human osteoblast differentiation. The liposome nanoparticles (LNPs) were fabricated by the thin film hydration method. Encapsulation of rhSLPI was performed by adding 0.33 μg/mL rhSLPI in ultrapure water to the thin film lipid, and then rhSLPI-LNPs formed into vesicles. The morphology of LNPs was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The morphology of both blank-LNPs and rhSLPI-LNPs showed a spherical shape and a rough surface. The shell of rhSLPI-LNPs showed an onion-like structure. The size, PDI, and zeta potential of blank-LNPs or rhSLPI-LNPs were measured by the Zetasizer. The results showed that the size of both blank-LNPs and rhSLPI-LNPs was approximately 120 nm, the PDI was 0.08 ± 0.008 and 0.075 ± 0.006, respectively and the zeta potentials were -64.47 ± 1.12 mV and -51.98 ± 0.90 mV, respectively. The rhSLPI encapsulation was confirmed by ELISA. The results showed that liposome nanoparticles could encapsulate rhSLPI. The %EE of rhSLPI-LNPs was 18.313 ± 0.24%. The percentage of release was determined by ELISA. The result showed that rhSLPI was released at less than 1%. Additionally, proteinase inhibition activity was investigated, and the experimental results demonstrated that it still possesses the ability to inhibit protease activity. The rhSLPI-LNPs increased cell proliferation at concentrations of 1 and 10 μg/mL. For cytotoxicity, rhSLPI-LNPs treatment did not cause any toxicity to the human osteoblast cell line (hFOB 1.19). In addition, pre-incubation of hFOB 1.19 with rhSLPI-LNPs could significantly enhance osteoblast adhesion when compared with the untreated group. Finally, osteoblast differentiation was observed by qRT-PCR. The results showed that rhSLPI-LNPs enhanced Runx2, Col1a1, and Ocn mRNA expression. In conclusion, this is the first study showing that rhSLPI-encapsulated liposome nanoparticles (rhSLPI-LNPs) are biocompatible with human osteoblast cells and could enhance human osteoblast cell adhesion and differentiation.