Selenium nanoparticles prepared by myco-fabrication : a novel bone-forming agent for managing/preventing postmenopausal osteoporosis

Abstract

Osteoporosis is a skeletal disease characterized by low bone mass and density as well as deterioration in bone microarchitecture, leading to high risk of bone fracture. With an increase in aging population, osteoporosis has become one of the major public health issues nowadays, causing significant medical and socioeconomic burdens. Selenium is an essential trace mineral to human health. Substantial evidences have demonstrated that selenium deficiency is detrimental to bone microarchitecture and even associated with osteopenia, Kashin-Beck osteoarthropathy and osteoporosis, suggesting its crucial role in bone metabolism. Recently, selenium nanoparticles (SeNPs) have become the new research target, since they were found to possess relatively low toxicity and remarkable anti-tumor efficacy compared to other organic and inorganic selenocompounds. Nevertheless, scientific research concerning their effects on bone health is currently very limited. By using the mushroom polysaccharide-protein complex (PSP) isolated from Pleurotus tuber-regium, our research team has successfully prepared novel SeNPs (PTR-SeNPs) under a simple redox system [US patent no.: 9,072,669]. We discovered that PTR-SeNPs existed as well-dispensed spherical particles in water with an average diameter of 91.3 ± 1.53 nm and was highly stable without significant increase in size after 13 weeks. Further characterization using TEM also showed that the particle size of SeNPs in PTR-SeNPs was around 20 nm. For individual SeNP, the clear lattice fringes (3.34Å), SAED pattern as well as EDX spectrum obtained by HR-TEM-EDX collectively indicated that the resulting nanoparticle possessed a polycrystalline structure with high level of Se (81.2%), implying a successfully fabrication of SeNPs using mushroom PSP. Endocytosis has been widely reported as the major cellular uptake mechanism for nanoparticles. Our study found that coumarin-6 labelled PTR-SeNPs mainly localized in lysosomes of the murine preosteoblast MC3T3-E1 subclone 4 cells (bone forming progenitor cells) as early as 5 min after cellular internalization. It is the first study of its kind to report the intracellular localization of SeNPs by MC3T3-E1 cells via endocytosis. Besides, substantial previous findings have demonstrated that promoting bone formation is one of the effective strategies to prevent and/or manage postmenopausal osteoporosis. Interestingly, our study indicated that PTR-SeNPs was found to exhibit a significant dose-dependent proliferation effect (range from 1.33 - 1.73 folds) on the MC3T3-E1 cells for 24, 48 and 72 hours with the most effect dosage of 10µM. More importantly, PTR-SeNPs (10µM) were found to markedly induce both osteoblast differentiation and bone mineral formation of the MC3T3-E1 cells as evidenced by a significant increase in ALP activity (1.36 ± 0.10 folds), as well as an enhancement of bone nodule formation (Von Kossa and Alizarin Red S staining). Further investigation on their possible interaction with osteoclasts (bone resorption cells) also showed that PTR-SeNPs could significantly up-regulate the gene expression ratio of the bone remodeling markers OPG/RANKL (2.11 ± 0.33 folds), suggesting their indirect inhibition effect on osteoclastogenesis in addition to promoting osteoblastogenesis. Osteoblast differentiation is a crucial step of bone formation. Previous studies have demonstrated that BMP-2 signaling via Smad-dependent and/or Smad-independent pathway(s) is one of the most important signaling transduction cascades for regulating osteoblast differentiation. Interestingly, our study discovered that PTR-SeNPs could trigger both BMP-2/Smad dependent and independent pathways simultaneously as evidenced by a significant upregulation of BMP-2 gene expression (1.64 ± 0.24 folds) as well as Smad 1/5/8 (1.31 ± 0.15 folds), ERK (3.67 ± 1.48 folds), p38 (2.86 ± 0.58 folds) and JNK (1.36 ± 0.07 folds) proteins phosphorylation in the MC3T3-E1 cells after 4-day treatment. Besides, PTR-SeNPs were found to significantly up-regulate the gene expression of those major downstream biomarkers in bone remodeling such as Dlx5 (1.54 ± 0.06 folds), Runx2 (2.11 ± 0.1 folds), Osx (1.87 ± 0.60 folds), ALP (2.09 ± 0.10 folds), and OCN (9.69 ± 1.07 folds), further supporting their active role in promotion of osteoblast differentiation and bone mineralization. Last but not least, by using Oryzias latipes (medaka) larvae, we have successfully established a brand new in vivo model to investigate the simulating effect of PTR-SeNPs on bone formation. And we found that PTR-SeNPs (10ppm) could significantly enhance the vertebrate development (both bone and cartilage) of medaka larvae after feeding for 11 days as visualized by Alizarin Red S and Alcian Blue double staining. Consistent with the findings of in vitro study, the gene expression level of those major osteogenic markers such as ALP (1.53 ± 0.1 folds), Runx2 (1.45 ± 0.10 folds), Osx (1.41 ± 0.15 folds) as well as OPG/RANKL (4.38 ± 1.31 folds) were all significantly upregulated. Findings of this study could provide significant insights into the in vitro and in vivo effects of PTR-SeNPs on bone formation. Besides, medaka larvae, as a novel non-mammalian animal model for bone formation study, could be further developed into a rapid and economical in vivo screening platform for bone formation prior to conducting traditional animal study using mice or rats. Our long-term goal is to develop an evidence-based bone-forming agent for promoting the bone health of postmenopausal patients in our community.