High Biocompatibility, MRI Enhancement, and Dual Chemo- and Thermal-Therapy of Curcumin-Encapsulated Alginate/Fe3O4 Nanoparticles.

(1) Background: Magnetite (Fe3O4) nanoparticles have great potential for biomedical applications, including hyperthermia and magnetic resonance imaging. In this study, we aimed to identify the biological activity of nanoconjugates composed of superparamagnetic Fe3O4 nanoparticles coated with alginate and curcumin (Fe3O4/Cur@ALG) in cancer cells. (2) Methods: The nanoparticles were evaluated for the biocompatibility and toxicity on mice. The MRI enhancement and hyperthermia capacities of Fe3O4/Cur@ALG were determined in both in vitro and in vivo sarcoma models. (3) Results: The results show that the magnetite nanoparticles exhibit high biocompatibility and low toxicity in mice at Fe3O4 concentrations up to 120 mg/kg when administered via intravenous injection. The Fe3O4/Cur@ALG nanoparticles enhance the magnetic resonance imaging contrast in cell cultures and tumor-bearing Swiss mice. The autofluorescence of curcumin also allowed us to observe the penetration of the nanoparticles into sarcoma 180 cells. In particular, the nanoconjugates synergistically inhibit the growth of sarcoma 180 tumors via magnetic heating and the anticancer effects of curcumin, both in vitro and in vivo. (4) Conclusions: Our study reveals that Fe3O4/Cur@ALG has a high potential for medicinal applications and should be further developed for cancer diagnosis and treatment.

Differential Cytotoxicity of Curcumin-Loaded Micelles on Human Tumor and Stromal Cells.

Although curcumin in the form of nanoparticles has been demonstrated as a potential anti-tumor compound, the impact of curcumin and nanocurcumin in vitro on normal cells and in vivo in animal models is largely unknown. This study evaluated the toxicity of curcumin-loaded micelles in vitro and in vivo on several tumor cell lines, primary stromal cells, and zebrafish embryos. Breast tumor cell line (MCF7) and stromal cells (human umbilical cord vein endothelial cells, human fibroblasts, and human umbilical cord-derived mesenchymal stem cells) were used in this study. A zebrafish embryotoxicity (FET) assay was conducted following the Organisation for Economic Co-operation and Development (OECD) Test 236. Compared to free curcumin, curcumin PM showed higher cytotoxicity to MCF7 cells in both monolayer culture and multicellular tumor spheroids. The curcumin-loaded micelles efficiently penetrated the MCF7 spheroids and induced apoptosis. The nanocurcumin reduced the viability and disturbed the function of stromal cells by suppressing cell migration and tube formation. The micelles demonstrated toxicity to the development of zebrafish embryos. Curcumin-loaded micelles demonstrated toxicity to both tumor and normal primary stromal cells and zebrafish embryos, indicating that the use of nanocurcumin in cancer treatment should be carefully investigated and controlled.

Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model.

Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl) under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs.

An adult osteopetrosis model in medaka reveals the importance of osteoclast function for bone remodeling in teleost fish.

Osteoclasts play important roles during bone growth and in maintaining bone health and bone homeostasis. Dysfunction or lack of osteoclasts leads to increased bone mass and osteopetrosis phenotypes in mouse and human. Here we report a severe osteopetrosis-like phenotype in transgenic medaka fish, in which membrane bound EGFP (mEGFP) was expressed in osteoclasts under control of the cathepsin K promoter (ctsk:mEGFP). In contrast to reporter lines with GFP expression in the cytoplasm of osteoclasts, adult fish of the mEGFP line developed bone defects indicative for an osteoclast dysfunction. Activity of tartrate-resistant acid phosphatase (TRAP) was down-regulated and excess bone was observed in most parts of the skeleton. The osteopetrotic phenotype was particularly obvious at the neural and haemal arches that failed to increase their volume in growing fish. Excess bone caused severe constriction of the spinal cord and the ventral aorta. The continuation of tooth development and the failure to shed teeth resulted in severe hyperdontia. Interestingly, at the vertebral column vertebral body arches displayed a severe osteopetrosis, while vertebral centra had no or only a mild osteopetrotic phenotype. This confirms previous reports from cichlids that, different from the arches, allometric growth of fish vertebral centra initially does not depend on the action of osteoclasts. Independent developmental mechanism that shapes arches and vertebral centra can also lend support to the hypothesis that vertebral centra and arches function as independent developmental modules. Together, this medaka osteopetrosis model confirms the importance of proper osteoclast function during normal skeletal development in teleost fish that requires bone modeling and remodeling.

A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization.

In teleosts, such as medaka, ossification of the vertebral column starts with the mineralization of the notochordal sheath in a segmental pattern. This establishes the chordal centrum, which serves as the basis for further ossifications by sclerotome derived osteoblasts generating the vertebral body. So far, it is unclear which cells produce the notochordal sheath and how a segmental pattern of mineralization is established in teleosts. Here, we use a transgenic medaka line that expresses nlGFP under the control of the col10a1 promoter for in vivo analysis of vertebral body formation. We show that col10a1:nlGFP expression recapitulates endogenous col10a1 expression. In the axial skeleton, col10a1:nlGFP cells appear prior to the mineralization of the notochordal sheath in a segmental pattern. These cells remain on the outer surface of the chordal centra during mineralization as well as subsequent perichordal ossification of the vertebral bodies. Using twist1a1:dsRed and osx:mCherry transgenic lines we show that a subset of col10a1:nlGFP cells is derived from sclerotomal precursors and differentiates into future osteoblasts. For the first time, this shows a segmental occurrence of putative osteoblast precursors in the vertebral centra prior to ossification of the notochordal sheath. This opens the possibility that sclerotome derived cells in teleosts are implicated in the establishment of the mineralized vertebral column in a similar manner as previously described for tetrapods.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model.

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

Pituitary-interrenal interaction in zebrafish interrenal organ development.

To further elucidate pituitary adrenal interactions during development, we studied the organogenesis of the interrenal organ, the teleost homolog of the mammalian adrenal gland, in zebrafish. To this end we compared wild-type zebrafish interrenal development with that of mutants lacking pituitary cell types including corticotrophs. In addition, we studied the effects of ACTH receptor (Mc2r) knockdown and dexamethasone (dex) on interrenal development and pituitary feedback. Until 2 d post fertilization (2 dpf) interrenal development assessed by transcripts of key steroidogenic genes (cyp11a1, mc2r, star) is independent of proopiomelanocortin (Pomc) as demonstrated in aal/eya1and lia/fgf3 mutants. However, at 5 dpf lack of pituitary cells leads to reduced expression of steroidogenic genes at both the transcriptional and the protein level. Pituitary control of interrenal development resides in corticotrophs, because pit1 mutants lacking pituitary cells except corticotrophs have a phenotype similar to that of wild-type controls. Furthermore, development in mc2r knockdown morphants does not differ from aal/eya1 and lia/fgf3 mutants. Inhibition of steroidogenesis by mc2r knockdown induces up-regulation of pomc expression in the anterior domain of pituitary corticotrophs. Accordingly, dex suppresses pomc in the anterior domain only, leading to impaired expression of steroidogenic genes commencing at 3 dpf and interrenal hypoplasia via reduced interrenal proliferation. In contrast, negative feedback on pituitary corticotrophs by dex is evident at 2 dpf and precedes effects of Pomc on the interrenal primordium. These data demonstrate a gradual transition from early pituitary-independent interrenal organogenesis to developmental control by the anterior domain of pituitary corticotrophs acting via Mc2 receptors.