Process analytical technology as in-process control tool in semi-continuous manufacturing of PLGA/PEG-PLGA microspheres.

Nowadays, among 3rd generation drug delivery systems, biodegradable polymeric based long-acting injectable depot has achieved tremendous success in clinical application. So far, there have been two dozen of commercial products of Poly (lactic-co-glycolic acid) microspheres available in the market. Recently, continuous manufacturing concept has been successfully applied on oral solid formulation from buzzword to reality. However, the polymeric injectable microspheres are still stayed at batch manufacturing phase due to the lack of understanding of knowledge matrix. In this study, micro-mixer as a plug-and-play emulsification modules, Raman spectroscopy and focused beam reflectance measurement as real-time monitoring modules are integrated into a novel semi-continuous manufacturing streamline to provides more efficient upscaling flexibility in microspheres production. In this end to end semi-continuous manufacturing process, amphiphilic block polymer monomethoxy-poly (ethylene glycol) modified PLGA (mPEG-PLGA) was used for encapsulating Gallic acid. Additionally, with guarantee of good robustness, the correlation relationship between critical process parameters, critical material attributes and critical quality attributes were investigated. The time-space evolution process and mechanism for formation of PEG-PLGA microsphere with particular morphology were elaborated. Altogether, this study firstly established semi-continuous manufacturing streamline for PLGA/PEG-PLGA microspheres, which would not only lower the cost of production, narrow process variability and smaller equipment/environmental footprint but also applied in-process control (IPC) and QbD principle on complicated production process of microspheres. Therefore, this study build confidence in the industrial development of PLGA/PEG-PLGA microspheres and establish best practice standards, which might be a quantum leap for developing PLGA microspheres in the future.

Glucocorticoids induce osteoporosis mediated by glucocorticoid receptor-dependent and -independent pathways.

Clinically, glucocorticoids (GCs) are widely used to treat inflammation-related diseases; however, their long-term use causes side effects, such as osteoporosis and predisposition to bone fractures, known as glucocorticoid-induced osteoporosis (GIOP). Nr3c1 is the major glucocorticoid receptor, and its downstream signaling pathway is involved in regulating various intracellular physiological processes, including those related to bone cells; however, its mechanism in glucocorticoid-induced osteoporosis (GIOP) remains unclear. In this study, a zebrafish nr3c1-mutant was successfully generated using CRISPR/Cas9 technology to investigate the role of nr3c1 in GIOP. Mutations in nr3c1 altered cartilage development and significantly decreased bone mineralization area. Additionally, qRT-PCR results showed that the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes was altered in the nr3c1-mutant. The GC-Nr3c1 pathway regulates the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes via Nr3c1-dependent and Nr3c1-independent pathways. A dual-luciferase reporter assay further revealed that GCs and Nr3c1 transcriptionally regulate matrix metalloproteinase 9 (mmp9), alkaline phosphatase (alp), and acid phosphatase 5a (acp5a). This study reveals that GCs/Nr3c1 affect the expression of genes involved in bone metabolism and provides a basis to determine the role of GIOP and Nr3c1 in bone metabolism and development. We also identified a new effector target for the clinical treatment of GIOP.

Upregulation of ATG4A promotes osteosarcoma cell epithelial-to-mesenchymal transition through the Notch signaling pathway.

Osteosarcoma is a malignant tumor in children and adolescents. Previous studies showed that ATG4A is an autophagy-related gene involved in cancers. In this study, we aimed to identify the biological role of ATG4A in osteosarcoma. The expression levels of ATG4A were analyzed in osteosarcoma tissues by using reverse transcription-quantitative polymerase chain reaction (qRT-PCR) and western blotting. ATG4A was knocked-down or overexpressed in SAOS2 and HOS cell lines by transfection. Cell counting kit-8 (CCK-8) and clone formation assay were used to assess the effects of ATG4A on cell proliferation. Wound healing and Transwell assays were performed to evaluate the effects of ATG4A on cell migration and invasion, respectively. Epithelial-mesenchymal transition (EMT) markers and Notch signaling pathway targeting molecules were examined by western blotting. The results indicated that ATG4A was up-regulated in osteosarcoma tissues. In SAOS2 cells, knockdown of ATG4A inhibited the proliferation, migration and invasion, up-regulated the expression of E-cadherin and down-regulated the expression of vimentin, Notch1 and Hes1. In HOS cells, overexpression of ATG4A promoted the proliferation, migration and invasion, up-regulated the expression of vimentin, Notch1 and Hes1 and down-regulated the expression of E-cadherin. In conclusion, these findings demonstrate that ATG4A is up-regulated in osteosarcoma tissues. In osteosarcoma cells, ATG4A promotes the EMT process partly by the Notch signaling pathway. These results suggest that ATG4A might represent a potential therapeutic target for patients with osteosarcoma.

miR-195 inhibits the proliferation and migration of chondrocytes by targeting GIT1.

Previous studies have demonstrated that G-protein coupled receptor kinase interacting protein-1 (GIT1) and microRNAs (miRNAs) serve an important role in chondrocyte proliferation and migration. However, a limited number of studies conducted thus far have investigated the association between GIT1 and miRNAs. In the present study, putative miR­195 binding sites in the GIT1 3'­untranslated region were identified using common bioinformatic algorithms (miRanda, TargetScan, miRBase and miRWalk), and it was demonstrated that they may be involved in regulating GIT1 expression. Following transfection of miR­195 mimics in chondrocytes, the expression of GIT1 was significantly reduced, whereas the expression was significantly increased following transfection with miR­195 inhibitors. In addition, the results of the current study demonstrated that increased miR­195 expression may downregulate chondrocyte proliferation and reduce cell migration. However, chondrocyte proliferation and migration was enhanced following suppression of miR­195 expression. Furthermore, upon co­transfection of miR­195 and GIT1 expression vectors, the inhibitory effect of miR­195 on chondrocyte proliferation and migration was attenuated. Therefore, miR­195 may affect chondrocyte proliferation and migration via targeted regulation of GIT1 expression. The results of the current study provide novel evidence for the regulatory mechanisms of miRNAs in bone and cartilage tissues, which may facilitate further research and provide a greater understanding of different osteoarticular diseases.