Etoposide Amorphous Nanopowder for Improved Oral Bioavailability: Formulation Development, Optimization, in vitro and in vivo Evaluation.

INTRODUCTION: Etoposide refers to a derivative of podophyllotoxin, which plays an important role in the treatment of cancer due to its prominent anti-tumor effect. As a BCS IV drug, etoposide exhibits insufficient aqueous solubility and permeability, thereby limiting its oral absorption. To enhance the oral bioavailability of etoposide, this study developed an amorphous nanopowder. METHODS: Based on preliminary screening and experimental design, the stabilizer and preparation process of etoposide nanosuspension were explored. Subsequently, using a Box-Behnken design, the effects of independent factors (ultrasonication time, ratio of two phases and stabilizer concentration) on response variables (particle size and polydispersity index) were studied, and then the formulation was optimized. Finally, nanosuspension was further freeze dried with 1% of mannitol resulting in the formation of etoposide amorphous nanopowder. RESULTS: The optimized etoposide nanopowder showed as spherical particles with an average particle size and polydispersity index of 211.7 ± 10.4 nm and 0.125 ± 0.028. X-ray powder diffraction and differential scanning calorimetry confirmed the ETO in the nanopowder was amorphous. Compared with coarse powder and physical mixture, etoposide nanopowder achieved significantly enhanced saturated solubility and dissolution in various pH environments. The Cmax and AUC0-t of etoposide nanopowder after oral administration in rats were respectively 2.21 and 2.13 times higher than the crude etoposide suspension. Additionally, the Tmax value of nanopowder was 0.25 h, compared with 0.5 h of reference group. DISCUSSION: In the present study, the optimized amorphous nanopowder could significantly facilitate the dissolution and oral absorption of etoposide and might act as an effective delivery method to enhance its oral bioavailability.

Knockdown of LncRNA H19 Relieves LPS-Induced Damage by Modulating miR-130a in Osteoarthritis.

PURPOSE: Osteoarthritis (OA) is a commonly occurring illness without a definitive cure, at present. Long non-coding RNAs (lncRNAs) have been widely confirmed to be involved in the modulation of OA progression. This study aimed to investigate the role and mechanism of lncRNA H19 in OA. MATERIALS AND METHODS: Abundances of H19 and microRNA-130a (miR-130a) in lipopolysaccharide (LPS)-treated C28/I2 cells were measured by reverse-transcription quantitative PCR (RT-qPCR). CCK-8 and flow cytometry analyses were carried out to assess cell viability and apoptosis. Starbase online software was used to predict the putative binding sites between H19 and miR-130a. Luciferase reporter, RNA pull down, and RT-qPCR were performed to analyze the true interaction between H19 and miR-130a. RESULTS: A notably dose-dependent elevation of H19 levels was observed in LPS-treated C28/I2 cells. Knockdown of H19 ameliorated the injury of LPS-induced C28/I2 cells, reflected by induced viability, decreased apoptosis, and reduced inflammatory factor secretions. Moreover, H19 negatively regulated the expression of miR-130a via acting as a molecular sponge for miR-130a. The stimulatory effects of H19 on cell damage were abolished following the restoration of miR-130a. CONCLUSION: LncRNA H19 aggravated the injury of LPS-induced C28/I2 cells by sponging miR-130a, hinting a novel regulatory mechanism and a potential therapeutic target for OA.

A Controlled Release Codelivery System of MSCs Encapsulated in Dextran/Gelatin Hydrogel with TGF-ß3-Loaded Nanoparticles for Nucleus Pulposus Regeneration.

Mesenchymal stem cell- (MSC-) based therapy is regarded as a potential tissue engineering strategy to achieve nucleus pulposus (NP) regeneration for the treatment of intervertebral disc degeneration (IDD). However, it is still a challenge to induce MSC differentiation in NP-like cells when MSCs are implanted into the NP. The purpose of this study was to construct poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles as carriers for TGF-ß3 controlled release and establish a codelivery system of a dextran/gelatin hydrogel with the nanoparticles for long-term processing of discogenesis differentiation. TGF-ß3-loaded PLGA nanoparticles were prepared by the double-emulsion solvent evaporation method and seeded uniformly into the hydrogel. Morphological observations, an assessment of the release kinetics of TGF-ß3, a cytotoxic assay, a cell proliferation test, a biochemical content assay, qRT-PCR, and immunohistological analyses of the codelivery system were conducted in the study. The results showed that the TGF-ß3-loaded nanoparticles could release TGF-ß3 gradually. The codelivery system exhibited favorable cytocompatibility, and the TGF-ß3 that was released could induce MSCs to NP-like cells while promoting ECM-related biosynthesis. These results suggest this codelivery system may be employed as a promising carrier for discogenesis of MSCs in situ.

Dynamic Compression Effects on Immature Nucleus Pulposus: a Study Using a Novel Intelligent and Mechanically Active Bioreactor.

BACKGROUND: Previous cell culture and animal in vivo studies indicate the obvious effects of mechanical compression on disc cell biology. However, the effects of dynamic compression magnitude, frequency and duration on the immature nucleus pulposus (NP) from an organ-cultured disc are not well understood. OBJECTIVE: To investigate the effects of a relatively wide range of compressive magnitudes, frequencies and durations on cell apoptosis and matrix composition within the immature NP using an intelligent and mechanically active bioreactor. METHODS: Discs from the immature porcine were cultured in a mechanically active bioreactor for 7 days. The discs in various compressive magnitude groups (0.1, 0.2, 0.4, 0.8 and 1.3 MPa at a frequency of 1.0 Hz for 2 hours), frequency groups (0.1, 0.5, 1.0, 3.0 and 5.0 Hz at a magnitude of 0.4 MPa for 2 hours) and duration groups (1, 2, 4 and 8 hours at a magnitude of 0.4 MPa and frequency of 1.0 Hz) experienced dynamic compression once per day. Discs cultured without compression were used as controls. Immature NP samples were analyzed using the TUNEL assay, histological staining, glycosaminoglycan (GAG) content measurement, real-time PCR and collagen II immunohistochemical staining. RESULTS: In the 1.3 MPa, 5.0 Hz and 8 hour groups, the immature NP showed a significantly increase in apoptotic cells, a catabolic gene expression profile with down-regulated matrix molecules and up-regulated matrix degradation enzymes, and decreased GAG content and collagen II deposition. In the other compressive magnitude, frequency and duration groups, the immature NP showed a healthier status regarding NP cell apoptosis, gene expression profile and matrix production. CONCLUSION: Cell apoptosis and matrix composition within the immature NP were compressive magnitude-, frequency- and duration-dependent. The relatively high compressive magnitude or frequency and long compressive duration are not helpful for maintaining the healthy status of an immature NP.

Osmolarity affects matrix synthesis in the nucleus pulposus associated with the involvement of MAPK pathways: A study of ex vivo disc organ culture system.

Matrix homeostasis within the nucleus pulposus (NP) is important for disc function. Unfortunately, the effects of osmolarity on NP matrix synthesis in a disc organ culture system and the underlying mechanisms are largely unknown. The present study was to investigate the effects of different osmolarity modes (constant and cyclic) and osmolarity levels (hypo-, iso-, and hyper-) on NP matrix synthesis using a disc organ culture system and determine whether ERK1/2 or p38MAPK pathway has a role in this process. Porcine discs were cultured for 7 days in various osmotic media, including constant hypo-, iso-, hyper-osmolarity (330, 430, and 550 mOsm/kg, respectively) and cyclic-osmolarity (430 mOsm/kg for 8 h, followed by 550 mOsm/kg for 16 h). The role of ERK1/2 and p38MAPK pathways were determined by their inhibitors U0126 and SB202190 respectively. The expression of SOX9 and downstream aggrecan and collagen II, biochemical content, and histology were used to assess NP matrix synthesis. The findings revealed that NP matrix synthesis was promoted in iso- and cyclic-osmolarity cultures compared to hypo- or hyper-osmolarity culture although the level of matrix synthesis in cyclic-osmolarity culture did not reach that in iso-osmolarity culture. Further analysis suggested that inhibition of the ERK1/2 or p38MAPK pathway in iso- and cyclic-osmolarity cultures reduced NP matrix production. Therefore, we concluded that the effects of osmolarity on NP matrix synthesis depend on osmolarity level (hypo-, iso-, or hyper-) and osmolarity mode (constant or cyclic), and the ERK1/2 and p38MAPK pathways may participate in this process. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1092-1100, 2016.