Dataset Information

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

ABSTRACT:

Aim

The aim of this study is to examine the elevation of MYOC in long-term treatment of human trabecular meshwork (HTM) cells using dexamethasone (DEX) encapsulated pentablock (PB) copolymer-based nanoparticles (NPs) (DEX-PB-NPs).

Materials & methods

PB copolymers and DEX-PB-NPs were synthesized and characterized using nuclear magnetic resonance, gel permeation chromatography, and X-ray diffraction analyses. MYOC levels secreted from HTM cells were measured by western blot (WB) analysis.

Results

DEX-PB-NPs were formulated in the size range of 109 ± 3.77 nm (n = 3). A long term DEX release from the NPs was observed over three months. Cell viability and cytotoxicity were not affected up to 12 weeks of treatment with PB-copolymer or DEX-PB-NPs. WB data from five HTM cell strains showed that MYOC levels increased by 5.2 ± 1.3, 7.4 ± 4.3, and 2.8 ± 1.1-fold in the presence of DEX-PB-NPs compared with 9.2 ± 3.8, 2.2 ± 0.5, and 1.5 ± 0.3-fold at 4, 8 and 12 weeks in control-DEX treatment group, respectively (n = 5). Based on the decline in MYOC levels after withdrawal of DEX from control wells, DEX-PB-NPs released the DEX for at least 10 weeks.

Conclusion

The treatment of HTM cells using DEX-PB-NPs were analyzed in this study. The in vitro cell-based system developed here is a valuable tool for determining the safety and effects of steroids released from polymeric NPs.

SUBMITTER: Agrahari V

PROVIDER: S-EPMC5551562 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

Agrahari Vibhuti V Li Guorong G Agrahari Vivek V Navarro Iris I Perkumas Kristin K Mandal Abhirup A Stamer W Daniel WD Mitra Ashim K AK

Nanomedicine (London, England) 20170731 16

<h4>Aim</h4>The aim of this study is to examine the elevation of MYOC in long-term treatment of human trabecular meshwork (HTM) cells using dexamethasone (DEX) encapsulated pentablock (PB) copolymer-based nanoparticles (NPs) (DEX-PB-NPs).<h4>Materials & methods</h4>PB copolymers and DEX-PB-NPs were synthesized and characterized using nuclear magnetic resonance, gel permeation chromatography, and X-ray diffraction analyses. MYOC levels secreted from HTM cells were measured by western blot (WB) an ...[more]

PMID: 28756742

Similar Datasets

Project description:The trabecular meshwork (TM) plays an essential role in the circulation of aqueous humor by sensing mechanical stretch. The balance between the outflow and inflow of aqueous humor is critical in regulating intraocular pressure (IOP). A dysfunctional TM leads to resistance to the outflow of aqueous humor, resulting in an elevated IOP, a major risk factor for glaucoma. It is widely accepted that mutant myocilin (MYOC) can cause damage to the TM. However, few studies have investigated how TM cells carrying mutant MYOC respond to cyclic mechanical stretch (CMS) and whether these cells are more sensitive to CMS under this genetic background. In this study, we applied mechanical stretch to TM cells using the Flexcell system to mimic physiological stress. In addition, we performed genome-wide transcriptome analysis and oxidized lipidomics to systematically compare the gene expression and oxylipin profiles of non-stretched control human primary TM cells, human primary TM cells under CMS (TM-CMS), and human primary TM cells overexpressing MYOCS341P under CMS (S341P-CMS). We found that TM cells that overexpressed MYOCS341P were more sensitive to mechanical stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that downregulated genes were most enriched in oxidative phosphorylation, indicating mitochondria dysfunction and the likelihood of oxidative stress. Oxidized lipidomics analysis revealed significant changes in oxylipin profiles between the S341P-CMS and TM-CMS groups. Through further genome-wide transcriptomic analysis, we identified several genes that may be involved in the sensitivity of TM cells that overexpressed MYOCS341P to mechanical stress, including SARM1, AHNAK2, NT5C, and SOX8. The importance of these genes was validated by quantitative real-time PCR. Collectively, our findings indicate that mitochondrial dysfunction may contribute to the damage that occurs to TM cells with a MYOCS341P background under mechanical stretch.

Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Dataset Information

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

Aim

Materials & methods

Results

Conclusion

Publications

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets