Research And Application Of Microfluidic And Mass-spectrometric Techniques In Drug Screening

Most of the drugs that are found effective outside the brain cannot permeate through the blood-brain barrier(BBB)and the complex brain tissues.Therefore,development of a reliable platform to evaluate the drug permeability will greatly promote the research and development of central nervous system(CNS)drugs.Moreover,the metabolomics technique has been widely used to investigate the mechanism of action of drugs,which is of great importance for drug screening.Therefore,we developed a microfluidic device and a metabolomics platform to investigate the pharmacological properties for drug screening.Firstly,an integrated microfluidic device was established to directly evaluate drug permeability across the BBB and cytotoxicity on target cells.hCMEC/D3 cells were dynamically cultured on a membrane to reconstitute the in vivo BBB properties for permeability assay.Glioma cells(U251)were cultured inside agarose gel in the downstream for cytotoxicity assay.For permeability assay,a solid-phase-extraction microchip(μSPE)was used to extract the permeated model drug,sunitinib,before direct detection by ESI-Q-TOF MS.The measured permeability was highly correlated with the value reported in literature,and the permeability analysis only required 30 min and 5 μL.The cytotoxicity results were in accordance with the previously reported studies.These results demonstrate that this platform is a valid model for CNS drug screening and corresponding cytotoxicity assays,which is crucial for drug discovery and development.Secondly,the previous studies of flavopiridol have shown that it could not only induce cell cycle arrest in proliferative cells but also toxicity in resting cells.Moreover,most of the studies only focused on specific kinases or genes.Therefore,a comprehensive study of the metabolic alterations will certainly assist in revealing the underlying mechanism of action of flavopiridol.In this part,an UPLC/Q-TOF MS platform was utilized to obtain the distinct metabolites in combination with the relevant biological verifications.In conclusion,flavopiridol induced cell cycle arrest and oxidative stress in MCF-7 cells,which subsequently disturbed several metabolic pathways and finally caused cell death.