Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells
| Autor: | Xin Chen, Xiaoxu Li, Shiqi Liao, Yunzhen Xu |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Clinical Biochemistry
Pharmaceutical Science Peptide Cell-Penetrating Peptides Proof of Concept Study 01 natural sciences Biochemistry Fluorescence Cell membrane chemistry.chemical_compound Hydrolysis Drug Discovery medicine Fluorescence microscope Humans Prodrugs Fluorescein Molecular Biology Fluorescent Dyes chemistry.chemical_classification Chromatography 010405 organic chemistry Organic Chemistry Esters Prodrug Flow Cytometry Fluoresceins 0104 chemical sciences Drug Liberation 010404 medicinal & biomolecular chemistry medicine.anatomical_structure Microscopy Fluorescence chemistry Molecular Medicine HeLa Cells Conjugate |
| Zdroj: | Bioorganic & Medicinal Chemistry. 27:851-858 |
| ISSN: | 0968-0896 |
| DOI: | 10.1016/j.bmc.2019.01.030 |
| Popis: | Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d -Val and l -Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l -Ala diester show strong fluorescence accumulation in 30 min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d -Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t1/2 for l -Ala diester is 15 min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings. |
| Databáze: | OpenAIRE |
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