Enhancement Of TAT Cell Membrane Penetration Efficiency By Dimethyl Sulphoxide (DMSO)

Objectives: Cell penetrating peptides (CPPs) are promising tools for transducing presynthesized therapeutic molecules which posses a low membrane permeability. The poor efficiency of cellular uptake is the main obstacles to the development of drug delivery by using CPPs. The present study is undertaken to investigate the possibility of improving TAT penetrating efficiency.Methods: 1) FITC-labeled peptides TAT-FITC and NCO-FITC (negative control) synthesized artificially were used in this study. Human cervical carcinoma cell lines Caski and Siha, human hepatocarcinoma cell lines HepG2, Human lung cancer cell lines A549 and African green monkey kidney cell lines COS7 cells were used in vitro. The penetrating efficiency of TAT and the distribution of fluorescence were observed under fluorescence microscope after the cells were exposed to 10% DMSO; fluorescence intensity in cell was also quantified by FACS and fluorescence spectrum analysis. 2) To analyze whether DMSO enhanced TAT penetration by affecting the integrity of the plasma membrane, hemolysis and lactate dehydrogenase (LDH) release assays were employed. Characterized inhibiting drugs were selected for their ability to inhibit specific steps in the endocytosis pathway. 3) Constructing recombinant prokaryotic expression plasmids expressing GFP,Apoptin,TAT-GFP and TAT-Apoptin. The recombinant plasmids were confirmed by restrictional enzymes analysis, PCR and DNA sequencing. E. coli BL21 (DE3) were transformed with the recombinant prokaryotic expression plasmids expressing GFP,Apoptin,TAT-GFP and TAT-Apoptin, expression were induced by adding IPTG into LB medium and the fusion proteins were purified by Ni-NTA His-Bind Resin and than were dialyed in PBS finally. The purified yields of prokaryotic expression were confirmed by SDS-PAGE and Western blot assay. 4) Caski cells incubated with TAT-GFP after pretreating by 10% DMSO, then fluorescence uptake into culturing cells was observed using fluorescence microscopy. Apoptotic Caski cells induced by TAT-Apoptin after pretreating with DMSO were detected by TUNEL assay.Results: 1) Pretreatment of cells by 10% DMSO significantly enhanced the penetrating efficiency of TAT-FITC, also flourescence was well-distributed in nucleus and cytosol, whereas no flourescence in the cells added NCO-FITC was observed. Quantification by fluorescence spectrum of TAT-FITC flourescence in cells also showed that fluorescence was enhanced by 10% DMSO pretreatment remarkably. After pretreated by 10% DMSO incubated with serum, quantification by fluorescence spectrum of TAT flourescence in cells showed that the enhancement effect of DMSO pretreatment on TAT penetrating was significantly decreased than that without serum culturing. 2) DMSO pretreatment enhanced cell membrane penetration of TAT was not through inducing pores on cell membrane. There was only very slight cytotoxicity to some cell lines up to 10% of DMSO treatment for 2 hours. TAT transduction levels were moderately inhibited by endocytic inhibitors of ammonium chloride and sodium azide with 10% DMSO pretreatment. 3) The constructs of recombinant plasmids expressing GFP,Apoptin,TAT-GFP and TAT-Apoptin were checked by double digestion, PCR and DNA Sequencing analysis. Western blotting revealed that the fusion proteins were successfully expressed in E. coli BL21 (DE3). 4) After co-incubation with 10% DMSO and Caski cells, Fusion protein TAT-GFP significantly enhanced and well-distributed, TUNEL assay revealed that TAT-apoptin can translocate into Caski cells functioning biologocal activity of inducing apoptosis.Conclusion: In the present study, we investigated the effect of a penetration enhancer, dimethylsulfoxide (DMSO), on the penetrating efficiency of TAT peptide or the TAT fusion protein. FITC-labeled TAT and TAT-GFP were added to 10% DMSO pretreated cells, fluorescence uptake into culturing cells was observed using fluorescence microscopy, FACS or quantitatively analyzed by a fluorescence spectrum. 10% DMSO treatment markedly increased internalization of TAT into cells and appeared in a well-distributed pattern throughout the cytosol and nucleus without membrane perforating or minimal cytotoxicity, the enhancement effect by 10% DMSO was reduced by endocytosis inhibitors including ammonium chloride and sodium azide. 10% DMSO also enhanced TAT-Apoptin induced apoptosis of carcinoma cells. These findings implicated that DMSO can be a novel delivery enhancer appropriate for CPPs penetration.