1 Preparation Of C₂₁steroidal MT2 Nanoemulsions And Its Reversal Of Paclitaxel Resistance In Tumor Cells 2 Synthesis Of A Novel Fluorescent Probe DCFHA And Its Application In The Rapid Detection Of Heme And Blood

Part ? Preparation of C21 Steroid MT2 Nanoemulsion and Its reversion to paclitaxel resistance in tumor cellsObjectiveBoth the anticancer drug paclitaxel(PTX)and a chemosensitizer MT2,which is 11 ?-O-2-methylbutanoyl-12?-O-(E)-tigloyl-tenacigenin B,a C21 steroidal ester derivativeisolated from Chinese herbal medicine Marsdenia tenacissima,have lower solubility in water.To solve the problem,the nanoemulsion systems of PTX and/or MT2 with distearylphosphatidylethanolamine-polyethylene glycol(DSPE-PEG)with high drug loading and good stability are designed and manufactured.And the reversal effect of MT2-loaded nanoparticle on PTX resistance was evaluated in multi-drug resistant Hela/taxol cells.Methods(1)MT2 and paclitaxel were wrapped with distearoylphosphatidylethanolamine-polyethylene glycol(DSPE-PEG),and the morphology of the resulting nanoemulsion spheres was measured by laser particle size analyzer(DLS)and transmission electron microscopy(TEM)Characterization.(2)The drug loading capacity and drug release in vitro of the nanoemulsion are determined by HPLC methods.(3)Evaluation of in vitro efficacy:Semi-inhibitory concentration(IC50)of drug substance,nanoemulsion and its combination on the proliferation of drug resistant tumor cells(Hela/taxol)measured by SRB assay was used to evaluate the effect of nanoemulsion on the antitumor synergistic.Results(1)A nanoemulsion ball with high drug loading was prepared.The MT2 encapsulation rate was(80.6±5.12)%,and the paclitaxel encapsulation rate was(83.7±4.23)%.The particle size of MT2 nanoemulsions is(114±8.0)nm,and the particle size of paclitaxel nanoemulsions is(73.45±5.35)nm(2)In Hela/taxol cells,when MT2 is combined with paclitaxel/paclitaxel nanospheres(PTX NP),the IC50 of paclitaxel nanospheres is 0.19 nM;while the IC50 of paclitaxel is 10.32 nM,the former is 54 times more effective than the latter!It shows that the growth inhibition effect of MT2+PTX NP on drug-resistant tumor cells HeLa/Tax is stronger than that of MT2+PTX;paclitaxel and MT2 were used in combination or in common to inhibit the growth of drug-resistant tumor cells.The IC50 value of(PTX-MT2)NP was 4.95?M,and the IC50 value of mixed PTX NP and MT2 NP is 6.04 ?M.ConclusionThis work created a paclitaxel-MT2 nanoemulsion[(PTX-MT2)NP]with high drug loading and good stability.The experiments showed that the nano-emulsion spheres had a loading capacity of paclitaxel and MT2 up to xx.The in vitro anti-tumor activity test showed that the growth inhibition rate of drug-resistant tumor cells was basically the same when the paclitaxel and MT2 were wrapped together or separately,and the(PTX-MT2)NP formed by the same package had slightly better anti-tumor effect than that of the alone package.Part ? Synthesis of Novel Fluorescent Probe DCFHA and Its Application in Rapid Detection of Hem in and Blood Stai nObjectiveAs an important protoporphyrin IX-Fe complex,hemin plays a very important role in many biological processes.In addition,hemin and its derivatives are considered biochemical markers that distinguish blood from other colored samples.The purpose of this study was to develop a new fluorescent probe for rapid,sensitive and visible fluorescence detection of heme.On this basis,a simple test strip of artemisinin/probe loading was prepared to achieve rapid identification of heme and blood stains.Methods(1)A fluorescent reporter was prepared by using 2',7'-dichlorofluorescein(DCF)as.a dye,and a new non-fluorescent dihydrofluorescein amide(DCFHA)was synthesized through two steps of reaction:simple condensation reaction and reduction reaction.The product is purified by column chromatography and analyzed by 1H NMR and 13C NMR.(2)Hemin catalyzes the production of superoxide radical anions(O2·-)from peroxides,and then superoxide radical anions oxidizes non-fluorescent dihydrofluorescein(DCFHA)to a fluorescent form.We used H2O2,TBHP,and ART as the oxide sources,respectively,to explore the catalytic reaction of heme,and record the fluorescence on of the probe molecules.The DCFHA/H2O2 system is used to distinguish blood stains from coffee stains.(3)Chinese traditional medicine-derived organic peroxide artemisinin(ART)has good stability and easy storage.By incorporating ART and DCFHA into filter paper(type:medium speed 102),a kind of simple test strip is prepared and used for blood stain detection.Results(1)Through analysis methods such as 1H NMR and 13C NMR,the structure of the reaction product was identified as:2-(2,7-dichloro-3,6-dihydroxy-9H-xanthine-9-yl)-N,N-Diethyl benzamide(DCFHA).(2)H2O2,TBHP and ART catalyze the generation of reactive radical superoxide radical anions(O2·-)on hemin to oxidize non-fluorescent dihydrofluorescein(DCFHA)to fluorescent form.After testing,the detection limit(LOD)of DCFHA can reach picomolar levels:DCFHA/H2O2,DCFHA/TBHP and DCFHA/ART systems are 64 pM,350 pM,420 pM,respectively.DCFHA/H202 system has significantly increased green fluorescence in the presence of trace blood stains,but no fluorescence in the presence of coffee stains.(3)The DCFHA/ART test paper shows good sensitivity and selectivity for detecting trace amounts of blood,and can discriminate hemin and detection based on visual observation of the fluorescence.Inaddition,DCFHA/ART test paper has good stability.ConclusionWe report a new type of fluorescence assay that can directly detect hemin.The detection is based on hemin-catalyzed production of the active oxygen free radicals,which can oxidize the non-fluorescent DCFHA into fluorescent DCFA.Three peroxides H202,TBHP and ART were mixed with DCFHA in BR solution to form detection reagents.All three systems have great potential for sensitive detection of hemin with detection limits down to picomolar levels.DCFHA/H2O2solutions were also evaluated to distinguish between blood and coffee stains.The significantly increased green fluorescence in the presence of trace blood not only provides an opportunity for quantitative evaluation,but also facilitates visual inspection of hemin or blood under ultraviolet light.In addition,ART and DCFHA can be incorporated into filter paper strips to prepare simple artemisinin/probe-loaded test strips to achieve rapid identification of hemin and blood stains.