Spectal Property And Photodynamic Effect Of New Hypocrellin Derivatives

Objective:To study the photodynamic activity of the five new hypocrellins derivatives in human lung cancer A549 cells line,to filtrate the hypocrellin derivatives and discuss the metabolism and transport in vivo,and to study the combination of hypocrellin derivative molecule with HSA for photosensitive drug development.Material and Methods:1.The spectral property of hypocrellin derivativesThe absorption spectra of THB,3HB,4HB,3SB,4SB and HB(8μM in PBS and 35μM in DMSO)were measured by UV-vis spectrophotometerThe absorption and emission spectra of fluorescence of 4SB and HpD,were measured in PBS or mixed 20μM HSA in PBS.2.Photodynamic activity of novel hypocrellin B derivatives in human lung cancer A549 cellsTreatment conditions:20mW/cm²,1000 s;A549 cells were incubated with drug for 4h was estimated.After the incubate cells were irradiated 24h,the cells survival was estimated.0.94nM HB and its derivatives were irradiated with 630 nm and 532 nm laser,the cells survival was estimated. A549 cells were incubated with 3SB,4SB and HpD for 24 hours and IC₅₀of cytotoxin was calculated.3SB,4SB and HpD were irradiated with 532nm laser and IC₅₀was calculated.4SB and HpD were irradiated with 630nm laser and IC₅₀was calculated.A safety factor was used as ratio of cytotoxic IC50 to phototoxic IC50 to compare the safety of hypocrellin derivatives and HpD under 532 nm and 630 nm irradiation.3.Interaction between 4SB and HSASoon after 4SB was added to excessive HSA,the absorbance intensity was measured at 293nm and fluorescence intensity was measured(λex=490 nm)at 660 nm.After 0,1.25,2.5,5.0,10,20μM HSA added to 5μM 4SB,the absorption spectra and the emission spectra(λex=493 nm)were measured.After 0,2.5,5.0,7.5,10,20,30,40μM 4SB were added to 10μM HSA,emission spectra(λex=493 nm)and emission spectra(λex=295 nm,fluorescence of TrP-214)were measured.The fluorescence intensity of TrP-214 was modified by F_cor=F_obsdantilog[(A_exc+A_em)/2].Result:1.The spectra property of hypocrellin derivativesCompared with the parent HB,the absorbance of each derivative in tumor phototherapy window(600-900 nm)was enhanced.The absorption spectra were all red shift(1-15 nm)after HB and its derivatives were bound to HSA.The absorbance intensity was increased.The absorbance of HB was increased 100%. The fluorescence peak of 45B was blue shift 20 nm and the intensity was 9 times higher than in PBS.However,the spectra of HpD had no difference in PBS or did not bind to HSA.The spectra of HB derivatives of C-2 were similar to those of C-17.2.The photodynamic activity of novel HB derivatives in human lung cancer A549 cellsThe cells death of 3SB,4SB was stronger than that of other HB derivatives under 630 nm or 532 nm laser irradiation.The dark-cytotoxin sequence was HpD＞3SB＞4SB(7.84,11.22,21.93 ug/ml),HpD was 2.79 times higher than 4SB,1.42 times higher than 3SB.The photo-cytotoxin sequence was 4SB＞3SB＞HPD(103.86,84.16,960.14ng/ml).3SB and 4SB was 10.53 and 11.4 times higher than HpD under 532nm laser irradiation(P＞0.05).The Photo-cytotoxin of 4SB(50.7ng/ml)was 21.2 times than HPD(1069.88)under 630nm laser irradiation(P＜0.05).The safety factor sequence was 4SB(260.6)＞3SB(108)＞HPD(8.2)under 532nm laser irradiation and was 4SB(432.5)＞HPD(7.3)under 630nm laser irradiation.The safety factor of HpD under 532nm between 630nm had no statistical difference(P＞0.05)opposite to 4SB(P＜0.05).3.The interaction between 4SB with HSAThe absorbance and fluorescence of 4SB were enhanced after binding to HSA.The saturation was observed in 30min and the absorption spectra were red shift.In HSA fluorescence quench experiment,when the 4SB and HSA molecule ratio was 1:1,acorrelation was abserved.After modified it was more obviously.Conculusion:1.Compared with biological photo-cytotoxin,cytotoxin and safety factor of five kinds of novel HB derivatives,4SB is perfect.2.Two strong absorbance peaks (493 nm,630 nm)can be found in CNV and tumor phototherapy window.The absorbance in the red area and PDT is better than HpD.The absorbance of 4SB in 630 nm is 5.56 times higher than HpD in 620 nm.The photo-cytotoxin of 4SB is 11.4 times higher than HpD under 532 nm and 21.2 times higher than HpD under 630 nm,but the cytotoxin of 4SB is 35%of HPD,indicating that 4SB is better in PDT for tumor and CNV.4SB as a novel HB derivative is a potential drug for future research.The fluorescence intensity of 4SB may change in different environments,suggesting that 4SB is fit in environment Probe.3.4SB can bind to TrP-214 of HSA at 1:1,which is benefical to its transport in blood.