Photofrin联合碘化钾抗白色念珠菌光动力效果的研究

Study on Photodynamic Effect and Mechanism of Photofrin plus Potassium Lodide on Candida Albicans

目的探讨光敏剂(photosensitizer,PS)Photofrin(PF)联合不同浓度碘化钾(potassium iodide,KI)介导的抗菌光动力疗法(antimicrobial photodynamic therapy,aPDT)抗白色念珠菌(Candida albicans,C.albicans)的效果。方法实验对象C.albicans(DAY286),进行三部分实验分别为:(1)PF及PF联合KI介导的aPDT对C.albicans的杀伤作用。实验分为PDT组(A组)和对照组(B组),A组处理分别为:(1)PF-PDT,取10^(7)CFU/ml的C.albicans菌悬液1 ml与PF(终浓度分别为0.1、0.2、1、2和4μM)1 ml混匀,室温避光孵育30 min,行能量密度10 J/cm^(2)的蓝光光照;(2)PF+KI-PDT,取菌悬液1 ml与PF(终浓度分别为5、10μM)1 ml混匀,室温避光孵育30 min,再添加KI(终浓度分别为0、1、10、25、50和100 mM)1 ml后,行能量密度10 J/cm^(2)的蓝光光照。B组处理分别为:(1)仅加入PF或仅加入PF+KI,光照或不光照;(2)仅加入菌悬液,不加入PF、KI也不光照。(2)单线态氧猝灭剂叠氮化钠(sodium azide,NaN_(3))对PF+KI-aPDT抗菌作用的影响。实验分为aPDT组(A组)和空白对照组(B组)。A组首先将107CFU/ml的C.albicans菌悬液1 ml与PF(终浓度为10μM)1 ml混匀后避光孵育30 min,然后分为4个小组:(1)不加入KI、NaN_(3),仅光照;(2)加入KI(终浓度为100 mM)后光照;(3)加入NaN_(3)(终浓度为50 mM)后光照;(4)加入KI、NaN_(3)后光照,4小组均采用能量密度10 J/cm^(2)光照。B组:空白对照组,仅接种菌悬液,不加入PF、KI、NaN_(3),也不光照。(3)两种荧光探针检测PF光化学特性。实验分为单线态氧荧光探针(singlet oxygen sensor green reagent,SOSG)组(A组)、羟基自由基荧光探针[3’-(p-hydroxyphenyl)-fluorescein,HPF]组(A0组)和对照组(B组)。A组先将SOSG(终浓度为10μM)与PF(终浓度为10μM)混匀,加或不加入KI(终浓度为100 mM),构成总体积200μl,光照;A0组将HPF(终浓度为10μM)与PF混匀,加入或不加入KI,光照;B1、B2组:不加PF及KI,分别加入SOSG和HPF,光照;B3、B4组:分别加入SOSG和HPF,两组均加入KI,不加PF,光照。此部分各组均予以能量密度为0、1、2、5、10、20、30、40、50、60、70、80 J/cm^(2)的蓝光照射,每次照射后利用分光光度计测量各组的荧光量。第(1)、(2)部分处理后的菌悬液经连续梯度稀释后接种于培养基,30℃恒温培养24~36 h后计数菌落数并计算存活率。结果(1)PF浓度为0.1~2μM时,比较PDT组与空白对照组的存活率,差异无统计学意义(P>0.05);当PF浓度为4μM时,PDT组存活率下降88.6111%,与空白对照组相比差异具有统计学意义(P<0.05),但未能有效杀菌。(2)当PF浓度为5、10μM时,PF-aPDT组存活率分别下降60.7365%、99.6030%,添加100 mM KI后,PF+KI-aPDT组存活率分别下降99.9853%、99.9996%,与PF-aPDT组相比差异均有统计学意义(P<0.001)。(3)单线态氧猝灭剂NaN_(3)显著降低了PF-aPDT组及PF+KIaPDT组的抗菌效果(P<0.01)。(4)在各个光能量密度下,SOSG组荧光量均大于HPF组,差异具有统计学意义(P<0.05)。加入KI后SOSG组及HPF组的荧光量均下降,与未加入前相比差异具有统计学意义(P<0.05)。结论KI可增强PF介导的光动力抗C.albicans效果,PF产生光动力抗菌效应主要经历Ⅱ型光化学反应。

Objective To explore the antimicrobial photodynamic therapy(aPDT)effect of photofrin(PF)combined with different concentrations of potassium iodide(KI)on Candida albicans(C.albicans)Methods C..albicans standard strain(DAY286)was prepared as the experimental subject.The experiment was divided into three parts.Part one:the PF-aPDT and PF+KI-aPDT killing effect on C.albicans.In this part,two groups were established,namely,the PDT group(group A)and control group(group B).The treatments for group A were as follows:firstly,1 ml of C.albicans suspension(10^(7) CFU/mL)was mixed with 1 ml of PF(final concentration:0.1,0.2,1,2 and 4μM),and incubated at room temperature for 30 min then irradiated with blue light of 10 J/cm^(2);secondly,1 ml of bacterial suspension was mixed with 1 ml of PF(final concentration:5 and 10μM),and incubated at room temperature for 30 min without light.The suspension was then exposed to 10 J/cm^(2)of blue light upon the addition of 1 ml of KI(final concentration:0,1,10,25,50 and 100 mM).The treatment for Group B was as follows:firstly,the suspension was mixed with PF with or without KI,and then exposed to light or not;secondly,neither PF nor light were given to the C.albicans suspension.Part two:the effect of sodium azide(NaN_(3))on the killing effect of PF+KI-aPDT.In this part of experiment,two groups were formed,i.e.,the aPDT group(group A)and blank control group(group B).For group A,1 ml of C.albicans suspension(107 CFU/ml)was mixed with 1 ml of PF(final concentration:10μM),then incubated for 30 min without light.The suspension was further divided into four groups.For the first group,neither KI nor NaN_(3)was added except of the illumination of10 J/cm^(2)blue light.For the second group,the suspension was illuminated after being added KI(final concentration:100 mM).For the third group,upon the addition of NaN_(3)(final concentration:50 mM),the suspension was illuminated with light.For the fourth group,the suspension was added with KI and NaN_(3)and then illuminated with light.For the blank control group,none of PF,KI and NaN_(3)was given to the bacterial suspension.The suspension was not exposed to the light.Part three:two fluorescence probes were used to detect the photochemical properties of PF.In this part of experiment,three groups were established:the singlet oxygen sensor green reagent(SOSG)group(group A),and 3’-(p-hydroxyphenyl)-fluorescein(HPF)group(group A0)and control group(group B).For group A,SOSG(final concentration:10μM)and PF(final concentration:10μM)were mixed with or without KI(final concentration 100 mM)to form the suspension of a total volume of 200μl,and then the suspension was exposed to the light.For group A0,HPF(final concentration:10μM)was mixed with PF with or without KI,and then illuminated with light.For group B1 and B2,SOSG and HPF were added respectively.Neither PF nor KI was added before the illumination with light.For group B3 and B4,SOSG and HPF were added respectively,and then KI was added to both groups without addition of PF,upon which light illumination was given.The subject in each group was irradiated respectively with the blue light with an optical energy density of 0,1,2,5,10,20,30,40,50,60,70 and 80 J/cm^(2).After each irradiation,a spectrophotometer was used to assess the fluorescence of each group.The survival fraction of Calbicans after the treatment was evaluated via counting CFU.Results(1)When the PF concentration was 0.1~2μM,there was no significant difference in survival fraction between the PDT group and blank control group(P>0.05).When the PF concentration was 4μM,the survival fraction of the PDT group decreased by88.6111%,which was apparently different from that of the blank control group(P<0.05),but no effective sterilization was found.(2)When the concentration of PF was 5μM and 10μM,the survival rate of the PF+KI-aPDT group decreased by 60.7365%and99.6030%respectively.When 100 mM KI was added,the survival fraction of the PF+KI-aPDT group decreased by 99.9853%and99.9996%respectively,and the differences were statistically significant when they were compared with that of the PF-aPDT group(P<0.001).(3)Singlet oxygen quench agent NaN_(3)significantly reduced the antibacterial effect of the PF-aPDT group and PF+KI-aPDT group(P<0.01).(4)The fluorescence amount in the SOSG group was higher than that in the HPF group at each light energy density and the differences were statistically significant(P<0.05).Upon the addition of KI,the fluorescence of both the SOSG group and HPF group decreased,and the difference was statistically significant(P<0.05).Conclusion The effect of PDT-killing of C.albicans mediated by PF will be enhanced by adding KI.The PDT-killing effect of PF is attributed mainly to typeⅡphotochemical reaction.