Fermentation Optimization Of Shiraia Bambusicola And The Photodynamic Antitumor Activity Of Two Metabolites Of Perylenequinone

Shiraia bambusicola,a rare medicinal fungus,only distributes in south of Yangtze River in China and in Japan.Hypocrellin family was the main active components of S.bambusicola.Hypocrellin A(HA)is the highest content and most well-studied compound among them.Elsinochrome A(EA)was recently detected in the metabolites of S.bambusicola strain.Before that,it was mainly obtained from the cultures of Elsinoe species.However,a tardiness of mycelium growth restricted EA accumulation.HA and EA are natural potential photosensitizers for photodynamic therapy(PDT)in treating various diseases,particularly in cancer treatment.However,their PDT applications were delayed due to their limited resources.With the depletion of wide S.bambusicola resource,the development of S.bambusicola strain is promoted.In this study,S.bambusicola ZH42 was isolated and identified by morphological characteristics and phylogenetic analysis.Two perylenequinones from S.bambusicola were purified then identified as HA and EA.The calibration curves for HA(y = 19.59 x + 52.99)and EA(y = 31.37 x-265.06)were developed with high performance liquid chromatography method.HA and EA production were 275.34 and 491.50?g/g by S.bambusicola ZH42 in solid-state fermentation.HA and EA production were 103.51 and 72.09 mg/L by S.bambusicola ZH42 in submerged fermentation.Culture conditions(temperature,shaker speed,and initial pH)and medium compositions(carbon source,nitrogen source,and growth factor)in submerged fermentation of S.bambusicola ZH42 were optimized with onefactor-at-a-time method.The concentrations of medium compositions were further optimized with Box-Behnken design.The optimal parameters were obtained as follows: temperature 28?,shaker speed 180 rpm,initial pH 6.5,glucose 24 g/L,NH4 Cl 2.2 g/L,potato infusion 260 g/L.Under the optimal conditions,HA production(365.14 mg/L)and EA production(340.85 mg/L)were 3.52 times and 4.73 times greater than their initial production,respectively.PDT has emerged as a potent novel therapeutic modality that induces cell death by excessive reactive oxygen species produced by light-induced activation of photosensitizer in the presence of oxygen.But some photosensitizers have characteristics of poor water-solubility and non-specific tissue distribution,such as HA.These characteristics of photosensitizers become main obstacles of their PDT application.The overexpression of transferrin receptors on the membrane surface of cancer cell should be considered to realize site-specific targeting drug delivery.In our study,a transferrin-modified Poly(Lactide-co-glycolide acid)and carboxymethyl chitosan nanoparticle was successfully synthesized to load HA,named TF-HA-CMCPLGA NPs.Morphology,size distribution,infrared spectra,encapsulation efficiency,and loading capacity of TF-HA-CMC-PLGA NPs were characterized.In vitro,TFHA-CMC-PLGA NPs presented targeting ability to A549 cells(transferrin receptor positive)with higher cell cytotoxicity,cell uptake,reactive oxygen species generation and cell apoptosis than HA-CMC-PLGA NPs(a nanoparticle without transferrin)treatment.In vivo photodynamic antitumor efficacy of TF-HA-CMC-PLGA NPs was investigated with an A549 tumor-bearing model in male athymic nude mice.TF-HACMC-PLGA NPs caused tumor delay of mice with a remarkable tumor inhibition rate of 63% after 15 days.TF-HA-CMC-PLGA NPs mostly accumulated in intestinal and tumor and it was quickly eliminated in normal organs.TF-HA-CMC-PLGA NPs caused extensive cell damage in tumor tissue.EA possesses high yield of singlet oxygen generation.However,there has been lack of research on PDT effects of EA to different types of cancer cells,especially for multidrug resistance cells.Curves of singlet oxygen generation indicated that 15 min was the suitable light irradiation period for further research.The photodynamic anticancer activity was measured and the half maximal inhibitory concentration(IC50,?g/L)of EA was 9.62 on Hela cell,15.51 on A549 cell,21.20 on MDA-MB-231 cell,22.95 on Hep G2 cell,and 51.75 on A549/Taxol cell,respectively.However,treated with doxorubicin or paclitaxel at a high 1 mg/L,cell survival rate of A549/Taxol was higher than 90% because of the multidrug resistance.Analysis of apoptosis using flow cytometry also demonstrated that EA induced significant cell apoptosis in tested A549/Taxol cell,A549 cell,and Hep G2 cell.Two types of nanoparticles were prepared to encapsulate EA for studying the PDT efficiency.One is amphipathic chitosan nanoparticle loaded EA and the other is TPGS&PEI-PLGA&PLGA&EA with positive charge.They would provide nanocarriers of EA for further research.In summary,S.bambusicola ZH42 would provide HA and EA for potential PDT applications.We developed a site-specific targeting drug delivery system to accumulate more drug and enhance the therapeutic efficacy.Furthermore,EA has great potential to overcome multidrug resistance of cancer cells.