Biosynthetic Melanin-Ce6 Liposome Mediated Photothermal-sonodynamic Synergistic Therapies For Triple Negative Breast Cancer Of Mice

BackgroundTriple negative breast cancer(TNBC)is a subtype of breast cancer that is aggressive and has a poor prognosis.Photothermal therapy(PTT)and sonodynamic therapy(SDT)have become promising therapeutic modalities for TNBC in recent years.However,single therapeutic modality with PTT or SDT is difficult to achieve satisfactory anti-tumor effects due to their own inherent limitations,such as poor tissue penetration for near infrared(NIR)laser and the limited cytotoxic reactive oxygen species(ROS)generated from conventional sonosensitizers irradiated by ultrasound(US).The combination of PTT and SDT can complement each other in the treatment of TNBC.In previous studies,the choice of sonosensitizer was mostly inorganic sonosensitizer that was difficult to degrade or organic sonosensitizer that lacked fluorescence property,while the photothermal agents were mostly metal nanoparticles with potential toxicity.Additionally,both of them have no tracer properties and cannot realize the visual treatment of tumors,so it is particularly important to seek a relatively safe and imaging performance of sonosensitizer and photothermal agent.In view of the fluorescent imaging properties of the new sonosensitizer Chlorin e6(Ce6)from plants and the photothermic agent melanin,which can be obtained in large quantities by genetically engineered bacteria and has photoacoustic(PA)imaging properties,both of them have good safety and imaging properties.Therefore,this study intends to conduct biosynthesis of melanin by modifying genetically engineered bacteria to express tyrosinase,and to explore the effect of PTT-SDT synergistic treatment for TNBC guided by dual-mode imaging with liposomes containing biosynthetic melanin and Ce6.Chapter One Synthesis and characterization of biosynthetic melaninObjectiveIn order to obtain a large amount of melanin with photothermal properties for PDT of TNBC,this chapter intended to transform a plasmid containing tyrosinase gene tyrl into Escherichia coli(E.coli),explore a biosynthetic pathway for obtaining photothermal agent of melanin through genetically engineered bacteria,and characterize it.MethodsThe tyrosinase gene named tyrl was transformed into E.coli.The expression of tyrosinase was induced by E.coli at different temperature and analyzed by SDS-PAGE.Melanin was subsequently synthesized by adding L-tyrosine and CuSO4 to the E.coli at 37℃.The morphology and particle size of melanin were characterized by transmission electron microscopy(TEM)and dynamic light scattering(DLS),respectively.Ultraviolet-visible-near-infrared(UV-Vis-NIR)absorbance spectra of melanin were measured.At last,fourier transform infrared spectroscopy(FTIR)was used to identify the functional groups of biosynthetic melanin.Results1.The protein band of tyrosinase at 28 kDa was observed in the gel of SDS-PAGE after 2 h culture at 37℃.When L-tyrosine and CuSO4 were added to the E.coli cultivated at 37℃,the suspension color turned black after 12 h.2.The incubation time of E.coli with L-tyrosine and CuSO4 prolonged,the particle size of melanin increased.3.The light absorption range of biosynthetic melanin were broad.The FTIR spectra revealed the main functional groups of melanin.Conclusions1.E.coli containing Tyrl gene could express tyrosinase at 37℃ after 2 h and biosynthesize melanin with particle size of about 8 nm after culturing with L-tyrosine and CuSO4 for 12 h.2.The light absorption spectra and FTIR spectra of biosynthetic melanin were consistent with those of melanin reported in previous studies.The research in this chapter proved that we could successfully prepare biosynthetic melanin nanoparticles by genetically engineering E.coli,which provided a new method for the synthesis of bioderived photothermal agents with controllable size.Chapter Two Fabrication and characterization of MC@LipObjectiveBased on the previous biosynthesis of photothermal agent melanin,this chapter intended to further explore the preparation method of integrated photothermal agent and sonosensitizer nanocarriers,characterize them and measure their encapsulation rate in order to realize the synergism of photothermal-sonodynamic and efficient treatment of TNBC.MethodsBiosynthetic melanin and Ce6 loaded liposomes(termed MC@Lip)were fabricated through the thin film hydration method.The morphology and structure of MC@Lip were examined by TEM.The particle size distribution and zeta potential were measured.The particle size of MC@Lip dispersed in water,phosphate buffered saline(PBS)and 10%fetal bovine serum(FBS)were recorded in 7 days,respectively.Results1.The biosynthetic melanin with 8 nm particle size and Ce6 were encapsulated into the liposome and successfully obtained the MC@Lip with an average diameter of about 100 nm by thin-film hydration.2.The encapsulation efficiencies of melanin and Ce6 in MC@Lip were 74.71%±0.54%and 94.52%± 0.78%,respectively.3.After being stored at 4℃ for seven days,MC@Lip in water,phosphate-buffered saline(PBS)and 10%fatal bovine serum(FBS)still displayed the negligible size change and low polydispersity index(PDI).Conclusions1.Liposomes containing melanin and Ce6 with a total particle size of about 8 nm(MC@Lip)could be successfully obtained by classical thin film hydration method,with an average particle size of about 100 nm,which was favorable for MC@Lip to aggregate to the tumor area through enhanced permeability and retention(EPR)effect.2.MC@Lip had good dispersibility in water,PBS and 10%FBS,and showed good stability in 7 days of storage at 4℃,which laid a good foundation for the subsequent PTT-SDT synergistic treatment of TNBC.Chapter Three Sonodynamic and photothermal effects of MC@LipObjectiveIn the chapter two,liposomes MC@Lip had been successfully constructed.In this chapter,the in vitro sonodynamic and photothermal effects of MC@Lip would be discussed,which would provide a reliable basis for the subsequent PTT-SDT synergistic killing of 4T1 breast cancer cells.MethodsThe 1,3-diphenylisobenzofuran(DPBF),the singlet oxygen sensor green(SOSG)and 2,2,6,6-tetramethylpiperidine(TEMP)were added into the MC@Lip solution,and then the mixture was irradiated by the US irradiation to evaluate the 1O2 generation.The photothermal effect of MC@Lip in vitro was evaluated by irradiating MC@Lip with 808 nm NIR laser.The temperature variations(ΔT)and infrared radiation thermal images were respectively recorded by an infrared thermal imaging camera.The SPSS 26.0 software was used to analyze the data.Significance was calculated using Student’s t test,one-way ANOVA and Mann-Whitney U test.Results1.There was about 47%of DPBF to be oxidized in the US-irradiated MC@Lip.The fluorescence signal of the typical SOSG was enhanced for the prolonged US irradiation duration of MC@Lip.The ESR analysis by the TEMP probe revealed the strong 1O2-induced characteristic signal peaks for MC@Lip exposed to US irradiation.2.The most remarkable ΔT was for the MC@Lip with 200 μg mL-1 melanin after 5-min NIR irradiation at 1.00 W cm-2 808 nm laser.3.MC@Lip could maintain the same level temperature elevation after repeated exposure to 808 nm NIR laser for five laser on/off cycles.The photothermal conversion efficiency(η)of MC@Lip was 42.47%.Conclusions1.In the sonodynamic effect,1O2 could be effectively produced by US(1.0 MHz,200 mvpp,50%duty cycle)irradiation of MC@Lip for 1 min.In the photothermal effect,the application of 1.00 W cm-2 808 nm NIR laser irradiation of MC@Lip(including melanin concentration of 200 μg mL-1)for 5 min could effectively generate heat energy(ΔT=23.4℃).2.MC@Lip had good sonodynamic and photothermal effects under US and laser irradiation,which laid a good foundation for subsequent in vitro PTT-SDT coordinated killing of 4T1 breast cancer cells.Chapter Four In vitro sonodynamic and photothermal 4T1 breast cancer cell-killing capabilities of MC@LipObjectiveIn this chapter,we mainly investigated the toxic effects of MC@Lip on 4T1 breast cells and explored whether effective reactive oxygen species could be produced in cells.In addition,we needed to further evaluate the efficacy of synergistic therapy(PTT-SDT)in killing 4T1 breast cancer cells.MethodsThe 4T1 and bEnd.3 cell were incubated with MC@Lip solution for 24 h to evaluate the cytotoxicity of MC@Lip by the cell counting kit-8(CCK-8)assay.4T1 breast cancer cells were incubated with MC@Lip for 4 h and imaged by the confocal laser scanning microscopy(CLSM).Moreover,intracellular 1O2 production was detected using 2,7-Dichloro-fluorescein diacetate(DCFH-DA)assay kit.4T1 cells were divided into six groups:Control group,MC@Lip group,US+NIR group,MC@Lip+US group,MC@Lip+NIR group and MC@Lip+US+NIR group,in which MC@Lip+US+NIR group,MC@Lip solution was added and irradiated with US for 1 min and then irradiated with NIR laser for 5 min.At last,CCK-8 and Calcein(calcein-AM)/propyl iodide(PI)stain were used for evaluation.Results1.After 4T1 breast cancer cells and bEnd.3 cells were incubated with MC@Lip for 24 h,the cell viabilities were above 80%.2.MC@Lip showed strong Ce6 red fluorescence in cytoplasm after co-incubation with 4T1 breast cancer cells.The strong green fluorescence of DCFH-DA appeared in the 4T1 cell after US irradiation for MC@Lip.3.The results of CCK8 showed that MC@Lip+US+NIR group had the strongest cytotoxicity to 4T1 cells,the Calcein AM/PI staining of which showed strong red dead cell fluorescence.Conclusions1.MC@Lip had high biocompatibility.MC@Lip could be phagocytized by 4T1 breast cancer cells,and effectively produced intracellular ROS under US irradiation,resulting in sonodynamic effects.2.The photothermal-sonodynamic synergistic therapies were confirmed to generate the best killing effect on 4T1 cells and effectively improved the efficiency of single SDT or PTT treatment,which provided a crucial reference for the subsequent in vivo animal therapy.Chapter Five In vivo study of MC@Lip mediated photothermalsonodynamic synergistic therapy for TNBCObjectiveThis chapter would investigate the PA and fluorescence imaging capabilities of MC@Lip in the tumor region of mice and evaluate the efficacy of MC@Lip mediated SDT and PTT synergistic therapy for TNBC under the guidance of bimodal imaging and the biosafety of MC@Lip in vivo.MethodsFor the establishment of the subcutaneous mice TNBC model,4T1 breast cancer cells were subcutaneously injected into the right flank of each female BALB/c mouse.4T1 tumor-bearing mice were injected with MC@Lip solution through the tail vein,PA and fluorescence information of tumor areas were collected at different time points.The 4T1 tumor-bearing mice were randomly divided into above-mentioned 6 groups,in which MC@Lip+US+NIR group,mice were injected with MC@Lip solution through tail vein,subcutaneous tumor was firstly irradiated with US for 5 min and then irradiated with NIR laser for 10 min.The effect of tumor treatment was evaluated and the lung metastasis of mice was observed.Results1.The PA and fluorescence imaging of the tumor area were the most obvious at 4 h after the injection of MC@Lip into the tail vein of mice.2.Ex vivo fluorescence revealed that a large number of MC@Lip gathered in the tumor area of mice.Moreover,fluorescence imaging of tumor sections showed that red fluorescence signal from MC@Lip could be seen in the tumor area of mice after the injection of MC@Lip through the tail vein.3.The tumor growth of mice in MC@Lip+US+NIR group was most significantly inhibited,and there was no obvious metastatic tumor in the lungs of mice in MC@Lip+US+NIR group.Conclusions1.MC@Lip had good PA and fluorescence dual-mode imaging performance and could visually trace the aggregation of MC@Lip in TNBC subcutaneous tumor of mice.2.The study confirmed that MC@Lip had good biosafety,and its mediated PTTSDT synergistic therapy was better than single PTT or SDT therapy in the treatment of mice TNBC.