Targeted Melanin/Hematoporphyrin Monomethyl Ether Nanoparticles For Sonodynamic Therapy Of Tumor

PART ? PREPARATION,CHARACTERIZATION AND TARGETING OF TARGETED PLGA NANOPARTICLESObjective To prepare a folic acid-targeted PLGA nanoparticles loaded with melanin/hematoporphyrin monomethyl ether(MPME),investigate its basic properties and observe its targeting to breast cancer MDA-MB-231 cells.Methods 1.FHMP NPs,folate-targeted hematoporphyrin monomethyl ether loaded PLGA nanoparticles(FHP NPs)and folate-targeted melanin loaded PLGA nanoparticles(FMP NPs)were prepared viaadouble emulsification methodand their morphologies were studied under a light or electron microscopy.The entrapment efficiency of melanin nanoparticles(MNPs)and hematoporphyrin monomethyl ether(HMME)were measured and calculated by a ultraviolet spectrophotometer method.The mean particle sizes of FHMP NPs dissolved in phosphate-buffered solution(PBS)or fetal bovine serum(FBS)were measured with prolonged time duration(1,2,3,4,5,6 and 7 days).2.FHMP NPs were co-incubated with folate(FA)receptor negative expression cells A-549 and FA receptor positive expression cells MDA-MB-231 for different times.The cell uptake was observed under laser confocal microscopy.Cell phagocytosis was observed by confocal laser microscopy after incubation of FHMP NPs and HMP NPs and MDA-MB-231 cells.Results 1.The obtained FHMP NPs displayed a well-defined spherical shape and homogenous size,as revealed by SEM and TEM images.In addition,the FHMP NPs exhibited strong red florescence,as detected by CLSM.The mean diameters of the FHMP NPs,FHP NPs and FMP NPs were 310.3 ± 10.6 nm,266.1 ± 2.4 nm and 270.1 ± 4.9 nm.The average zeta potentials of FHP NPs,FMP NPs and FHMP NPs were-21.4 ± 1.7 m V,-21.2 ± 0.8 m V and-23.03 ± 1.33 m V.The encapsulation efficiencies of HMME and MNPs in FHMP NPs were 80.13 ± 4.81% and 47.72 ± 4.26%,respectively.Meanwhile,the mean particle size of FHMP NPs did not show appreciable change when dissolved in PBS or FBS,and the particle suspension did not aggregate or precipitate within 7 days,revealing the excellent long-term stability of the FHMP NPs.2.FHMP NPs aggregated around MAD-MB-231 cells,but not in A-549 cells.After incubation with MDA-MB-231 cells for several hours,there were only a few HMP NPs can be seen.Conclusion Folic acid targeting nanoparticles(FHMP NPs)were successfully prepared in this study.The nanoparticles have specific targeting effect on MDA-MB-231 cells.PART ? THE BIOSAFETY EVALUATION AND DISTRIBUTION OF TARGETED PLGA NANOPARTICLESObjective To evaluate the safety of FHMP NPs in vitro and in vivo,and to observe the targeting distribution of FHMP NPs in breast cancer nude-mice xenograft.Methods 1.The cells were seeded in 96-well culture plates at a density of 1 × 104 cells per well in RPMI-1640 medium at 37 ?C in the presence of 5% CO2 for 24 h to allow the cells to adhere.Then,the above culture medium was replaced with fresh culture medium containing FHMP NPs at different concentrations(0,2.5,5,7.5 and 1 mg/m L).After 3 h,6 h and 24 h of incubation,the CCK-8 assay was used to evaluate the viability of cells.The optical density(OD)at 450 nm was read with an EL× 800 Universal Microplate Reader(Bio-Tek Instrument Inc.,USA).Twenty-four healthy mice were randomly divided into blank control group(200 ?Lofsaline)and FHMP NPs group(200 ?Lof FHMP NPs,10 mg/m L).The mice were killed after intravenous injection for 1 hour,6 hours,24 hours and 48 hours.Blood samples were collected for blood test and blood biochemical test.2.When the tumor volume reached to 0.8 mm3,the mice were administrated with Di R labeled FHMP NPs or HMP NPs and the fluorescence images were acquired at various times post injection(0 h,0.5 h,2 h,4 h,6 h and 24 h).In the group of free folate+FHMP NPs,the mice were administrated with free folate before the injection of FHMPNPs.Then,major organs of the mice were collected for fluorescence imaging after 24 hours.The corresponding fluorescence signals were calculated by the fluorescence analysis system.Results1.FHMP NPs had no effect on cell viability.FHMP NPs did not change the hematological test results of mice.2.The fluorescence signal value in the tumor area reached its peak 2 hours after injection of FHMP NPs.Ex vivofluorescence imaging confirmed that fluorescent signals could be detected in the liver,spleen and tumors of the mice.Conclusion FHMP NPs havelow cytotoxicity and high biological safety,and can be enriched in the tumor area.PART ? TARGETED PLGA NANOPARTICLES ENHANCED PHOTOACOUSTIC IMAGING AND SONODYNAMIC THERAPYObjective To observe the effect of nanoparticles enhanced photoacoustic(PA)imaging and its SDT effect,and to discussits related mechanism.Methods 1.FHMP NPs resuspension was prepared,and the photoacoustic intensity was scanned by a photoacoustic laser in the wavelength range of 700-950 nm to select the appropriate excitation wavelength.The PA values of the FHMP NPs,FMP NPs and FHMP NPs at concentrations of 5 mg/m L,17.5 mg/m L,30 mg/m L,respectively,were recorded in vitro.After intravenously injecting 200 ?L(10 mg/m L)of HMP NPs or FHMP NPs emulsions at different time points(0 h,1 h,2 h,4 h,6 h and 24 h),the corresponding PA images were simultaneously recorded by the VEVO LSER PA imaging system.Especially,the mice in group of Free FA + FHMP NPs were injected with free FA solution before the administration of FHMP NPs.2 In vitro SDT 2.1 The ROS generation activated by US The experimental groups: Control group(Control),US only group(US),FMP NPs combined with US group(FMP NPs + US)and FMHP NPs combined with US(FMHP NPs + US).The ROS generation of the nanoparticles was detected by DPBF method after US irradiation.Detection of ROS produced by FHMP NPs at different time after ultrasound irradiation by singlet oxygen sensor green(SOSG).2.2 Intracellular ROS production following US MDA-MD-231 cells were divided into seven groups: Control group(Control),US only group(US),FMP NPs combined with US group(FMP NPs + US),FHMP NPs group(FHMP NPs),HMP NPs combined with US group(HMP NPs + US),NAC + FHMP NPs combined with US(NAC + FHMP NPs)and FMHP NPs combined with US(FMHP NPs + US).After incubation with 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA),the cells in each group were irradiated by ultrasound,and the production of reactive oxygen species was observed by confocal laser microscopy.2.3 Detection of stability of targeted nanoparticles DPBF was used to compare the ROS output of FHMP NPs before and after irradiation by photoacoustic laser(PA laser).The experimental groups were: Control group,FHMP NPs + PA laser + US group,FHMP NPs + US group.The ROS production among different groups was compared.2.4 Detection of SDT-producing cytotoxicity The cell viability of control group,US group,FMP NPs + US group,FHMP NPs group,HMPNPs + US group and FHMPNPs + US group were measured by CCK-8 method.Calein-AM(CAM)and Pyridinium iodide(PI)were used to co-stain the living and dead cells.The survival status of the cells in the above groups was observed under confocal laser microscopy.3.In vivo SDT MDA-MB-231 nude mice were randomly divided into the following six groups: control group,US group,FMP NPs + US group,FHMP NPs group,HMPNPs + US group,FHMP NPs + US group and FHMP NPs + US group.The above treatment was repeated every three days for 15 days.The weight and tumor volume of nude mice were recorded daily,and digital photos of BALB/C mice were taken.At the end of treatment,one BALB/C mouse was executed in each group,and the tumor tissue was analyzed by pathological section.Results 1.FHMP NPs had the strongest PA signal at 700 nm.the PA values of FHMP NPs were substantially higher than those of FHP NPs or FMP NPs alone under the same condition.The PA signal in the tumor region reached maximum value approximately 2 h post-injection.2.FHMP NPs produced a large number of ROS after ultrasound irradiation,and photoacoustic laser irradiation had no effect on the production of reactive oxygen species.The production of reactive oxygen species increased with the increase of ultrasound time.FHMP NPs had no effect on cell viability,but after ultrasound irradiation,a large number of dead cells(red)were observed under confocal microscopy.3.After the first treatment,there was no significant change in the tumor volume of mice in each group.In the later treatment,the growth of tumors in FHMP NPs + US group was slow,while the volume of tumors in other control groups increased gradually.After treatment,HE staining of the tumors in FHMP NPs + US group showed that only a few normal cells remained,a large number of cells could not be observed,and nuclear lysis appeared,indicating a large number of necrotic cells.TUNEL immunoassay showed that the number of dark brown nuclei increased in the treatment group(FHMP NPs + US),indicating that apoptosis was serious,while the other control groups could not observe this phenomenon.PCNA results showed that FHMP NPs + US had the least brown granules,indicating that cell proliferation was significantly inhibited,while the other control groups had no significant inhibition.Conclusion FHMP NPs can not only enhance PA imaging,but also generate a large number of ROS after the irradiation of US,which can significantly inhibit the growth of breast cancer xenografts in nude mice by targeting SDT.