Nanoagent Loaded UCNPs-RB For Multimodal Imaging And Therapy On Mice Xenograft Skov3 Tumor

PART?THE PREPARATION AND CHARACTERIZATION OF NANOPARTICLES LOADED UCNPs-RB(FURH-PFH-NPS)Objectives To provide a novel nanoagent(denoted as FURH-PFH-NPs),whose core is perfluorohexane(PFH)and the shell is the lipid modified by folic acid.The shell contains 10-hydroxycamptothecin(HCPT)and the compound of self-fluorescent photosensitizer Bengal Rose(RB)and up-conversion nanoparticles(UCNPs).Three imagings(ultrasound,photoacoustic and fluorescence imaging)can guide the visualized process of concentration of FURH-PFH-NPs in tumor in vivo,Then,980 nm laser irradiation was used to irradiate the local area of the tumor after LIFU to enhance the synergistic effect of photodynamic therapy and chemotherapy.Methods Firstly,covalently coupled product of photosensitizer Bengal Rose and UCNPs was composed and was verified to undergo fluorescence resonance energy transfer function(FRET).The lipid film was composed by rotary evaporation-?ltrasonic methods,Then the multifunctional nanoparticle was tested with the light microscope,electron microscope,Malvern particle size analyzer and other instruments.Results FURH-PFH-NPs was observed with electron microscopy.The size of FURH-PFH-NPs was uniform and and the shape was spherical.UCNPs-RB nanoparticles were uniformly distributed.The particle size was 220.2 ±76 nm by Malvern laser particle size analyzer;the polydispersity index(PDI)was 0.093 and the zeta potential was-0.24 mV.The element peaks of Na and La were tested in the energy spectrum EDS of FURH-PFH-NPs.The nanoparticles triggered by LIFU showed the phase transition.The single-strand oxygen production test of the novel nanoparticles showed indirect detection of high-efficiency singlet oxygen production.Conclusion We successfully synthesized the contrast agent FA/UCNPs-RB/HCPT/PFH@Lipid,which has a spherical shape with UCNPs-RB particles embedded in it and has the ability to induce liquid-gas phase transition and to produce singlet oxygen.PART II THE IN VITRO STUDY OF PHASE-CHANGEABLE NANOPARTICLES LOADED UCNPs-RBObjective To observe the cytotoxicity of FURH-PFH-NPs and screen the appropriate parameters of ultrasound and laser irradiation with FURH-PFH-NPs on ovarian cancer cell SKOV3.The targetable SKOV3 cell ability and photodynamic therapeutic properties of FURH-PFH-NPs were studied in vitro.Enhanced ultrasound,photoacoustic and fluorescence imagings of FURH-PFH-Nps were finally investigated in vitro.Methods(1)The cytotoxicity of FURH-PFH-NPs was detected by MTT method to screen the concentration of FURH-PFH-NPs administration,followed by ultrasound and laser parameters screening,and the targetable SKOV3 cell ability and the effect of photodynamic therapy were evaluated by flow cytometry and fluorescence microscopy.(2)In vitro ultrasound imaging study of FURH-PFH-NPs was performed under different parameters of LIFU.(3)Photoacoustic imaging of FURH-PFH-NPs was analysed.Finally,fluorescence imaging of FURH-PFH-NPs before and after LIFU was studied.Results The concentration of FURH-PFH-NPs administration was 100?L/ml,the LIFU parameter was 2W/cm2,2min,and the laser parameter was 0.2W/cm2,120 s.The different factor groups with FURH-PFH-NPs influenced on the cell viability.The inhibition rate of cells was significantlyhigher than that of FURH-PFH-NPs free group,suggesting that FURH-PFH-NPs inhibited cell proliferation.The results of flow cytometry shows early(lower right)and late(upper right)apoptosis.The right apoptotic rate is its expression;after immunofluorescence,the nucleus volume increases after FURH-PFH-NPs treatment,chromatin concentrated,chromatin distribution in the nucleus is uneven,marginalized,part of the nucleus broke,and the cells underwent different apoptosis stages.It suggested that FURH-PFH-NPs plus laser and LIFU may significantly promote the apoptosis of tumor cells and inhibit the growth of tumors.The photodynamic effect in vitro was evaluated by the fluorescence intensity after incubation with DCFH-DA to determine the ability of singlet oxygen production : intracellular ROS production of FURH-PFH-NPs+ultrasound+laser was observed to be the highest among the three groups;Confocal laser results showed that folic acid has the ability to efficiently bind its receptors,and the specificity of folate binding to SKOV3 cells is shown in the picture.Ultrasonic echo intensity of FURH-PFH-NPs was the strongest under 2.0W/cm2 of LIFU,and there was significant difference compared with other powers.There was no significant difference among different action time 1min,2min,4min both gray-scale mode and contrast mode under 2W/cm2 of LIFU.have the highest average echo intensity.FURH-PFH-NPs can be fluorescently imaged from a fluorescence imaging system with anexcitation wavelength of 980 nm and an emission wavelength of 650 nm.The fluorescence imaging can be performed from 0.125 mg/ml to 2 mg/ml with a positive concentration correlation.FURH-PFH-NPs has a photoacoustic imaging capability with a specific absorption peak at 705 nm and a PA value of 3.4 a.u.The data shows that the photoacoustic intensity is linearly positively correlated with the concentration of FURH-PFH-NPs.Through the observation of tumor-bearing null mice,we analyzed the intensity of contrast-enhanced imaging to determine that the peak nanoparticles began at 6h and last for 12 hours.The conclusion were verified by photoacoustic imaging and fluorescence imaging.The photoacoustic imaging results were 6h.At the peak time,the fluorescence imaging can enhance the targeted aggregation of nanoparticles,and the distribution in the body in vivo can be studied by fluorescence imaging and quantitative analysis of isolated organs and tumors.Results In either vivo or vitro,FURH-PFH-NPs plays an important role in enhancing ultrasound,photoacoustic and fluorescence imaging properties for multimodality.PART ? FURH-PFH-NPs SYNERGISTIC TREATMENT ON NULL MICE'S OVARIAN CANCER XENOGRAFT TUMOR TREATMENT EXPERIMENTAL STUDYObjectives To investigate the distribution of FURH-PFH-NPs contrast agent and drug HCPT in null mice and the targeting ability of FURH-PFH-NPs on SKOV3 cell.Methods Concentration of contrast agent and drug in vivo: 30 null mice bearing tumors were divided into two groups randomly.The FURH-PFH-NPs was injected into the the null mice through tail vein and the null mice were executed from the neck at 0.5h,1h,6h,12 h and 24 h after injection.Ultrathin sections of the tumors were taken and the distribution of contrast agents in the sections were observed under a microscope.50 null mice were randomly divided into two groups.HCPT injection and FURH-PFH-NPs were injected into the mice.Blood samples were taken from the right atrium of the heart after anesthesia 0.5h,1h,6h,12 h,24h after injection.Then the concentration of the drug in the serum was measured respectively.Furthermore,the important organs of the null mice taken apart from the corpse including heart,liver,spleen,kidneys,lungs were measured by imaging.And after homogenizatio,the concentration of the drug in each tissue was detected by high performance liquid chromatography.Targeted treatment experiments in mice: 30 null mice were randomly divided into 4 groups,which were treated with different treatment of FURH-PFH-NPs+US+laser group,FURH-PFH-NPs+US group,HCPT group and saline group.Each group receieved the respectively treatmentmentioned above every other day during 14 days.The size of the tumors were measured every other day also.The tumor growth curve and tumor inhibition rate were obtained.Immunohistochemistry of PCNA and HIF-1? was detected to evaluate the proliferation and apoptosis of tumor cells.Results Contrast agent and drug in vivo distribution experiment: In the targeted group,ultrathin sections of the tumor showed fluorescence at each time point,and the fluorescence was most obvious at the point of 6h.The non-targeted group showed few of fluorescence.At each time point,the blood concentration of the FURH-PFH-NPs group was higher than that of the HCPT group.Targeted treatment experiments: The null mice in the FURH-PFH-NPs group had the lowest tumor growth index and the highest tumor inhibition rate.Immunohistochemical results showed that the FURH-PFH-NPs group had the lower expression in both PCNA and HIF-1? compared with contrast group.Conclusion Nanoparticle FURH-PFH-NPs can prolong the retention time of HCPT in vivo.The nanoparticles bound to tumor cells under ultrasound can have a good therapeutic effect on SKOV3 ovarian cancer in null mice.It is expected to develop new therapeutic methods on tumors.