Perfluorooctyl Bromide And Indocyanine Green Co-Loaded Nanoliposomes For Enhanced Multimodal Imaging-Guided Phototherapy Of Breast Cancer

PART ? PREPARATION AND CHARACTERIZATION OF PERFLUOROOCTYL BROMIDE AND INDOCYANINE GREEN CO-LOADED NANOLIPOSOMESObjective To prepare perfluorooctyl bromide and indocyanine green co-loaded nanoliposomes(LIP-PFOB-ICG)and characterize their basic properties,the ability of loading oxygen,photothermal and photodynamic effect.Methods The liposomes encapsulating ICG and PFOB were synthetized through film hydration and two-step emulsion method.Their physical and chemical properties including size distribution,zeta potential,morphology,UV-vis-NIR absorption spectrum and the capacity of loading oxygen were investigated.The photothermal properties of LIP-PFOB-ICG with various ICG concentrations were tested by exposing the liposomes to 808 nm laser at different power intensities.And under both normal and hypoxia conditions,the photodynamic effect was measured with different ICG concentrations under different irradiation time.The intracellular ROS generation was also evaluated.Results The LIP-PFOB-ICG nanoliposomes were successfully prepared.As shown in TEM images,the liposomes dispersed well with core-shell spherical structure and uniform size.The average diameter size of LIP-PFOB-ICG was measured to be 319.1 nm,which increased to 348.7 nm after 808 nm laser irradiation.And the zeta potential changed from-37.33 m V to-36 m V.No significant size and zeta potential change of LIP-PFOB-ICG before and after laser irradiation was observed after storing in water at room temperature over 7 days.Compared to PFOB-loaded nanoliposomes(LIP-PFOB),LIP-PFOB-ICG displayed a strong absorption peak at 810 nm in its UV-vis-NIR adsorption spectrum.The ICG loading efficiency and loading content of LIP-PFOB-ICG were calculated about 80% and 7.41 wt %,respectively.And obvious colocalization of green fluorescence of ICG and red fluorescence of Di I labeled LIP-PFOB was observed,suggesting successful encapsulation of ICG in LIP-PFOB-ICG liposomes.Besides,due to the excellent oxygen dissolving capacity of PFOB,the dissolved oxygen concentration in the degassed water increased rapidly and maintained at a high saturated concentration after addition of oxygen saturated LIP-PFOB-ICG and LIP-PFOB liposomes.As for the photothermal effect,the temperature of LIP-PFOB-ICG rose up rapidly under 808 nm laser irradiation,linearly increasing with ICG concentration and laser power intensity.And the photostability of ICG in LIP-PFOB-ICG liposomes was greatly improved compared with free ICG.Besides,obvious concentration-dependent enhancement of ROS generation from LIP-PFOB-ICG nanoliposomes in both normal and hypoxic conditions were observed.Conclusions The LIP-PFOB-ICG nanoliposomes with regular morphology,uniform size,improved photostability,high ICG loading efficiency,excellent oxygen loading capacity as well as excellent photothermal/photodynamic effect were successfully prepared,displaying the potential of application to the image and treatment of breast cancer.PART ? PERFLUOROOCTYL BROMIDE AND INDOCYANINE GREEN CO-LOADED NANOLIPOSOMES FOR FL/PA/CT MULTIMODAL IMAGINGObjective To investigate the potential of LIP-PFOB-ICG nanoliposomes as fluorescent imaging(FL),photoacoustic imaging(PA)and Computed tomography(CT)triple-modality contrast agent through both in vitro and in vivo experiments.Methods The LIP-PFOB-ICG liposomes solution was diluted according to the concentration of ICG from 1.5 ?g/m L to 2.5 ?g/m L and FL imaging of LIP-PFOB-ICG was performed on the Xenogen IVIS spectrum imaging system.And PA imaging experiments were performed on the Vevo Laser imaging system with the LIP-PFOB-ICG liposomes diluting to a series of ICG concentration from 0 ?g/m L to 400 ?g/m L.CT imaging was conducted on a micro-CT scanner system and the samples were imaged with the concentration varied from 0.5 mg/m L to 8 mg/m L.And the multimodal imaging in vivo was performed on MDA-MB-231-tumor-bearing mice when the tumor volume reached about 100 mm3.For the in vivo FL and PA imaging,mice were randomly allocated to three groups: free ICG,LIP-ICG and LIP-PFOB-ICG groups(the concentration of ICG were all 800 ?g/m L)and the images gained at 0,6,24 and 48 h after injection.As for the CT imaging,the tumor-bearing mice were injected with LIP-PFOB-ICG or LIP-PFOB at the same dose(the concentrations of the liposomes were 10 mg/m L).CT images were acquired at 0,6,24,48 and 96 h after injection.Results In vitro fluorescent imaging showed that when the concentration of ICG was over 3.5 mg/m L,self-quenching was observed with diminished fluorescent intensity.And it was obvious to observe that the PA signals of LIP-PFOB-ICG liposomes were linearly enhanced with increased ICG concentrations.The CT signal intensity of LIP-PFOB-ICG also followed a linear enhancement along with the increased concentration of liposomes.As for the in vivo FL and PA imaging,the signal intensities in tumor region treating with LIP-PFOB-ICG and LIP-ICG liposomes strengthened gradually over time through enhanced permeability and retention effect,reaching peak enhancement at 24 h post injection with high contrast and resolution.And there was no significant difference between the FL/PA signal intensities of LIP-PFOB-ICG and LIP-ICG groups.However,no obvious FL/PA signal change was noted in free ICG group.And CT imaging in vivo showed that the tumor site was discernible and distinguished from other tissues with high spatial resolution and clear borderline 24 h post injection of LIP-PFOB-ICG and LIP-PFOB liposomes.Besides,the distinctive CT contrast imaging effect could prolong to 48 h post injection,a longer guidance window than that of PA and FL imaging.No significant difference between the CT signal intensities of LIP-PFOB-ICG and LIP-PFOB groups was observed.Conclusions The LIP-PFOB-ICG liposomes could be used as triple-modal contrast agents,which accumulated at tumor region through EPR effect and enhanced the FP/PA/CT imaging in vivo effectively.PART ? PERFLUOROOCTYL BROMIDE AND INDOCYANINE GREEN CO-LOADED NANOLIPOSOMES FOR THE TREATMENT OF BREAST CANCERObjective To investigate the therapeutic efficacy of photothermal therapy,photodynamic therapy and photothermal & photodynamic synergistic therapy of LIP-PFOB-ICG liposomes as well as its therapeutic mechanism.And the biosafety of LIP-PFOB-ICG liposomes was also evaluated.Methods The cytotoxicity of LIP-PFOB-ICG nanoliposomes in vitro was measured through incubation with MDA-MB-231 cells for 24 h in dark and the cell viabilities were determined by CCK-8 assay.To test the therapeutic effects of LIP-PFOB-ICG,MDA-MB-231 cells were exposed to laser irradiation after treating with LIP-PFOB-ICG liposomes at normal and hypoxia conditions respectively.Then the therapeutic effect of PTT,PDT and synergistic PTT&PDT were measured by CCK-8 assay and CAM/PI co-staining.And the phototherapy in vivo was performed on MDA-MB-231-tumor-bearing mice when the tumor volume reached about 100 mm3.Mice were randomly divided into ten groups: 1: control,2: Laser,3: ICG,4: ICG + continuous laser,5: LIP-ICG,6: LIP-ICG + interval laser(30 s on,30 s off),7: LIP-ICG + continuous laser,8: LIP-PFOB-ICG,9: LIP-PFOB-ICG + interval laser(30 s on,30 s off),10: LIP-PFOB-ICG + continuous laser.The mice in 2,4,6,7,9 and 10 groups were exposed to laser exposure 24 h post injection.The temperature changes at tumor region were monitored by infrared thermal imaging camera.Tumor sizes and body weights were recorded every 2 days for 18 days.On the 3rd day after treatments,tumors were collected for HE and PCNA,TUNEL immunohistochemical staining to evaluate histological changes and apoptotic levels.The tumor oxygenation status was monitored by PA imaging system in oxy-hem mode and HIF-1? staining before and 24 h post injection of various materials including saline,LIP-ICG,LIP-PFOB,LIP-PFOB-ICG.The HSP expression was evaluated by immunofluorescent staining and western blot before and 24 h post injection of various materials including saline,LIP-ICG,LIP-ICG with continuous laser,LIP-PFOB-ICG and LIP-PFOB-ICG with continuous laser.On the 3rd day after various treatments,major organs were collected for Hematoxylin and Eosin(HE)staining to observe the change of morphology.And to examine the toxicity of LIP-PFOB-ICG,blood samples were collected at 1,7,14 and 28 d post injection of LIP-PFOB-ICG liposomes for biochemical examinations.Results Inconspicuous cytotoxicity of the LIP-PFOB-ICG nanoliposomes on MDA-MB-231 was observed without laser irradiation.However,in the presence of irradiation,an obvious dose dependent photo-cytotoxicity of LIP-PFOB-ICG liposomes was shown.When the concentration of ICG was 12.5 ?g/m L,cells maintained about 25% viability after pure PDT therapy,18% for PTT therapy and only 7% viability after synergistic PTT&PDT therapy.As for the phototherapy in vivo,in mice treated with LIP-ICG and LIP-PFOB-ICG plus continuous laser,significant elevations of local temperature at tumor region were noted and the maximum temperature reached about 47.4 ? and 48.1? respectively.And in groups of LIP-ICG and LIP-PFOB-ICG for photodynamic therapy,the temperature was kept below 43 ? to avoid photothermal effect.18 days after various treatment,LIP-ICG plus interval laser irradiation showed no obvious tumor inhibition efficiency,the tumor growth was partially suppressed in the LIP-PFOB-ICG plus interval laser group,and tumors received pure PTT(LIP-ICG+continuous laser)showed significant tumor inhibition during the first 8 d but regrew later.Only the mice treated with LIP-PFOBICG plus continuous laser irradiation achieved complete tumor regression without recurrence.And HE staining showed that severe necrosis appeared in the tumors treated with LIP-PFOB-ICG plus photoirradiation,accompanying with the largest apoptotic index and lowest proliferative index.Moreover,as shown in the PA imaging and HIF-1? staining,the tumor treated with LIP-PFOB and LIP-PFOB-ICG liposomes showed obviously increased oxygen saturation and decreased HIF-1? expression.And both HSP immunofluorescent staining and WB results showed that lower HSP expression was detected in the group of LIP-PFOB-ICG with laser irradiation compared to that of LIP-ICG with laser exposure,demonstrating that the introduction of PFOB could inhibit HSP expression to some extent to sensitize cancer cells to hyperthermiaHE staining of major organs exhibited no apparent physiological abnormalities,suggesting the negligible toxicity of LIP-PFOB-ICG.And the various blood indexes showed that there was no significant difference among the groups under different stages,indicating undetectably toxic to mice at our tested dose no matter in short or in long terms.Conclusions The LIP-PFOB-ICG liposomes could effectively relive tumor hypoxia condition,which resulted in enhanced PDT effect of ICG.And such intensified photodynamic effect further induced reduced expression of HSP,leaving cancer cells more sensitive to photothermal effect.Through enhanced PDT&PTT synergistic therapy,LIP-PFOB-ICG liposomes completely inhibited tumor growth,offering a novel approach to treat breast cancer.