Laser-activatable And Targeted Phase-changeable Nanoprobes For Multimodal Imaging And Breast Cancer Therapy

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PREPARATION AND CHARACTERIZATION OF LASER-ACTIVATABLE AND TARGETED PHASE-CHANGEABLE NANOPROBESObjective To prepare the laser-activatable and targeted phase-changeable nanoprobes(HER-DIR-SPIO-PLGA/PFP)and investigate their basic physicochemical characteristics,liquid-gas phase-transformation ability and photothermal ability in vitro.To observe the photothermal ability and specifically targeting ability of HER-DIR-SPIO-PLGA/PFP towards the HER2-positive breast cancer cells.Methods HER-DIR-SPIO-PLGA/PFP targeted nanoprobes were prepared by double emulsion method combined with carbodiimide method.Their properties including surface morphology,size distribution,zata potential,ultraviolet absorption spectrum,SPIO loading efficency were detected.Immunofluorescence and flow cytometry were used to detect the attachment of HER2 antibody(Herceptin)to the nanoprobes.The liquid-gas phase-transformation ability and photothermal ability of DIR-SPIO-PLGA/PFP nanoprobes were investigated under the NIR laser irradiation.The cell-killing effect of different concentrations of HER-DIR-SPIO-PLGA/PFP,DIR-SPIO-PLGA/PFP and Herceptin on breast cancer cell lines MDA-MB-231 and SKBR3 were detected by CCK-8 assay.Under the NIR laser irradiation,the photothermal effects of different concentrations of HER-DIR-SPIO-PLGA/PFP on breast cancer cell lines MDA-MB-231 and SKBR3 were also detected.The specifically targeting ability of the nanoprobe to HER2-positive human breast cancer cell line SKBR3 was observed by confocal laser scanning microscopy.Results The brown HER-DIR-SPIO-PLGA/PFP nanoprobes were prepared by double emulsion method combined with carbodiimide method.The nanoprobes with core-shell structure were observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The average diameter of DIR-SPIO-PLGA/PFP and HER-DIR-SPIO-PLGA/PFP was 278±83.29 nm and 298±109.5 nm,respectively.And the relative average potential was-1.78±3.57 mV and-2.61±3.53 mV,respectively.The absorption spectrum of HER-DIR-SPIO-PLGA/PFP showed a slight absorption peak at 764 nm.The incorporation rate of SPIO was 93%,which was detected by ICP-OES.The flow cytometry revealed that 99% of the Herceptin was bound to the nanoprobes.The BCA curve further demonstrated that Herceptin was linked with 0.28 mg/mL.No significant size change of HER-DIR-SPIO-PLGA/PFP was observed after storing at 4? over 7 days.Under the NIR laser irradiation,DIR-SPIO-PLGA/PFP phase-changed into microbubbles.In vitro heating experiment showed that DIR-SPIO-PLGA/PFP has good photothermal efficiency and has the potential for photothermal therapy.There were no significant cytotoxicity of HER-DIR-SPIO-PLGA/PFP,DIR-SPIO-PLGA/PFP and free Herceptin on MDA-MB-231 cells.A dose dependent cytotoxicity was detected when SKBR3 cells were incubated with HER-DIR-SPIO-PLGA/PFP or free Herceptin.After laser irradiation,HER-DIR-SPIO-PLGA/PFP showed conspicuous cytotoxicity on SKBR3 cells with increased concentration,but no obvious cytotoxicity was observed on MDA-MB-231 cells.In vitro targeting experiments showed that amount of HER-DIR-SPIO-PLGA/PFP specifically aggregated around the membrane of HER2-positive human breast cancer cell line SKBR3,but none aggregated around the membrane of HER2-negative human breast cancer cell line MDA-MB-231,further confirming the excellent targeting ability of HER-DIR-SPIO-PLGA/PFP to HER2-positive cells.Conclusions HER-DIR-SPIO-PLGA/PFP nanoprobes were synthesized successfully using double emulsion method with carbodiimide method.The nanoprobe exhibited uniform size distribution,good dispersion,high SPIO and Herceptin loading efficiencies as well as excellent stable ability.At the same time,HER-DIR-SPIO-PLGA/PFP can be activated by the NIR laser and phase-shifted into microbubbles through liquid-gas transformation.The nanoprobe has high efficiency in targeting HER2-positive breast cancer cells,was an ideal nanoprobe for the imaging and treatment of HER2-positive breast cancer.PART ? LASER-ACTIVATABLE AND TARGETED PHASE-CHANGEABLE NANOPROBES FOR MULTIMODAL IMAGINGObjective To investigate the magnetic resonance imaging(MRI),photoacoustic(PA)imaging and ultrasound(US)imaging ability of DIR-SPIO-PLGA/PFP nanoprobes in vitro;using breast cancer-bearing mice to observe the ability and principles of HER-DIR-SPIO-PLGA/PFP for MRI/PA/US/Near-infrared fluorescence(NIRF)imaging in vivo.Methods In vitro agar-based phantom was prepared.Different concentrations of DIR-SPIO-PLGA/PFP were applied for MRI and PA imaging using the magnetic resonance and photoacoustic imaging system,respectively.And the relative signal intensities were detected.Under the NIR laser irradiation,the US imaging ability of DIR-SPIO-PLGA/PFP,PLGA/PFP and saline were performed using ultrasonic diagnostic system.The gray scale signals of US images were acquired by using the ultrasound analysis software.In vivo multi-modal imaging were conducted when the SKBR3 xenograft of nude mice were established and the tumor volume reached 200 mm~3.The tumor-bearing mice were radomly divided into 3 groups,each group received tail vein injection with HER-DIR-SPIO-PLGA/PFP,DIR-SPIO-PLGA/PFP or saline.MR/PA imaging was performed before injection and at different time point post-injection of nanoprobes.And the MR/PA signals were measured by a magnetic resonance scanner and Vevo Laser imaging system,respectively.For US imaging in vivo,a NIR laser irradiation was performed 6 h post tail vein administration of different nanoprobes.After that,the ultrasound images and signal intensities of the tumor before and after laser irradiation were collected and analyzed.For NIRF imaging,the fluorescence images and signal intensities of the tumor were collected after tail vein administration of different nanoprobes.Results As shown,the T2*WI MR and PA images of DIR-SPIO-PLGA/PFP were enhanced significantly with increased concentration in vitro.And the 1/T2* signals and PA signals were linearly enhanced with increased concentration of DIR-SPIO-PLGA/PFP.In vitro ultrasound imaging results showed that DIR-SPIO-PLGA/PFP exhibited a slightly high echo intensity before laser irradiation,and the echo intensities were significantly improved in both ultrasound B mode and CEUS mode after laser irradiation.In vivo multi-modal imaging showed that,the signal intensities of MR,PA and NIRF in tumor region strengthened over time using HER-DIR-SPIO-PLGA/PFP,peaked at 6 h post injection.However,no obvious MR/PA/NIRF signals were observed in the tumor region of the control groups.In vivo ultrasound imaging showed that the B-mode and CEUS mode of ultrasound images were significantly enhanced after the mice receiving HER-DIR-SPIO-PLGA/PFP plus laser irradiation.While no significant changes of ultrasound signals were detected at the tumor site after injection of non-targeted nanoprobes DIR-SPIO-PLGA/PFP and saline.Conclusions The DIR-SPIO-PLGA/PFP could be used as multi-modal imaging agent in agar-based phatom.The in vivo breast cancer xenograft model further demenstrate that HER-DIR-SPIO-PLGA/PFP can be effectively accumulated and accurately used for multimodal imaging,and has the potential for the diagnosis of HER2-positive breast cancer.PART ? LASER-ACTIVATABLE AND TARGETED PHASE-CHANGEABLE NANOPROBES FOR HER2 POSITIVE BREAST CANCER THERAPYObjective To investigate the therapeutic efficacy of HER-DIR-SPIO-PLGA/PFP combined with NIR laser on breast cancer as well as the relative mechanism.And to evaluate the biosafety and biodistribution of HER-DIR-SPIO-PLGA/PFP.Methods In vivo targeted therapy was performed on SKBR3 xenograft tumor model.The mice were randomly divided into 5 groups when the tumor volume reached 200 mm3: group 1.HER-DIR-SPIO-PLGA/PFP+NIR,group 2.DIR-SPIO-PLGA/PFP+NIR,group 3.HER-DIR-PLGA/PFP+NIR,group 4.Saline+NIR,group 5.HER-DIR-SPIO-PLGA/PFP.The mice in groups 1,2,3 and 4 were exposed to a NIR laser(2 W/cm2,10 min)after 6 h administration of nanoprobes.And the temperature changes within the tumor region were monitored using infrared thermal imager.Nude mice in each group were observed for 21 days,and their tumor volumes and body weights were monitored every 2-3 days.Two days after treatment,one nude mouse in each group was randomly sacrificed and the tumors were collected for H&E,PCNA and TUNEL staining.The proliferation and apoptosis levels of tumor cells were evaluated through microscope.To detect the penetration distance of HER-DIR-SPIO-PLGA/PFP in the tumor,9 tumor-bearing nude mice were randomly divided into 3 groups: group 1.HER-DIR-SPIO-PLGA/PFP+NIR,group 2.HER-DIR-SPIO-PLGA/PFP,group 3.HER-DIR-SPIO-PLGA+NIR.The mice in group 1 and 3 were exposed to a NIR laser(2 W/cm2,10 min)6 h after administration,and the penetration distance at the tumor site in each group was examined.To evaluate the biosafety of HER-DIR-SPIO-PLGA/PFP,21 days after different treatment,the mice were sacrificed and the main organs were collected for H&E staining in order to observe the morphological changes.HER-DIR-SPIO-PLGA/PFP(10 mg/m L,200 mL)was intravenously injected into the Kunming mice,then the blood samples were collected at different time points after treatment,liver/kidney function and fluorescence signals were detected.ICP-OES was used to detect the content of Fe in the main organs of tumor-bearing mice injected with nanoprobes at different time points.Results In vivo photothermal results showed that after laser irradiation,the temperature at the tumor site in the HER-DIR-SPIO-PLGA/PFP+NIR group rapidly increased to 54.1°C,which can effectively destroy tumor cells.The tumor temperature in the DIR-SPIO-PLGA/PFP+NIR group only increased to 47.9°C.The tumor temperature in HER-DIR-PLGA/PFP+NIR group and the saline+NIR group were 44.2°C and 43.9°C,respectively.21 days after various treatments,the tumor volume in the control HER-DIR-SPIO-PLGA/PFP group without laser irradiation increased 2.9-fold,which showed no obvious tumor inhibition efficiency.The DIR-SPIO-PLGA/PFP+NIR,HER-DIR-PLGA/PFP+NIR and saline+NIR groups showed similar increases in tumor volume of 1.4-fold,1.9-fold and 2.1-fold,reflecting the tumor growths were partially suppressed.Only the mice in the HER-DIR-SPIO-PLGA/PFP+NIR group showed a significantly inhibited tumor growth.H&E-slices of tumor tissues showed that in the HER-DIR-SPIO-PLGA/PFP+NIR group,the cell structure of the tumor disappeared and the morphology was unclear,and severe necrosis appeared.PCNA and TUNEL results showed that the proliferation index of this group was significantly lower than the other groups,and the apoptotic index was the highest in all the groups(*P<0.05).Moreover,as shown in the immunofluorescence images,a large number of blood vessels were destroyed in the HER-DIR-SPIO-PLGA/PFP+NIR group,which promoted the penetration of nanoprobe fragments into the tumor tissue.In the HER-DIR-SPIO-PLGA/PFP group without laser irradiation,nanoprobes were mainly localized near vascular areas.HER-DIR-SPIO-PLGA+NIR group showed moderate penetration,indicating that the blasting effect of PFP under laser irradiation was the main reason promoting the nanoprobes diffusion.During the observation period,no significant difference in hepatic-renal function of nude mice was observed.H&E staining of the main organs also exhibited no apparent abnormality in each group,confirming the good biosafety of the nanoprobe.The nanoprobes had a long blood circulation time and was mainly distributed in the reticuloendothelial system in vivo.Conclusions HER-DIR-SPIO-PLGA/PFP plus NIR laser can effectively inhibit tumor growth of HER2-positive breast cancer,and has good biosafety in vivo,providing a new strategy for the diagnosis and treatment of HER2-positive breast cancer.