Peptide-functionalized HMME-Gd-loaded Phase Change Nanoparticles For Multimodal And Multifunctional Diagnosis And Treatment Of Breast Cancer

PART I PREPRATION AND CHARACTERIZATION OF PEPTIDE-FUNCTIONALIZED HMME-GD-LOADED PHASE CHANGE NANOPARTICLESObjective To prepare peptide-functionalized HMME-Gd-loaded phase change nanoparticles(PFP@t Ly P-1-LIP-H(Gd)),and to investigate their basic properties including size,surface potential and encapsulation efficiency etc.Methods 1.Peptide-functionalized HMME-Gd-loaded phase change nanoparticles(PFP@t Ly P-1-LIP-H(Gd))were prepared by membrane hydration and emulsification.The size and surface potential of the nanoparticles were analyzed by Malvern size analyzer.The general physical properties were measured by transmission electron microscopy and the encapsulation efficiency was determined by UV-Vis.2.The targeting abilities towards MDA-MB-231 and HUVEC by PFP@LIP-H(Gd)and PFP@t Ly P-1-LIP-H(Gd)were observed by flow cytometer and CLSM.The MDA-MB-231 tumor-bearing mice model was first established and then mice were sacrificed and the tumor tissues were reserved at different time points(1,2,4,6,12,24h)after injecting Di I-labeled PFP@LIP-H(Gd)and PFP@t Ly P-1-LIP-H(Gd).The tumor tissues were made into frozen sections,which were then observed under florescent microscope to examine the distribution of nanoparticles.3.After incubating with MDA-MB-231 cells for 24 h,the cytotoxicities of different concentrations of PFP@LIP,PFP@LIP-H(Gd)and PFP@t Ly P-1-LIP-H(Gd)were assessed by Cell Counting Kit-8(CCK-8).Balb/c mice were divided into control group and experimental group.Mouse blood was collected at different time points(1,5,7,14d)after injecting NPs in experimental group,while mouse blood was collected at 14 d after injecting normal saline in control group.The blood normal examination and blood biochemical examination were performed after the blood was collected for both groups.Results 1.TEM showed that PFP@t Ly P-1-LIP-H(Gd)were generally spherical.The size and the zeta potential of the NPs were measured by Malvern Instruments and determined to be(260.93±5.28)nm and(-15.7±2.646)m V,respectively.The encapsulation efficiency of HMME-Gd determined by UV-Vis was about 93.33%.2.The red fluorescence of the Di I-labeled PFP@t Ly P-1-LIP-H(Gd)localized around the MDA-MB-231 cells was more than that of the non-targeted NPs observed by CLSM,while there was no obvious aggregation around HUVEC.Meanwhile,Di I-labeled PFP@LIP-H(Gd)didn't localize around MDA-MB-231 cells due to the lack of the targeting and transmembrane function of t Ly P-1.The outcomes determined by flow cytometer and CLSM were consistent.A large quantity of red fluorescence was observed in tumor tissue after injecting targeted NPs 1h,which was correlated with time(P<0.05);minor red fluorescence was observed in tumor tissue after injecting non-targeted NPs,and there was no distribution after 24h(P>0.05).3.There was no obvious cytotoxicity in MDA-MB-231 cells at different concentration of each NPs,which was measured by CCK-8(P<0.05).Mouse blood tests showed that there were no significant differences between experimental group and control group(P>0.05).Conclusion PFP@t Ly P-1-LIP-H(Gd)were successfully prepared.The nanoparticles were in uniform size and good dispersion with high encapsulation efficiency.The targeting abilities of NPs towards MDA-MB-231 cells and MDA-MB-231 tumor-bearing mouse were obvious.Such nanoparticles demonstrated favorable biosafety in both vitro and vivo.PART II MULTIMODALITY IMAGING OF PEPTIDE-FUNCTIONALIZED HMME-GD-LOADED PHASE CHANGE NANOPARTICLESObjective To observe the ultrasound imaging,photoacoustic imaging,fluorescent imaging and MRI imaging effect of PFP@t Ly P-1-LIP-H(Gd)in vitro and in vivo.Methods 1.Ultrasound imaging in vitro and in vivo 1)Ultrasound imaging in vitro: the diluted PFP@t Ly P-1-LIP-H(Gd)were irradiated by LIFU(1.6W/cm2,pulse mode)in different time points(0,1,2,3,4min)and then the acoustic phase-change of the nanoparticles was observed by optical microscope.The diluted PFP@t Ly P-1-LIP-H(Gd)were dropped into hydrogel and were irradiated by LIFU(0.8,1.6,3.2W/cm2;pulse mode)in different time points(0,1,2,3,4min)as experimental group,and t Ly P-1-LIP-H(Gd)without PFP were irradiated by LIFU(1.6W/cm2;pulse mode)in different time points(0,1,2,3,4min)as control group.Both groups were observed by ultrasonic diagnostic apparatus in US-mode and CEUS-mode.The gray values of all ultrasound graphs were quantitatively analyzed by DFY.2)Ultrasound imaging in vivo: MDA-MB-231 tumor-bearing mice model was established,and mice were randomly divided into control group(PFP@LIP-H(Gd))and experimental group(PFP@t Ly P-1-LIP-H(Gd)).The gray values of all ultrasound graphs were collected after injecting nanoparticles via tail vein at different time points(pre,2h,2h and sonicated by LIFU(1.6W/cm2,2min,pulse mode)).The results were quantitatively analyzed by DFY.2.Photoacoustic imaging in vitro and in vivo 1)Photoacoustic imaging in vitro: Photoacoustic imaging was detected after PFP@t Ly P-1-LIP-H(Gd)NPs were diluted in different concentrations(0.15625-5mg/ml).The correlation between the concentrations and the photoacoustic signal intensity was evaluated.2)MDA-MB-231 tumor-bearing mice model was established,and mice were randomly divided into control group(PFP@LIP-H(Gd))and experimental group(PFP@t Ly P-1-LIP-H(Gd)).The photoacoustic images were collected after injecting nanoparticles via tail vein at different time points(pre,2,4,6,8,12,24h),and photoacoustic signal intensity of each graph was quantitatively analyzed.3.Fluorescent imaging in vitro and in vivo 1)Fluorescent imaging in vitro: Di R-labeled PFP@t Ly P-1-LIP-H(Gd)were diluted in different concentrations(0.009765625-5mg/ml),and NPs were dropped in 96-well plates 100?L per well with three wells in every concentration.The fluorescent intensity of each graph was analyzed by fluorescence imaging system.2)Fluorescent imaging in vivo: MDA-MB-231 tumor-bearing mice model was established,and mice were randomly divided into control group(PFP@LIP-H(Gd))and experimental group(PFP@t Ly P-1-LIP-H(Gd)).The fluorescent images were collected after injecting NPs via tail vein at different time points(pre,2,4,6,8,12,24h),and fluorescent signal intensity of each graph was quantitatively analyzed.After 24 h,mice were sacrificed and tumors were excised.Simultaneously,the major organs(heart,liver,spleen,lung,kidney and brain)of the mice were collected and used for ex vivo fluorescence imaging.4.Magnetic resonance imaging(MRI)in vitro and in vivo 1)MRI in vitro: PFP@t Ly P-1-LIP-H(Gd)were diluted in different concentrations(0.015-0.1m M),and nanoparticles were dropped into 2ml centrifuge tube as a contrast agent for T1-weighted MRI.The correlation between the dose of Gd3+ and relaxation rate was evaluated.2)MRI in vitro: MDA-MB-231 tumor-bearing mice model was established,and mice were randomly divided into control group(PFP@t Ly P-1LIP)and experimental group(PFP@t Ly P-1-LIP-H(Gd)).The T1-weighted images were collected after injecting nanoparticles via tail vein at different time points(pre,2,6,12,24h),and T1-weighted signal intensity of each image was quantitatively analyzed.Results 1.Ultrasound imaging in vitro and in vivo 1)Ultrasound imaging in vitro: The volume of nanoparticles was positively correlated with LIFU irradiating time under optical microscope.The ultrasound signal intensity of experimental group was positively correlated with LIFU power and irradiation time,while the intensity of control group was no significant change(P>0.05).The result of DFY analyzer was consistent with ultrasound image.2)The tumor areas of both groups were low echo area before injecting NPs.The US-mode and CEUS-mode signal intensity of experimental group enhanced a little after 2h injection of NPs.However,the signal intensity was enhanced greatly after irradiation by LIFU(P<0.05).The signal intensity of control group was no significant change(P>0.05).The result of DFY analyzer was consistent with ultrasound image.2.Photoacoustic imaging in vitro and in vivo 1)The photoacoustic signal intensity was in a linear relation with the concentration of nanoparticles.2)There was no significant photoacoustic signal in tumor areas of either group before injecting nanoparticles.The photoacoustic signal of experimental group could be detected after 2h injection of nanoparticles. There was still photoacoustic signal after 24 h injection of nanoparticles(P<0.05).While The photoacoustic signal of control group couldn't be detected after injecting nanoparticles at any time(P>0.05).The result of quantitative analysis was consistent with that of photoacoustic image.3.Fluorescent imaging in vitro and in vivo 1)Fluorescent imaging in vitro: The fluorescent signal intensity of Di R-labeled PFP@t Ly P-1-LIP-H(Gd)increased as the concentration of nanoparticles increased,but ceased to increase when concentration reached to a certain level.2)Fluorescent imaging in vivo: There was no significant fluorescent signal in tumor areas of either group before injecting nanoparticles.The signal intensity of experimental group enhanced greatly after 2h injection of nanoparticles.There was still fluorescent signal after 24 h injection of nanoparticles(P<0.05).The signal intensity of control group was no significant change(P>0.05).Ex vivo fluorescence imaging showed that the fluorescent signal of control group was distributed mostly in liver,spleen and little in tumor area,while the signal of experimental group was distributed mostly in liver,spleen and tumor area.The result of quantitative analysis was consistent with that of fluorescent image.4.Magnetic resonance imaging in vitro and in vivo 1)MRI in vitro: The relaxation rate of PFP@t Ly P-1-LIP-H(Gd)was in a linear relation with the dose of Gd3+.2)MRI in vivo: The T1-weighted signal intensity of tumor area in both groups was low before injecting nanoparticles.The signal intensity of experimental group enhanced greatly after 2h injection of nanoparticles and still could be observed after 24h(P<0.05),while the signal intensity of control group was no significant change after injection(P>0.05).The result of quantitative analysis was consistent with that of T1-weighted image.Conclusion PFP@t Ly P-1-LIP-H(Gd)could enhance obviously ultrasound imaging,photoacoustic imaging,fluorescent imaging and magnetic resonance imaging ability in vitro and in vivo and could target effectively to the tumor area.PART III THE MULTIFUNCTIONAL SYNERGISTIC TREATMENT OF PEPTIDE-FUNCTIONALIZED HMME-GD-LOADED PHASE CHANGE NANOPARTICLESObjective To measure the SDT ability of the nanoparticles and the treatment effect towards breast cancer with Cavitation effect and SDT effect under the condition of normoxia or hypoxia.Methods 1.The detection of SDT effect 1)The detection of ROS produced in NPs: ROS produced by different concentrations of PFP@t Ly P-1-LIP-H(Gd)under irradiation of LIFU was measured by SOSG.2)The detection of ROS produced in cells: MDA-MB-231 cells were divided into(1)Control group,(2)t Ly P-1-LIP(hypoxia)group,(3)t Ly P-1-LIP-H(Gd)(hypoxia)group,(4)t Ly P-1-LIP-H(Gd)(normoxia)group,(5)PFP@t Ly P-1-LIP-H(Gd)(hypoxia)group,(6)Control+LIFU group,(7)t Ly P-1-LIP(hypoxia)+LIFU group,(8)t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group,(9)t Ly P-1-LIP-H(Gd)(normoxia)+LIFU group and(10)PFP@t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group.All groups were irradiated by LIFU(1.6W/cm2,pulse mode)after incubating with DCFH-DA,and the ROS was observed by CLSM.2.The treatment effect in vitro 1)Cell Counting Kit-8 method:(1)MDA-MB-231 cells were divided into PFP@LIP+LIFU group,PFP@LIP-H(Gd)+LIFU group and PFP@t Ly P-1-LIP-H(Gd)+LIFU group.The cell viability of all groups was detected by CCK-8 method.(2)The cells were divided into t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group,t Ly P-1-LIP-H(Gd)(normoxia)+ LIFU group and PFP@t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group.The cell viabilities of all groups were detected by CCK-8 method.2)Live/Dead Cell Double Staining Kit method: The cells were divided into(1)Control group,(2)t Ly P-1-LIP(hypoxia)group,(3)t Ly P-1-LIP-H(Gd)(hypoxia)group,(4)t Ly P-1-LIP-H(Gd)(normoxia)group,(5)PFP@t Ly P-1-LIP-H(Gd)(hypoxia)group,(6)Control+LIFU group,(7)t Ly P-1-LIP(hypoxia)+LIFU group,(8)t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group,(9)t Ly P-1-LIP-H(Gd)(normoxia)+LIFU group and(10)PFP@t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group.The cells were stained by calcein-AM(CAM)and pyridinium iodide(PI)and observed by CLSM.3.The treatment effect in vivo The tumor-bearing mice were randomly divided into(1)Control group,(2)LIFU group,(3)t Ly P-1-LIP+LIFU group,(4)t Ly P-1-LIP-H(Gd)+LIFU group,(5)PFP@t Ly P-1-LIP-H(Gd)group and(6)PFP@t Ly P-1-LIP-H(Gd)+ LIFU group.Mice were injected nanoparticles via tail vein,while the control group was injected the same amount of normal saline.Mice were irradiated by LIFU(1.6W/cm2,2 min,pulse mode)after 4h.The tumor volume and body weight of mice were recorded every two days.Mice were treated every three days.Every time one mouse of each group was sacrificed randomly after 24 h treatment and the major organs(heart,liver,spleen,lung,kidney and brain)and tumor of the mouse were collected and used for histological and biosafety analysis.Results 1.The detection of SDT effect 1)The detection of ROS produced in nanoparticles: the ability of PFP@t Ly P-1-LIP-H(Gd)NPs generating ROS was correlated with the concentration of nanoparticles and the irradiation time of LIFU.With the increase of nanoparticles concentration and irradiation time,the generation of ROS increased gradually.2)The detection of ROS produced in cells: through the detection of DCFH-DA,the green fluorescence of t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group was obviously more than that of t Ly P-1-LIP(hypoxia)+LIFU group,whereas obviously less than that of t Ly P-1-LIP-H(Gd)(normoxia)+LIFU group.In addition,PFP@t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group contained much more green fluorescence than that of t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group.However,little green fluorescence was observed in the groups lacking irradiation by LIFU.2.The treatment effect in vitro 1)Cell Counting Kit-8 method:(1)The cell viability decreased as the increase of concentration of nanoparticles irradiated by LIFU,of which PFP@t Ly P-1-LIP-H(Gd)decreased the most(P<0.05).(2)The cell viability under normoxia condition was lower than that of hypoxia condition after irradiation at different concentration of t Ly P-1-LIP-H(Gd).After the irradiation of LIFU,the cell viability of PFP@t Ly P-1-LIP-H(Gd)under hypoxia condition was slightly higher than that of(1)under normoxia condition.2)Live/Dead Cell Double Staining Kit method: the quantity of dead cells(red fluorescence)of t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group was obviously more than that of t Ly P-1-LIP(hypoxia)+LIFU group,whereas obviously less than that of t Ly P-1-LIP-H(Gd)(normoxia)+LIFU group.In addition,PFP@t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group contained a much larger quantity of dead cells than that of t Ly P-1-LIP-H(Gd)(hypoxia)+LIFU group.However,very few dead cells were observed in the groups lacking irradiation by LIFU.The cell viability was the worst when PFP@t Ly P-1-LIP-H(Gd)were irradiated under the condition of normoxia or hypoxia,which was consistent with the outcome of CCK-8 method.3.The treatment effect in vivo The PFP@t Ly P-1-LIP-H(Gd)+LIFU group showed the most significant inhibiting effect until the end of the treatment.Meanwhile,there was no obvious weight change during the treatment.There were a lot of apoptosis and necrosis in PFP@t Ly P-1-LIP-H(Gd)+LIFU group comparing with the control group in tumor tissue test of H&E and PCNA.The main organs(heart,liver,spleen,lung,kidney and brain)were not injured under the observation of H&E staining.Conclusion The SDT effect and Cavitation effect could be generated by PFP@t Ly P-1-LIP-H(Gd)under the irradiation of LIFU,which could significantly inhibit the growth of hypoxic tumor cells in vitro and the growth of tumor in tumor bearing nude mice in vivo.