Peptide Functionalized Phase-change Nanoenzyme Loaded With HMME-Gd For Precise Diagnosis And Therapy Of Breast Cancer

PARTⅠPREPARETION AND CHARACTERIZATION OF PEPTIDE FUNCTIONALIZED PHASE-CHANGE NANOENZYME LOADED WITH HMME-GDObjective To Prepare peptide functionalized phase-change nanoenzyme loaded with HMME-Gd(t Ly P-1@H(Gd)-GOD@PFP NPs),and the basic characteristics of the NPs,including particle size,potential,entrapment efficiency,drug loading rate,catalytic ability and phase transition ability,were detected.The biosafety and ability of targeting MDA-MB-231 cells in vitro and in vivo were tested.Methods t Ly P-1@H(Gd)-GOD@PFP NPs was prepared by double emulsion method.The morphology and size were observed by transmission electron microscopy.The particle size and potential were measured by Malvern size analyzer.The encapsulation efficiency and drug loading rate of H(Gd)were detected by UV spectrophotometer.The encapsulation efficiency and drug loading rate of GOD were detected by high performance liquid chromatography.The phase transition of nanoenzyme after LIFU irradiation(1.6 W/cm~2,pulse mode,4 min)was observed under light microscopy.The p H change of nanoenzyme with glucose solution was detected by p H meter.The enzyme activity kinetics of the nanoenzyme was detected by microplate.CCK-8 assay was used to detect the biosafety of nanoenzyme in vitro.Confocal laser scanning microscopy(CLSM)and flow cytometry were used to analyze the targeting effect of nanoenzyme in vitro.After injecting nanoenzyme into the tail vein of nude mice with ectopic xenografts tumor,tumor tissues were taken at different time points to evaluate the targeting efficacy in vivo,and the orbital vein was taken for blood routine test and blood biochemical examination,which were performed to evaluate the in vivo safety.Results The structure of t Ly P-1@H(Gd)-GOD@PFP NPs was generally spherical.The particle size was(266.7±10.1)nm and the potential was(-16.23±0.45)m V.The encapsulation efficiency and drug loading rate of H(Gd)were 94.8%and 9.5%,respectively.The encapsulation efficiency and drug loading rate of GOD were 15.1%and 1.51%,respectively.Under the light microscopy,with the increase of irradiation time,the size of nanoenzyme increased and tended to rupture,and the size of nanoenzyme decreased when the irradiation time arrived at 4 min.There was no significant difference in p H between t Ly P-1@H(Gd)-GOD@PFP NPs group and GOD group(P>0.05).In addition,The Michaelis constant(Km)and maximum reaction velocity(Vmax)of the nanoenzyme were(16.97±2.04)mm and(5.31×10-~7±2.13×10-~8)m/s,respectively.CCK-8 assay in vivo showed that the biological safety of nanoenzyme was excellent.In the targeting experiment in vitro,t Ly P-1@H(Gd)-GOD@PFP NPs gathered around MDA-MB-231 cells more than H(Gd)-GOD@PFP NPs(P<0.05)under CLSM and there was no obvious aggregation around human umbilical vein endothelial cells(HUVEC).The quantitative analysis by flow cytometry was also consistent with the results.In the targeting experiment in vivo,CLSM was used to observe tumor tissue sections,t Ly P-1@H(Gd)-GOD@PFP NPs was more concentrated in tumor tissue than H(Gd)-GOD@PFP NPs(P<0.05).Conclusion Peptide functionalized phase-change nanoenzyme loaded with HMME-Gd were successfully prepared,which were in typical shell core structure,uniform spherical shape,good dispersion and high drug entrapment efficiency.Under the irradiation of LIFU,the nanoenzyme showed the ability of phase-change and burst to release GOD to catalyze glucose effectively.In addition,it was biosecurity and could precisely target to MDA-MB-231 tumor cells in vitro and in vivo.PART Ⅱ EXPERIMENTAL STUDY ON MULTIMODAL IMAGING OF BREAST CANCER BY PEPTIDE FUNCTIONALIZED PHASE-CHANGE NANOENZYME LOADED WITH HMME-GDObjective To observe the fluorescence imaging,ultrasound imaging,photoacoustic imaging and magnetic resonance imaging of t Ly P-1@H(Gd)-GOD@PFP NPs in vitro and in MDA-MB-231 xenograft-bearing nude mice models.Methods The agarose gel model was established and the ultrasound imaging and photoacoustic imaging of t Ly P-1@H(Gd)-GOD@PFP NPs were performed and signal intensity was quantitatively analyzed by ultrasonic diagnostic apparatus and photoacoustic imager in vitro.Fluorescence imaging model was established in 96 well plate,and in vitro fluorescence imaging was performed by small animal fluorescence imager.In vitro magnetic resonance imaging was performed by 1.5 m L centrifuge tube under T1 weighted magnetic resonance imaging system.The imaging experiments of t Ly P-1@H(Gd)-GOD@PFP NPs,H(Gd)-GOD@PFP NPs or t Ly P-1@GOD@PFP NPs in vivo were performed with ultrasound diagnostic system,photoacoustic imaging system,small animal fluorescence imaging system and magnetic resonance imaging system by injection via tail vein of xenograft-bearing nude mice model.Results In vitro imaging showed that t LyP-1@H(Gd)-GOD@PFP NPs showed excellent multimodal imaging,and the imaging intensity was proportional of the concentration of nanoenzyme.In vivo imaging showed that t Ly P-1@H(Gd)-GOD@PFP NPs,compared to H(Gd)-GOD@PFP NPs,could specifically aggregate and target tumor tissue,and showed good multimodal imaging performance in tumor tissue in situ.Conclusion The t Ly P-1@H(Gd)-GOD@PFP NPs in vitro and ectopic xenografts showed significant fluorescence imaging,ultrasound imaging,photoacoustic imaging and magnetic resonance imaging.PART Ⅲ EXPERIMENTAL STUDY ON MULTIFUNCTIONAL PRECISION THERAPY OF BREAST CANCER BY PEPTIDE FUNCTIONALIZED PHASE-CHANGE NANOENZYME LOADED WITH HMME-GDObjective To research the sonodynamic and therapeutic effect of t LyP-1@H(Gd)-GOD@PFP NPs on breast cancer in vitro and in vivo.Methods The production of reactive oxygen species(ROS)after different irradiation time of LIFU with different concentrations of t Ly P-1@H(Gd)-GOD@PFP NPs was detected by Singlet Oxygen Sensor Green(SOSG)via fluorescence spectrophotometer.After the nanoenzyme of all groups were incubated with MDA-MB-231 cells,CCK-8 assay and flow cytometry were used to evaluate the killing effect on tumor cells.CLSM was used to observe the cell survival after staining.In addition,the cells treated with nanoenzyme were co-incubated with DCFH-DA,and then the effect of reactive oxygen species was observed by CLSM.After the nanoenzyme was injected into tail vein of nude mice and combined with LIFU irradiation,the nude mice in each group were killed,and the heart,liver,spleen,lung,kidney and tumor tissues were made into sections and stained with H&E.And the tumor tissues were made into sections and stained with PCNA and TUNEL to analyze the therapeutic effect.Results The ability of t Ly P-1@H(Gd)-GOD@PFP NPs to produce ROS under LIFU irradiation was concentration-dependent and time-dependent.CCK-8 assay and staining of living and dead cells,and flow cytometry showed that the killing effect of t Ly P-1@H(Gd)-GOD@PFP NPs + LIFU group was higher than that of other groups.And the ability of producing ROS in t Ly P-1@H(Gd)-GOD@PFP NPs + LIFU group observed by CLSM observation was also significantly better than that of other groups.The tumor growth of t Lyp-1@H(Gd)-GOD@PFP NPs + LIFU group was completely inhibited,and there was no significant change in the weight of nude mice during the treatment.H&E staining of the tumor tissue showed that the number of necrotic cells and apoptotic cells in t Lyp-1@H(Gd)-GOD@PFP + LIFU group was more than that in other groups.PCNA and TUNEL staining also showed that the cell proliferation index was significantly decreased and the apoptosis index was increased significantly(P <0.05).The main organs(heart,liver,spleen,lung and kidney)of tumor bearing nude mice showed no obvious damage.Conclusion Under the specific targeting effect of t Ly P-1,H(Gd)and GOD could reach the tumor tissue accurately.Under the irradiation of LIFU,the drug can be released by phase transition,and the combination of ROS and "starvation" therapy can achieve multifunctional therapeutic effect.