The Study Of Ultrasound Molecular Imaging And Therapy For Breast Cancer In Vitro Using Drug-loaded Phase-transformation Nanparticles Mediated By Tumor Homing-penetrating Peptide

PART? THE STUDY OF SYNTHESIS AND ACTIVITYIN VITRO OF TUMOR HOMING-PENETRATINGPEPTIDEObjective To synthesize a new tumor homing-penetrating peptide t Ly P-1(CGNKRTR)with fluorescein isothiocyanate(FITC)by solid-phase synthesis method.To investigate tumor targeting and cell-penetrating properties of FITC-t Ly P-1 in vitro.Methods The FITC-t Ly P-1 was solid-phase synthesized by the Na-fluorene methoxyl carbonyl method(Fmoc),and its N-terminal was labeled by FITC.The purity and molecular weight of the FITC-t Ly P-1 were determined using HPLC and MALDI-TOF-MS.The tumor homing and cell-penetrating properties in vitro of FITC-t Ly P-1 were examined by confocal laser scanning microscopy(CLSM)and flow cytometry(FCM).Three-dimensional tumor spheroids were established and the tumor homing-penetrating property of FITC-t Ly P-1 in three-dimensional tumor spheroid was examined by CLSM.The cytotoxicity of the FITC-t Ly P-1 was evaluated by CCK8 assay.Results The purity of FITC-t Ly P-1 was 99.14%,and molecular weight was 1334.7.CLSM showed that FITC-t Ly P-1 was aggregated to cell membrane of MDA-MB-231 and penetrate into the cell,but not to HUVEC.FCM showed that the intracellular fluorescence intensity in MDA-MB-231 group was higher than that of HUVEC group.Three-dimensional spheroids of MDA-MB-231 cells were penetrated deeply about 120 ?m by FITC-t Ly P-1.The CCK8 assay indicated that different concentrations of FITC-t Ly P-1 had no significant effects on cell activity(P>0.05).Conclusion A new tumor homing-penetrating peptide with FITC(FITC-t Ly P-1)was successfully synthesized by solid-phase synthesis method.FITC-t Ly P-1 had the tumor homing and cell-penetrating properties in vitro,which could be used as a potential carrier for tumor targeting and drug transporting.PART? THE STUDY OF PREPARATION,TUMORTARGETING AND CELL-PENETRATING PROPERTIESIN VITRO OF DRUG-LOADEDPHASE-TRANSFORMATION NANOPARTICLESMEDIATED BY TUMOR HOMING-PENETRATINGPEPTIDEObjective To prepare a novel ultrasound molecular probe,10-HCPT-loaded phase-transformation nanoparticles mediated by t Ly P-1 peptide(t Ly P-1-10-HCPT-PFP NPs).To detect characteristics and evaluate tumor targeting and cell-penetrating properties of t Ly P-1-10-HCPT-PFP NPs in vitro.Methods t Ly P-1-10-HCPT-PFP NPs were prepared by filming-rehydration and acoustic-vibration methods.The morphology,distribution,particle size and zeta potential were detected.The tumor homing and cell-penetrating properties of t Ly P-1-10-HCPT-PFP NPs were examined by CLSM and FCM.Three-dimensional tumor spheroids were established and the tumor homing-penetrating properties of t Ly P-1-10-HCPT-PFP NPs in three-dimensional tumor spheroids were examined by CLSM.Results The size and distribution of t Ly P-1-10-HCPT-PFP NPs were uniformed.The size and zetapotential of t Ly P-1-10-HCPT-PFP NPs were(366.30±11.50)nm and(5.70±3.70)m V,respectively.CLSM showed that t Ly P-1-10-HCPT-PFP NPs were aggregated to cell membrane of MDA-MB-231 and penetrate into the cell,but not to HUVEC.10-HCPT-PFP NPs were not aggregated obviously to cell membrane of MDA-MB-231 or HUVEC.FCM showed that intracellular fluorescence intensity of t Ly P-1-10-HCPT-PFP NPs in MDA-MB-231 group was higher than other groups(P<0.05).CLSM also showed that three-dimensional spheroid of MDA-MB-231 cells was penetrated deeply about 80 ?m by t Ly P-1-10-HCPT-PFP NPs.While only a little of 10-HCPT-PFP NPs were around the surface of spheroid,and penetrated only about 25 ?m.Conclusions A novel ultrasound molecular probe,10-HCPT-loaded phase-transformation nanoparticles mediated by t Ly P-1 peptide(t Ly P-1-10-HCPT-PFP NPs)was prepared successfully.It could target to MDA-MB-231 cell and penetrate into the cell and three-dimensional spheroids in vitro,which laid a solid foundation for subsequent studies.PART ? THE STUDY OF ULTRASOUND IMAGINGAND TUMOR THERAPY IN VITRO USINGDRUG-LOAED PHASE-SHIFT NANOPARTICLESMEDIATED BY TLYP-1 PEPTIDEObjective To prepare a novel ultrasound molecular probe,10-HCPT-loaded phase-transformation nanoparticles mediated by t Ly P-1 peptide(t Ly P-1-10-HCPT-PFP NPs).To observe enhancement effect for ultrasound imaging in vitro and evaluate the treatment effect on breast cancer MDA-MB-231 cells of t Ly P-1-10-HCPT-PFP NPs.Methods The envelopment rate and 10-HCPT-loading rate of t Ly P-1-10-HCPT-PFP NPs were detected by HPLC.After heating by heating plate and irradiating by low intensity focused ultrasound(LIFU),the phase-transformation characteristic and the enhancement effect for ultrasound imaging in vitro were observed.The early and late apoptosis of MDA-MB-231 cells treated by nanoparticles were observed by FCM.The cytotoxicity of the nanoparticles on MDA-MB-231 cells was evaluated by CCK8 assay.Results The envelopment rate and 10-HCPT-loading rate of t Ly P-1-10-HCPT-PFP NPs were 86.04±4.27% and 7.82±0.38%,respectively.When t Ly P-1-10-HCPT-PFP NPs were heated by heating plate at 45 ? or irradiated by LIFU,nanoparticles generated phase-transformation and enhanced ultrasound imaging in vitro(P<0.05).Combined with LIFU,the early and late apoptosis of MDA-MB-231 cells were enhanced obviously(P<0.05),the cytotoxicity of t Ly P-1-10-HCPT-PFP NPs on MDA-MB-231 cells also increased obviously(P<0.05),which were higher than that of other groups(P<0.05).Conclusion Combined with LIFU,t Ly P-1-10-HCPT-PFP NPs enhanced the ultrasound imaging in vitro.The drugs in the nanoparticles were released by LIFU,which achieved effective treatment effect.