Preparation Of EGFR-targeted Nanoprobes And Application In The Diagnosis Of Breast Cancer Brain Metastases

Part I Characterization of GE11 peptide with affinity for EGFR-overexpressing human breast cancer brain metastasesObjective To investigate GE11 peptide targeting property of human epidermal growth factor receptor(EGFR)positive breast cancer brain metastases,and find suitable target groups for the construction of molecular nanoprobe specific targeting breast cancer brain metastases.Methods The EGFR expression level of triple negative breast cancer cell lines(MDA-MB-231-BR)was detected using Western blot and flow cytometry.GE11 peptide targeting property was tested using cell immunofluorescence staining and flow cytometry detection in MDA-MB-231-BR cell and in the subcutaneous tumor model.Results Western blot and flow cytometry examination results showed that EGFR cell binding percent in MDA-MB-231-BR cell was 88.6%,significantly higher expressed than other cells.Cell flow cytometry test results indicated that the cell binding percent with 10?mol/L concentration GE11 peptide was 38.4%,significantly higher than control peptide with same concentration.Cell immunofluorescence staining results showed GE11 peptides can be detected stronger signal after incubation with cells,and its combination with the energy and dose dependence.Experiment results show that the GE11 peptide has stronger signal in tumor site,other organizations signal is weak.Conclusion GE11 peptide can specific target with EGFR positive breast cancer brain metastases,laid the foundation of building targeting molecular nanoprobe for breast cancer brain metastases.Part II Construction,characterization and targeting property verification of EGFR-targeted molecular imaging nanoprobes Objective Breast cancer brain metastases is one of the most serious outcomes with high mortality and poor prognosis,especially the triple negative breast cancer.It is difficult to diagnosis of brain metastases in the early stage or smaller lesions with the existing clinical diagnostic methods.This study aims to synthesis an EGFR-targeted and blood-brain-barrier permeable dual-modality molecular imaging nanoprobes as the real time and noninvasive imaging for accurate detection of early breast cancer brain metastases.Methods Designing and synthesising of an EGFR-targeted molecular imaging nanoprobes used PAMAM dendrimers as the carrier,GE11 peptide as EGFR targeting peptide,Angiopep2 for brain targeting peptide,Gd3+-DTPA as MR imaging agent,IR783 for near-infrared fluorescence imaging agents.Using triple negative breast cancer brain metastasis cell line(MDA-MB-231-BR),human glioma cell line(U87)and breast ductal cancer cell(BT-474)to verify the specificity of molecule nanoprobe targeting EGFR in vivo.Using triple negative breast cancer brain metastasis cell line(MDA-MB-231-BR)to establish a mouse model of breast cancer brain metastases and image with magnetic resonance imaging and near infrared imaging in vivo.Results(1)Cell immunofluorescence staining results showed that Den-Angio-GE11 nanoprobe group can be detected stronger signal after incubation with cells,and its combination with the energy and dose dependence;MD-MB-231-BR and U87 cell lines can be detected stronger signal than the BT-474 cell after incubation with Den-Angio-GE11 nanoprobe.(2)Flow cytometry examination results showed that Den-Angio-GE11 nanoprobe binding percent in MDA-MB-231-BR cell was 64.2%with the concentration of 5?mol/L and was significantly higher expressed than other nanoprobes,Den-Angio was 22.7%,Den-PEG was 21.6%(P<0.05).The binding percent of U87 cell was 47.5%after incubation with Den-Angio-GE11 nanoprobe,BT-474 cell was 0.7%.(3)The magnetic resonance and near infrared imaging of MDA-MB-231-BR cells results showed that Den-Angio-GE11 group signal intensity was significantly higher than control nanoprobes.(4)After injecting nanoprobes 2h,the near infrared imaging of breast cancer brain metastases-bearing mouse model showed that Den-Angio-GE11 group's fluorescence signal was significantly higher than control nanoprobes.(5)After injecting Den-Angio-GE11 nanoprobe 5min,the T1-weighted magnetic resonance imaging of metastases-bearing mouse model showed the signal enhancement in the brain metastases,none of control nanoprobe groups.Conclusion The EGFR-targeted molecular imaging nanoprobe can realize the metastases lesions of breast cancer brain metastases-bearing mouse model with magnetic resonance imaging and near infrared imaging in vivo.As a real time and noninvasive imaging,this nanoprobe has potential in the early diagnosis of breast cancer brain metastases.