Design,Synthesis And Imaging Of Optical-Magnetic Resonance Dual-Mode Targeting Probes For Colorectal Cancer

Colorectal cancer（CRC）is the third most common type of tumor and the second most common cause of cancer death worldwide.However,the difficulty of early and accurate diagnosis is a huge challenge facing by the clinical diagnostic techniques of CRC.It is of great significance to achieve early and precise diagnosis of CRC to improve survival rate of patients.Molecular imaging,as an emerging technology in recent years,has opened up new perspectives for diagnosis of cancer,providing more accurate diagnosis strategy.The precise targeting strategy of CRC bio-markers is the key to achieve early diagnosis of CRC,which needs to carefully examine the in vivo properties of molecular probes,such as,targeting ability,binding efficiency to bio-marker,stability,clearance time by metabolic system,toxic side effects,etc.Meanwhile,it is also necessary to combine different imaging techniques in molecular imaging,using their benefits to design corresponding targeted imaging strategies for CRC.Lastly,molecular imaging technology is efficiently used to significantly improve the sensitivity and specificity of the diagnosis of CRC.In this study,three different molecular imaging strategies have been investigated and effectively validated in cell and animal models.The main research contents and conclusions are as follows.First,we synthesized four NaErF₄:Yb@NaGdF₄:Yb core@shell nanoprobes（the doping ratio of Er and Yb elements,80%:20%,85%:15%,90%:10%and 95%:5%,respectively）,in which the upconversion nanoparticles（UCNPs）（Er:Yb,85%:15%）provided higher upconversion luminescence（UCL）signal in the red region.The prepared UCNPs were transferred from the organic phase to the aqueous phase by coating the Si O₂ shell and coupled with two polypeptide ligands derived from the L-SP5 polypeptide（i.e.,L-SP5-H and L-SP5-C）,respectively.As the PSP motif in the polypeptide ligand has high affinity for tumor targeting,the prepared polypeptide-functionalized UCNPs（UCNP@Si O₂-L-SP5-H and UCNP@Si O₂-L-SP5-C）can be used as in vivo tumor-targeting contrast agents for UCL and magnetic resonance（MR）dual-modality imaging.The results of in vitro and in vivo experiments showed that the nanoprobe UCNP@Si O₂-L-SP5-C modified by L-SP5 peptide had relatively high affinity with HCT116 CRC subtype,and it achieved in vivo imaging of ultra-small CRC（HCT116）subcutaneous tumors（c.a.13 mm³ in volume）.In the second work,based on UCNP@SiO₂@L-SP5-C probe,we successfully synthesized a fluorescence resonance energy transfer（FRET）sensing platform,namely UCNP@Si O₂@L-SP5-C/MMP-7-Cy5 nanoprobe,using MMP-7,which is highly expressed in CRC.In the probe,UCNP@Si O₂-COOH serves as an energy donor,the Cy5 serves as an energy acceptor,and the probe was used for detecting the activity of MMP-7 of HCT116 tumor in vitro and in vivo.In addition to L-SP5-C tumor-targeting peptide,adding MMP-7 protease-responsive peptide significantly improved the detection sensitivity.The probe also clearly distinguished different level of MMP-7expression in different number of HCT116 cells with decent accuracy.Finally,the probe was proved to achieve noninvasive monitoring the MMP-7 activity in the HCT116orthotopic CRC mouse model by UCL-MR dual modality imaging.Lastly,in order to solve the problem of organ accumulation and potential toxicity of rare earth nanoparticles,this research used the second near infrared window（NIR-Ⅱ）dye,indocyanine green（ICG）and TQT1009.They were conjugated with the classic triphenylphosphonium（TPP）cation derivative,which is known for its mitochondria targeting ability.ICG-TPP was obtained by direct coupling TPP with ICG,while TQP-2TPP was obtained by conjugation TQT1009 with two TPP molecules.The specific targeting ability of ICG-TPP and TQP-2TPP to CRC HCT116 cells in vitro was proven by the uptake experiment of colon cancer cells and laser confocal microscopy imaging.The results showed ICG-TPP and TQP-2TPP specifically accumulated in the mitochondria of HCT116 cells.It was proven that both probes showed no obvious cytotoxicity and in vivo toxicity.Compared with ICG-TPP,TQP-2TPP presented better metabolic characteristics through in vivo experiment,and it had higher accumulation and longer retention in HCT116 tumors,which indicates TQP-2TPP has better targeting ability for HCT116 colon cancer.The study demonstrates a new method for the early diagnosis of CRC using NIR-Ⅱ organic small molecule probes.In summary,aiming at the difficulties in the early and precise diagnosis of CRC,three different molecular imaging probes have been developed based on L-SP5-C tumor-targeting peptide,MMP-7 protease-responsive peptide and mitochondrial targeting molecule TPP,and they have been used to image HCT116 CRC in vitro and in vivo.The research provides new strategies for early and accurate diagnosis of CRC.