Studies On Tumor-Targeting Agents For Fluorescence Imaging And Positron Emission Computed Tomography

In recent years,the malignant tumors have become one of the serious problems to the human health.The early accurate screening and diagnosis of precancerous lesions is crucial to the treatment of cancers.However,most of the early symptoms of cancer are not obvious and the scope of the disease is small,and then the existing medical techniques are often difficult to achieve accurate diagnostic results.It is the key to improve the early diagnosis of cancer and reduce the mortality by looking for a diagnostic method with high specificity and sensitivity.Positron emission computed tomography?PET?can provide more detailed anatomic information,such as receptor function,metabolism,and so on.Fluorescence imaging?FI?possesses the high detection sensitivity and then can effectively distinguish between tumor and normal tissue.Therefore,PET and FI technology have become a research hotspot in the tumor detection and diagnosis.However,the current clinically used of the PET tracers(such as¹⁸F-FDG and¹⁸F-Al-NOTA)and fluorescence imaging probes?such as fluorescein and rhodamine B?distribute in the body according to the different needs of the metabolism.They do not have the tissue or organ selective or targeting property,especially no specific selective to tumors.Moreover,these agents possess the poor imaging effect on tumor,fast metabolism,short retention time and low utilization rate in vivo.Therefore,the tumor-targeting imaging agents will be an important development trend of cancer imaging diagnosis by using PET and FI techniques.In this thesis,a series of novel tumor-targeting PET tracers and FI probes were designed and synthesized.The working principle of PET and FI and the application of imaging agents were reviewed herein.The research progress of PET tracer and FI probe were discussed in detail.The tumor specific affinity and targeting properties of polypeptides and the research progress of current bifunctional imaging technology were introduced briefly.YIGSR?Tyr-Ile-Gly-Ser-Arg?is a pentpolypeptide as a core sequence located at the?-1 chain that can promote cell adhesion and inhibit the growth and metastasis of cancer cells.So YIGSR can be potentially used as a good tumor-targeting group in anti-tumor drugs.A pentpolypeptide-rhodamine B derivative?YIGSR-RhB?was synthesized by the bound of rhodamine B?RhB?to YIGSR as a tumor-targeting group using the solid phase synthesis method with Fmoc protected amino acids as the raw materials.The derivative was further characterized by Mass spectrometry?MS?,Nuclear magnetic resonance spectroscopy?¹H NMR?,Fourier transform infrared spectroscopy?FT-IR?,Ultraviolet-visible spectroscopy?UV?,and so on.The biological experiments including the cell cytotoxicity,cell uptake and fluorescence imaging of tumor cells,and fluorescence imaging in BALB/c-nu female mice with 4T1 tumors in vivo were also measured.Experiment results demonstrated that YIGSR-RhB possessed the low cell cytotoxicity,good tumor-targeting property and fluorescence properties similar to rhodamine B.Moreover,YIGSR-RhB can be taken up highly by the B16F10 melanoma cells and 4T1breast cancer cells,achieve the good red fluorescence imaging in tumor cells and also enhance the contrast of fluorescence images of 4T1 tumors in mice.Therefore,YIGSR-RhB can be used as a potential tumor-targeting probe for fluorescent imaging in vivo.Tripeptide?Arg-Gly-Asp,RGD?is a common amino acid sequence of the extracellular matrix?ECM?and adhesion proteins in cells.It can be taken up by tumor cells through the receptor-mediated mechanism.A tripeptide-rhodamine B derivative?RGD-RhB?was synthesized by the attachment of rhodamine B?RhB?to RGD as a tumor-targeting group using the solid phase synthesis method with Fmoc protected amino acids as the raw materials.The derivative was further characterized by MS,¹H NMR,FT-IR,UV,etc..Its cell cytotoxicity,cell uptake of tumor cells and fluorescence imaging were also measured.Compared with rhodamine B,RGD-RhB possessed the low cell cytotoxicity,good tumor-targeting property and similar fluorescence properties.Moreover,RGD-RhB can be taken up highly by the B16F10 melanoma cells and 4T1 breast cancer cells,achieve the good red fluorescence imaging in B16F10 melanoma cells and enhance the contrast of fluorescence images of 4T1 tumors in mice.A porphyrin-1,8-naphthalimide derivative was synthesized by the attachment of1,8-naphthalimide to 5-?4''-aminophenyl?-10,15,20-[tri?4''-sulfonic acid?-phenyl]porphyrin?APTSPP?as a tumor-targeting group.The derivative was further characterized by MS,¹H NMR,FT-IR,UV,etc..Its cell cytotoxicity,cell uptake of tumor cells and fluorescence imaging were also measured.Experiment results indicated that porphyrin-1,8-naphthali-mide possessed the low cell cytotoxicity,and good tumor-targeting property.Moreover,porphyrin-1,8-naphthalimide can be taken up highly by the B16F10 melanoma cells and then achieve the good green and red fluorescence imaging in B16F10 melanoma cells.Therefore,porphyrin-1,8-naphthalimide can be used as a potential tumor-targeting probe for fluorescent imaging in vivo.A PET ligand?NOTA-YIGSR?was synthesized by the incorporation of 1,4,7-triazacyclodecane-1,4,7-triacetic acid?NOTA?to YIGSR as a tumor-targeting group.Subsequently,⁶⁸Ga-NOTA-YIGSR was prepared by the chelation reaction of NOTA-YIGSR with⁶⁸Ga.These compounds were characterized by¹H NMR,UV,FT-IR,MS,etc.⁶⁸Ga-NOTA-YIGSR was further analyzed and purified by HPLC and its cell cytotoxicity was also measured.These results indicated ⁶⁸Ga-NOTA-YIGSR possessed the low cell cytotoxicity and its⁶⁸Ga-labeled ratio was 92.3%,radiochemical purity was over 95%.So these properties have met the requirements for the purity and dosage of radioactive tracers.Therefore,⁶⁸Ga-NOTA-YIGSR can be further used as a PET tracer to do the cell experiments in vitro and PET imaging in vivo.A PET ligand compound?NOTA-RGD?was synthesized by the condensation reaction between 1,4,7-triazacyclodecane-1,4,7-triacetic acid?NOTA?and RGD as a tumor-targeting group.The compound was also characterized by ¹H NMR,FT-IR,MS,etc.Its cell cytotoxicity,particle size and Zeta potential in aqueous solution were further measured.These results indicated NOTA-RGD possessed the low cell cytotoxicity to B16F10melanoma cells.NOTA-RGD was expected to chelate with⁶⁸Ga and¹⁸F to make⁶⁸Ga,¹⁸F-labeled NOTA-RGD,which can be further used as the PET tracers to do the cell experiments in vitro and PET imaging in vivo.A water-soluble dextran PET ligand?NOTA-Dextran-YIGSR?was synthesized by the incorporation of NOTA and YIGSR to dextran as a polymer carrier.This compound was also characterized by¹H NMR,UV,FT-IR,etc.its cell cytotoxicity assay,particle size and Zeta potential in aqueous solution were further measured.Experimental data showed that NOTA-Dextran-YIGSR possessed the good water solubility and low cell cytotoxicity to B16F10 melanoma cells.NOTA-Dextran-YIGSR was expected to chelate with ⁶⁸Ga and¹⁸F to produce⁶⁸Ga,¹⁸F-labeled NOTA-Dextran-YIGSR,which can be further used as macromolecular PET tracer to do the cell experiments in vitro and PET imaging in vivo.A water-soluble hyaluronic acid PET ligand?NOTA-HA-YIGSR?was synthesized by the attachment of NOTA and YIGSR to hyaluronic acid?HA?as a polymer carrier.This compound was also characterized by ¹H NMR,UV,FT-IR,etc.Its cell cytotoxicity assay,particle size and Zeta potential in aqueous solution were further measured.Experimental data indicated that NOTA-HA-YIGSR possessed the good water solubility and low cell cytotoxicity to B16F10 melanoma cells.NOTA-HA-YIGSR was expected to chelate with⁶⁸Ga and¹⁸F to make⁶⁸Ga,¹⁸F-labeled complex,which can be further used as a macromolecular PET tracer to do the cell experiments in vitro and PET imaging in vivo.A water-soluble polyaspartamide FI/PET dual-mode ligand?NOTA-PHEA-RhB-YIGSR?was synthesized by the incorporation of NOTA,RhB and YIGSR as a tumor-targeting group to poly-?,?-[N-?2-hydroxyethyl?-L-aspartamide]?PHEA?.This compound was characterized by IR,¹H NMR,UV,and so on.Its cell cytotoxicity,fluorescence property,particle size and Zeta potential in aqueous solution were further measured.Experiment results demonstrated that NOTA-HA-RhB-YIGSR possessed the low cell cytotoxicity to B16F10 melanoma cells,fluorescence properties similar to rhodamine B and homogeneous dispersion in aqueous solution.NOTA-PHEA-RhB-YIGSR was expected to chelate with⁶⁸Ga and¹⁸F to produce⁶⁸Ga,¹⁸F-labeled complex,which can be further used as macromolecular dual-mode imaging agent to do the cell experiments in vitro and FI and PET imaging in vivo.