Attenuated Salmonella Typhimurium Mediated Tumor Targeting Imaging Based On Peptides

Nowadays,cancer（malignant tumor）is one of the major threats to human health.Surgery,radiotherapy,chemotherapy and traditional Chinese medicine treatment are often used in the clinical cancer treatment.Although these methods have partly solved the need for cancer treatment,cancer still worries people because of the difficulty in early detection and eradication.The early detection of tumor is crucial for its treatment.The therapeutic efficiency of cancer will be greatly improved if cancer could be diagnosed early upon the onset.Therefore,it is very important to develop an early diagnosis method for malignant tumors.In recent years,with the continuous development of biomedical engineering technology,biological materials and biological methods have been widely used in the diagnosis and treatment of tumors.In this dissertation,based on the previous studies on the specific proliferation of some bacteria in the hypoxic tumor microenvironment,we developed an innovative strategy for the early detection of tumors using bacterial targeting peptides carrying imaging agents(Cy5.5 or ¹²⁵I).In this paper,attenuated salmonella typhimurium is selected as the tumor-targeting bacteria.The antibacterial peptide Ubiquicidine 29-41（UBI）which could target attenuated salmonella typhimurium was labeled with the fluorescent dye Cy5.5 or the radionuclide iodine-125 for dual-modal fluorescence and nuclear imaging of tumors.The main results of this dissertation are summarized as following:Chapter 1:This chapter is an introductive overview that summarizes the current progress of tumor imaging technology,the application of bacteria in the treatment of tumor and the anti-tumor mechanism of attenuated salmonella typhimurium.Moreover,the biological mechanism of antimicrobial peptides and research progress of antimicrobial peptide UBI are also summarized.Chapter 2:Verification of the targeting ability of antibacterial peptide UBI to attenuated salmonella typhimurium in vitro.Cy5.5-UBI was prepared by coupling the antibacterial peptide UBI with the fluorescent dye Cy5.5 through amidation reaction,and then purified through a Sephadex^TMG-25M dextran gel column.The Cy5.5-UBI solution was co-cultured with attenuated salmonella typhimurium in vitro,and the targeting ability of the antibacterial peptide UBI to attenuated salmonella typhimurium in vitro was evaluated.It was found that the antibacterial peptide UBI could significantly enhance the efficiency of fluorescent dye to label attenuated salmonella typhimurium compared with the control group.In addition,antibacterial peptide UBI was labeled with radioisotope ¹²⁵I in the presence of Iodogen.The obtained ¹²⁵I-UBI solution was purified through Sephadex^TM G-25M dextran gel column for the following experiments.Chapter 3:Investigation of the tumor targeting ability of attenuated salmonella typhimurium.We discovered that the attenuated salmonella typhimurium could selectively proliferate in the tumor sites after intravenous injection based on the in vivo autofluorescence experiments of attenuated salmonella typhimurium and Gram staining of tissue sections.The toxicity of attenuated salmonella typhimurium in Balb/c mice was investigated by bacteria plating experiments and hematoxylin-eosin（H&E）staining.Chapter 4:Attenuated salmonella typhimurium mediated tumor targeting imaging based on peptides.On the basis of the targeting ability of attenuated Salmonella typhimurium to tumor hypoxia microenvironment and antibacterial peptide UBI to attenuated Salmonella typhimurium,we conducted the in vivo fluorescence imaging and SPECT imaging using Cy5.5 or ¹²⁵I labeled UBI as contrast agents in two-independent tumor models（4T1 and CT 26）.In summary,we proposed a strategy for tumor-targeted imaging using fluorescent dye or radioisotope-labeled antimicrobial peptides in combination with attenuated salmonella typhimurium.Attenuated salmonella typhimurium with facultative anaerobic properties could specifically proliferate in tumor sites after intravenous injection,while does not proliferate in normal tissues of mice.The antibacterial peptide UBI could target to the attenuated salmonella typhimurium both in vitro and in vivo.After the treatment of attenuated salmonella typhimurium and antibacterial peptide UBI labeled with fluorescent dye Cy5.5 or radionuclide ¹²⁵I,in vivo fluorescence imaging and SPECT/CT imaging showed that peptide UBI could specifically target the two independent tumor models of mice with infection of attenuated salmonella typhimurium,achieving multimodal imaging of tumor.Therefore,this strategy（bacteria-mediated tumor targeting）can remarkably improve the imaging sensitivity for the early detection of tumors.We hope that this strategy can be further extended to the field of cancer treatment in the future and bring good news to cancer patients.