In Vivo Fluorescence Imaging Of Solid Tumors Using Aptamer

In malignant tumors with high incidence and mortality, solid tumors account forthe majority, which are seriously endangering human health and life. The explorationof novel methods and techniques for early diagnosis and accurate classification ofsolid tumors has now become one of the focused problems to be urgently solved intumor medicine research field. In recent years, the emergence of molecular imagingtechnology, which generally adopts specific molecular probes to perform the in vivo,real-time and in-situ imaging studies, provides an opportunity to realize thequalitative and quantitative analysis of molecular-level biology behaviors in tumoroccurrence and development. This holds great potentials to advance the tumordetection level to the molecular abnormity stage so as to satisfy requires for earlydiagnosis and classification. As a novel and promising kind of tumor-targetedrecognition molecule, aptamer has a series of advantages like high affinity, excellentspecificity, facile synthesis, easy modification and little immunogenicity, thusaffording an ideal molecular probe for developing novel in vivo tumor imagingtechniques. Aiming at this frontier study direction, this thesis selected solid tumorsincluding lung cancer and liver cancer as research objects and constructed severaltumor imaging probes using aptamers generated by cell-SELEX as target recognitionmolecules. Through combination with the in vivo fluorescence imaging technique, thereal-time and in-situ fluorescence imaging study of solid tumors inside mice wassystematically conducted. The two parts discussed in this thesis are as follows:1. Cy5dye-labeled “always on” aptamer for in vivo fluorescence imaging ofseveral solid tumorsConsidering that the in vivo tumor imaging research of aptamer evolved bycell-SELEX mainly focuses on blood tumors like B lymphoma cells and humanleukemia cells, A549non-small-cell lung cancer was firstly selected as the model. Byadopting its specific aptamer S6as the target recognition molecule, S6was directlylabeled with the NIR fluorescent dye Cy5so as to successfully construct the "alwayson" imaging probe Cy5-S6for in vivo A549tumor detection. Results of flowcytometry analysis showed that Cy5-S6exhibited specific detection performance forboth in vitro cultured A549cells in buffer and ex vivo cultured A549cells in mouseserum. In vivo fluorescence imaging results also revealed that Cy5-S6not only held the targeted imaging ability for A549tumors inside mice, but also exhibited excellentsequence specificity and tumor selectivity. On this basis, in order to further expandapplications of aptamer generated by cell-SELEX for in vivo imaging other tumors,Bel-7404and SMMC-7721liver cancers were chosen as the study objects. Using thespecific aptamers screened in our laboratory, Cy5-LS2probe for Bel-7404tumorimaging and Cy5-ZY8for SMMC-7721tumor imaging were successfully constructed.Through a series of in vivo and in vitro investigations, it was found that these probescould not only achieve the specific detection and imaging of target tumor cells in vitroand in vivo, but also effectively distinguish different types of liver cancer cells in thesame mouse body. This study not only extends the application of aptamer generatedby cell-SELEX as novel in vivo tumor imaging probeto the solid tumor research field,but also strongly supports the application of cell-SELEX to screen specific molecularimaging probes.2. Acid-activatable aptamer probe for in vivo fluorescence imaging of A549non-small-cell lung cancerAiming at the drawbacks of above "always on" probes for in vivo fluorescencetumor imaging, such as high background signal, limited imaging contrast and longdetection time, the acidic environment of tumor sites and lysosomes in tumor cellswere selected as the target stimulation. Using A549non-small-cell lung cancer as themodel, an acid-activatable aptamer probe (AAAP) simultaneously with targetrecognition capability and signal swithable mode was successfully constructed byadopting an acid-labile molecule2,4,8,10-tetraoxaspiro undecane-3,9-dipropanamine (ATU) to connect the Cy5-S6and BHQ3quencher. At the neutralcondition, the fluorescence signal of AAAP is highly quenched, and after incubationin pH4.5buffer for24hours, its fluorescence recovery efficiency could reach over90%. Through laser confocal microscopy imaging investigation, it was found thatAAAP could achieve the specifically activated fluorescence imaging in comparisonwith negative control probe and negative tumor cells. In vivo fluorescence imagingresults also showed that after injected into A549tumor site, AAAP exhibited theactivatable imaging function with the fluorescence signal changed from weak tostrong and substantially decreased the fluorescence background in comparison with"always on" probe. However, a further investigation of AAAP for in vivo activatablefluorescence imaging through the intravenous injection revealed that AAAP couldachieve the specific imaging of A549tumors inside mice, but the imaging contrastwas still on demand of improvement resulting from its quick excretion from the body.