Functional DNA Nanostructures As Dual-modality Imaging Probes: Study On The Preparation And Biological Properties

The method of molecular imaging is a class of non-invasive technology that couldachieve in situ observation, identification and measurement of biological processes atthe molecular or cellular level in vivo. The diagnostic and visualization of diseases onthe molecular level could be understood as detecting the diseases at their infancy, whichcould greatly enhance the efficiencies of the treatment and healthcare of patients. Nowmost commonly used molecular imaging probes includes small molecules, peptides andproteins, and recently nanoparticles gradually attracted researchers’ attention, mainlydue to their great biocompatibility, excellent function ability, and good capacity forcoupling a variety of function imaging modalities.Among different nanoparticles, DNA nanoparticles with excellent spatialaddressability, could be functionalized at designated direction or orientation in3dimensional space, therefore particular needs of platform of molecular imaging probescould be met by DNA nanoparticles. Thus we aimed to develop one of DNAnanoparticles as dual-modality imaging probes.We first prepared a series of tetrahedral DNA nanoparticles, and we developed twodifferent HPLC (High performance liquid chromatography) methods for thepurification of the as-prepared DNA nanoparticles. Taking advantages of the HPLCmethods, we have readily purified all7kinds of tetrahedral DNA nanoparticles. Thenthe UV quantification method of purified DNA nanoparticles have been developed, andwe have constructed a series of DNA complexes by a side chain hybridization strategybased on the purified DNA nanostructures. We have compared the differences ofconstructing bigger DNA nanostructures before of after the HPLC purification process, and results suggested that only purified DNA nanocage monomers could be well suitedfor bottom-up construction of bigger DNA nanocomplexes, and the as-preparedstructures would find potential applications in the field of biosensing.Then we have labeled tetrahedral DNA nanostructures (TDN) with near infrared(NIR) fluorophores (Dylight755) and radioactive isotopes (Technetium-99m). With thehelp of agarose gel electrophoresis (AGE) and fluorescent energy resonance transfer(FRET), we have proved that TDN with side chains on the edges could survive for atleast12hours in the environment with high concentration of mice serum. Then usingtwo labeling methods (NIR and radio-labeling methods), we examined the biologicalproperties of TDN, such as the plasma half-life and in vivo pharmacokinetics; resultsshowed that TDN and double-strand (ds) DNA shared a quite different in vivo behaviors,the plasma half-life of TDN in mice was about6min,2times longer that dsDNA; andTDN in mice was mainly accumulated in the liver, kidney and spleen, and excretedprimarily in the urine. Finally, with the use of folic acids as tumor targeting groups, wehave successfully achieved the fluorescent imaging and SPECT/CT imaging of TDN intumor-bearing mice, results suggested that TDN would exhibited better accumulationwithin the tumor site when conjugated with folic acids.In addition, graphene oxide (GO), as a kind of carbon-based nanostructures, haveexhibited great prospects in the field of biomedicine, therefore we have used radioactiveisotopes (technetium-99m) to radiolabel GO nanosheets in order to develop GO as aneffective platform for molecular imaging, as well as to pave the path for subsequentbiological researches. We first synthesized graphene oxide nanosheets with a plane sizeof about500nm, following the click chemistry between GO nanosheets with a bi-functional chelator (DOTA in this case), the labeling of GO nanosheets with Tc-99mcould be achieved by a reduction reaction. The whole labeling process would take about30min, and the radiochemistry yield was higher than90%. We hope the99mTc-DOTA-GO nanosheets prepared by this method could be further developed for potentialclinical applications. In the final part, we compared two F-18labeling methods of peptides, and hope toprovide a reference for the subsequent F-18radiolabeling of peptide. We have used[18F]SFB (N-succinimidyl4-[18F]fluorobenzoate) and [18F]AlF for labeling of RGDfkpeptides, respectively. Then labeling results, in vitro and in vivo stability, andbiodistribution profiles in tumor-bearing mice of two labeling peptides have beencompared. Two labeling peptides showed few differences in their stability andbiological properties in vitro or in vivo. Two methods could both achieve F-18labelingof peptides with high efficiency. With further development, We believe that bothmethods could provide more possibilities for molecular imaging.