Abstract
1069
Background: DNA nanostructures, with the advantages of structural designability and spatial addressability, have shown great potential in the field of bio-sensing, drug delivery, and bio-medicine.
Methods: Equimolar quantities (1 μM) of six DNA single strand oligonucleotides (A, B, C, D, E, and F) were mixed in TM buffer, heated to 95 oC for 10 min, and then cooled to 4 oC for 20 min. PAGE was conducted to analyze the products of the single-step annealing process of DCNs. DCNs were purified by HPLC with a size exclusive chromatography (SEC) column. S-acetyl-MAG3-NHS was conjugated with NH2-A(20) ssDNA, and the product MAG3-A(20) was radiolabeled by technetium-99m with a single reduction reaction to obtain 99mTc-A(20) ssDNA. 99mTc-DCNs were obtained by mixing T20-DCNs with 99mTc-A(20) ssDNA in PBS. Radio-HPLC was used for analysis and purification of radiolabeled DCNs. The biodistribution and plasma half-life of DCNs was conducted by injecting 200 uL 99mTc-DCNs (~ 10 μCi) into KM mice (n=5 in each group) via tail vein. Interested tissues or organs were collected to measure radioactivity by a γ-counter at 60 sec counting periods. KM mice (n=5) were injected with 99mTc-DCNs (~ 500 μCi/ each mouse) without anesthesia via the tail vein, and carried out SPECT/CT imaging. The animal study protocol had been approved by the Medical Ethics Committee of Inner Mongolia Medical University. Results: DCNs were successfully prepared in a single annealing procedure with six single-stranded oligonucleotides. The process and results of DCNs preparation could be characterized by PAGE. With the addition of each single-stranded oligonucleotide in the annealing process, the moving of as-formed structure in each lane would gradually slow down until the final formation of DCNs. 99mTc-A(20) ssDNA was obtained by radiolabeled MAG3-A(20) with technetium-99m, and the radiochemistry purity of ssDNA prepared was about 92%. 99mTc-DCNs were obtained by mixing T20-DCNs with 99mTc-A(20) ssDNA. The retention time (RT) of radiolabeled A(20) ssDNA was at ~22 min, and the RT of as-prepared 99mTc-DCNs was at ~12 min, indicative of the successful preparation of radiolabeled DCNs, 99mTc-DCNs. From biodistribution study, 99mTc-DCNs in the blood pool peaked at 5 min after injection, then rapidly decreased later on, suggesting a rapid distribution of DCNs in vivo. This was in accordance with the calculated half-life in vitro. Within 4 hours post injection (p.i.), the tracer uptake in the heart, lung, intestine, stomach and brain were never higher than 3 %ID/g, while accumulation in the liver was constantly at about 15 %ID/g. In the kidneys, 99mTc-DCNs had reached about 27 ± 3.54 %ID/g at 15 min p.i., and then decreased rapidly to about 5.77 ± 4.25 and 0.59 ± 0.08 %ID/g at 30 min and 4 h p.i., respectively. As for the tracer uptake in the spleen, 99mTc-DCNs dropped down from 14.23 ± 0.98 %ID/g at 15 min to 0.59 ± 0.27 %ID/g at 4 h %ID/g. From SPECT/CT images of 99mTc-DCNs 1 h p.i., a significant amount of DCNs was seen in the bladder. Moreover, the gallbladder showed a high uptake of 99mTc-DCNs, which was in agreements with the biodistribution study, suggesting that a significant proportion of DCNs was indeed metabolized within the liver. Conclusions: We have successfully radiolabeled DNA cube nanoparticles with Tc-99m and prepared 99mTc-DCNs as a SPECT/CT imaging probe via the side chain hybridization strategy.