Radiolabeling and Preliminary Evaluation of DNA Cube Nanoparticles in Vivo & in Vitro

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.