Fig 3 -

Cell binding assay of ErbB2 aptamers using flow cytometry and fluorescence spectroscopy (A) The specific binding capacity of ErbB2 aptamers to human breast cancer cells was investigated by flow cytometry. ErbB2-positive SK-BR-3 and KPL4, and ErbB2-negative MCF-7 cells were stained with Cy5-labeled ErbB2 aptamer, hyErbB2-idT aptamer, scrambled ErbB2 (ScrErbB2) aptamer or cODN. (B) Confocal microscopy analysis with Cy5-ErbB2 or Cy5-hyErbB2-idT on SK-BR-3, KPL4, and MCF-7 cells at 4°C. Aptamers are visualized in red. (C) The binding properties of Cy5-ScrErbB2 aptamer were also measured on KPL4 and MCF-7 cells for comparison. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue).</p

Representative <i>in vivo</i> PET images of ¹⁸F-hyErbB2, ¹⁸F-hyErbB2-idT and ¹⁸F-hyScrErbB2 aptamer in athymic nude mice bearing KPL4 tumors.

Coronal images were acquired at 1 h after injection. The white arrows indicate the location of the tumor. Uptake values are shown as mean %ID/g.</p

Hybridization efficiency and serum stability of ErbB2 aptamer.

(A) Hybridization efficiency of Cy5-labeled complementary oligonucleotide (Cy5-cODN, lane 1), mixture of Cy5-cODN and reverse complement (RC) sequence containing ErbB2-idT aptamer (RC-ErbB2-idT, lane 2), Cy5-cODN hybridized ErbB2-idT aptamer (hyErbB2-idT, lane 3), Cy5-cODN hybridized ErbB2 aptamer (hyErbB2, lane 4) and Cy5-cODN hybridized scrambled ErbB2 aptamer (hyScrErbB2, lane 5) (B) In vitro serum stability analysis of ErbB2, RC-ErbB2-idT and hyErbB2-idT aptamers in human serum. Aptamers were incubated with an equal volume of serum for 0, 3, 6, 12, 24, 48, and 72 h at 37°C and analyzed by using urea polyacrylamide gel electrophoresis. M: DNA size marker, bp: base pair.</p

S1 Raw images -

Aptamers have great potential for diagnostics and therapeutics due to high specificity to target molecules. However, studies have shown that aptamers are rapidly distributed and excreted from blood circulation due to nuclease degradation. To overcome this issue and to improve in vivo pharmacokinetic properties, inverted deoxythymidine (idT) incorporation at the end of aptamer has been developed. The goal of this study was to evaluate the biological characterization of 3’-idT modified ErbB2 aptamer and compare with that of unmodified aptamer via nuclear imaging. ErbB2-idT aptamer was labeled with radioisotope F-18 by base-pair hybridization using complementary oligonucleotide platform. The hyErbB2-idT aptamer demonstrated specific binding to targets in a ErbB2 expressing SK-BR-3 and KPL4 cells in vitro. Ex vivo biodistribution and in vivo imaging was studied in KPL4 xenograft bearing Balb/c nu/nu mice. 18F-hyErbB2-idT aptamer had significantly higher retention in the tumor (1.36 ± 0.17%ID/g) than unmodified 18F-hyErbB2 (0.98 ± 0.19%ID/g) or scrambled aptamer (0.79 ± 0.26% ID/g) at 1 h post-injection. 18F-hyErbB2-idT aptamer exhibited relatively slow blood clearance and delayed excretion by the renal and hepatobiliary system than 18F-hyErbB2 aptamer. In vivo PET imaging study showed that 18F-hyErbB2-idT aptamer had more stronger PET signals on KPL4 tumor than 18F-hyErbB2 aptamer. The results of this study demonstrate that attachment of idT at 3’-end of aptamer have a substantial influence on biological stability and extended blood circulation led to enhanced tumor uptake of aptamer.</div

Characterization of ErbB2 aptamer.

Secondary structure predictions of ErbB2, RC-ErbB2-idT and hyErbB2 aptamer. The equilibrium dissociation constant (Kd) values of ErbB2 aptamer to recombinant ErbB2 protein were determined by ELONA assay.</p

<i>Ex vivo</i> biodistribution study of ¹⁸F-hyErbB2-idT and ¹⁸F-hyErbB2 aptamer in a subcutaneous KPL4 tumor xenograft model.

(A) Quantitative analysis of biodistribution of 18F-hyErbB2-idT and 18F-hyErbB2 at 30 min post-injection (n = 4). (B) Xenograft mice were sacrificed at 1 h after injection of 18F-hyErbB2-idT, 18F-hyErbB2 and 18F-hyScrErbB2 aptamer (n = 5–6). Tumor-to-blood ratio was derived from the biodistribution data. Data are expressed as a percentage of injected activity per gram of tissue (%ID/g). Each bar represents the mean %ID/g ± standard deviation (SD). S.I.: small intestine, L.I.: large intestine. *P P < 0.001.</p