SL2B aptamer and folic acid dual-targeting DNA nanostructures for synergic biological effect with chemotherapy to combat colorectal cancer

Abstract

DNA nanostructures prepared by self-assembly possess good stability, high biocompatibility, and low immunogenicity as drug delivery vehicles. In this work, DNA tetrahedron (TD) was constructed and modified with SL2B aptamer (S) and folic acid (F). TD possessed a small diameter (~6 nm) and entered into the nucleus quickly. SL2B aptamer can inhibit cancer cell growth by disturbing vascular endothelial growth factor/Notch signaling pathways. To explore the effect of SL2B number on colorectal cancer inhibition, SL2B multimers (dimer, trimer, and tetramer) were constructed by functionalization of TD with different numbers of SL2B. One SL2B per TD was the most efficient anticancer strategy and showed significantly better anticancer efficacy than SL2B, probably due to the enhanced stability of SL2B by TD. Doxorubicin (DOX) is a potent anticancer agent that can intercalate into DNA double strands. Results showed that TD could facilitate DOX entrance into the nucleus and the intracellular delivery of DOX was further enhanced by functionalization of SL2B and F. DOX-intercalated TD modified with two F and two S (DOX@TD-2F2S) could cause sufficient HT-29 cell inhibition at a much lower DOX concentration. In sum, DOX@TD-2F2S exhibited a synergic anticancer biological effect with chemotherapy and can be a promising strategy for treating colorectal cancer.

Keywords: DNA tetrahedron; SL2B aptamer; VEGF/Notch; colorectal cancer; doxorubicin; folic acid; synergic biological effect with chemotherapy.

Figure 1

(A) Gel electrophoretic analysis of DNA TD (1, 50 bp marker; 2, TD; 3, 1F-TD; 4, 2F-TD; 5, 1SL2B-TD; 6, 2SL2B-TD; 7, 1F/1SL2B-TD; 8, 2F/2SL2B-TD); (B) AFM image of DNA TD (green triangles outline the TD); (C) concentration of loaded DOX by DNA TD with increasing added DOX; (D) concentration of loaded DOX by DNA TD with increasing incubation time. Abbreviations: TD, tetrahedron; bp, base pair; F, folic acid; AFM, atom force microscopy; DOX, doxorubicin.

Figure 2

(A) Gel electrophoretic analysis of DOX-loaded DNA TD in McCoy’s 5A medium plus 10% FBS (1, 50 bp marker; 2, TD control; 3, 0 h; 4, 0.5 h; 5, 3 h; 6, 6 h; 7, 15 h; 8, 24 h; 9, 48 h; 10, 72 h); (B) the relative stability of DOX in McCoy’s 5A medium plus 10% FBS; (C) release profiles of DOX, DOX@TD, and DOX@TD-2F2S. Abbreviations: TD, tetrahedron; bp, base pair; DOX, doxorubicin; FBS, fetal bovine serum; F, folic acid; S, SL2B.

Figure 3

In vitro HT-29 cell inhibition analysis at varied concentrations of the listed treatments at (A) 24 h, (B) 48 h, (C) 72 h, and (D) IC₅₀ values of HT-29 cells by the listed treatments. Notes: Data presented as mean value ± SD (n=3). *Free DOX vs DOX@TD-2F2S, P<0.01. Abbreviations: IC₅₀, inhibitory concentration of 50%; DOX, doxorubicin; TD, tetrahedron; F, folic acid; S, SL2B; SD, standard deviation.

Figure 4

(A) Apoptotic statistics of HT-29 cells after incubation with listed treatments for 48 h; (B) flow cytometry histograms of HT-29 cell cycle after incubation with listed treatments for 48 h. Abbreviations: DOX, doxorubicin; TD, tetrahedron; F, folic acid; S, SL2B.

Figure 5

(A) HT-29 cell inhibition by the listed SL2B treatment; (B) HT-29 cell inhibition by the combined DOX and SL2B treatment; (C) Western blotting results for the downregulation of jagged-1 that plays a key role in the Notch signaling pathway; (D) binding affinity curve of SL2B-FITC and TD-1SL2B-FITC with HT-29 cells. Abbreviations: DOX, doxorubicin; FI, fluorescence intensity; TD, tetrahedron; FITC, fluorescein isothiocyanate.

Figure 6

(A) Confocal microscopy images of HT-29 cells after incubation with FITC-labeled TD for 4 h (green FITC represented the location of TD); (B) confocal microscopy images of intracellular uptake of DOX by HT-29 cells after incubation with listed treatments for 2 h; (C) flow cytometry histograms of intracellular DOX uptake after incubation with listed treatments for 1 h. Abbreviations: DOX, doxorubicin; TD, tetrahedron; FITC, fluorescein isothiocyanate; F, folic acid; S, SL2B.

Scheme 1

Compositions of multifunctional DNA nanostructures and schematic illustration of combined biological effect and chemotherapy for colorectal cancer. Notes: SL2B binds to VEGF165 and inhibits colorectal cancer cells by interfering subsequent VEGF/Notch signaling pathways. DNA TD can enter the nucleus due to its small diameter. Folate interacts with its receptor and enhanced intracellular delivery of DNA nanostructures by receptor-mediated endocytosis. TD releases DOX in the cytoplasm and nucleus to inhibit cell proliferation. The combined therapy of SL2B and DOX ultimately caused enhanced cell death. Abbreviations: DOX, doxorubicin; TD, tetrahedron; VEGF, vascular endothelial growth factor.