Table 1.

Overview of different DNA nanostructures and their applications.

	Structure Characterization								Biological activity
N°	DNA Nanostructure	Characterization	Size (nm)	Cargo (Loading)	Stability	Targeting	Loading efficiency	Release	Internalization	In vitro tests		In vivo tests		Biodistribution	Ref
										Cells	Activity	Cells	Activity
1	Tetrahedral	AFM DLS PAGE (6%)	AFM: height 2-3 nm DLS: 9.08 nm ± 3nm	Doxo	> 4 < 23 hrs Solution: 10% human serum Temp: 37°C	N/A	26 molecules/structure Time: 1 hr Temp: N/A	100% in 10hrs. <10% in 3hrs. Solution: PBS pH 7.4 Temp: 37°C 60% in 10 hrs Solution: PBS pH 5 Temp: 37°C	Macropinocytosis and caveolae-mediated endocytosis pathways	Breast cancer MCF7 (drug sensitive) and MCF7-ADR (Doxo resistant).	Doxo-tetrahedral is more effective on cell viability of MCF7-ADR than free doxo. Same efficacy on MCF7 cells.	N/A	N/A	N/A	[63]
2	Icosahedral: five- (120 bp) and six- (144 bp) (aptamer) point-star structure	AGE (2%) DLS TEM	DLS: - Five stars (28.2 nm ± 3nm) - Six stars (28.6 nm ± 5nm). TEM: 25 nm	Doxo	>30min Solution: cell culture medium Temp: 37°C	Mucin 1 (MUC1). Tumor surface marker	1200 molecules/structure Time: 1h incubation Temp: room temperature	N/A	Dynamin-dependent and clathrin-mediated endocytosis. Degradation in lysosomes	Breast cancer MCF7 (MUC1+), CHO-K1 (MUC1-)	Doxo-Aptamer six-point-star structure is more effective on cell viability of MUC1+ cell than free doxo. Same efficacy on MUC- cells.	N/A	N/A	N/A	[64]
3	Tetrahedral (30bp/edge, Total 6 edges)	AFM DLS PAGE (5%)	AFM: height 7.5 nm. DLS: 28.6 nm ± 2.38 nm	siRNA against GFP gene. DNA structure conjugated with folic acid.	N/A	Folic acid receptor (FAR)	1-6 siRNA per tetrahedral (1 siRNa/edge)	N/A	N/A	HeLa cells (LUC+). KB cells (HeLa cell contaminant overexpressing folate receptor)	HeLa: LUC expression < 50%. KB: GFP expression <40%	KB xenograft tumours	siRNA no effect on LUC. siRNA-DNA- tetrahedral: IC 50 LUC expression: 1.8 mg/kg. Inj: intratumor or tail-vein Stability: siRNA 6 min; siRNA-DNA tetrahedral 24 min.	Tumor, Kidney Time: 12 hours post injection	[65]
4	Tetrahedral	PAGE (3.5%);	N/A	Straptavidin (STV) and CpGs	5hrs Solution: FBS 50% Temp: room temperature	N/A	N/A	N/A	Endocytosis. Antigen localized in lysosomes after 2 hours	RAW264.7 (macrophage, Abelson murine leukemia virus transformed).	Increased internalization of the complex tetrahedron-STV-CpG by APC cells	BALB/c immunocompetent mice	Mice immunized with the complex tetrahedron-STV-CpG developed a stronger and a longer immunitary response.	N/A	[66]
5	Aptamer-tethered DNA nanotrain	AFM AGE (3%) TEM	AFM: length 100 nm.	Doxo, epirubicin, daunorubicin	>45 hrs Solution: PBS + 5mM Mg2+ Temp: N/A	Protein Tyrosin Kinase 7 (PTK7)	N/A	N/A	Endocytosis	CEM cells (human T-cell acute lymphocytic leukemia PTK7+) and Ramos (human B lymphocyte Burkitt's lymphoma PTK7-)	Drug-Aptamer-DNA-drug is more cytotoxic on PTK7+ cells than free drug. Same efficacy on PTK7- cells.	CEM (PTK7+) xenograft mouse model	Increased antitumor efficacy and reduced side effects of doxo delivered via Aptamer-DNA nanotrain. Inj: I.V.		[67]
6	Open Caged DNA (pyramidal), 408 bp	PAGE (7%)	N/A	Doxo	35 hrs (half-life) Solution: cell culture medium+10% FBS Temp: 37°C	N/A	172 molecules doxo/structure (at 15% of loading efficiency)	50% of Doxo release from py-Doxo in PBS in 5 hrs and 3 hrs in FBS. Free Doxo in 20 minutes	py-Doxo is able to penetrate inside MDA-MB-231, release Doxo in the nucleus	MDA-MB-231, HepG2	Decrease cell viability compared to free doxo	N/A	N/A	N/A	[68]
7	Tube with different global twist (Straight (S) 10.5 bp/turn and Twist (T) tube 12 bp/turn)	AGE (2%) TEM	TEM: length: 138 nm, diameter: 13 nm	Doxo	48hr (T-tube) Solution: 10% FBS Temp: 37°C	N/A	N/A	80% (T-tube) and 90% (S-tube) in 10 hrs Solution: PBS pH 7.4 Temp: 37°C	N/A	Breast cancer MDA-MB-231; MDA-MB-468; MCF-7	In all the cell lines tested the T-DNA IC50 is ≈2 times lower than free Doxo. (calculated by our group)	N/A	N/A	N/A	[70]
8	Tube	AGE (2%) TEM	TEM: length: 80 nm, diameter: ≈20 nm	62 CpG sequence specific for mouse Toll like receptor 9	6 hrs Solution: cell culture medium Temp: 37°C	Toll like receptor 9 (TLR9).	62 binding sites per tube	N/A	N/A	Splenocytes from female C57BL/6 mice	N/A Immunoresponse through the TLR9. Nontoxic	N/A	N/A	N/A	[71]
9	Hexagonal barrel with aptamer-based lock (antigen keys).	AFM AGE (2%) DLS TEM	DLS: 90 nm TEM: 35nm x 35 nm x 45 nm	Fab antibody fragments	N/A	CD33, CDw328	N/A	N/A	N/A	NKL	Increase apoptosis	N/A	N/A	N/A	[72]
10	Triangle and tube	AFM AGE (1%)	AFM: -Triangle: edge ≈ 150nm -Tube: length ≈ 183 nm	Doxo	N/A	N/A	>200000 molecules (calculated by our group) Time: 24 hrs Temp: room temperature.	≈15% pH 7.4, 35% pH 5.5 in 48 hrs ≈25% in MCF7 cell lysate >40% with 50U DNAaseI Solution: PBS Temp: 37°C	Endocytosis and localization of origami in lysosomes after 6 hours of treatment.	Breast cancer MCF7 and MCF7 resistant	MCF-7: 2.5µM and MCF-7 resistant >100μM. DNA origami loaded with doxo enhanced the cells death compared to free doxo on MCF7 doxo resistant cells. No differences between free doxo and origami doxo in regular MCF7.	N/A	N/A	N/A	[73]
11	Tube, triangle and square	AFM AGE (1%) DLS	AFM: -Tube: height: 7 nm, diameter: 380 nm, -Triangle: edge 120 nm, -Square: length*width 90nmx60nm DLS: -Triangle: 59 nm -Square: 80.9 nm -Tube: 98.6 nm.	Doxo	24 hrs Solution: serum Temp: 37°C	N/A	>200000 molecules (calculated by our group) Time: 24 hrs Temp: room temperature.	≈20% of Doxo is released in 48 hours at pH 7.4, ≈35% is released at pH 5.5 Solution: PBS Temp: 37°C	N/A	Breast cancer MDA-MB-231	No significant difference compared to the free doxo.	MDA-MB-231 cells Orthotopic breast cancer model	Increased EPR effect of DNA origami. Significant tumor reduction in mice treated with doxo-origami compared to free doxo Inj: I.V	Tumor: Triangle > tube >square Tumor > liver > kidney > spleen Time: 24 hrs	[74]
12	Tube (Tile)	AGE (2%) TEM	TEM: Length: 27 nm and diameter: 8 nm	siRNA (GFP)	8hrs Solution: cell culture medium Temp: 37 °C (Degradation depends on Mg2+ concentration, oligonucleotide sequences, salt concentration, structural extension)	Folic acid receptor (FAR)	N/A	N/A	Endosomal trapping (no release)	N/A	No effects on GFP expression	N/A	NA	NA	[86]

AGE: Agarose gel electrophoresis. PAGE: Polyacrylamide gel electrophoresis. TEM: Transmission electron microscopy. DLS: Dynamic light scattering. AFM: Atomic force microscopy. N/A: Not Applicable. Temp: Temperature. Inj: Injection. I.V.: Intravenous. Luc: Luciferase