One nanometer self-assembled aptamer-DNA dendrimers carry 350 doxorubicin: Super-stability and intra-nuclear DNA comet tail

Highlights

• The DNA dendrimers display super-stability in serum after annealing.

• The sgc8-DNA dendrimers show specific binding to target cells in blood.

• The sgc8-DNA dendrimers-Dox specifically induce DNA damage of the target cells evidenced by the comet tail, and can trace the target cells in mice.

Abstract

Recent development of aptamer-nanomaterial assemblies witnesses a great success. However, the instability and off-target imperfection of the aptamer-nanomaterial assemblies still need to be improved for clinical theranostic development. Here, we show the aptamer sgc8-hybridized DNA dendrimers with engineering simplicity, robust biostability and target specificity. We introduced a trigger to a mixture of intelligently-designed oligonucleotides to initiate programmable hybridization/polymerization process that was controlled by substrates/byproducts equilibration cycles, yielding nick-sealed sgc8DNA dendrimers after ligation with sgc8-Linkers. The molecular entity and biostability, targeting specificity and theranostic efficacy of the sgc8-DNA dendrimers were characterized by physicochemistry, molecular and cellular biology and in vivo models. The DNA dendrimers showed super-stability in FBS-containing culture medium or in serum for more than 36 h and were resistant to 100 °C-annealing and physiological DNase. The sgc8-DNA dendrimers specifically distinguished target CCRF-CEM cells from the cognate ones, and bound to CCRF-CEM even in the presence of many interfering cells or in blood. The highly-branched dendrimers provided huge surface interfaces to load doxorubicin by G-C hybrids at a molar ratio over 350, and specifically delivered doxorubicin to nuclei of CCRF-CEM evidenced by DNA synthesis arrest and comet tail, thus preventing doxorubicin’s non-selective cytotoxicity. The sgc8-DNA dendrimers showed specific capturing of circulating CCRF-CEM cells and in vivo theranostic effects on implanted tumors. The novel and stable sgc8-DNA dendrimers with high pay-load may be best-suited for cancer theranostics.

Introduction

The self-assembly is a simple, reliable and practical assembly technique for the spontaneous construction of basic molecular units into ordered geometric structures via Van der Waals' force, hydrogen bond, hydrophobic force and electrostatic interaction [1], [2]. Various biomolecules can be used as a basic unit, such as peptides, liposomes and DNAs to achieve their self-assemblies under given conditions [3], [4], [5], [6], [7], [8]. The assembled products have been extensively used in many fields, including biomedical materials, biosensors, and molecular devices. Among them, DNA self-assembly has drawn much attention because of their precise and rigorous base pairing, predictable spatial structures, fixed pitch and double-strand height together with simple synthesis and flexible sequence design. The DNA self-assembly technique has been rapidly developed especially since the DNA origami was put forward [8]. The representative DNA assemblies include DNA tetrahedron, DNA nanotubes, DNA nanowire, DNA dendrimer, and DNA nanomachine [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22].

DNA dendrimers, a class of polymer that is completely assembled by DNA, have the characteristics of high branch, easy to be functionalized together with a large number of sticky ends on its surface. The DNA dendrimers were first reported in 1997 by Nilsen et al. [17]. They synthesized dendritic DNA nanostructures with layer-by-layer assembly via employing a double-strand DNA monomer containing four “arm” chains. Later, a novel DNA dendrimer assembly strategy has been developed through hybridization chain reaction (HCR), which becomes a commonly-used method in the field [18], [23]. Recently, Hsing’s group presented a nonlinear HCR system, in which a trigger DNA initiates six different non-hairpin nucleic acid probes for self-assembly to form DNA dendrimer in a cascade way [24]. Compared with the previously reported DNA dendrimers, this assembly process needs no additional probes for each generation growth. This probe design is relatively easy and the assembly process is also brief and easy to be completed. The nonlinear HCR system has been used to detect DNA sensitively in enzyme-free surface plasmon resonance biosensing and electrochemical biosensor [25], [26]. Although the nonlinear HCR system allows chain-branching growth of the assembled DNA dendrimers at higher-order growth kinetics, there are many unsealed nicks existed in the final DNA dendrimer products after the self-assembly process. These unsealed nicks have a great impact on the stability of the final products [12]. Based on our previous studies, we consider biostability as a prerequisite for a success in developing meaningful and useful biotechnology products [27], [28], [29], [30], [31]. It is well-known that natural nucleic acids are vulnerable to biological media, particularly to serum. Many developing biomaterials have been abandoned due to their blood instability. Currently, the DNA dendrimers prepared from the nonlinear HCR system contain numerous sticky ends and unsealed nicks that remain problematical in terms of biostability. In addition, biochemical modifications on these nonlinear HCR DNA dendrimers have not been explored, and we hypothesized that aptamers, which have high binding affinity and high specificity, might be introduced into the DNA dendrimers to enhance the latter’s target specificity without compromising the biostability [30], [32], [33], [34].

To test the above hypothesis, we employed a trigger and six non-hairpin substrate strands and assistants (Table S1), which can be self-assembled into a DNA dendrimer via nonlinear HCR method [24]. We then treated the 5′ end of the partially-assembled sequences with a phosphorylase to seal the nicks in the self-assembled DNA dendrimers via ligation. We also mixed the aptamer sgc8-Linker with the nick-containing DNA dendrimers to seal the nicks by integral ligase reaction. The aptamer sgc8 can specifically bind to human protein tyrosine kinase 7 (PTK7) that is highly expressed on the CCRF-CEM cells (CCL-119, T-cell line, human acute lymphoblastic leukemia), but not on Ramos cells (CRL-1596, B-cell line, human Burkitt’s lymphoma). To make the DNA dendrimers tumor-theranostic, we intercalated the cytotoxic drug doxorubicin (Dox) to thousands of “G-C” bases on the sgc8-DNA dendrimers, and applied many assays, in particular, the DNA comet tail assay for the first time, to evaluate the stability, payload, intracellular and intranucleus theranostic effects of the sgc8-DNA dendrimers in the present study.