Hu et al. J Nanobiotechnol (2020) 18:109 https://doi.org/10.1186/s12951-020-00667-6 Journal of Nanobiotechnology RESEARCH Open Access Loop-armed DNA tetrahedron nanoparticles for delivering antisense oligos into bacteria Yue Hu1†, Zhou Chen1†, Xinggang Mao2†, Mingkai Li1, Zheng Hou1, Jingru Meng1, Xiaoxing Luo1* and Xiaoyan Xue1* Abstract Background: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bac- teria efectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efciency in bacteria. Results: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more efectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 μL/μg ratio to Td, but the same concentration of LP2000 had no apparent efect on linear DNA. Furthermore, linkage modes between ASOs and Td infuenced gene-knockdown efciency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efciency than the overhung structure. Conclusions: This study established an efective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics. Keywords: DNA tetrahedron, Antisense antibiotics, Nanoparticles, Drug delivery system Background hardly enter bacterial cells because of their high molecu- Infections caused by antibiotic-resistant bacteria have lar weight [6] and the complex structure of the bacterial raised public concerns worldwide. Among the assorted cell wall [7], which are the main obstacles for ASOs in solutions, such as antibiotic combination [1] and small- therapeutic feld. Tus far, a multitude of materials have molecular compound screening [2], the antisense anti- been studied for ASOs delivery, including cell-penetrat- bacterial strategy has drawn considerable attention due ing peptides (CPPs) [4], vitamin B 12 [8], liposomes [9] to its convenient target selection, safety and decreased and other polymers [10]. Among these materials, CPPs likelihood of inducing antibiotics resistance [3]. By spe- are the most widely used and have proven to be efective cifcally blocking the expression of targeted genes in when covalently linked with ASOs, but their utilization is bacteria, antisense oligonucleotides (ASOs) have shown impeded by their cytotoxicity at high concentrations and great potential to kill bacteria [4] or reverse the resist- potential immunogenicity to the host [11]. Terefore, the ance of bacteria [5]. However, without vectors, ASOs can development of alternative carriers is essential. Recently, DNA nanomaterials have ofered entirely new avenues for drug delivery systems [12]. Based on *Correspondence: [email protected]; [email protected] †Yue Hu, Zhou Chen and Xinggang Mao contributed equally to this work Watson–Crick base pairing, DNA nanoparticles exhibit 1 Department of Pharmacology, Fourth Military Medical University, excellent advantages over traditional nanoparticles, No. 169, Changle West Road, Xi’an 710032, Shaanxi, People’s Republic including precise manipulation of shape and size, bio- of China Full list of author information is available at the end of the article compatibility, nontoxicity and increased likelihood of © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hu et al. J Nanobiotechnol (2020) 18:109 Page 2 of 12 intelligent modifcation [13]. Particularly, DNA tetra- Ten, we studied the uptake features of Td in dif- hedra (Td) is widely used due to its simple preparation, ferent bacteria. E. coli or S. aureus bacterial cells were rigid structure and fexible optimization [14]. In previous incubated with FAM-labeled Td at various concentra- studies, it has been verifed that Td can enter live HEK tions (0.1, 0.5, and 1 μM) for 1.5 h, and the number of cells without transfection agents [15] and deliver small- FAM-positive bacteria was analyzed by fow cytometry. molecule compounds [16] or nucleic acid drugs [17, 18] As a result, the positive ratios of E. coli incubated with into eukaryotic cells. Furthermore, Td has great fexibility FAM-labeled Td at concentrations of 0.1, 0.5, and 1 μM for structural modifcation by aptamers [19], folate acids were 5%, 35%, and 49%, respectively (Fig. 2a), while the [17] or tumor-penetrating peptides [20], exhibiting con- corresponding positive ratios of S. aureus were 5%, 24%, siderable potential for facilitating versatile drug delivery. and 56% (Additional fle 1: Figure S1a). To determine Nevertheless, only a few studies have focused on the whether the observed fuorescence signals represented application of Td as a delivery system for antibacterial the uptake of the Td into bacteria or simple adherence to agents. Leong reported that Td intercalated with actino- the bacterial membrane, the bacterial cells were treated mycin D could be internalized efciently by Escherichia with DNase before fow cytometry analysis [15]. And we coli (E. coli) and Staphylococcus aureus (S. aureus) and demonstrated that DNase had no efect on FAM fuores- showed stronger antibiotic efects than free actinomy- cence intensity (Additional fle 1: Figure S2). We found cin D in vitro [21]. Other studies demonstrated that Td that after DNase treatment, the positive ratios of both incorporated with peptide nucleic acids targeting blaCTX- tested strains decreased to approximately 20% when M-group 1 in cefotaxime-resistant E. coli [22] or ftsZ in treated with 1 μM Td (Fig. 2a and Additional fle 1: Fig- methicillin-resistant Staphylococcus aureus (MRSA) [23] ure S1a). However, the positive ratios of single-strand could enter bacteria to restore sensitivity to cefotaxime or S1 labeled with FAM were lower than 5% in both tested to inhibit bacterial growth by inhibiting targeted genes. bacteria whether treated with DNase or not (Fig. 2a Tese results imply that Td could be a carrier to deliver and Additional fle 1: Figure S1a). Furthermore, confo- ASOs into bacteria. However, the delivery efciency of cal laser scanning microscopy (CLSM) also confrmed Td in diferent strains and the factors that infuence Td that the fuorescence intensity of the tested bacteria was into bacteria, as well as the type of Td structure or link- reduced signifcantly after treatment with DNase (Fig. 2b age modes with ASOs remain unclear. and Additional fle 1: Fig. S1b). Similar results were also In this study, we investigated the uptake characteris- observed in other bacterial strains, including S. fexneri, tics and efciency of Td by diferent bacterial strains, NDM1-K. pneumoniae, multiple-drug resistant P. aerugi- including S. aureus, E. coli, Shigella fexneri (S. fexneri), nosa and A. baumannii (Additional fle 1: Figure S3). All NDM1-Klebsiella pneumoniae (K. pneumoniae), mul- the data demonstrated that Td had a tendency to bond tiple-drug resistant Pseudomonas aeruginosa (P. aer- with the bacterial membrane and only a fraction of Td uginosa) and Acinetobacter baumannii (A. baumannii). entered into bacterial cells successfully. Next, we designed two types of linkages modes between Next, Lipofectamine 2000 (LP2000) was used to Td and ASOs targeting gfp, encoding green fuorescent improve the uptake efciency of the Td by bacteria protein (GFP), or acpP, encoding the acyl carrier protein because of the following reasons: (1) We have demon- (Acp), and assessed the efciency of delivery and gene strated that lipofectamine 2000 (LP2000) could deliver knockdown in E. coli. oligonucleotides into bacteria efectively [25], and showed no toxicity when the concentration was lower than 10 μL/mL (Additional fle 1: Figure S4). (2) Lipo- Results and discussion fectamine could interact with DNA by electrostatic Td was prepared according to methods described previously force, and could be used as a modifer to increase the [24] (Fig. 1a) and characterized by agarose gel, atomic force liposolubility of Td [26], which may help the DNA microscope (AFM) and dynamic light scattering (DLS). To nanomaterials penetrate into bacterial cells more eas- verify the formation of Td, the four strands of the Td (S1, S2, ily. Terefore, we explored the uptake efciency of Td S3, S4) were added one-by-one, and the gradually formed mixed with LP2000 (LP2000/Td: 0.0025, 0.0125, 0.025, complex presented distinct bands with slower mobility as and 0.125 μL/μg) for initial optimization. Te Td mixed one more strand was added, indicating the successful step- with LP2000 (LP-Td) showed the same size as the Td wise assembly of the Td (Fig. 1b). AFM images showed when the LP2000/Td ratio was 0.0025 (Additional fle 1: that the Td exhibited an average diameter of approximately Figure S5a), while larger nanoparticles were formed 10 nm, and a few aggregates were observed (Fig.