Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment.

Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization and editable functionality, tetrahedral DNA nanostructures show promising potential as an alternative vehicle for drug delivery and biomedical treatment. Here, we describe the design, fabrication, purification, characterization and potential biomedical applications of a self-assembling tetrahedral DNA nanostructure (TDN)-based multifunctional delivery system. First, relying on Watson-Crick base pairing, four single DNA strands form a simple and typical pyramid structure via one hybridization step. Then, the protocol details four different modification approaches, including replacing a short sequence of a single DNA strand by an antisense peptide nucleic acid, appending an aptamer to the vertex, direct incubation with small-molecular-weight drugs such as paclitaxel and wogonin and coating with protective agents such as cationic polymers. These modified TDN-based complexes promote the intracellular uptake and biostability of the delivered molecules, and show promise in the fields of targeted therapy, antibacterial and anticancer treatment and tissue regeneration. The entire duration of assembly and characterization depends on the cargo type and modification method, which takes from 2 h to 3 d.

Aptamer-targeted DNA nanostructures with doxorubicin to treat protein tyrosine kinase 7-positive tumours.

OBJECTIVES: Aptamer sgc8c is a short DNA sequence that can target protein tyrosine kinase 7 (PTK7), which was overexpressed on many tumour cells. This study aimed to fabricate a novelty DNA nanostructure drug delivery system target on PTK7-positive cells-CCRF-CEM (human T-cell ALL). METHODS: Aptamer-modified tetrahedron DNA was synthesized through one-step thermal annealing process. The sgc8c-TDNs (s-TDNs) loading DOX complexes were applied to investigate the effect to PTK7-negative and -positive cells. RESULTS: When s-TDN:DOX acted on PTK7-positive and -negative cells respectively, the complexes exhibited specific toxic effect on PTK7-positive cells but not on PTK7-negative Ramos cells in vitro research. CONCLUSIONS: In this work, we successfully constructed a PTK7-targeting aptamer-guided DNA tetrahedral nanostructure (s-TDN) as a drug delivery system via a facile one-pot synthesis method. The results showed that s-TDN:DOX exhibited enhanced cytotoxicity against PTK7-positive CCRF-CEM cells, with a minor effect against PTK7-negative Ramos cells. Hence, this functionalized TDNs drug delivery system displayed its potential application in targeting PTK7-positive tumour T-cell acute lymphoblastic leukaemia.

Tetrahedral DNA Nanostructure Promotes Endothelial Cell Proliferation, Migration, and Angiogenesis via Notch Signaling Pathway.

The problem of tissue vascularization is one of the obstacles that currently restricts the application of tissue engineering products to the clinic. Achieving tissue vascularization and providing adequate nutrients for tissues are an urgent problem to build complex and effective tissue-engineered tissues and organs. Therefore, the aim of this study was to investigate the effect of tetrahedral DNA nanostructures (TDNs), a novel and biocompatible nanomaterial, on angiogenesis. The results showed that TDNs can enter into endothelial cells (ECs) and promote EC proliferation, migration, tube formation, and expressions of angiogenic growth factors at the concentration of 250 nmol L-1, which was accompanied by activation of the Notch signaling pathway. These results provided a theoretical basis for the further understanding and potential use of TDNs in tissue engineering vascularization.

Tetrahedral DNA nanostructures facilitate neural stem cell migration via activating RHOA/ROCK2 signalling pathway.

OBJECTIVES: The main purpose of current study was to explore the effects of tetrahedral DNA nanostructures (TDNs) on neuroectodermal (NE-4C) stem cells migration and unveil the potential mechanisms. MATERIALS AND METHODS: The successfully self-assembled TDNs were also determined by dynamic light scattering (DLS). A bidirectional wound-healing assay and transwell chamber assay were employed to test the migrating behaviour of NE-4C stem cells cultured under different conditions. RESULTS: Through an in vitro study, we found that stem cells could internalize TDNs quickly, and the cells' parallel and vertical migration was promoted effectively. Besides, the effects of TDNs were found being exerted by upregulating the gene and protein expression levels of RhoA, Rock2 and Vinculin, indicating that the RHOA/ROCK2 pathway was activated by the TDNs during the cell migration. CONCLUSIONS: In conclusion, TDNs could enter NSCs without the aid of other transfection reagents in large amounts, whereas only small amounts of ssDNA could enter the cells. TDNs taken up by NSCs activated the RHOA/ROCK2 signalling pathway, which had effects on the relevant genes and proteins expression, eventually promoting the migration of NE-4C stem cells. These findings suggested that TDNs have great potential in application for the repair and regeneration of neural tissue.

Inhibiting Methicillin-Resistant Staphylococcus aureus by Tetrahedral DNA Nanostructure-Enabled Antisense Peptide Nucleic Acid Delivery.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Neuroprotective Effect of Tetrahedral DNA Nanostructures in a Cell Model of Alzheimer's Disease.

Accumulating evidence supports the abnormal deposition of amyloid ß-peptide (Aß) as the main cause of Alzheimer's disease (AD). Therefore, fighting against the formation, deposition, and toxicity of Aß is a basic strategy for the treatment of AD. In the process of in vitro nerve cell culture, screening out drugs that can antagonize a series of toxic reactions caused by ß-amyloid deposition has become an effective method for the follow-up treatment of AD. Our previous studies showed that tetrahedral DNA nanostructures (TDNs) had good biocompatibility and had some positive effects on the biological behavior of cells. In this study, the main aim of our work was to explore the effects and potential mechanism of TDNs in protecting neuronal PC12 cells from the toxicity of Aß. Our study demonstrated that TDNs can protect and rescue PC12 cell death through Aß25-35-induced PC12 cell apoptosis. Further studies showed that TDNs significantly improved the apoptosis by affecting the abnormal cell cycle, restoring abnormal nuclear morphology and caspase activity. Western blot analysis showed that TDNs could prevent the damage caused by Aß deposition by activating the ERK1/2 pathway and thus be a potential therapeutic agent with a neuroprotective effect in Alzheimer's disease. Our finding provides a potential application of TDNs in the prevention and treatment of AD.

Tea Polyphenol-Reduced Graphene Oxide Deposition on Titanium Surface Enhances Osteoblast Bioactivity.

Graphene, a novel carbon-based material, has been widely used as osteogenic agent for the potential effect on the promotion of osteoblast proliferation. Tea polyphenol-reduced graphene oxide (TPG) is a simple and environmental-friendly raw material to obtain graphene. In this study, TPG was deposited on the Ti substrate to promote the bone regeneration. We prepared a honeycomb-like structure by acid and alkali pretreatment and immobilized the TPG layer (Ti-TPG) on the surface via electrochemical deposition. Scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) were used to identify the immobilization of TPG on the titanium (Ti) successfully. Furthermore, the biological response of the Ti-TPG surface to rat osteoblast was evaluated. We also studied the cell adhesion, proliferation and expression of ossification genes on the sample. The results revealed that Ti-TPG had an advantage over Ti alloys in modulating cellular activity and Ti-TPG may be a promising coating for biological materials.

Substrate stiffness regulated migration and invasion ability of adenoid cystic carcinoma cells via RhoA/ROCK pathway.

OBJECTIVES: Human salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumours of the salivary gland and has strong migratory and invasive ability, which often lead to poor prognosis and lower survival rate. Tumour tissue tends to stiffen during solid tumour progression. This study aimed to investigate the influence of various substrate stiffness on the migration and invasion of SACC. METHODS: Salivary adenoid cystic carcinoma cell line ACC2 cells were cultured on polydimethylsiloxane substrates (PDMS) with varying stiffness for investigating the effects of substrate stiffness on the activities of MMPs and TIMPs. The underlying mechanism was also explored. RESULTS: When ACC2 cells were cultured on various stiffness of PDMS, the expressions of matrix metalloproteinases 2 (MMP2), MMP9, MMP14, RhoA, Rac1, Rho-associated protein kinase 1 (ROCK1) and ROCK2 were up-regulated with increasing substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 and TIMP4 were down-regulated with increasing substrate stiffness. CONCLUSIONS: Our results showed that substrate stiffness regulated the activities of MMPs and TIMPs and then modulate migratory and invasive ability of ACC2 cells via RhoA/ROCK pathway. This work indicate that matrix stiffness played an important role in progression of SACC, which not only can help understand the strong invasive ability of SACC, but also suggested that therapeutically targeting matrix stiffness may help reduce migration and invasion of SACC and improve effective therapies.

Self-Assembled Tetrahedral DNA Nanostructures Promote Neural Stem Cell Proliferation and Neuronal Differentiation.

Stem cell-based therapy is considered a promising approach for the repair of nervous tissues. Neural stem cells (NSCs) cannot proliferate or differentiate efficiently; hence, different biomaterials have been explored to improve NSC proliferation and differentiation. However, these agents either had low bioavailability or poor biocompatibility. In this work, our group investigated the effects of tetrahedral DNA nanostructures (TDNs), a novel DNA biological material, on the self-renew and differentiation of neuroectodermal (NE-4C) stem cells. We observed that TDN treatment promoted self-renew of the stem cells via activating the Wnt/ß -catenin pathway. In addition, our findings suggested that NE-4C stem cells' neuronal differentiation could be promoted effectively by TDNs via inhibiting the notch signaling pathway. In summary, this is the first report about the effects of TDNs on the proliferation and differentiation of NE-4C stem cells and the results demonstrate that TDNs have a great potential in nerve tissue regeneration.

Effects of tetrahedral DNA nanostructures on autophagy in chondrocytes.

Tetrahedral DNA nanostructures (TDNs) have gathered great attention and are being widely used in biomedicine. We demonstrated that autophagy increased after exposure to TDNs (250 nM) along with the up-regulation of several autophagy-related genes and proteins. TDNs enhanced cell autophagy through the PI3K/AKT/mTOR signaling pathway.

Substrate stiffness regulated migration and angiogenesis potential of A549 cells and HUVECs.

Tumor tissue tends to stiffen during solid tumor progression. Substrate stiffness is known to alter cell behaviors, such as proliferation and migration, during which angiogenesis is requisite. Mono- and co-culture systems of lung cancer cell line A549 and human umbilical vein endothelial cells (HUVECs), on polydimethylsiloxane substrates (PDMS) with varying stiffness, were used for investigating the effects of substrate stiffness on the migration and angiogenesis of lung cancer. The expressions of matrix metalloproteinases (MMPs) and angiogenesis-related growth factors were up-regulated with the increase of substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase (TIMPs) were down-regulated with increasing substrate stiffness. Our data not only suggested that stiff substrate may promote the migration and angiogenesis capacities of lung cancer, but also suggested that therapeutically targeting lung tumor stiffness or response of ECs to lung tumor stiffness may help reduce migration and angiogenesis of lung tumor.

Aptamer-Modified Tetrahedral DNA Nanostructure for Tumor-Targeted Drug Delivery.

Tetrahedral DNA nanostructures (TDNs) are considered promising drug delivery carriers because they are able to permeate cellular membrane and are biocompatible and biodegradable. Furthermore, they can be modified by functional groups. To improve the drug-delivering ability of TDNs, we chose anticancer aptamer AS1411 to modify TDNs for tumor-targeted drug delivery. AS1411 can specifically bind to nucleolin, which is overexpressed on the cell membrane of tumor cells. Furthermore, AS1411 can inhibit NF-κB signaling and reduce the expression of bcl-2. In this study, we compared the intracellular localization of AS1411-modified TDNs (Apt-TDNs) with that of TDNs in different cells under hypoxic condition. Furthermore, we compared the effects of Apt-TDNs and TDNs on cell growth and cell cycle under hypoxic condition. A substantial amount of Apt-TDNs entered and accumulated in the nucleus of MCF-7 cells; however, the amount of Apt-TDNs that entered L929 cells was comparatively less. TDNs entered in much lower quantity in MCF-7 cells than Apt-TDNs. Moreover, there was little difference in the amount of TDNs that entered L929 cells and MCF-7 cells. Apt-TDNs can inhibit MCF-7 cell growth and promote L929 cell growth, while TDNs can promote both MCF-7 and L929 cell growth. Thus, the results indicate that Apt-TDNs are more effective tumor-targeted drug delivery vehicles than TDNs, with the ability to specifically inhibit tumor cell growth.

Modulation of chondrocyte motility by tetrahedral DNA nanostructures.

OBJECTIVES: Contemporarily, a highly increasing attention was paid to nanoconstructs, particularly DNA nanostructures possessing precise organization, functional manipulation, biocompatibility and biodegradability. Amongst these DNA nanomaterials, tetrahedral DNA nanostructures (TDNs) are a significantly ideal bionanomaterials with focusing on the property that can be internalized into cytoplasm in the absence of transfection. Therefore, the focus of this study was on investigating the influence of TDNs on the chondrocytes locomotion. MATERIALS AND METHODS: Tetrahedral DNA nanostructures was confirmed by 6% polyacrylamide gel electrophoresis (PAGE) and dynamic light scattering (DLS). Subsequently, the effect of TDNs on chondrocyte locomotion was investigated by real-time cell analysis (RTCA) and wound healing assay. The variation of relevant genes and proteins was detected by quantitative polymerase chain reaction (qPCR), western blotting and immunofluorescence respectively. RESULTS: We demonstrated that tetrahedral DNA nanostructures have positive influence on chondrocytes locomotion and promoted the expression of RhoA, ROCK2 and vinculin. Additionally, upon exposure to TDNs with the concentration of 250 nmol L-1 , the chondrocytes were showed the highest motility via both RTCA and wound healing assay. Meanwhile, the mRNA and protein expression of RhoA, ROCK2 and vinculin were also significantly enhanced with the same concentration. CONCLUSIONS: It can be concluded that the TDNs with the optimal concentration of 250 nmol L-1 could extremely promoted the chondrocytes locomotion through facilitating the expression of RhoA, ROCK2 and vinculin. These results seemed to reveal that this special three-dimensional DNA tetrahedral nanostructures may be applied to cartilage repair and treatment in the future.

The Effect of shape on Cellular Uptake of Gold Nanoparticles in the forms of Stars, Rods, and Triangles.

Gold nanomaterials have attracted considerable interest as vehicles for intracellular drug delivery. In our study, we synthesized three different shapes of methylpolyethylene glycol coated-anisotropic gold nanoparticles: stars, rods, and triangles. The cellular internalization of these nanoparticles by RAW264.7 cells was analyzed, providing a parametric evaluation of the effect of shape. The efficiency of cellular uptake of the gold nanoparticles was found to rank in the following order from lowest to highest: stars, rods, and triangles. The possible mechanisms of cellular uptake for the three types of gold nanoparticles were examined, and it was found that different shapes tended to use the various endocytosis pathways in different proportions. Our study, which has demonstrated that shape can modulate the uptake of nanoparticles into RAW264.7 cells and that triangles were the shape with the most efficient cellular uptake, provides useful guidance toward the design of nanomaterials for drug delivery.

Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co-Culture Model.

This study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model with co-culture of adipose-derived stromal cells (ASCs) and endothelial cells (ECs). A 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein-labeled mouse and ECs from red fluorescent protein-labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS, respectively were observed by confocal laser scanning microscopy. Semi-quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis-related genes and proteins. Angiogenesis in the co-culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co-culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1,and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs. These results revealed the VEGFA/B-Notch1/2-Hes1/Hey1- VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co-cultured in a 3D collagen gel model. J. Cell. Physiol. 232: 1548-1558, 2017. © 2016 Wiley Periodicals, Inc.

Self-Assembled Tetrahedral DNA Nanostructures Promote Adipose-Derived Stem Cell Migration via lncRNA XLOC 010623 and RHOA/ROCK2 Signal Pathway.

Self-assembled tetrahedral DNA nanostructures (TDNs) with precise sizes have been extensively applied in various fields owing to their exceptional mechanical rigidity, structural stability, and modification versatility. In addition, TDNs can be internalized by mammalian cells and remain mainly intact within the cytoplasm by escaping degradation by nucleases. Here, we studied the effects of TDNs on cell migration and the underlying molecular mechanisms. TDNs remarkably enhanced the migration of rat adipose-derived stem cells and down-regulated the long noncoding RNA (lncRNA) XLOC 010623 to activate the mRNA expression of Tiam1 and Rac1. Furthermore, TDNs highly up-regulated the mRNA and protein expression of RHOA, ROCK2, and VCL. These results indicate that TDNs suppressed the transcription of lncRNA XLOC 010623 and activated the TIAM1/RAC1 and RHOA/ROCK2 signaling pathways to promote cell migration. On the basis of these findings, TDNs show a high potential for application in tissue repair and regenerative medicine as a functional three-dimensional DNA nanomaterial.