Methods to Improve the Stability of Nucleic Acid-Based Nanomaterials.

Nucleic acid strands can be synthesized into various nucleic acid-based nanomaterials (NANs) through strict base pairing. The self-assembled NANs are programmable, intelligent, biocompatible, non-immunogenic, and non-cytotoxic. With the rapid development of nanotechnology, the application of NANs in the biomedical fields, such as drug delivery and biological sensing, has attracted wide attention. However, the stability of NANs is often affected by the cation concentrations, enzymatic degradation, and organic solvents. This susceptibility to degradation is one of the most important factors that have restricted the application of NANs. NANs can be denatured or degraded under conditions of low cation concentrations, enzymatic presence, and organic solvents. To deal with this issue, a lot of methods have been attempted to improve the stability of NANs, including artificial nucleic acids, modification with specific groups, encapsulation with protective structures, etc. In this review, we summarized the relevant methods to have a deeper understanding of the stability of NANs.

Tetrahedral framework nucleic acids alleviate irradiation-induced salivary gland damage.

In this study, we investigated the role of tetrahedral framework nucleic acids (tFNAs) in irradiation-induced salivary gland damage in vitro and in vivo. Irradiation-damaged submandibular gland cells (SMGCs) were treated with different concentrations of tFNAs. Cell activity was measured by CCK-8 assay. Cell death was detected by Calcein-AM/PI double staining. Cell apoptosis was assessed by flow cytometry. The expression of apoptosis proteins and inflammatory cytokines were detected by western blot. Body weight, drinking volume, saliva flow rate and lag time was measured 8 weeks after irradiation. Micromorphological changes of submandibular gland were assessed by haematoxylin-eosin and masson staining. Cell proliferation, apoptosis and microvessel density of submandibular gland were evaluated by immunohistochemical staining. tFNAs could promote cell proliferation, inhibit cell apoptosis of irradiation-damaged SMGCs and reduce irradiation induced cell death. Mechanism studies revealed that tFNAs inhibited cell apoptosis through regulating the Bcl-2/Bax/Caspase-3 signalling pathway and inhibited the release of TNF-α, IL-1ß and IL-6 to reduce cell damage caused by inflammation. Animal experiments showed that tFNAs could alleviate irradiation-induced weight loss, increased water intake, decreased saliva production and prolonged salivation lag time and could ameliorate salivary gland damage. tFNAs have a positive effect on alleviating irradiation-induced salivary gland damage and might be a promising agent for the treatment of this disease.

Delayed ileal neobladder fistula caused by bladder stones: a case report.

BACKGROUND: Ileal neobladder fistula is a rare complication after radical cystectomy, with an incidence of approximately 0.7%. At present, there are scattered reports of vesicoileal fistula, but there are no reports of ileal neobladder fistula (INF) caused by bladder stones. In this paper, a case of ileal neobladder fistula caused by chronic stimulation of bladder stones was successfully diagnosed and treated. CASE PRESENTATION: A 68-year-old man who had undergone radical cystectomy and an orthotopic ileal neobladder procedure 10 years prior presented with refractory diarrhoea and oliguria and was diagnosed with ileal neobladder fistula caused by chronic stimulation of bladder stones. We performed fistulectomy, cystotomy, partial ileectomy, and end-to-end ileal anastomosis, and the patient recovered and was discharged after the operation. CONCLUSION: Urinary calculi are delayed complications of orthotopic neobladder construction after total cystectomy. Bladder stones are a rare complication of ileal neovesical fistula, which can cause neovesical cutaneous fistula. It is difficult to diagnose through routine examination and easily misdiagnosed as acute gastroenteritis. Surgery is an effective treatment for INF and can achieve a good prognosis.

The biological applications of DNA nanomaterials: current challenges and future directions.

DNA, a genetic material, has been employed in different scientific directions for various biological applications as driven by DNA nanotechnology in the past decades, including tissue regeneration, disease prevention, inflammation inhibition, bioimaging, biosensing, diagnosis, antitumor drug delivery, and therapeutics. With the rapid progress in DNA nanotechnology, multitudinous DNA nanomaterials have been designed with different shape and size based on the classic Watson-Crick base-pairing for molecular self-assembly. Some DNA materials could functionally change cell biological behaviors, such as cell migration, cell proliferation, cell differentiation, autophagy, and anti-inflammatory effects. Some single-stranded DNAs (ssDNAs) or RNAs with secondary structures via self-pairing, named aptamer, possess the ability of targeting, which are selected by systematic evolution of ligands by exponential enrichment (SELEX) and applied for tumor targeted diagnosis and treatment. Some DNA nanomaterials with three-dimensional (3D) nanostructures and stable structures are investigated as drug carrier systems to delivery multiple antitumor medicine or gene therapeutic agents. While the functional DNA nanostructures have promoted the development of the DNA nanotechnology with innovative designs and preparation strategies, and also proved with great potential in the biological and medical use, there is still a long way to go for the eventual application of DNA materials in real life. Here in this review, we conducted a comprehensive survey of the structural development history of various DNA nanomaterials, introduced the principles of different DNA nanomaterials, summarized their biological applications in different fields, and discussed the current challenges and further directions that could help to achieve their applications in the future.

Broadening the biocompatibility of gold nanorods from rat to Macaca fascicularis: advancing clinical potential.

BACKGROUND: The biomedical field has used gold nanorods (GNRs) for decades; however, clinical trials and translation is limited except gold nanoshells. The preparation of gold nanoshells is more complex than that of polyethylene glycol-modified GNRs (PEG-GNRs), and it is difficult to ensure uniform thickness. It is important to encourage and broaden the use of the star member (PEG-GNRs) of gold nanoparticles family for clinical translation. Existing studies on PEG-GNRs are limited with no relevant systematic progression in non-human primates. Herein, we assessed the systematic biocompatibility of PEG-GNRs in rats and clinically relevant Macaca fascicularis. RESULTS: In this small animal study, we administrated multiple doses of PEG-GNRs to rats and observed good biocompatibility. In the non-human primate study, PEG-GNRs had a longer blood half-life and produced a negligible immune response. Histological analysis revealed no significant abnormality. CONCLUSIONS: PEG-GNRs were well-tolerated with good biocompatibility in both small animals and large non-human primates. The information gained from the comprehensive systemic toxicity assessment of PEG-GNRs in M. fascicularis will be helpful for translation to clinical trials.

Tetrahedral Framework Nucleic Acids Loading Ampicillin Improve the Drug Susceptibility against Methicillin-Resistant Staphylococcus aureus.

The overuse of antibiotics has led to the emergence of multidrug-resistant pathogens. There is an urgent need to develop alternative therapeutic strategies to reduce mortality and morbidity related to drug-resistant bacterial infections. Self-synthesized tetrahedral framework nucleic acids (tFNAs) are used as the drug loading platform to deliver ampicillin to combat methicillin-resistant Staphylococcus aureus (MRSA) infection. The results of average dimension, zeta potential, transmission electron microscopy, and ultraviolet spectrophotometry showed that tFNAs-ampicillin combined with a sufficient encapsulation rate and good stability. tFNAs-ampicillin had a better affinity to MRSA than free ampicillin because it had a better uptake by MRSA cells. Additionally, tFNAs-ampicillin had a better antibacterial effect and lower levels of resistance development than free ampicillin. The downregulation of genes related to bacterial cell wall synthesis (murA and murZ) and upregulation of a gene related to antibiotic sensibility (PBP2) were responsible for the enhanced killing effect of tFNAs-ampicillin against MRSA.

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.

Treatment of Alzheimer's disease with framework nucleic acids.

OBJECTIVES: To provide a new research direction for nerve regeneration and strategy for Alzheimer's disease treatment, tetrahedral DNA nanostructures (TDNs)-novel tetrahedral framework nucleic acid molecule nanoparticles (tFNA) that can inhibit the apoptosis of nerve cells are employed in the experiment. MATERIALS AND METHODS: To verify the successful preparation of TDNs, the morphology of TDNs was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The expression of apoptosis-related genes and proteins was investigated by confocal microscope, flow cytometry, PCR and Western blot to detect the impact of TDNs on the Alzheimer's model. And finally, Morris water maze experiment was used to test behavioural changes and Nissl stain was detected to observe the morphology and quantity of neurons in the hippocampus. Immunofluorescence stain was used to observe the Aß stain, and TUNEL dyeing was utilized to observe neuronal apoptosis. RESULTS: In vitro and in vivo experiments confirm that TDNs, in a specific concentration range, have no toxic or side effects on nerve cells, can effectively inhibit apoptosis in an Alzheimer's disease cell model and effectively improve memory and learning ability in a rat model of Alzheimer's disease. CONCLUSIONS: These findings suggest that TDNs may be a promising drug for the treatment of Alzheimer's disease.

Multi-targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence.

Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge. Bacteria and the extracellular polysaccharides (EPS) cause biofilms to become adherent, toxic, resistant to antibiotics, and ultimately difficult to remove. Inhibition of EPS synthesis can prevent the formation of bacterial biofilms, reduce their robustness, and promote removal. Here, we have developed a framework nucleic acid delivery system with a tetrahedral configuration. It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes. We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site. Once delivered to bacterial cells, they significantly decreased EPS synthesis and biofilm thickness. Compared to existing approaches, this system is highly efficacious because it simultaneously reduces the expression of all targeted genes (gtfBCD, gbpB, ftf). We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.

Enhanced Neural Regeneration with a Concomitant Treatment of Framework Nucleic Acid and Stem Cells in Spinal Cord Injury.

Spinal cord injury (SCI), began with a primary injury including contusion and compression, is a common disease caused by various pathogenesis. Characterized disruption of axons and irreversible loss of neurons in SCI, and further damage in spinal cord tissue caused by following secondary injuries, such as the formation of glial scar and inflammation, makes it even harder to recover for affected patients. Tetrahedral framework nucleic acid (tFNA), which possesses the capability of promoting neuroprotection and neuroregeneration in vitro, might alleviate the injuries, and facilitate the neural tissue regeneration in experimental animal models of SCI. Here, we developed a concomitant treatment of tFNA and neural stem cells (NSCs) for the synergistic therapy in treating the injury of the spinal cord. We first observed that tFNA could promote cell proliferation of NSCs then verified that the concomitant treatment of tFNA and NSCs showed the neuroprotective actions by increasing the survival of transplanted NSCs. Furthermore, the recovery of motor function and the tissue regeneration in the lesion site of the spinal cord achieved the best performance in the SCI rats treated with the combination of tFNA and NSCs than others, and the formation of glial scar was the least. Our findings provide novel evidence of a promising strategy for synergistic treatment of SCI in the future.

Diversity of DNA Nanostructures and Applications in Oncotherapy.

DNA nanotechnology is a new frontier in the field of tumor biotherapy. Simple DNA strands can be precisely constructed for integration into nanostructures of desired shapes and sizes, with excellent stability and biocompatibility. In this review, an account of the wide range of nanostructures composed of DNA sequences and related advances in oncotherapy using aptamers and chemical drugs is given. Functional ligands, including enzymes, antibodies, and agents, have been appended to DNA frameworks based on their external and internal modifiability. Hence, additional functionalities, such as immunogenicity and enzymatic activity, have been obtained, which extend their practical applications. Importantly, aptamers and drugs can be attached to or incorporated into the wireframes, bringing in highly selective targeting and killing abilities for the modified DNA nanostructures (DNs). In conclusion, distinct DNA sequences, various functional molecules, and different interactions and modifications lead to the diversity of DNs. Currently, one of the leading areas is their applications in tumor therapy. But beyond that, DNs should have much wider application prospects.

Review of craniofacial regeneration in China.

AIM: Tissue engineering has been recognised as one of the most effective means to form a new viable tissue for medical purpose. Tissue engineering involves a combination of scaffolds, cells, suitable biochemical and physicochemical factors, and engineering and materials methods. This review covered some biomedicine, such as biomaterials, bioactive factors, and stem cells, and manufacturing technologies used in tissue engineering in the oral maxillofacial region, especially in China. MATERIALS AND METHODS: Data for this review were identified by searches of Web of Science and PubMed, and references from relevant articles using the search terms "biomaterials", "oral tissue regeneration", "bioactive factors" and "stem cells". Only articles published in English between 2013 and 2018 were included. CONCLUSION: The combination of stem cells, bioactive factors and 3D scaffolds could be of far-reaching significance for the future therapies in tissue repair or tissue regeneration. Furthermore, the review also mentions issues that need to be solved in the application of these biomedicines.

Potent anti-angiogenesis and anti-tumour activity of pegaptanib-loaded tetrahedral DNA nanostructure.

OBJECTIVES: Pegaptanib might be a promising anti-tumour drug targeting VEGF to inhibit tumour vascular endothelial cell proliferation. However, the poor biostability limited its application. In this study, we took tetrahedron DNA nanostructures (TDNs) as drug nanocarrier for pegaptanib to explore the potent anti-angiogenesis and anti-tumour activity of this drug delivery system. MATERIALS AND METHODS: The successful synthesis of TDNs and pegaptanib-TDNs was determined by 8% polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis and dynamic light scattering (DLS). The cytotoxicity of pegaptanib alone and pegaptanib-TDNs on HUVECs and Cal27 was evaluated by the cell count kit-8 (CCK-8) assay. The effect of pegaptanib and pegaptanib-TDNs on proliferation, migration and tube formation of HUVECs induced by VEGF was examined by CCK-8 assay, wound healing assay and tubule formation experiment. The cell binding capacity and serum stability were detected by flow cytometry and PAGE, respectively. RESULTS: Pegaptanib-TDNs had stronger killing ability than pegaptanib alone, and the inhibiting effect was in a concentration-dependent manner. What's more, pegaptanib-loaded TDNs could effectively enhance the ability of pegaptanib to inhibit proliferation, migration and tube formation of HUVECs induced by VEGF. These might attribute to the stronger binding affinity to the cell membrane and greater serum stability of pegaptanib-TDNs. CONCLUSIONS: These results suggested that pegaptanib-TDNs might be a novel strategy to improve anti-angiogenesis and anti-tumour ability of pegaptanib.

An Intelligent DNA Nanorobot with in Vitro Enhanced Protein Lysosomal Degradation of HER2.

DNA nanorobots have emerged as new tools for nanomedicine with the potential to ameliorate the delivery and anticancer efficacy of various drugs. DNA nanostructures have been considered one of the most promising nanocarriers. In the present study, we report a DNA framework-based intelligent DNA nanorobot for selective lysosomal degradation of tumor-specific proteins on cancer cells. We site-specifically anchored an anti-HER2 aptamer (HApt) on a tetrahedral framework nucleic acid (tFNA). This DNA nanorobot (HApt-tFNA) could target HER2-positive breast cancer cells and specifically induce the lysosomal degradation of the membrane protein HER2. An injection of the DNA nanorobot into a mouse model revealed that the presence of tFNA enhanced the stability and prolonged the blood circulation time of HApt, and HApt-tFNA could therefore drive HER2 into lysosomal degradation with a higher efficiency. The formation of the HER2-HApt-tFNA complexes resulted in the HER2-mediated endocytosis and digestion in lysosomes, which effectively reduced the amount of HER2 on the cell surfaces. An increased HER2 digestion through HApt-tFNA further induced cell apoptosis and arrested cell growth. Hence, this novel DNA nanorobot sheds new light on targeted protein degradation for precision breast cancer therapy.

DNA-Based Nanomedicine with Targeting and Enhancement of Therapeutic Efficacy of Breast Cancer Cells.

Recently, a DNA tetrahedron has been reported to be a novel nanomedicine and promising drug vector because of its compactness, biocompatibility, biosafety, and editability. Here, we modified the DNA tetrahedron with a DNA aptamer (AS1411) as a DNA-based delivery system, which could bind to nucleolin for its cancer cell selectivity. Nucleolin is a specific biomarker protein overexpressed on membranes of malignant cancer cells and its deregulation is implicated in cell proliferation. The antimetabolite drug 5-fluorouracil (5-FU) is an extensively used anticancer agent; however, its major limitation is the lack of target specificity. Cyanine 5 (Cy5), a fluorescent probe, can be used to label DNA tetrahedron and enhance photostability with minimal effects on its basic functions. In this study, we additionally attached 5-FU to the DNA-based delivery system as a new tumor-targeting nanomedicine (AS1411-T-5-FU) to enhance the therapeutic efficacy and targeting of breast cancer. We examined the difference of the cellular uptake of AS1411-T-5-FU between breast cancer cells and normal breast cells and concluded that AS1411-T-5-FU had a better targeting ability to kill breast cancer cells than 5-FU. We further evaluated the expressions of cell apoptosis-related proteins and genes, which are associated with the mitochondrial apoptotic pathway. Ultimately, our results suggest the potential of DNA tetrahedron in cancer therapies, and we develop a novel approach to endow 5-FU with targeting property.

Over-the-scope-clip applications for perforated peptic ulcer.

AIM: To investigate the effectiveness of over-the-scope-clip (OTSC)-based endoscopic closure in patients with perforated peptic ulcer (PPU). METHODS: One hundred six patients diagnosed with PPU were treated with either OTSC (n = 26) or conservative treatments (n = 80), respectively. The outcome assessments included technical success rate, clinical success rate, post-treatment complications after 1 month, mortality rate, time to resume oral feeding, length of hospital stay, and the administration of antibiotics. RESULTS: In the OTSC group, technical and clinical success was achieved in 100% of patients without any complications, including death, incomplete closure, duodenal obstruction, and gastrointestinal bleeding, with a median operation time of 10 min. All patients in the OTSC group were discharged, while the mortality rate in the control group was 13.8%. Subsequent surgeries were required in 30% of patients in the control group. The median times to resume oral feeding were 3.5 (interquartile range [IQR] 2.0-5.25) days in the OTSC group and 7.0 (IQR 5.0-9.0) days in the control group (p < 0.001). One month post-procedure, 30% (24/80) of patients in the control group and 0 (0/26) in the OTSC group required additional operations (p < 0.001). No significant difference was found in the length of the hospital stay and the administration of antibiotics between the two groups (p > 0.05). CONCLUSIONS: OTSC-based endoscopic technique, with a high clinical success rate and a shorter time to resume oral feeding, was effective in achieving closure of PPU with a diameter < 15 mm.

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.

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.

The Review of Nanomaterials Inducing the Differentiation of Stem Cells into Chondrocyte Phenotypes in Cartilage Tissue Engineering.

Cartilage, as a nanostructured tissue, because of its awfully poor capacity for inherent regeneration and complete hierarchical structure, is severely difficult to regenerate after damages. Tissue engineering methods have provided a great contribution for cartilage repair. Nanomaterials have special superiority in regulating stem cell behaviors due to their special mechanical and biological properties and biomimetic characteristics. Therefore, they have been given great attention in tissue regeneration. Nanomaterials are divided into organic and inorganic nanomaterials. They provide the microenvironment to support differentiation of stem cells. Nanomaterials inducing stem cells to differentiate into chondrocyte phenotypes would be a benefit for cartilage tissue regeneration, then promoting the development of cartilage tissue engineering. In this review, we summarized the important roles of nanomaterials in chondrogenic differentiation of stem cells.

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.