Covalent Organic Framework-Derived Carbonous Nanoprobes for Cancer Cell Imaging.

Covalent organic frameworks (COFs) have emerged as promising materials for biomedical applications, but their functions remain to be explored and the potential toxicity concerns should be resolved. Herein, it is presented that carbonization significantly enhances the fluorescence quenching efficiency and aqueous stability of nanoscale COFs. The probes prepared by physisorbing dye-labeled nucleic acid recognition sequences onto the carbonized COF nanoparticles (termed C-COF) were employed for cell imaging, which could effectively light up biomarkers (survivin and TK1 mRNA) in living cells. The C-COF has enhanced photothermal conversion capacity, indicating that the probes are also promising candidates for photothermal therapy. The potential toxicity concern from the aromatic rigid building units of COFs was detoured by carbonization. Overall, carbonization is a promising strategy for developing biocompatible and multifunctional COF-derived nanoprobes for biomedical applications. This work may inspire more versatile COF-derived nanoprobes for bioanalysis and nanomedicine.

Improved Treatment Options for Glaucoma with Brimonidine-Loaded Lipid DNA Nanoparticles.

Glaucoma is the second leading cause of irreversible blindness worldwide. Among others, elevated intraocular pressure (IOP) is one of the hallmarks of the disease. Antiglaucoma drugs such as brimonidine can lower the IOP but their adherence to the ocular surface is low, leading to a low drug uptake. This results in a frequent dropping regime causing low compliance by the patients. Lipid DNA nanoparticles (NPs) have the intrinsic ability to bind to the ocular surface and can be loaded with different drugs. Here, we report DNA NPs functionalized for loading of brimonidine through specific aptamers and via hydrophobic interactions with double stranded micelles. Both NP systems exhibited improved affinity toward the cornea and retained release of the drug as compared to controls both in vitro and in vivo. Both NP types were able to lower the IOP in living animals significantly more than pristine brimonidine. Importantly, the brimonidine-loaded NPs showed no toxicity and improved efficacy and hence should improve compliance. In conclusion, this drug-delivery system offers high chances of an improved treatment for glaucoma and thus preserving vision in the aging population.

DNA Nanostructures for Targeted Antimicrobial Delivery.

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.

Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors.

Recently, pH-sensitive polymers have received extensive attention in tumor therapy. However, the rapid response to pH changes is the key to achieving efficient treatment. Here, a novel shielding system with a rapidly pH-responsive polymer (PAMT) is synthesized by click reaction between poly(γ-allyl-l-glutamate) and thioglycolic acid or 2-(Boc-amino)ethanethiol. The zwitterionic biodegradable polymer PAMT, which is negatively charged at physiological pH, can be used to shield positively charged nanoparticles. PAMT is electrostatically attached to the surface of the positively charged PEI/pDNA complex to form a ternary complex. The zwitterionic PAMT-shielded complex exhibits rapid charge conversion when the pH decreases from 7.4 to 6.8. For the in vivo tumor inhibition experiment, PAMT/PEI/shVEGF injected intravenously shows a more significant inhibitory effect on tumor growth. The excellent results are mainly attributed to introduction of the zwitterionic copolymer PAMT, which can shield the positively charged PEI/shVEGF complex in physiological conditions, while the surface potential of the shielded complexes changes to a positive charge in the acidic tumor environment.

Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Cancer Cell Membrane Vesicle for Multiplex MicroRNA Imaging in Living Cells.

Highly efficient cellular transfection and intracellular signal amplification is a prerequisite for low-abundant microRNA (miRNA) imaging and biomedical application. Herein, we report a functional cancer cell membrane (CM) vesicle, Au-P/DSN@CM (DSN, double-specific nucleases), which consists of Au nanoparticles modified with three types of fluorescent miRNA detection probes (Au-P) and DSN that simultaneously encapsulate in cancer CM. We find that the Au-P/DSN@CM could specifically target the cancer cell and transfect the cell with higher efficiency than Au nanoparticles. The internalized Au-P/DSN@CM could further specifically recognize the target miRNA and induce DSN-assisted target recycle signal amplification, leading to multiple miRNA simultaneous detection with high sensitivity. It successfully detects oncogenic miRNAs in MCF-7 cells with high sensitivity and is amenable to monitor the dynamic expression change of oncogenic miRNAs in cancer cells. Our study represents a promising gene delivery vector for cancer diagnosis and potential therapy.

Quantitative and specific detection of cancer-related microRNAs in living cells using surface-enhanced Raman scattering imaging based on hairpin DNA-functionalized gold nanocages.

Variations in the intracellular expression level of cancer-related microRNAs (miRNAs) are connected with worsening tumor progression. A simple, accurate, and sensitive analytical method for the imaging and detection of intracellular miRNA is still a great challenge due to the low abundance of miRNAs and the complexity of intracellular environments. In this work, target miRNA (miRNA)-mediated catalytic hairpin assembly (CHA)-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC nanostructures were designed for surface-enhanced Raman scattering (SERS) detection and imaging of the specific miR-125a-5p in the normal lung epithelial cell line (BEAS-2B cells) and lung cancer cell line (A549 cells). The finite difference time domain (FDTD) simulations showed that the polymer of GNCs possessed a much stronger electromagnetic field in nanogaps than that of single GNC, theoretically confirming the rational design of the CHA assembly strategy. Using this method, miR-125a-5p can be detected in a wide linear range with a detection limit of 43.96 aM and high selectivity over other miRNAs in vitro. Moreover, SERS imaging successfully detected and distinguished the expression levels of intracellular miR-125a-5p in BEAS-2B cells and A549 cells. The results obtained by the SERS assay were consistent with those obtained by the real-time quantitative polymerase chain reaction (qRT-PCR). This method can offer a powerful strategy for the imaging and quantitative detection of various types of biomolecules in vitro as well as in living cells.

Ultrasensitive Fluorometric Angling Determination of Staphylococcus aureus in Vitro and Fluorescence Imaging in Vivo Using Carbon Dots with Full-Color Emission.

Rapid, accurate, and safe screening of foodborne pathogenic bacteria is essential to effectively control and prevent outbreaks of foodborne illness. Fluorescent sensors constructed from carbon dots (CDs) and nanomaterial-based quenchers have provided an innovative method for screening of pathogenic bacteria. Herein, an ultrasensitive magnetic fluorescence aptasensor was designed for separation and detection of Staphylococcus aureus (S. aureus). Multicolor fluorescent CDs with a long fluorescent lifetime (6.73 ns) and high fluorescence stability were synthesized using a facile hydrothermal approach and modified cDNA as a highly sensitive fluorescent probe. CD fluorescence was quenched by Fe3O4 + aptamer via fluorescence resonance energy transfer (FRET). Under optimal conditions, the FRET-based aptasensor can detect S. aureus accompanied by a wide linear range of 50-107 CFU·mL-1 and a detection limit of 8 CFU·mL-1. Compared with other standard methods, this method was faster and more convenient, and the entire test was finished within 30 min. The capability of the aptasensor was simultaneously investigated on food samples. Additionally, the developed CDs exhibited excellent biocompatibility and were thus applied as fluorescent probes for bioimaging both in vitro and in vivo. This new platform provided an excellent application of the CDs for detecting and bioimaging pathogenic bacteria.

Evaluation and comparison of cytotoxicity, genotoxicity, and apoptotic effects of poly-l-lysine/plasmid DNA micro- and nanoparticles.

The principal impediment to gene therapy is the development of efficient, nontoxic gene carriers that can handle and deliver foreign genetic materials into various cell types, including healthy and cancerous cells. Poly-l-lysine (PLL) polymers are one of the most favorable gene carriers among nonviral vectors, and PLL had low transfection and safety issues. The purpose of this study was to measure cellular toxicity, DNA damage, and apoptotic effects of PLL nanoparticles. Neuro2A mammalian cells were cultured and exposed to PLL/DNA complexes at different polymer/DNA ratios (C/P ratio 2 and 6) for 24 h. To evaluate metabolic activity, genotoxicity, and apoptotic influences of PLL nanoparticle, the following experimental methods were employed, in order: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), DNA damage (COMET analysis) assay, and sub-G1 peak apoptosis assay. Our data indicate that toxicity is concentration dependent and a high concentration of polymer declined the metabolic activity. In addition, largest complexes (C/P 6 in HEPES buffered saline buffer) have slighter negative impact on metabolic activity. In agreement with our cytotoxicity data, apoptotic assay result represented that increase in size of PLL/DNA complexes decrease the number of apoptotic cells. Also, there was a remarkable increase in percent tail DNA of Neuro2A cells treated with higher concentration of PLL and its polyplexes. The present study demonstrated that PLL/DNA complexes caused cytotoxic, apoptotic, and genotoxic effects in a dose-dependent and weight ratio-dependent manner, which also affected the size of polyplexes.

Dual Energy Transfer-Based Fluorescent Nanoprobe for Imaging miR-21 in Nonalcoholic Fatty Liver Cells with Low Background.

Nonalcoholic fatty liver disease (NAFLD) can progress gradually to liver failure, early warning of which is critical for improving the cure rate of NAFLD. In situ imaging and monitoring of overexpressed miR-21 is an advanced strategy for NAFLD diagnosis. However, this strategy usually suffers from the high background imaging in living cells owing to the complexity of the biological system. To overcome this problem, herein, we have developed a one-donor-two-acceptor nanoprobe by assembling gold nanoparticles (AuNPs) coupled with BHQ2 (AuBHQ) and quantum dots (QDs) through DNA hybridization for imaging of miR-21 in living cells. The fluorescence of QDs was quenched up to 82.8% simultaneously by the AuNPs and the BHQ2 via nanometal surface energy transfer and fluorescence resonance energy transfer, reducing the background signals for target imaging. This low background fluorescent nanoprobe was successfully applied for imaging the target miR-21 in nonalcoholic fatty liver cells by catalyzing the disassembly of QDs with the AuBHQ and the fluorescence recovery of QDs. In addition, the sensitivity of this nanoprobe has also been enhanced toward detecting miR-21 in the range of 2.0-15.0 nM with the detection limit (LOD, 3σ) of 0.22 nM, which was 13.5 times lower than that without BHQ2. The proposed approach provides a new way for early warning, treatments, and prognosis of NAFLD.

Visualizing the down-regulation of hTERT mRNA expression using gold-nanoflare probes and verifying the correlation with cancer cell apoptosis.

The human telomerase reverse transcriptase catalytic subunit (hTERT) is the rate-limiting subunit of the telomerase holoenzyme. Down-regulating the expression of hTERT mRNA by antisense oligonucleotides would reduce the expression of hTERT, inhibit telomerase activity, and impair the growth of cancer cells in vitro. In this work, we propose a locked nucleic acid-functionalized gold nanoparticle flare probe (AuNP-probe). After transferring these probes into cells by endocytosis of the gold nanoparticles, the binding process of the antisense locked nucleic acid with hTERT mRNA along with gene regulation can be visualized by fluorescence recovery of flare-sequences. A significant decline in hTERT mRNA levels and the hTERT content occurred in cancer cells after treatment with the AuNP-probes, and only approximately 25% of the original level of hTERT mRNA remained after 72 h. AuNP-probe treated cancer cells were arrested in the G1 phase of the cell cycle and underwent apoptosis; cell viability decreased obviously compared with that of telomerase-negative normal cells.

Impact of DNA Oxidation on Toxicology: From Quantification to Genomics.

Understanding the toxicological implications of deoxyribonucleic acid (DNA) oxidation arising from cellular oxidative stress depends on identifying DNA oxidation products, their location in the genome, and their interaction with repair, replication, and gene expression.

Multivalent Self-Assembled DNA Polymer for Tumor-Targeted Delivery and Live Cell Imaging of Telomerase Activity.

The efficient detection and in situ monitoring of telomerase activity is of great importance for cancer diagnosis and biomedical research. Here we report for the first time that the development of a novel multivalent self-assembled DNA polymer, constructed through telomerase primer sequence (ITS) triggered hybridization chain assembly using two functional hairpin probes (tumor-trageting aptamer modified H1 and signal probe modified H2), for sensitive detection and imaging of telomerase activity in living cells. After internalizing into the tumor cells by multivalent aptamer targeting, the ITS on DNA polymers can be elongated by intracellular telomerase to generate telomere repeat sequences that are complementary with the signal probe, which can proceed along the DNA polymers, and gradually light up the whole DNA polymers, leading to an enhanced fluorescence signal directly correlated with the activity of telomerase. Our results demonstrated that the developed DNA polymer show excellent performance for specifically detecting telomerase activity in cancer cells, dynamically monitoring the activity change of telomerase in response to telomerase-based drugs, and efficiently distinguishing cancer cells from normal cells. The proposed strategy may afford a valuable tool for the monitoring of telomerase activity in living cells and have great implications for biological and diagnostic applications.

Assembly of DNA Probes into Superstructures for Dramatically Enhancing Enzymatic Stability and Signal-to-Background Ratio.

DNA fluorescent probes are versatile tools that are widely used for biological detection in tubes. Using DNA probes in living systems, however, represents a significant challenge because of the endogenous nuclease-induced DNA degradation and strong background fluorescence in complex biological environments. Here, we show that assembling DNA probes into core-satellite gold nanoparticle (AuNP) superstructures could unprecedentedly enhance enzymatic stability and reduce background interference. The embedded DNA probes are protected from interaction with nuclease, eliminating the enzymatic degradation. In the meantime, the AuNP superstructures show extremely high quenching efficiency (>98%) toward the embedded DNA probes, whose fluorescence can be instantly turned on by the target binding, resulting in high signal-to-background ratio. To demonstrate these distinct properties, we made use of the assembled nanoprobes to monitor the ATP levels under different stimuli in living cells. The assembly strategy leads to a new opportunity for accurately sensing targets in living systems.

A novel nucleic acid aptamer tag: a rapid fluorescence strategy using a self-constructing G-quadruplex from AGG trinucleotide repeats.

Herein, we have developed a novel fluorescence labeling strategy for nucleic acid aptamers based on self-assembling between AGG tri-nucleotide repeats and a pyrene-modified oligonucleotide. This strategy could be an effective tool for developing targeting-imaging systems and biosensor systems to detect target molecules.

Rox-DNA Functionalized Silicon Nanodots for Ratiometric Detection of Mercury Ions in Live Cells.

A ratiometric fluorescent sensor for mercury ions (Hg2+) has been constructed via covalent functionalization of silicon nanodot (SiND) with Hg2+-specific 6-carboxy-X-rhodamine (Rox)-tagged DNA. For the Rox-DNA functionalized SiND, the red fluorescence of Rox can be quenched by the blue-emitting SiND in the presence of Hg2+ due to structural change in DNA, which serves as the response signal. Meawhile, the fluorescence of SiND is insensitive to Hg2+ and acts as the reference signal. The wavelength difference in the optimal emission peak is as large as 190 nm between SiND (422 nm) and Rox (612 nm), which can efficaciously exclude the interference of the two emission peaks, and facilitates dual-color visualization of Hg2+ ions. The biofunctionalization of SiND improves the acid-base stability of SiND significantly, which is favorable for its application in the intracellular environment. Accordingly, a sensitive, simple, precise and rapid method for tracing Hg2+ was proposed. The limit of detection and precision of this method for Hg2+ was 9.2 nM and 8.8% (50 nM, n = 7), respectively. The increase of Hg2+ concentration in the range of 10-1500 nM was in accordance with linearly increase of the I422/ I612 ratio. As for practical application, the recoveries in spiked human urine and serum samples were in the range of 81-107%. Moreover, this fluorescent nanosensor was utilized to the ratiometric detection of Hg2+ in HeLa cells.

Fluorescence and photoacoustic dual-mode imaging of tumor-related mRNA with a covalent linkage-based DNA nanoprobe.

We developed a dual-mode DNA nanoprobe based on covalent linkage for fluorescence imaging and photoacoustic imaging of tumor-related mRNA.

Editor's Highlight: Formulation and Toxicology Evaluation of the Intrathecal AYX1 DNA-Decoy in Sprague Dawley Rats.

The longevity of pain after surgery is debilitating and limits the recovery of patients. AYX1 is a double-stranded, unprotected, 23 base-pair oligonucleotide designed to reduce acute post-surgical pain and prevent its chronification with a single intrathecal perioperative dose. AYX1 mimics the DNA sequence normally bound by EGR1 on chromosomes, a transcription factor transiently induced in the dorsal root ganglia-spinal cord network following a noxious input. AYX1 binds to EGR1 and prevents it from launching waves of gene regulation that are necessary to maintain pain over time. A formulation suitable for an intrathecal injection of AYX1 was developed, including a specific ratio of AYX1 and calcium so the ionic homeostasis of the cerebrospinal fluid is maintained and no impact on neuromuscular control is produced upon injection. A GLP toxicology study in naïve Sprague Dawley rats was conducted using 3 dose levels up to the maximum feasible dose. Clinical observations, neurobehavioral observations, clinical pathology and histopathology of the nervous system and peripheral tissues were conducted. An additional nonGLP study was conducted in the spared nerve injury model of chronic neuropathic pain in which EGR1 is induced in the dorsal root ganglia and spinal cord. Similar testing was performed, including a modified Irwin test to assess a potential impact of AYX1 on autonomic nervous system responses, locomotion, activity, arousal, sensorimotor, and neuromuscular function. No AYX1-related adverse events were observed in any of the studies and the no-observed-adverse-effect-level was judged to be the maximum feasible dose.

Effect of Eudragit on In Vitro Transfection Efficiency of PEI-DNA Complexes.

AIM: Eudragit® E 100 (EE100) was used to improve the transfection efficiency of polyethylenimine (PEI). MATERIALS AND METHODS: Mobility of PEI-DNA complexes with and without EE100 were visualized by agarose gel electrophoresis and their transfection efficiencies were investigated in KB human oral carcinoma cells by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of transfected cells. RESULTS: Gel electrophoresis illustrated formation of complete complexes at N/P ratios above 5. PEI had the highest transfection efficiency at an N/P ratio of 15, whereas in combination with EE100, the transfection efficiency was highest at an N/P ratio of 7.5. High concentrations of EE100 in combination with PEI were found to reduce cell viability. CONCLUSION: The results show a synergistic action of EE100 in transfection of DNA at low N/P ratios compared to PEI alone.

Reversibly cross-linked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity.

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy.