Recent Advancements in Ultrasound Contrast Agents Based on Nanomaterials for Imaging.

Ultrasound (US) is a type of mechanical wave that is capable of transmitting energy through biological tissues. By utilization of various frequencies and intensities, it can elicit specific biological effects. US imaging (USI) technology has been continuously developed with the advantages of safety and the absence of radiation. The advancement of nanotechnology has led to the utilization of various nanomaterials composed of both organic and inorganic compounds as ultrasound contrast agents (UCAs). These UCAs enhance USI, enabling real-time monitoring, diagnosis, and treatment of diseases, thereby facilitating the widespread adoption of UCAs in precision medicine. In this review, we introduce various UCAs based on nanomaterials for USI. Their principles can be roughly divided into the following categories: carrying and transporting gases, endogenous gas production, and the structural characteristics of the nanomaterial itself. Furthermore, the synergistic benefits of US in conjunction with various imaging modalities and their combined application in disease monitoring and diagnosis are introduced. In addition, the challenges and prospects for the development of UCAs are also discussed.

Thermal-Responsive Antibacterial Hydrogel with Photothermal Therapy and Improving Wound Microenvironment for Promote Healing.

Skin damage is one of the most prevalent human injuries, which affects the health of human beings. However, skin damage is often accompanied by bacterial infection and wound microenvironment changes, causing damage to normal cells and inhibiting wound healing. Herein, we designed a thermal-responsive antibacterial hydrogel (GAG hydrogel) loaded with catalase (CAT)-like Au@Pt@MgSiO3 nanoparticles (APM NPs) and gentamicin (GM) to promote wound healing. The GAG hydrogel was used in a photothermal therapy (PTT)/antibiotic combination to kill bacteria, reduce the use of antibiotics, improve the wound microenvironment, promote cell proliferation, and accelerate wound healing. Under near-infrared laser irradiation, APM NPs in the hydrogel generated local hyperthermia to kill bacteria. Meanwhile, the generated heat led to a change in the hydrogel's morphology, enabling it to release GM and APM NPs to prevent the overuse of antibiotics. Subsequently, the CAT-like ability of the APM NPs decreased the oxidative stress caused by hydrogen peroxide (H2O2), thus remodeling the wound microenvironment. Then, the weakly acidic microenvironment of the wound caused the decomposition of the APM NPs and the release of magnesium ions (Mg2+), promoting the growth and migration of cells for wound healing. Therefore, the studied thermal-responsive antibacterial (GAG) hydrogel has potential in the field of wound healing.

Construction of a Functional Nucleic Acid-Based Artificial Vesicle-Encapsulated Composite Nanoparticle and Its Application in Retinoblastoma-Targeted Theranostics.

Retinoblastoma (RB) is an aggressive tumor of the infant retina. However, the ineffective targeting of its theranostic agents results in poor imaging and therapeutic efficacy, which makes it difficult to identify and treat RB at an early stage. In order to improve the imaging and therapeutic efficacy, we constructed an RB-targeted artificial vesicle composite nanoparticle. In this study, the MnO2 nanosponge (hMNs) was used as the core to absorb two fluorophore-modified DNAzymes to form the Dual/hMNs nanoparticle; after loaded with the artificial vesicle derived from human red blood cells, the RB-targeted DNA aptamers were modified on the surface, thus forming the Apt-EG@Dual/hMNs complex nanoparticle. The DNA aptamer endows this nanoparticle to target the nucleolin-overexpressed RB cell membrane specifically and enters cells via endocytosis. The nanoparticle could release fluorophore-modified DNAzymes and supplies Mn2+ as a DNAzyme cofactor and a magnetic resonance imaging (MRI) agent. Subsequently, the DNAzymes can target two different mRNAs, thereby realizing fluorescence/MR bimodal imaging and dual-gene therapy. This study is expected to provide a reliable and valuable basis for ocular tumor theranostics.

Recent theranostic applications of hydrogen peroxide-responsive nanomaterials for multiple diseases.

It is well established that hydrogen peroxide (H2O2) is associated with the initiation and progression of many diseases. With the rapid development of nanotechnology, the diagnosis and treatment of those diseases could be realized through a variety of H2O2-responsive nanomaterials. In order to broaden the application prospects of H2O2-responsive nanomaterials and promote their development, understanding and summarizing the design and application fields of such materials has attracted much attention. This review provides a comprehensive summary of the types of H2O2-responsive nanomaterials including organic, inorganic and organic-inorganic hybrids in recent years, and focused on their specific design and applications. Based on the type of disease, such as tumors, bacteria, dental diseases, inflammation, cardiovascular diseases, bone injury and so on, key examples for above disease imaging diagnosis and therapy strategies are introduced. In addition, current challenges and the outlook of H2O2-responsive nanomaterials are also discussed. This review aims to stimulate the potential of H2O2-responsive nanomaterials and provide new application ideas for various functional nanomaterials related to H2O2.

Simultaneous Fe2+/Fe3+ imaging shows Fe3+ over Fe2+ enrichment in Alzheimer's disease mouse brain.

Visualizing redox-active metal ions, such as Fe2+ and Fe3+ ions, are essential for understanding their roles in biological processes and human diseases. Despite the development of imaging probes and techniques, imaging both Fe2+ and Fe3+ simultaneously in living cells with high selectivity and sensitivity has not been reported. Here, we selected and developed DNAzyme-based fluorescent turn-on sensors that are selective for either Fe2+ or Fe3+, revealing a decreased Fe3+/Fe2+ ratio during ferroptosis and an increased Fe3+/Fe2+ ratio in Alzheimer's disease mouse brain. The elevated Fe3+/Fe2+ ratio was mainly observed in amyloid plaque regions, suggesting a correlation between amyloid plaques and the accumulation of Fe3+ and/or conversion of Fe2+ to Fe3+. Our sensors can provide deep insights into the biological roles of labile iron redox cycling.

Monitoring leaching of Cd2+ from cadmium-based quantum dots by an Cd aptamer fluorescence sensor.

Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd2+. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd2+-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd2+ and is able to measure the Cd2+ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd2+ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

Aptamer-Based Probes for Cancer Diagnostics and Treatment.

Aptamers are single-stranded DNA or RNA oligomers that have the ability to generate unique and diverse tertiary structures that bind to cognate molecules with high specificity. In recent years, aptamer researches have witnessed a huge surge, owing to its unique properties, such as high specificity and binding affinity, low immunogenicity and toxicity, and simplicity of synthesis with negligible batch-to-batch variation. Aptamers may bind to targets, such as various cancer biomarkers, making them applicable for a wide range of cancer diagnosis and treatment. In cancer diagnostic applications, aptamers are used as molecular probes instead of antibodies. They have the potential to detect various cancer-associated biomarkers. For cancer therapeutic purposes, aptamers can serve as therapeutic or delivery agents. The chemical stabilization and modification strategies for aptamers may expand their serum half-life and shelf life. However, aptamer-based probes for cancer diagnosis and therapy still face several challenges for successful clinical translation. A deeper understanding of nucleic acid chemistry, tissue distribution, and pharmacokinetics is required in the development of aptamer-based probes. This review summarizes their application in cancer diagnostics and treatments based on different localization of target biomarkers, as well as current challenges and future prospects.

DNAzyme-Based Lithium-Selective Imaging Reveals Higher Lithium Accumulation in Bipolar Disorder Patient-Derived Neurons.

Lithium has been a drug for bipolar disorders (BD) for over 70 years; however, its usage has been limited by its narrow therapeutic window (between 0.6 and 1.2 mM). Understanding the cellular distribution of lithium ions (Li+) in patient cells will offer deep insight into this limitation, but selective imaging of Li+ in living cells under biomedically relevant concentration ranges has not been achieved. Herein, we report in vitro selection and development of a Li+-specific DNAzyme fluorescent sensor with >100-fold selectivity over other biorelevant metal ions. This sensor allows comparative Li+ visualization in HeLa cells, human neuronal progenitor cells (NPCs), and neurons derived from BD patients and healthy controls. Strikingly, we detected enhanced accumulation of Li+ in cells derived from BD patients compared with healthy controls in differentiated neurons but not NPCs. These results establish the DNAzyme-based sensor as a novel platform for biomedical research into BD and related areas using lithium drugs.

Recent progress in developing fluorescent probes for imaging cell metabolites.

Cellular metabolites play a crucial role in promoting and regulating cellular activities, but it has been difficult to monitor these cellular metabolites in living cells and in real time. Over the past decades, iterative development and improvements of fluorescent probes have been made, resulting in the effective monitoring of metabolites. In this review, we highlight recent progress in the use of fluorescent probes for tracking some key metabolites, such as adenosine triphosphate, cyclic adenosine monophosphate, cyclic guanosine 5'-monophosphate, Nicotinamide adenine dinucleotide (NADH), reactive oxygen species, sugar, carbon monoxide, and nitric oxide for both whole cell and subcellular imaging.

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells.

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+ -specific 10-23 or Zn2+ -specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells.

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+-specific 10-23 or Zn2+-specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.

Folic acid-modified fluorescent dye-protein nanoparticles for the targeted tumor cell imaging.

Serum albumin has a wide range of applications in biochemical experiments and pharmaceutical field. We found that a cyanine dye, dimethylindole red (Dir), could selectively interact with bovine serum albumin (BSA). Dir exhibited very weak red fluorescence, while the fluorescence intensity at 630 nm was enhanced up to 130-fold upon noncovalently interacting with 30 µM BSA. Besides, Dir showed a highly selective response to BSA over human serum albumin (HSA). For the detection of BSA, a limit of detection as low as 23 nM was obtained. Then biocompatible Dir-BSA nanoparticles were prepared by the desolvation technique. The Dir-BSA nanoparticles possess excellent fluorescence properties with a quantum yield of 32%. Furthermore, folic acid as a targeting group was conjugated to Dir-BSA nanoparticles and these nanoparticles were characterized by TEM and laser particle analyzer, etc. Folic acid-modified Dir-BSA nanoparticles were successfully used for tumor cell-targeted imaging.

Aptamer-integrated α-Gal liposomes as bispecific agents to trigger immune response for killing tumor cells.

A novel bispecific α-Gal liposome was constructed by self-assembling AS1411 aptamers into the α-Gal containing liposomes. The α-Gal liposomes were prepared using cell membranes of red blood cells from rabbit, which are composed of cholesterol, phospholipids, and α-Gal glycolipids. AS1411 is a DNA aptamer with high specificity and affinity for nucleolin and could integrate into liposomes by the modification of cholesterol. The bispecific α-Gal liposomes surface-functionalized by α-Gal and AS1411 aptamer could recognize anti-Gal antibodies and nucleolin overexpressed by tumor cells simultaneously, followed by activating the immune system to attack the tumor cells, resulting in the lysis of the tumor cells by antibody dependent cell-mediated cytotoxicity. Under simulated tumor environment, the lysis rate of MCF-7 cells treated by the AS1411 modified α-Gal liposomes drastically increased compared to the liposomes without AS1411 aptamer. This study suggests that the AS1411 modified α-Gal liposomes can recognize nucleolin-overexpressing tumor cells selectively, subsequently improve the effect of the immunotherapy with high specificity. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1176-1183, 2019.

A photo-regulated aptamer sensor for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells.

Fluorescent aptamer sensors have shown enormous potential for intracellular imaging of small molecule metabolites. Since metabolites distribute differently at different subcellular locations and their concentrations and locations fluctuate with time, methods are needed for spatiotemporally controlled monitoring of these metabolites. Built upon previous success in temporal control of aptamer-based sensors, we herein report an aptamer sensor containing a photocleavable linker and using DQAsomes to target mitochondria for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells. The photocleavable modification on the DNA ATP aptamer sensor can prevent sensor activation before reaching mitochondria and the sensor can then be activated upon light irradiation. The sensor has a detection limit of 3.7 µM and high selectivity against other nucleotides, allowing detection of ATP concentration fluctuations in mitochondria induced by Ca2+ or oligomycin. This work represents the first successful delivery of a DNA aptamer sensor to mitochondria, providing a new platform for targeted delivery to subcellular organelles for monitoring energy producing processes, as well as mitochondrial dysfunction-related diseases in different cells.

Self-Assembled saRNA Delivery System Based on Rolling Circle Transcription for Aptamer-Targeting Cancer Therapy.

With the capacity of gene promotion, RNA activation (RNAa) has been supposed to be a powerful technique in the field of biomedicine, especially in an antitumor aspect. However, one of the pressing challenges for clinical application is how to efficiently deliver therapeutic probes to cancer. Herein, we synthesized a carrier through rolling circle transcription (RCT) to deliver p21 saRNA with high loading rate and targeting capacity. This carrier could be condensed from a micron dimension to about 200 nm by polyethylenimine (PEI). In addition, the trait of robust tumor targeting and improved cytotoxicity was demonstrated when the aptamer was modified through layer-by-layer self-assembly. Moreover, the 4-fold activation of the p21 gene could be obviously detected in a targeting group. Meanwhile, the cell apoptosis induced by p21 promotion was also exhibited, which indicated that this highly efficient saRNA delivery carrier had a specific antitumor effect and could reduce side effects to normal cells. Therefore, this delivery system had the potential to be used in RNAa applications and cancer-targeted treatment.

Self-assembled RNAi nanoflowers via rolling circle transcription for aptamer-targeted siRNA delivery.

To deliver siRNA efficiently, prevailing conventional lipid or polymer encapsulation often needs multi-step compounding methods, which may inevitably introduce cationic or other components and may lead to cytotoxicity or an immune response. Herein, we present a novel enzymatic synthetic approach to produce tumor-targetable RNAi nanoflowers. The RNAi nanoflowers are mainly composed of multiple tandem copies of siRNA precursors by rolling circle transcription (RCT), and produce large amounts of siRNA to silence Bcl-2 gene expression after cellular uptake, which can overcome the problem of low loading capacity. In particular, the RNAi microspheres (RNAi-MS) were condensed into nanosized complexes (RNAi nanospheres, RNAi-NS) by cholesterol-modified DNA strands without the assistance of polycationic agents. RNAi-NS are entirely composed of nucleic acid, giving them lower cytotoxicity and immunogenicity, which can be caused by synthetic polycationic reagents. In addition, the RNAi nanoflowers can also integrate DNA aptamers that bind specifically to target membrane proteins for cell-targeting. Therefore, thousands of copies of siRNA will be delivered to cells specifically, and this RNAi nanoflower system will have great potential for siRNA delivery and biomedical applications.

In vitro selection of DNA aptamers against renal cell carcinoma using living cell-SELEX.

Renal cell carcinoma (RCC) is the most common form of kidney cancer with poor prognosis. Early diagnosis of RCC would significantly improve patient prognosis and quality of life. In this work, we developed new aptamer probes for RCC by using cell-SELEX (systematic evolution of ligands by exponential enrichment) only after 12 rounds of selection, in which a clear cell renal cell carcinoma (ccRCC) cell line 786-O was used as target cell, and embryonic kidney cell line 293T as negative control cell. The selected aptamers were subjected to flow cytometry and laser confocal fluorescence microscopy to evaluate their binding affinity and selectivity. The dissociation constant Kd values of four selected aptamers are all in the nanomolar range. Aptamer W786-1 with the best binding affinity and a Kd value of 9.4 ± 2.0nM was further optimized and its truncated sequence W786-1S showed considerable affinity to 786-O cells. The proteinase and temperature treatment experiment indicated that W786-1 could recognize the target 786-O cells through surface proteins, and remain good binding affinity and excellent selectivity under physiological conditions. Therefore, on the basis of its excellent targeting properties and functional versatility, W786-1 holds great potential to be used as a molecular probe for identifying and targeting RCC.

Label-free DNA-based biosensors using structure-selective light-up dyes.

Label-free biosensors (LFBs) have demonstrated a great potential in cost-effective applications, and most of the DNA-based LFBs are based on the principle of binding-induced structural transformation. This review is a collection of the latest reported studies, which have employed structure-selective nucleic acid dyes for the development of DNA-based LFBs. The collections in this review have been structured based on the selective binding of dyes towards specific DNA conformations, including single-stranded DNA, double-stranded DNA, triplex DNA, i-motifs and G-quadruplexes. The newest studies of employing versatile nucleases, fascinating nanomaterials, logic gates and cascades, DNA junctions and nanostructures have also been collected as examples. It is predicted that the imperative requirement for ultrahigh sensitivity, intelligent analysis, reliable detection, portable and fast assay would force the LFB development in a stimulative and continuous way.

Gd2O3 and GH combined with red blood cells to improve the sensitivity of contrast agents for cancer targeting MR imaging.

Herein, we fabricated efficient MR imaging probes by incorporating gadolinium oxide nanoparticles (Gd2O3) and gadolinium hybrid nanoparticles (GH) within RBCs. The Gd2O3 and GH encapsulated in the RBCs exhibited high relaxation rates and revealed high sensitivity for T1 MR imaging.

DNA Triplexes-Guided Assembly of G-Quadruplexes for Constructing Label-free Fluorescent Logic Gates.

Assembly of G-quadruplexes guided by DNA triplexes in a controlled manner is achieved for the first time. The folding of triplex sequences in acidic conditions brings two separated guanine-rich sequences together and subsequently a G-quadruplex structure is formed in the presence of K(+) . Based on this novel platform, label-free fluorescent logic gates, such as AND, INHIBIT, and NOR, are constructed with ions as input and the fluorescence of a G-quadruplex-specific fluorescent probe NMM as output.