Bacterial Drug Delivery Systems for Cancer Therapy: "Why" and "How".

Cancer is one of the major diseases that endanger human health. However, the use of anticancer drugs is accompanied by a series of side effects. Suitable drug delivery systems can reduce the toxic side effects of drugs and enhance the bioavailability of drugs, among which targeted drug delivery systems are the main development direction of anticancer drug delivery systems. Bacteria is a novel drug delivery system that has shown great potential in cancer therapy because of its tumor-targeting, oncolytic, and immunomodulatory properties. In this review, we systematically describe the reasons why bacteria are suitable carriers of anticancer drugs and the mechanisms by which these advantages arise. Secondly, we outline strategies on how to load drugs onto bacterial carriers. These drug-loading strategies include surface modification and internal modification of bacteria. We focus on the drug-loading strategy because appropriate strategies play a key role in ensuring the stability of the delivery system and improving drug efficacy. Lastly, we also describe the current state of bacterial clinical trials and discuss current challenges. This review summarizes the advantages and various drug-loading strategies of bacteria for cancer therapy and will contribute to the development of bacterial drug delivery systems.

Self-Priming DNA Polymerization-Propelled Stochastic Walkers on Magnetic Microbeads for Amplified Detection of miRNA.

Sensitive detection of miRNA targets in complex biological samples possesses great value in biopsy analysis and disease diagnosis but is still challenging because of low abundance and nonspecific interferences. In this work, self-primer DNA polymerization-propelled stochastic walkers (SWs) were proposed to detect miRNA-24 by combining magnetic microbeads (MMBs) and flow cytometry. The MMBs not only provide a three-dimensional interface for DNA walkers but also facilitate the enrichment and isolation of RNA targets from complex biological samples such as serum. The SWs can be initiated to walk through the entire surface of MMBs and transduce RNA walking into amplified fluorescence signals, with the detection limit of miRNA-24 at 0.95 pM. Moreover, this strategy integrating with flow cytometry was demonstrated to have good specificity with other homologous miRNAs. This platform offers promising applications in RNA biosensing and biomedical diagnostics.

Dual amplification of a bio-barcode and auto-cycling primer extension for highly sensitive detection of miRNA.

MicroRNAs (miRNAs) can be used as biomarkers for the diagnosis and therapy of cancers. However, their low abundance and the complex environment in biological samples hinder miRNA detection. A dual amplification strategy based on the bio-barcode technique (BCA) and auto-cycling primer extension (APE) is proposed to detect miRNA targets in complex biological samples. The strategy shows a good sensitivity for miRNA-19a with a detection limit of 50 fM, and can effectively distinguish other similar miRNAs. It provides a new idea to combine nanoparticle-based amplification with nucleic acid-based amplification together for the sensitive detection of nucleic acid targets.

Amplified FRET Nanoflares: An Endogenous mRNA-Powered Nanomachine for Intracellular MicroRNA Imaging.

It is of great value to detect biological molecules in live cells. However, probes for imaging low-abundance targets in live cells are limited by the one-to-one signal-triggered model. Here, we introduce the concept of the amplified FRET nanoflare, which employs high-abundance endogenous mRNA as fuel strands to amplify the detection of low abundance intracellular miRNA. As far as we know, this is the first report of an endogenous mRNA-powered nanomachine for intracellular molecular detection. We experimentally prove the mechanism of the nanomachine and demonstrate its specificity and sensitivity. The proposed amplified FRET nanoflare can act as an excellent intracellular molecular detection strategy that is promising for biological and medical applications.

Dual-microRNA-controlled double-amplified cascaded logic DNA circuits for accurate discrimination of cell subtypes.

Accurate discrimination between different cells at the molecular level is particularly important for disease diagnosis. Endogenous RNAs are such molecular candidates for cancer cell subtype identification. But the key is that there is often low abundance of RNAs in live cells, or some RNAs are often shared by multiple types of cells. Thus, we have designed dual-microRNA-controlled double-amplified cascaded logic DNA circuits for cancer cell subtype identification. The basic idea is to improve sensitivity by cascading DNAzyme and hybridization chain reaction (HCR), and improve accuracy by simultaneous detection of miR-122 and miR-21. The in-tube and in-cell experimental results show that the cascaded logic DNA circuits can work and serve to differentiate the liver cancer cells Huh7 from other normal cells and cancer cells. We anticipate that this design can be widely applied in facilitating basic biomedical research and accurate disease diagnosis.

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and "DD-A" FRET.

Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and "DD-A" FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.

Gold nanoparticle-based 2'-O-methyl modified DNA probes for breast cancerous theranostics.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulated diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways. An increasing number of data suggested that miRNA-21 could be identified as diagnostic and therapeutic biomarker for breast cancer. Meanwhile, inhibiting the function of miRNA-21, resulting in cells growth inhibition and apoptotic cells death. To realize miRNA-21detection and inhibition to diagnostic and therapeutic breast cancer cells, we developed gold nanoparticle-based 2'-O-methyl modified DNA probes (AuNP-2'-OMe-DNA probes) for diagnostic and therapeutic breast cancer. Gold nanoparticles were functionalized with chemically modified miRNA-21 inhibitor to suppress the function of miRNA-21 for the therapeutic breast cancer, at the same time, fluorophore-labeled DNA molecules were hybridized with antimiRNA-21 for diagnostic breast cancer. The results showed that the 2'-O-methyl modified DNA can improve stability, increase binding affinity to target strands and enhance the therapeutic effects. The experimental results also demonstrated that antimiR-21 were efficiently introduced into the cells and knocked down miRNA-21 to inhibit its function, leading to growth inhibition and apoptotic cells death. We prospected that chemically modified miRNA-21 inhibitor based on gold nanoparticles would be as a promising diagnostic and therapeutic platform for breast cancer clinically.

Two-Color-Based Nanoflares for Multiplexed MicroRNAs Imaging in Live Cells.

MicroRNAs (miRNAs) have become an ideal biomarker candidate for early diagnosis of diseases. But various diseases involve changes in the expression of different miRNAs. Therefore, multiplexed assay of miRNAs in live cells can provide critical information for our better understanding of their roles in cells and further validating of their function in clinical diagnoses. Simultaneous detection of multiple biomarkers could effectively improve the accuracy of early cancer diagnosis. Here, we develop the two-color-based nanoflares for simultaneously detecting two distinct miRNA targets inside live cells. The nanoflares consist of gold nanoparticles (AuNPs) functionalized with a dense shell of recognition sequences hybridized to two short fluorophore-labeled DNA molecules, termed "flares". In this conformation, the close proximity of the fluorophore to the AuNPs surface leads to quenching of the fluorescence. However, when target miRNAs bind to the recognition sequence, the concomitant displacement of the flare can be detected as a corresponding increase in fluorescence. The results demonstrate that the two-color-based nanoflares can simultaneously detect miR-21 and miR-141 expression levels in various live cancer cells successfully. Compared to the traditional single-color-based nanoflares, the two-color-based nanoflares could offer more reliable and practical information for cancer detection, improving the accuracy of early disease diagnosis.

Live-Cell MicroRNA Imaging through MnO2 Nanosheet-Mediated DD-A Hybridization Chain Reaction.

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live-cell miRNA imaging using a biodegradable MnO2 nanosheet-mediated DD-A FRET hybridization chain reaction (HCR). The MnO2 nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO2 nanosheets are degraded by cellular GSH. Then, the target miR-21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (donor) and one TAMRA (acceptor) into close proximity to generate significantly enhanced DD-A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies.

Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing.

Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature's examples of "protective and reversible responses" exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the "On" to "Off" signal is reversibly responding to "below" and "over" the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

DNA tetrahedron nanostructures for biological applications: biosensors and drug delivery.

With the rapid development of DNA nanotechnology, various DNA nanostructures with different shapes and sizes have been self-assembled using "bottom-up" fabrication strategies and applied to a wide range of fields such as biosensors, drug delivery and tools for molecular biology. As a classical and simple polyhedron, DNA tetrahedron can be easily synthesised by a one-step assembly. Due to the excellent biocompatibility and cellular permeability, it provides a universal and promising platform to construct a series of biosensors and drug delivery systems for living cells studies. Moreover, the high programmability of DNA tetrahedron determines its capability to perform artful design and combine with other materials. Herein, we review and summarise the development and applications of DNA tetrahedron in living cell studies. We mainly focus on two parts, cellular biosensors for the detection of nucleic acids, proteins, small molecules and cancer cells and drug delivery systems for chemotherapy, immunotherapy, photodynamic therapy and gene silencing. With the rapid progress in DNA tetrahedron as well as DNA nanotechnology, new avenues and opportunities have opened up in analytical chemistry, molecular biology and medicine.

Gold Nanoparticle Loaded Split-DNAzyme Probe for Amplified miRNA Detection in Living Cells.

A new class of intracellular nanoprobe, termed AuNP loaded split-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and substrates hybridized with two half of split DNAzymes. In the absence of target miRNA, the split DNAzymes form an inactive DNAzyme motif with their substrate through partial paring at the end of each strand, and the fluorescence is quenched. Inside the cells, the target miRNA binds with both of the two half of split DNAzymes, forming the active secondary structure in the catalytic cores, which can cleave the substrates, resulting in the rupture of the substrate and recovery of the fluorescence. Meanwhile, the target is released and binds to another inactive DNAzyme motif to drive another cycle of activation. During the cyclic process, a very small number of target miRNAs can initiate the cleavage of many fluorophore-labeled substrate strands from AuNP surface, providing an amplified fluorescent signal of the target miRNA and, thus, offering high detection sensitivity.

Gold Nanoparticle Based Hairpin-Locked-DNAzyme Probe for Amplified miRNA Imaging in Living Cells.

A new class of intracellular nanoprobe, termed AuNP-based hairpin-locked-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and hairpin-locked-DNAzyme strands. In the absence of target miRNA, the hairpin-locked-DNAzyme strand forms a hairpin structure by intramolecular hybridization, which could inhibit the catalytic activity of DNAzyme strand and the fluorescence is quenched by the AuNP. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active secondary structure in the catalytic cores to yield an "active" DNAzyme, which then cleaves the self-strand with the assist of Mg2+. The cleaved two shorter DNA fragments are separated with the target. As a result, the fluorophores are released from the AuNP and the fluorescence is enhanced. Meanwhile, the target is also released and binds to another hairpin-locked-DNAzyme strand to drive another cycle of activation. In such a way, the target-recycling amplification leads to significant signal enhancement and thus offers high detection sensitivity.

MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn2+ ions by intracellular GSH. The results indicated that the MnO2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

Aptamer-based FRET nanoflares for imaging potassium ions in living cells.

Due to the effective properties of the FRET signal and K+-sensitive recognition of G-quadruplex, aptamer-based FRET nanoflares were developed to sense intracellular potassium ions.

Aptazyme-Gold Nanoparticle Sensor for Amplified Molecular Probing in Living Cells.

To date, a few of DNAzyme-based sensors have been successfully developed in living cells; however, the intracellular aptazyme sensor has remained underdeveloped. Here, the first aptazyme sensor for amplified molecular probing in living cells is developed. A gold nanoparticle (AuNP) is modified with substrate strands hybridized to aptazyme strands. Only the target molecule can activate the aptazyme and then cleave and release the fluorophore-labeled substrate strands from the AuNP, resulting in fluorescence enhancement. The process is repeated so that each copy of target can cleave multiplex fluorophore-labeled substrate strands, amplifying the fluorescence signal. Results show that the detection limit is about 200 nM, which is 2 or 3 orders of magnitude lower than that of the reported aptamer-based adenosine triphosphate (ATP) sensors used in living cells. Furthermore, it is demonstrated that the aptazyme sensor can readily enter living cells and realize intracellular target detection.

Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits.

Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cells.

Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration.

A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells.

We report a supersandwich fluorescence in situ hybridization (SFISH) strategy for highly sensitive and selective in situ visualization of mRNA expression patterns at the single-cell level. This strategy uses two fluorophore-labeled signal probes to generate a supersandwich product, which in turn generates numerous signal probes located at the target mRNA position, resulting in the in situ fluorescence signal amplification.

Fluorescence resonance energy transfer-based hybridization chain reaction for in situ visualization of tumor-related mRNA.

The ability to visualize tumor-related mRNA in situ in single cells would distinguish whether they are cancer cells or normal cells, which holds great promise for cancer diagnosis at an early stage. Fluorescence resonance energy transfer (FRET) and hybridization chain reactions (HCRs) were combined with amplified sense tumor-related mRNA (TK1 mRNA) in situ with high sensitivity in single cells and tissue sections. Using this strategy, each copy of the target mRNA can propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked duplex that contains repeated FRET units, amplifying the fluorescent signal. The detection limit of 18 pM is about three orders of magnitude lower than that of a non-HCR method (such as the binary-probe-system). Meanwhile, due to the FRET strategy, complicated washing steps are not necessary and experimental time is sharply reduced. As far as we know, this is the first report of a fluorescence in situ hybridization (FISH) strategy that can simultaneously fulfil signal amplification and is wash-free. We believe that this FRET-based HCR strategy has great potential as a powerful tool in basic research and clinical diagnosis.