Hong-Min Meng

A Controlled‐Release Nanocarrier with Extracellular pH Value Driven Tumor Targeting and Translocation for Drug Delivery

This pHLIP is no flop: Functionalizing mesoporous silica nanoparticles (MSNs) with pHLIPss peptide provides a controlled-release nanoparticle drug delivery system targeting the acidic tumor microenvironment. At low pH values, pHLIPss inserts into the cell membrane and translocates carriers into cells, where the cargo is released by the cleavage of the pHLIPss disulfide bonds (see scheme).

A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing†

DNAzymes hold promise for gene-silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme-MnO2 nanosystem for gene-silencing therapy. MnO2 nanosheets adsorb chlorin e6-labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit (1)O2 generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO2 is reduced to Mn(2+) ions, which serve as cofactors of 10-23 DNAzyme for gene silencing. The release of Ce6 generates (1)O2 for more efficient photodynamic therapy. The Mn(2+) ions also enhance magnetic resonance contrast, providing GSH-activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.

DNA Dendrimer: An Efficient Nanocarrier of Functional Nucleic Acids for Intracellular Molecular Sensing

Functional nucleic acid (FNA)-based sensing systems have been developed for efficient detection of a wide range of biorelated analytes by employing DNAzymes or aptamers as recognition units. However, their intracellular delivery has always been a concern, mainly in delivery efficiency, kinetics, and the amount of delivered FNAs. Here we report a DNA dendrimer scaffold as an efficient nanocarrier to deliver FNAs and to conduct in situ monitoring of biological molecules in living cells. A histidine-dependent DNAzyme and an anti-ATP aptamer were chosen separately as the model FNAs to make the FNA dendrimer. The FNA-embedded DNA dendrimers maintained the catalytic activity of the DNAzyme or the aptamer recognition function toward ATP in the cellular environment, with no change in sensitivity or specificity. Moreover, these DNA dendrimeric nanocarriers show excellent biocompatibility, high intracellular delivery efficiency, and sufficient stability in a cellular environment. This FNA dendrimeric nanocarrier may find a broad spectrum of applications in biomedical diagnosis and therapy.

Unimolecular Catalytic DNA Biosensor for Amplified Detection of l-Histidine via an Enzymatic Recycling Cleavage Strategy

Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.

Gold nanorod-photosensitizer conjugate with extracellular pH-driven tumor targeting ability for photothermal/photodynamic therapy

AbstractChlorin e6-pHLIPss-AuNRs, a gold nanorod-photosensitizer conjugate containing a pH (low) insertion peptide (pHLIP) with a disulfide bond which imparts extracellular pH (pHe)-driven tumor targeting ability, has been successfully developed for bimodal photodynamic and photothermal therapy. In this bimodal therapy, chlorin e6 (Ce6), a second-generation photosensitizer (PS), is used for photodynamic therapy (PDT). Gold nanorods (AuNRs) are used as a hyperthermia agent for photothermal therapy (PTT) and also as a nanocarrier and quencher of Ce6. pHLIPss is designed as a pHe-driven targeting probe to enhance accumulation of Ce6 and AuNRs in cancer cells at low pH. In Ce6-pHLIPss-AuNRs, Ce6 is close to and quenched by AuNRs, causing little PDT effect. When exposed to normal physiological pH 7.4, Ce6-pHLIPss-AuNRs loosely associate with the cell membrane. However, once exposed to acidic pH 6.2, pHLIP actively inserts into the cell membrane, and the conjugates are translocated into cells. When this occurs, Ce6 separates from the AuNRs as a result of disulfide bond cleavage caused by intracellular glutathione (GSH), and singlet oxygen is produced for PDT upon light irradiation. In addition, as individual PTT agent, AuNRs can enhance the accumulation of PSs in the tumor by the enhanced permeation and retention (EPR) effect. Therefore, as indicated by our data, when exposed to acidic pH, Ce6-pHLIPss-AuNRs can achieve synergistic PTT/PDT bimodality for cancer treatment.

A label-free electrochemical biosensor for highly sensitive and selective detection of DNA via a dual-amplified strategy.

In this work, by combining the enzymatic recycling reaction with the DNA functionalized gold nanoparticles (AuNPs)-based signal amplification, we have developed an electrochemical biosensor for label-free detection of DNA with high sensitivity and selectivity. In the new designed biosensor, a hairpin-structured probe HP was designed to hybridize with target DNA first, and an exonuclease ExoIII was chosen for the homogeneous enzymatic cleaving amplification. The hybridization of target DNA with the probe HP induced the partial cleavage of the probe HP by ExoIII to release the enzymatic products. The enzymatic products could then hybridize with the hairpin-structured capture probe CP modified on the electrode surface. Finally, DNA functionalized AuNPs was further employed to amplify the detection signal. Due to the capture of abundant methylene blue (MB) molecules by both the multiple DNAs modified on AuNPs surface and the hybridization product of capture DNA and enzymatic products, the designed biosensor achieved a high sensitivity for target DNA, and a detection limit of 0.6 pM was obtained. Due to the employment of two hairpin-structured probes, HP and CP, the proposed biosensor also exhibited high selectivity to target DNA. Moreover, since ExoIII does not require specific recognition sequences, the proposed biosensor might provide a universal design strategy to construct DNA biosensor which can be applied in various biological and medical samples.

Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection and Imaging of Ascorbic Acid in Living Cells and Tissues

Ascorbic acid (AA) serves as a key coenzyme in many metabolic pathways, and its abnormal level is found to be associated with several diseases. Therefore, monitoring AA level in living systems is of great biomedical significance. In comparison with one-photon excited fluorescent probes, two-photon (TP) excited probes are more suitable for bioimaging, as they could afford higher imaging resolution with deeper imaging depth. Here, we report for the first time an efficient TP fluorescence probe for turn-on detection and imaging of AA in living cells and tissues. In this nanosystem, the negatively charged two-photon nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with a two-photon dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge by electrostatic force, leading to a remarkable decrease in their fluorescence intensity. However, the introduction of AA could induce the fluorescence recovery of the nanoprobe because it could reduce CoOOH into Co(2+) and result in the destruction of the CoOOH nanoflakes. The nanosystem exhibits a high sensitivity toward AA, with a LOD of 170 nM observed. It also shows high selectivity toward AA over common potential interfering species. The nanoprobe possessed both the advantages of TP imaging and excellent membrane-permeability and good biocompatibility of the silica nanoparticles and was successfully applied in TP-excited imaging of AA in living cells and tissues.

Multiple functional nanoprobe for contrast-enhanced bimodal cellular imaging and targeted therapy.

Many one-photon fluorescence-based theranostic nanosystems have been developed for simultaneous therapeutic intervention/monitoring for various types of cancers. However, for early diagnosis of cancer, two-photon fluorescence microscopy (TPFM) can realize deep-tissue imaging with higher spatial resolution. In this study, we first report a multiple functional nanoprobe for contrast-enhanced bimodal cellular imaging and targeted therapy. Components of the nanoprobe include (1) two-photon dye-doped mesoporous silica nanoparticles (TPD-MSNs); (2) MnO2 nanosheets that act as a (i) gatekeeper for TPD-MSNs, (ii) quencher for TP fluorescence, and (iii) contrast agent for MRI; (3) cancer cell-targeting aptamers. Guided by aptamers, TPD-MSNs are rapidly internalized into the target cells. Next, intracellular glutathione reduces MnO2 to Mn(2+) ions, resulting in contrast-enhanced TP fluorescence and magnetic resonance signal for cellular imaging. Meanwhile, preloaded doxorubicin and Chlorin e6 are released for chemotherapy and photodynamic therapy, respectively, with a synergistic effect and significantly enhanced therapeutic efficacy.

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor

Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer-target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer-target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5 μM to 1000 μM, with a detection limit of 500 nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.

Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase Detection and Imaging in Drug-Induced Liver Injury Sample

Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe P-GST for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2,4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, P-GST was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI.