Highly focused beam generated with a height tuned micro-optical structure for high contrast microscopic imaging.

Light sheet illumination technology improves the signal-to-noise ratio, resolution, and reduces scattered backgrounds for biological microscopic detection system. Here, we developed a novel micro-optical structure to produce a focused and uniform beam for the enhancement of imaging contrast. The beam intensity and working distance can be modified by adjusting the height and period of the structure. Our experiments successfully recorded structured light illumination, demonstrating the ability of the structure to capture high-contrast imaging data. We compared the light fields generated with and without the structure to assess the imaging quality, revealing a maximum 4.78-fold improvement in the signal-to-noise ratio. This work provides a potential method for high-resolution and high-contrast light sheet fluorescence microscopic detection.

Cascaded Nanozyme Based pH-Responsive Oxygenation for Targeted Eradication of Resistant Helicobacter Pylori.

Nanozymes, as substitutes for natural enzymes, are constructed as cascade catalysis systems for biomedical applications due to their inherent catalytic properties, high stability, tunable physicochemical properties, and environmental responsiveness. Herein, a multifunctional nanozyme is reported to initiate cascade enzymatic reactions specific in acidic environments for resistant Helicobacter pylori (H. pylori) targeting eradication. The cobalt-coated Prussian blue analog based FPB-Co-Ch NPs displays oxidase-, superoxide dismutase-, peroxidase-, and catalase- mimicking activities that trigger • O 2 - ${\mathrm{O}}_2^ - {\bm{\ }}$ and H2O2 to supply O2, thereby killing H. pylori in the stomach. To this end, chitosan is modified on the surface to exert bacterial targeted adhesion and improve the biocompatibility of the composite. In the intestinal environment, the cascade enzymatic activities are significantly inhibited, ensuring the biosafety of the treatment. In vitro, sensitive and resistant strains of H. pylori are cultured and the antibacterial activity is evaluated. In vivo, murine infection models are developed and its success is confirmed by gastric mucosal reculturing, Gram staining, H&E staining, and Giemsa staining. Additionally, the antibacterial capacity, anti-inflammation, repair effects, and biosafety of FPB-Co-Ch NPs are comprehensively investigated. This strategy renders a drug-free approach that specifically targets and kills H. pylori, restoring the damaged gastric mucosa while relieving inflammation.

Template-Free Self-Assembly of Hollow Microtubular Covalent Organic Frameworks for Oral Delivery of Insulin.

Covalent organic frameworks (COFs) have demonstrated versatile application potential since their discovery. Although the structure of COFs is orderly arranged, the synthesis of controllable macrostructures still faces challenges. Herein, we report, to our knowledge, the first template-free self-assembled COF-18 Å hollow microtubule (MT-COF-18 Å) structure and its use for insulin delivery that exhibits high loading capacity, gastroresistance, and glucose-responsive properties. The hollow MT-COF-18 Å was achieved by a template-free method benefiting from the mixed solvents of mesitylene and dioxane. The formation mechanism and morphology changes with insulin loading and release were observed. In Caco-2 cells, the transferrin-coated system demonstrated enhanced insulin cellular uptake and transcellular transport, which indicated great potential for oral applications. Additionally, the composites presented sustained glycemic control and effective insulin blood concentrations without noticeable toxicity in diabetic rats. This work shows that hollow microtubular COFs hold great promise in loading and delivery of biomolecules.

Synergetic Pyroptosis with Apoptosis Improving Phototherapy of Mitochondria-Targeted Cyanines with Superior Photostability.

Pyroptosis has been reported to improve the antitumor effect by evoking a more intense immune response and a therapeutic effect. For phototherapy, several photosensitizers have been found to initiate pyroptosis. However, the effect of pyroptosis associated with apoptosis in enhancing the antitumor therapy needs sufficient characterization, especially under long-term treatment. As a NIR photosensitizer, heptamethine cyanines have been discovered for anticancer phototherapy for deep tissue penetration and inherent tumor-targeted capability. However, they are not quite stable for long-term performance. To investigate the effect of pyroptosis along with apoptosis on the anticancer immune responses and phototherapy, here, we chemically modulate the cyanine IR780 to regulate hydrophobicity, stability, and intracellular targeting. Two photosensitizers, T780T-TPP and T780T-TPP-C12, were finally optimized and showed excellent photostability with high photothermal conversion efficiency. Although the cellular uptake of the two molecules was both mediated by OATP transporters, T780T-TPP induced tumor cell death via pyroptosis and apoptosis and accumulated in tumor accumulation, while T780T-TPP-C12 was prone to accumulate in the liver. Ultimately, via one injection-multiple irradiation treatment protocol, T780T-TPP displayed a significant antitumor effect, even against the growth of large tumors (200 mm3).

Mucus-Penetrating Nanoassembly as Potential Oral Phototherapeutic Formulation against Multi-Drug Resistant Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) infection presents increasing challenges to antibiotic therapies in limited penetration through gastric mucus, multi-drug resistance (MDR), biofilm formation, and intestinal microflora dysbiosis. To address these problems, herein, a mucus-penetrating phototherapeutic nanomedicine (RLs@T780TG) against MDR H. pylori infection is engineered. The RLs@T780TG is assembled with a near-infrared photosensitizer T780T-Gu and an anionic component rhamnolipids (RLs) for deep mucus penetration and light-induced anti-H. pylori performances. With optimized suitable size, hydrophilicity and weak negative surface, the RLs@T780TG can effectively penetrate through the gastric mucus layer and target the inflammatory site. Subsequently, under irradiation, the structure of RLs@T780TG is disrupted and facilitates the T780T-Gu releasing to target the H. pylori surface and ablate multi-drug resistant (MDR) H. pylori. In vivo, RLs@T780TG phototherapy exhibits impressive eradication against H. pylori. The gastric lesions are significantly alleviated and intestinal bacteria balance is less affected than antibiotic treatment. Summarily, this work provides a potential nanomedicine design to facilitate in vivo phototherapy in treatment of H. pylori infection.

Mitochondria-Mediated HSP Inhibition Strategy for Enhanced Low-Temperature Photothermal Therapy.

Low-temperature photothermal therapy (PTT) has the advantage of causing less damage to normal tissues and has attracted great attention in recent years. However, the efficacy of low-temperature PTT is restricted by the overexpression of heat shock proteins (HSPs), specifically HSP70 and HSP90. Inhibiting the function of these HSPs is a major strategy used in the development of new cancer therapies. Herein, we designed four T780T-containing thermosensitive nanoparticles to interrupt the energy supply for HSP expression using their TPP-based mitochondrial targeting action. The reversal behavior of the nanoparticles on the gambogic acid (GA)-induced compensatory increase of HSP70 was investigated in vitro by Western blot and in vivo by immunohistochemistry. The in vivo anticancer efficacy of the low-temperature PTT based on these thermosensitive nanoparticles was also systematically examined. The design proposes for the first time to utilize and elucidate the mechanism of the mitochondrial targeting of T780T-containing NPs in synergy with the HSP90 inhibition of GA to achieve an effective low-temperature PTT. This work not only provides a novel pathway for the dual inhibition of HSP70 and HSP90 but also opens up a new approach for low-temperature PTT of tumors.

Reversible Recognition-Based Boronic Acid Probes for Glucose Detection in Live Cells and Zebrafish.

Glucose, a critical source of energy, directly determines the homeostasis of the human body. However, due to the lack of robust imaging probes, the mechanism underlying the changes of glucose homeostasis in the human body remains unclear. Herein, diboronic acid probes with good biocompatibility and high sensitivity were synthesized based on an ortho-aminomethylphenylboronic acid probe, phenyl(di)boronic acid (PDBA). Significantly, by introducing the water-solubilizing group -CN directly opposite the boronic acid group and -COOCH3 or -COOH groups to the ß site of the anthracene in PDBA, we obtained the water-soluble probe Mc-CDBA with sensitive response (F/F0 = 47.8, detection limit (LOD) = 1.37 µM) and Ca-CDBA with the highest affinity for glucose (Ka = 4.5 × 103 M-1). On this basis, Mc-CDBA was used to identify glucose heterogeneity between normal and tumor cells. Finally, Mc-CDBA and Ca-CDBA were used for imaging glucose in zebrafish. Our research provides a new strategy for designing efficient boronic acid glucose probes and powerful new tools for the evaluation of glucose-related diseases.

An MMP-2 sensitive and reduction-responsive prodrug amphiphile for actively targeted therapy of cancer by hierarchical cleavage.

A hierarchically cleaved amphiphile, mPEG-pep-etcSS-CPT, was synthesized to pursue actively targeted cancer therapy through self-assembly. This micelle can respond to MMP-2 achieving dePEGylation and releasing RGD peptides to be internalized into targetable tumor cells. Inside the cell, free CPT could be released by reduction-response leading to cytotoxicity.

A visual diagnostic detection of Helicobacter pylori and the gastric carcinoma-related virulence genes (cagA and vacA) by a fluorescent loop-mediated isothermal amplification (LAMP).

Helicobacter pylori (H. pylori) infection has increasingly been a serious problem worldwide. The H. pylori infection can result in a series of stomach diseases including gastric carcinoma. There are two specific virulence genes (cagA and vacA) of H. pylori that are closely related to the occurrence of gastric cancer, and the common molecular detection methods (PCR, qPCR) are not suitable for high-screening test due to the requirement of expensive instruments and well-trained personals. Herein, we develop a rapid visual assay based on loop-mediated isothermal amplification (LAMP) for detecting H. pylori and its major virulence genes (cagA, vacAs1 and vacAm1) to guide clinical treatment for H. pylori infection. In this research, a fluorescent LAMP assay was established by optimizing the indicator of MnCl2-Calcein, so that the resulted color and fluorescence changes could be utilized to perform the visual detection for H. pylori and its virulence genes with high sensitivity (10-3 ng/µL). The proposed LAMP assay is simple, fast (30 min) and capable in providing more sensitive results than traditional methods in the test of 46 clinical biopsy samples. By detecting the three virulence genes together, we can profile the infection risk of the patients, and discuss the correlation among the genes. Moreover, the method could be used to diagnose virulently infected individuals and benefit the eradication of H. pylori in early warning for gastric cancer.

Phototherapy and Mechanism Exploration of Biofilm and Multidrug-Resistant Helicobacter pylori by Bacteria-Targeted NIR Photosensitizer.

Helicobacter pylori (H. pylori) infection has been the leading cause of gastric cancer development. In recent years, the resistance of H. pylori against antibiotic treatment has been a great challenge for most countries worldwide. Since biofilm formation is one of the reasons for the antibiotic resistance of H. pylori, and phototherapy has emerged as a promisingly alternative antibacterial treatment, herein the bacteria-targeted near-infrared (NIR) photosensitizer (T780T-Gu) by combining positively-charged guanidinium (Gu) with an efficient phototherapeutic agent T780T is developed. The proposed molecule T780T-Gu exhibits synergistic photothermal therapy/photodynamic therapy effect against both H. pylori biofilms and multidrug-resistant (MDR) clinical strains. More importantly, the phototherapy mechanism of T780T-Gu acquired by the RNA-seq analysis indicates that structural deficiency as well as a decrease in metabolism and defense activity are the possible reasons for the efficient H. pylori phototherapy.

Synergistic Release of Photothermal Molecules from Nanocarriers Induced by Light and Hyperthermia Benefits Efficient Anticancer Phototherapy.

Recently, nanoformulations have been widely applied in the delivery of organic photothermal agents (OPTAs) for cancer therapy to prolong blood circulation or improve tumor-targeting capacity. However, the systematic evaluations of their effects on the photothermal behavior of OPTAs are limited, especially for different types of nanoparticle systems. Herein, we prepared two kinds of nanoparticles (BSA and PEG nanoparticles (NPs)) to load an OPTA, a cyanine photosensitizer (IR780-O-TPE), and investigated their photothermal response, organelle targeting, and in vivo therapeutic efficacy. Due to different assembly forms, the two NPs showed distinct morphological changes after exposure to laser or hyperthermia. Under laser irradiation at 808 nm, BSA NPs could release IR780-O-TPE more efficiently than PEG NPs. We speculate that this phenomenon is probably caused by dual-responsive release of IR780-O-TPE from BSA NPs against light and hyperthermia. Moreover, IR780-O-TPE/BSA NPs were highly mitochondria-targeting and therefore displayed significant inhibition of cell viability. In contrast, IR780-O-TPE/PEG NPs were "shell-core" nanostructures and more stable under laser stimulation. As a consequence, the mitochondria-targeting and anticancer photothermal therapy by IR780-O-TPE/PEG NPs was less obvious. This study revealed the significance of nanocarrier design for OPTA delivery and demonstrated that BSA NPs could release IR780-O-TPE more effectively for efficient photothermal therapy. We also believe that the dual-responsive release of OPTAs from NPs can provide an effective strategy to promote anticancer photothermal treatment.

Size-Controllable DNA Origami-Stacked Gold Nanoparticles for Deep Tumor-Penetrating Therapy.

With the rapid development of nanotechnology, researchers have designed a variety of intelligent nanodelivery systems to enhance tumor targeting of anticancer drugs. However, increased tumor accumulation does not indicate deeper penetration in the tumor tissue, without which the tumor cells in the core area cannot be sufficiently killed. Herein, we develop a size-controllable nanoparticle system for deep-penetrating cancer therapy, which will be programmably disassembled with the decrease of the pH from the normal tissue to the tumor microenvironment and to the intracellular area. The integrated nanoparticle is composed of a gold nanoparticle (GNP, ∼30 nm) and a tetrahedral DNA nanostructure (TDN, ∼25 nm) loaded with doxorubicin (DOX). Initially, the nanoparticles maintain a larger size (∼100 nm) to accumulate in the tumor through the enhanced permeability and retention effect. At a pH of about 6.5 at the tumor microenvironment, with the linkage of DNA sequences converting into a triplex structure, the TDNs detach from the GNP and penetrate deeply into the tumor interstitium and then are internalized into the cells. Finally, in acidic lysosomes with pH 5.0, the TDNs release DOX by forming an i-motif structure. This nanosmart delivery system thus shows effective deep penetration into the tumor core with good antitumor efficacy and satisfactory biocompatibility and provides new insights into the development of intelligent nanosystems for anti-cancer treatment.

A Tumor-Targeting Near-Infrared Heptamethine Cyanine Photosensitizer with Twisted Molecular Structure for Enhanced Imaging-Guided Cancer Phototherapy.

In recent years, cancer phototherapy has been extensively studied as noninvasive cancer treatment. To present efficient recognition toward cancer cells, most photosensitizers (PSs) are required to couple with tumor-targeted ligands. Interestingly, the heptamethine cyanine IR780 displays an intrinsic tumor-targeted feature even without modification. However, the photothermal efficacy and photostability of IR780 are not sufficient enough for clinical use. Herein, we involve a twisted structure of tetraphenylethene (TPE) between two molecules of IR780 to improve the photothermal conversion efficiency (PCE). The obtained molecule T780T shows strong near-infrared (NIR) fluorescence and improved PCE (38.5%) in the dispersed state. Also, the photothermal stability and ROS generation capability of T780T at the NIR range (808 nm) are both promoted. In the aqueous phase, the T780T was formulated into uniform nanoaggregates (∼200 nm) with extremely low fluorescence and PTT response, which would reduce in vivo imaging background and side effect of PTT response in normal tissues. After intravenous injection into tumor-bearing mice, the T780T nanoaggregates display high tumor accumulation and thus remarkably inhibit the tumor growth. Moreover, the enhanced photostability of the T780T allows for twice irradiation after one injection and leads to more significant tumor inhibition. In summary, our study presents a tumor-targeted small-molecule PS for efficient cancer therapy and brings a new design of heptamethine cyanine PS for potential clinical applications.

A novel electrochemical aptasensor for exosomes determination and release based on specific host-guest interactions between cucurbit [7]uril and ferrocene.

The superior supramolecular recognition ability of macrocyclic compounds will enhance the sensitivity and selectivity of electrochemical detection, which has a great application potential in electrochemical sensing. Herein, we developed a novel electrochemical aptasensor based on the specific host-guest interactions between cucurbit [7]uril and ferrocene (Fc) for capture, determination and release of exosomes. Macrocyclic compounds, cucurbit [7]uril is modified on the surface of the gold nanoparticles composed electrode by self-assembling. CD63 aptamer linked ferrocene is introduced into this platform to capture exosomes specifically by CD63 protein on the exosomes. The dual specificity of macrocyclic compounds and aptamers enables highly selective and sensitive electrochemical detection of exosomes. The limit of detection (LOD) was 482 particles µL-1. In addition, the captured exosomes could be released on demand in a very mild manner through aminoferrocene (NH2-Fc) because of its higher affinity to cucurbit [7]uril. The proposed electrochemical aptasensor showed good performance in detecting exosomes even in plasma samples, thus demonstrating its great potential in early clinical diagnosis. Simultaneously, exosomes could be released undamaged by this protocol, exhibiting good applicability in comprehensive studies of exosomes. Moreover, this strategy can be applied to other target biomolecules by changing the recognition pairs.

DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations.

Extensive attention has been recently focused on designing signal adjustable biosensors. However, there are limited approaches available in this field. In this work, to visually track lysosomes with high contrast, we used the i-motif structure as a pH-responsive unit and proposed a novel strategy to regulate the fluorescence resonance energy transfer (FRET) response of the pH sensor. By simply splitting the i-motif into two parts and modulating the split parameters, we can tune the pH transition midpoint (pHt) from 5.71 to 6.81 and the signal-to-noise ratio (S/N) from 1.94 to 18.11. To facilitate the lysosome tracking, we combined the i-motif split design with tetrahedral DNA (Td). The obtained pH nanosensor (pH-Td) displays appropriate pHt (6.12) to trace lysosomes with high S/N (10.3). Benefited from the improved stability, the superior cell uptake and lysosomal location of pH-Td, the visualization of the distribution of lysosomes, the lysosome-mitochondria interaction, and the pH changes of lysosomes in response to different stimuli were successfully achieved in NIH 3T3 cells. We believe that the design concept of controlling the split sequence distance will provide a novel insight into the design of i-motif-based nanosensors and even inspire the construction of smart DNA nanodevices for sensing, disease diagnosis, and controllable drug delivery.

Fluorogenic Biosensors Constructed via Aggregation-induced Emission Based on Enzyme-catalyzed Coupling Reactions for Detection of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) is a main reactive oxygen by-product produced in the metabolism of organisms and a common biomarker of oxidative stress. Aggregation-induced emission (AIE) probes for H2O2 have been proposed. Such AIEgens mostly use benzeneboronic acid as a recognition group. Recently, a strategy involving enzyme-catalyzed polymerization of AIE compounds shows great potential in AIEgens design. We herein modify the AIE motif, tetraphenylethene (TPE) with o-phenylenediamine (TPE-TAF), which can be oxidated by H2O2 in HRP to form an intramolecular phenazine structure. Compared with a similar approach, the proposed strategy is simple and the TPE-TAF showed a sensitive "turn-on" fluorescence with H2O2. The detection limit (LOD) is 3.39 µM and the probe is highly specific against H2O2. We further verified the reaction mechanism of the enzyme-catalyzed coupling reaction. The probe is a promising candidate as a stable and safe fluorescent substrate in H2O2 sensing.

Synthesis of Diboronic Acid-Based Fluorescent Probes for the Sensitive Detection of Glucose in Aqueous Media and Biological Matrices.

Reliable and accurate glucose detection in biological samples is of great importance in clinical diagnosis and medical research. Chemical probes are advantageous in simple operation and flexible design, especially for the development of fluorescent probes. Anthracene-based diboronic acid (P-DBA) has shown potential in glucose probing because of its high sensitivity. However, poor solubility limits its applications in aqueous media. In this work, we systemically modify P-DBA by introducing fluoro (F-), chloro (Cl-), methoxyl (MeO-), or cyano (CN-) substituents. Among these probes, the cyano-substituted probe (CN-DBA) displays the highest glucose-binding constant (6489.5 M-1, 33% MeOH). More importantly, it shows good water solubility in the aqueous solution (0.5% MeOH), with ultrasensitive recognition with glucose (LOD = 1.51 µM) and robust sensing from pH 6.0 to 9.0. Based on these features, the CN-DBA is finally applied to detect glucose in cell lysates and plasma, with satisfactory recovery and precision. These results demonstrate that CN-DBA could serve as an accurate, sensitive fluorescent probe for glucose assays in biological samples.

Improved cancer phototheranostic efficacy of hydrophobic IR780 via parenteral route by association with tetrahedral nanostructured DNA.

As a photosensitizer with effective photothermal (PTT) and photodynamic (PDT) response, IR780 has been widely explored as promising cancer phototheranostic molecule. However, the systematic administration of IR780 usually suffers from poor water solubility and low photostability, so that it cannot be administrated by parenteral route. In this study, we design a tetrahedral DNA (Td)-based nanosystem to load IR780 (IR780@Td) via electrostatic interaction and π-π stacking. After encapsulation, the water solubility and photostability of IR780 have been greatly improved, and the IR780@Td shows an appropriate nanoformulated size (224 nm) to facilitate hyperthermia-mediated tumor targeting by EPR effect. The nanostructure of Td is proved to be crucial for the proper size and good stability of IR780@Td nanoformulation for in vivo application. The in vitro and ex vivo PTT/PDT efficiencies of IR780 are improved in IR780@Td group. In the tumor-bearing mice, the accumulation of IR780 in tumor site is significantly high in IR780@Td group. Under near-infrared laser irradiation, the intravenous administration of IR780@Td promotes the tumor imaging and enhances anti-tumor effect than IR780 treatment. In summary, the proposed strategy shows promising effect in facilitating intravenous injection of IR780 and enhancing the phototheranostic efficacy for cancer treatment.

AIEgens Conjugation Improves the Photothermal Efficacy and Near-Infrared Imaging of Heptamethine Cyanine IR-780.

Near-infrared (NIR) fluorescent probes can deeply penetrate through tissues with little damage. To facilitate image-guided theranostics, researchers usually apply a desired amount of photosensitizers to achieve effective photothermal responses. However, these probes could easily suffer from low photostability and aggregated-caused quenching effect in high concentrations. In this paper, the rational incorporation of an aggregated-induced emission (AIE) unit into the structure of heptamethine cyanine IR-780 is reported. Using tetraphenylethene (TPE) as an AIE core, we synthesize three TPE-modified IR-780 probes (IR-780 AIEgens) via different linkages. The IR-780 derivatives all show enhanced AIE features, in which the probe with an ether linkage (IR780-O-TPE) is superior in rapid cell uptake, high targeting capacity, and good photostability. Moreover, IR780-O-TPE exhibits the strongest cytotoxicity to HeLa cells (IC50 = 3.3 µM). The three IR-780 derivatives displayed a photothermal response in a concentration-dependent manner, in which IR-780 AIEgens are more cytotoxic than IR-780, with IC50 of 0.3 µM under 808 nm laser irradiation. In tumor-bearing mice, the optimal probe IR780-O-TPE also showed a more effective photothermal response than IR-780. By illustrating the relationship between aggregation state with photophysical properties, cell imaging, and cytotoxicity, this work is helpful in modulating NIR-based photosensitizers into AIE features for efficient image-guided theranostics.

Photostable pH-Sensitive Near-Infrared Aggregation-Induced Emission Luminogen for Long-Term Mitochondrial Tracking.

Mitochondria are crucial in the process of oxidative metabolism and apoptosis. Their morphology is greatly associated with the development of certain diseases. For specific and long-term imaging of mitochondrial morphology, we synthesized a new mitochondria-targeted near-infrared (NIR) fluorescent probe (TPE-Xan-In) by incorporating TPE with a NIR merocyanine skeleton (Xan-In). TPE-Xan-In displayed both absorption (660 nm) and emission peaks (743 nm) in the NIR region. Moreover, it showed aggregation-induced emission properties at neutral pH and specifically illuminated mitochondria with good biocompatibility, superior photostability, and high tolerance to mitochondrial membrane potential changes. With a pH-responsive unit, hydroxyl xanthene (Xan), the probe exhibited a pH-sensitive fluorescence emission in the range of pH 4.0-7.0, which indicated its potential in long-term tracking of pH and morphology changes of mitochondria in the biomedical research studies.