Microneedle System with Biomarker-Activatable Chromophore as Both Optical Imaging Probe and Anti-bacterial Agent for Combination Therapy of Bacterial-Infected Wounds and Outcome Monitoring.

Wounds infected with bacteria, if left untreated, have the potential to escalate into life-threatening conditions, such as sepsis, which is characterized by widespread inflammation and organ damage. A comprehensive approach to treating bacterial-infected wounds, encompassing the control of bacterial infection, biofilm eradication, and inflammation regulation, holds significant importance. Herein, a microneedle (MN) patch (FM@ST MN) has been developed, with silk fibroin (SF) and tannic acid-based hydrogel serving as the matrix. Encapsulated within the MNs are the AIEgen-based activatable probe (FQ-H2O2) and the NLRP3 inhibitor MCC950, serving as the optical reporter/antibacterial agent and the inflammation regulator, respectively. When applied onto bacterial-infected wounds, the MNs in FM@ST MN penetrate bacterial biofilms and gradually degrade, releasing FQ-H2O2 and MCC950. The released FQ-H2O2 responds to endogenously overexpressed reactive oxygen species (H2O2) at the wound site, generating a chromophore FQ-OH which emits noticeable NIR-II fluorescence and optoacoustic signals, enabling real-time imaging for outcome monitoring; and this chromophore also exhibits potent antibacterial capability due to its dual positive charges and shows negligible antibacterial resistance. However, the NLRP3 inhibitor MCC950, upon release, suppresses the activation of NLRP3 inflammasomes, thereby mitigating the inflammation triggered by bacterial infections and facilitating wound healing. Furthermore, SF in FM@ST MN aids in tissue repair and regeneration by promoting the proliferation of epidermal cells and fibroblasts and collagen synthesis. This MN system, free from antibiotics, holds promise as a solution for treating and monitoring bacterially infected wounds without the associated risk of antimicrobial resistance.

Injectable-Hydrogel-Based Tissue Sealant for Hemostasis, Bacteria Inhibition, and Pro-Angiogenesis in Organ Bleeding Wounds and Therapeutic Outcome Monitoring Via NIR-II Optical Imaging.

Sudden hemorrhage stemming from internal organ wounds poses a grave and potentially fatal risk if left untreated. Injectable-hydrogel-based tissue sealants featuring multiple actions, including fit-to-shape in situ gelation, rapid hemostasis, pro-angiogenic, anti-bacterial and outcome tracking, are ideal for the management of organ trauma wounds. Herein, an injectable-hydrogel tissue sealant AN@CD-PEG&TQ which consists of four-arm 4-arm poly(ethylene glycol) (PEG-SC) succinimidyl carbonate), AN@CD nanoprobe, and two bioactive peptides (anti-microbial peptide Tet213 and pro-angiogenic peptide QK) is developed. Among them, AN@CD nanoparticles form through host/guest complexation of amino-group-containing ß-cyclodextrin and adamantyl group, enabling in situ biomarker (NO)-activatable optoacoustic/NIR-II: Near-infrared second biological window fluorescent imaging. The ample ─NH2 groups on the surface of AN@CD readily engage in rapid cross-linking with succinimidyl ester groups located at the ends of four-arm PEG-SC. This cross-linking expedites the gelation process without necessitating additional initiators or cross-linking agents; thus, significantly enhancing both hydrogel's application convenience and biocompatibility. Bioactive peptides (Tet213 and QK) safeguard against possible bacterial infections, facilitate angiogenesis, and eventually, improve organ wounds healing. This hydrogel-based tissue sealant demonstrates superior therapeutic and bioimaging performance in various mouse models including liver hemorrhage, gastric perforation, and bacterial-infected skin wound mouse models, highlighting its potential as a high-performance wound sealant for organ bleeding wound management.

Cucurbit[8]uril-based water-dispersible assemblies with enhanced optoacoustic performance for multispectral optoacoustic imaging.

Organic small-molecule contrast agents have attracted considerable attention in the field of multispectral optoacoustic imaging, but their weak optoacoustic performance resulted from relatively low extinction coefficient and poor water solubility restrains their widespread applications. Herein, we address these limitations by constructing supramolecular assemblies based on cucurbit[8]uril (CB[8]). Two dixanthene-based chromophores (DXP and DXBTZ) are synthesized as the model guest compounds, and then included in CB[8] to prepare host-guest complexes. The obtained DXP-CB[8] and DXBTZ-CB[8] display red-shifted and increased absorption as well as decreased fluorescence, thereby leading to a substantial enhancement in optoacoustic performance. Biological application potential of DXBTZ-CB[8] is investigated after co-assembly with chondroitin sulfate A (CSA). Benefiting from the excellent optoacoustic property of DXBTZ-CB[8] and the CD44-targeting feature of CSA, the formulated DXBTZ-CB[8]/CSA can effectively detect and diagnose subcutaneous tumors, orthotopic bladder tumors, lymphatic metastasis of tumors and ischemia/reperfusion-induced acute kidney injury in mouse models with multispectral optoacoustic imaging.

Renal-Clearable Probe with Water Solubility and Photostability for Biomarker-Activatable Detection of Acute Kidney Injuries via NIR-II Fluorescence and Optoacoustic Imaging.

Acute kidney injuries (AKI) have serious short-term or long-term complications with high morbidity and mortality rate, thus posing great health threats. Developing high-performance NIR-II probes for noninvasive in situ detection of AKI via NIR-II fluorescent and optoacoustic dual-mode imaging is of great significance. Yet NIR-II chromophores often feature long conjugation and hydrophobicity, which prevent them from being renal clearable, thus limiting their applications in the detection and imaging of kidney diseases. To fully exploit the advantageous features of heptamethine cyanine dye, while overcoming its relatively poor photostability, and to strive to design a NIR-II probe for the detection and imaging of AKI with dual-mode imaging, herein, we have developed the probe PEG3-HC-PB, which is renal clearable, water soluble, and biomarker activatable and has good photostability. As for the probe, its fluorescence (900-1200 nm) is quenched due to the existence of the electron-pulling phenylboronic group (responsive element), and it exhibits weak absorption with a peak at 830 nm. Meanwhile, in the presence of the overexpressed H2O2 in the renal region in the case of AKI, the phenylboronic group is converted to the phenylhydroxy group, which enhances NIR-II fluorescent emission (900-1200 nm) and absorption (600-900 nm) and eventually produces conspicuous optoacoustic signals and NIR-II fluorescent emission for imaging. This probe enables detection of contrast-agent-induced and ischemia/reperfusion-induced AKI in mice using real-time 3D-MSOT and NIR-II fluorescent dual-mode imaging via response to the biomarker H2O2. Hence, this probe can be used as a practicable tool for detecting AKI; additionally, its design strategy could provide insight into the design of other large-conjugation NIR-II probes with multifarious biological applications.

A biomarker-responsive nanoprobe for detecting hepatic ischemia-reperfusion injury via optoacoustic/NIR-II fluorescence imaging.

A nanoprobe for detecting hepatic ischemia-reperfusion injury has been developed. Apparent optoacoustic and NIR-II fluorescent signals are given out upon the nanoprobe's response to the in situ biomarker H2O2 in the liver in the case of ischemia-reperfusion injury.

An NO-responsive probe for detecting acute inflammation using NIR-II fluorescence/optoacoustic imaging.

An NO-responsive probe for imaging acute inflammation was developed. The probe responds to in situ NO in acute inflammation sites such as LPS-induced acute dermatitis and MIA-induced acute joint inflammation with turn-on NIR-II fluorescence and optoacoustic signals.

A Biomarker-Responsive Nanosystem with Colon-Targeted Delivery for Ulcerative Colitis's Detection and Treatment with Optoacoustic/NIR-II Fluorescence Imaging.

Ulcerative colitis (UC) is a prevalent idiopathic inflammatory disease which causes such complications as intestinal perforation, obstruction, and bleeding, and thus deleteriously impacting people's normal work and quality of life. Hence, accurate diagnosis of UC is crucial in terms of planning optimal treatment plan. Herein, a pH/reactive oxygen species (ROS) dual-responsive nanosystem (BM@EP) is developed for UC's detection and therapy. BM@EP is composed of a chromophore-drug dyad and the enteric coating. The chromophore-drug dyad (BOD-XT-DHM) is synthesized by linking the chromophore (BOD-XT-BOH) and a flavonoid drug (dihydromyricetin DHM) through boronate ester bond. The enteric coating includes Eudragit S100 and poly(lactic-co-glycolic acid) (PLGA), the former is commonly employed as a pH-dependent polymer coating excipient so as to attain colon-targeted delivery, and the latter has been widely used as an excipient for the controlled-extended release. After oral administration, BM@EP delivers the dyad (BOD-XT-DHM) into the colon and releases the dyad molecules by being triggered by the alkaline pH in t colon, thereafter upon being stimulated by overexpressed H2 O2 in the inflamed colon, the boronate bond in the dyad is broken down and correspondingly the drug DHM is released for UC therapy, simultaneously the chromophore is released for near-infrared second window (NIR-II) fluorescence and optoacoustic imaging for UC diagnosis and recovery evaluation.

An activatable probe for detection and therapy of food-additive-related hepatic injury via NIR-II fluorescence/optoacoustic imaging and biomarker-triggered drug release.

Food additives are essential to guarantee processed foods' safety throughout its journey from workshops or factories to shops or catering establishment and eventually to consumers. As one of the commonly-used food additives, nitrites upon reaction with amines would generate highly toxic nitrosamines (e.g., N,N-diethylnitrosamine, DEN) as inadvertent byproducts resulted from food processing or preparation which are known to cause hepatotoxicity and even cancer. Hence detecting nitrosamine-induced acute liver injury accurately would be conducive to planning optimal treatment and avoid any further deterioration. Herein we design an activatable probe (BHC-Lut) that can release the drug luteolin for therapy and the chromophore (BHC-OH) for NIR-II fluorescence/optoacoustic imaging upon being triggered by hepatic biomarker hydrogen peroxide. In the probe BHC-Lut, benzoindolium heptamethine cyanine with NIR-II fluorescent emission is adopted as the chromophore scaffold, the incorporation of triethylene glycol into benzoindolium ensures sufficient water solubility and enhances biocompatibility of the probe, and luteolin is coupled onto the chromophore via boronate linkage that acts as both H2O2-responsive unit and the fluorescence quencher. The probe itself is weakly emissive. In the presence of H2O2, the boronate bond is cleaved, and the chromophore BHC-OH and the drug luteolin are released, which produces evident NIR-II fluorescent/optoacoustic signals for imaging and wields therapeutic effect respectively. The probe BHC-Lut has been used in DEN-induced hepatic injury model in mice, and the results evince BHC-Lut's capability for in-situ biomarker-activatable detection and imaging of the acute liver injury site as well as in-situ biomarker-triggered drug release for therapy.

An AIEgen-based oral-administration nanosystem for detection and therapy of ulcerative colitis via 3D-MSOT/NIR-II fluorescent imaging and inhibiting NLRP3 inflammasome.

Ulcerative colitis is the most prevalent forms of inflammatory bowel diseases and a refractory autoimmune disease and affects millions of people worldwide. Herein, we develop an oral-administration nanosystem (QM@EP) for colitis detection, targeted drug delivery/release to colon and therapy. QM@EP consists of a molecular probe QY-SN-H2O2, a NLRP3 inhibitor MCC950 and enteric polymers. QY-SN-H2O2 is based on the AIE-active chromophore QY-SN-OH with pentafluorobenzenesulfonate moieties as the recognition moiety for the biomarker H2O2 and the fluorescence quencher. H2O2 can cleave the pentafluorobenzenesulfonate moieties in QY-SN-H2O2 and thus generating the AIE-active chromophore Q-SN-OH. Two biocompatible polymers were employed in the nanosystem, in which poly(lactic-co-glycolic acid) (PLGA) serves as the sustained release excipient and the Eudragit® S100 acts as the excipient for controlled release of drug formulations in colonic pH to prevent premature drug release in stomach. Our experiments demonstrate that, upon oral administration the nanosystem effectively delivers the probe and drug into colon and release them therein upon being triggered by colonic pH. Then the released probe is activated and turned into the AIE-active chromophore upon being triggered by the pathological level of colonic ROS, thereby bringing about strong fluorescence and optoacoustic signals for NIR-II fluorescence and 3D multispectral optoacoustic tomography (MSOT) imaging for diagnosis and therapeutic outcome monitoring; and the released drug exerts high therapeutic efficacy against ulcerative colitis through inhibiting NLRP3 inflammasome formation.

Rational design of stable heptamethine cyanines and development of a biomarker-activatable probe for detecting acute lung/kidney injuries via NIR-II fluorescence imaging.

Developing high-quality dyes to construct activatable probes for analyte sensing via NIR-II fluorescence is critical for attaining enhanced imaging depths and resolution. Heptamethine cyanines can serve this purpose; however, they usually have poor stability and a tendency to self-aggregate. Herein, we present a design strategy involving the installation of pyridinium and tert-butyl groups onto the central cyclohexenyl core to increase steric crowding, enhance water solubility, and provide a site for the incorporation of analyte-responsive elements. The resulting NP-N dyes emit NIR-II light and can outperform benchmark heptamethine cyanines such as ICG. Using HP-N1, we developed HP-H2O2 and showed that NIR-II fluorescence signals could be enhanced when treating with H2O2. HP-H2O2 was subsequently evaluated in murine models of acute lung injury and acute kidney injury. This strategy unlocks the potential of heptamethine cyanines and is applicable to examples with extended conjugation.

Targeted and activatable nanosystem for fluorescent and optoacoustic imaging of immune-mediated inflammatory diseases and therapy via inhibiting NF-κB/NLRP3 pathways.

Immune-mediated inflammatory diseases (IMIDs) represent a diverse group of diseases and challenges remain for the current medications. Herein, we present an activatable and targeted nanosystem for detecting and imaging IMIDs foci and treating them through blocking NF-κB/NLRP3 pathways. A ROS-activatable prodrug BH-EGCG is synthesized by coupling a near-infrared chromophore with the NF-κB/NLRP3 inhibitor epigallocatechin-3-gallate (EGCG) through boronate bond which serves as both the fluorescence quencher and ROS-responsive moiety. BH-EGCG molecules readily form stable nanoparticles in aqueous medium, which are then coated with macrophage membrane to ensure the actively-targeting capability toward inflammation sites. Additionally, an antioxidant precursor N-acetylcysteine is co-encapsulated into the coated nanoparticles to afford the nanosystem BH-EGCG&NAC@MM to further improve the anti-inflammatory efficacy. Benefiting from the inflammation-homing effect of the macrophage membrane, the nanosystem delivers payloads (diagnostic probe and therapeutic drugs) to inflammatory lesions more efficiently and releases a chromophore and two drugs upon being triggered by the overexpressed in-situ ROS, thus exhibiting better theranostic performance in the autoimmune hepatitis and hind paw edema mouse models, including more salient imaging signals and better therapeutic efficacy via inhibiting NF-κB pathway and suppressing NLRP3 inflammasome activation. This work may provide perceptions for designing other actively-targeting theranostic nanosystems for various inflammatory diseases.

A Targeted Nanosystem for Detection of Inflammatory Diseases via Fluorescent/Optoacoustic Imaging and Therapy via Modulating Nrf2/NF-κB Pathways.

Inflammatory diseases are sometimes devastating and notoriously difficult to treat. Precisely modulating inflammatory signaling pathways is a promising approach for treating inflammatory diseases. Herein, a multifunctional nanosystem is developed for active targeting, activatable imaging and on-demand therapy against inflammatory diseases through modulating inflammatory pathways. A chromophore-drug dyad (QBS-FIS) is synthesized by linking a chromophore and a Nrf2 (nuclear factor E2-related factor) activator fisetin through boronate bond which serves as fluorescence quencher and ROS (reactive oxygen species)-responsive linker. QBS-FIS molecules form nanoparticles in water and are coated with macrophage cell membrane to ensure active targeting toward inflammation site. To further improve therapeutic efficacy, a NF-kB (nuclear-factor kappa-light-chain-enhancer of activated B cells) inhibitor thalidomide is co-encapsulated to afford the nanosystem (QBS-FIS&Thd@MM). Upon administration into mice, the nanosystem migrates to inflammatory site and pathological ROS therein cleaves the boronate bonds, thereby activating the chromophore for imaging liver/kidney inflammatory diseases for disease diagnosis and recovery evaluation via fluorescence and optoacoustic imaging as well as releasing the active drugs for treating acute liver inflammation through activating Nrf2 pathway and inhibiting NF-kB pathway. The 3D multispectral optoacoustic tomography imaging is applied to precisely locate the inflammatory foci in a spatiotemporal manner.

ALP-activated probe for diagnosis of liver injury by multispectral optoacoustic tomography.

In this chapter, we highlight the advantages of multispectral optoacoustic tomography (MSOT) technique and the activatable photoacoustic probes in the biomedical field, and give a brief introduction to enzyme-activated probes for disease diagnosis and therapeutic outcome evaluation. We also present a detailed description of the procedures for the synthesis of an activatable small molecule probe C1X-OR1 and confirmation of its specific response to alkaline phosphatase in solution and cells. With MSOT, the liposomal C1X-OR1 can be utilized for detection of hepatic ALP as well as for in vivo diagnosis of drug-induced liver injury in a three-dimensional manner.

An Activatable Probe with Aggregation-Induced Emission for Detecting and Imaging Herbal Medicine Induced Liver Injury with Optoacoustic Imaging and NIR-II Fluorescence Imaging.

Whilte herbal medicines are widely used for health promotion and therapy for chronic conditions, inappropriate use of them may cause adverse effects like liver injury, and accurately evaluating their hepatotoxicity is of great significance for public health. Herein, an activatable probe QY-N for diagnosing herbal-medicine-induced liver injury by detecting hepatic NO with NIR-II fluorescence and multispectral optoacoustic tomography (MSOT) imaging is demonstrated. The probe includes a bismethoxyphenyl-amine-containing dihydroxanthene serving as electron donor, a quinolinium as electron acceptor, and a butylamine as recognition group and fluorescence quencher. The hepatic level of NO reacts with butylamine, thereby generating the activated probe QY-NO which exhibits a red-shifted absorption band (700-850 nm) for optoacoustic imaging and generates strong emission (910-1110 nm) for NIR-II fluorescence imaging. QY-NO is aggregation-induced-emission (AIE) active, which ensures strong emission in aggregated state. QY-N is utilized in the triptolide-induced liver injury mouse model, and experimental results demonstrate the QY-N can be activated by hepatic NO and thus be used in detecting herbal-medicine-induced liver injury. The temporal and spatial information provided by three-dimensional MSOT images well delineates the site and size of liver injury. Moreover, QY-N has also been employed to monitor rehabilitation of liver injury during treatment process.

An activatable probe for detecting alcoholic liver injury via multispectral optoacoustic tomography and fluorescence imaging.

A probe has been developed for imaging alcoholic liver injury through detecting the overexpressed cytochrome P450 reductase in hypoxia in the hepatic region. Upon response to the enzyme, the activated probe displays turn-on fluorescence and near-infrared absorption and generates prominent optoacoustic signals.

Activatable fluorescent probe based on aggregation-induced emission for detecting hypoxia-related pathological conditions.

Hypoxia, as a condition in which a region of body has low oxygen tension, is closely related to a variety of pathological conditions, and in many diseases local hypoxia occurs that would further increase the severity of diseases. Hence the extent of hypoxia could reflect the related pathological conditions and diseases, and the detection of hypoxia is of great significance. In hypoxia, the elevated level of nitroreductase (NTR) usually occurs, which could serve as a biomarker for hypoxia and thus the related diseases. Herein, an activatable fluorescent probe TPAQS-NO2 based on aggregation-induced emission (AIE) was designed for hypoxia detection via responding to NTR. The probe consists of an electron acceptor quinolinium and an electron donor triphenylamine group. The activated probe shows a large Stokes shift (186 nm). The probe TPAQS-NO2 was successfully used for detecting the early-stage and the advanced-stage tumors via NTR detection in 4T1 tumor-bearing mouse model. Furthermore, the probe TPAQS-NO2 was applied for detecting NTR in the cerebral ischemia (CIS) mouse model. The probe could offer an effective approach for detecting hypoxia-related pathological conditions.

Activatable Nanocomposite Probe for Preoperative Location and Intraoperative Navigation for Orthotopic Hepatic Tumor Resection via MSOT and Aggregation-Induced Near-IR-I/II Fluorescence Imaging.

The precise location of tumor and completeness of surgical resection are critical to successful tumor surgery; thus, the method capable of preoperatively locating a tumor site and intraoperatively determining tumor margins would be highly ideal. Herein, an activatable nanocomposite probe was developed for preoperatively locating orthotopic hepatic tumor via multispectral optoacoustic tomography imaging and for intraoperative navigation via near-IR-1 (NIR-I) and NIR-II fluorescence imaging. The molecular probe comprises an electronic donor, an acceptor, and a recognition moiety and forms the nanocomposite probe with bovine serum albumin. The probe specifically responds to nitroreductase overexpressed in tumor cells, which transforms the aromatic nitro group into an electron-donating amino group and thus activates the probe. The activated probe with the aggregation-induced emission feature generates strong NIR-I/NIR-II fluorescence and optoacoustic signals for dual-mode imaging. Owing to the in situ response toward nitroreductase in tumor cells in the hepatic region, the probe is found capable of detecting early stage orthotopic liver tumors. Furthermore, with the nanocomposite probe, we can obtain the 3D MSOT images to accurately locate orthotopic liver tumors preoperatively and the NIR-I/NIR-II fluorescence images to provide intraoperative guidance for tumor resection surgery.

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation-Induced NIR-I/II Fluorescence Imaging.

An activatable nanoprobe for imaging breast cancer metastases through near infrared-I (NIR-I)/NIR-II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D-π-A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation-induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.

An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging.

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.

A fluorescent probe based on aggregation-induced emission for hydrogen sulfide-specific assaying in food and biological systems.

A fluorescent probe based on a triphenylamine benzopyridine platform for hydrogen sulfide (H2S) assaying has been designed and synthesized. As a result of the H2S-triggered cleavage reaction, the disappearance of the quenching effect of dinitrophenyl and the increased hydrophobicity in a poor solvent lead to the aggregation-induced emission (AIE) effect; consequently an obvious 'turn-on' fluorescence signal can be observed in this process. The probe TPANF features high selectivity towards H2S, low detection limit (0.17 µM), and good photostability and biocompatibility. Moreover, it has been successfully utilized to monitor H2S in food samples to distinguish the extent of food deterioration and to identify the H2S concentration variation in living cells. In addition, endogenous H2S in HCT-116 xenograft tumor tissues was imaged by using this probe. The approach could provide useful insight for the development of other activatable AIE-based probes that are potentially helpful for specific assaying in food chemistry and biological systems.