Organ-Targeted Ionizable Lipid Nanoparticles Facilitate Sequence-Activated Fluorogenic Probe for Precise Imaging of Inflammatory Liver Disease.

Activatable near-infrared (NIR) fluorogenic probes offer a potent tool for real-time, in situ detection of hepatic biomarkers, significantly advancing the precision in diagnosing inflammatory liver disease (ILD). However, the limited distribution of small molecule fluorogenic probes in the liver and their rapid clearance impair the accuracy of fluorescence imaging and in ILD diagnosis. In this study, an effective utilization of ionizable lipid nanoparticles (iLNPs) is presented as liver-targeted carriers for efficient delivery of fluorogenic probes, aiming to overcome biodistribution barriers and achieve accurate detection of hepatic biomarkers. Based on this strategy, a liver-targeted NIR fluorogenic nanoprobe hCy-H2O2@iLNP is prepared using hCy-H2O2 as a small molecule reporter for visualizing the over-produced hydrogen peroxide (H2O2) in situ of liver. Notably, iLNPs not only significantly enhance probe accumulation in the liver, but also enable sequence activation of fluorescent nanoprobes. This response is achieved through primary liposome-dissociation release and secondary hCy-H2O2 response with pathological H2O2, enabling high-precision detection of oxidative stress in hepatocytes. These distinctive features facilitate accurate early diagnosis of acetaminophen (APAP)-induced inflammatory liver injury as well as lipopolysaccharide (LPS)-induced hepatitis. Therefore, the organ-targeted nanoprobe design strategy showcasts great potential for early and accurate diagnosis of lesions in situ in different organs.

An Efficient Strategy for Constructing Fluorescent Nanoprobes for Prolonged and Accurate Tumor Imaging.

Activatable near-infrared (NIR) fluorescent probes possess advantages of high selectivity, sensitivity, and deep imaging depth, holding great potential in the early diagnosis and prognosis assessment of tumors. However, small-molecule fluorescent probes are largely limited due to the rapid diffusion and metabolic clearance of activated fluorophores in vivo. Herein, we propose an efficient and reproducible novel strategy to construct activatable fluorescent nanoprobes through bioorthogonal reactions and the strong gold-sulfur (Au-S) interactions to achieve an enhanced permeability and retention (EPR) effect, thereby achieving prolonged and high-contrast tumor imaging in vivo. To demonstrate the merits of this strategy, we prepared an activatable nanoprobe, hCy-ALP@AuNP, for imaging alkaline phosphatase (ALP) activity in vivo, whose nanoscale properties facilitate accumulation and long-term retention in tumor lesions. Tumor-overexpressed ALP significantly increased the fluorescence signal of hCy-ALP@AuNP in the NIR region. More importantly, compared with the small-molecule probe hCy-ALP-N3, the nanoprobe hCy-ALP@AuNP significantly improved the distribution and retention time in the tumor, thus improving the imaging window and accuracy. Therefore, this nanoprobe platform has great potential in the efficient construction of biomarker-responsive fluorescent nanoprobes to realize precise tumor diagnosis in vivo.

Enhancing Liver Delivery of Gold Nanoclusters via Human Serum Albumin Encapsulation for Autoimmune Hepatitis Alleviation.

Peptide-protected gold nanoclusters (AuNCs), possessing exceptional biocompatibility and remarkable physicochemical properties, have demonstrated intrinsic pharmaceutical activity in immunomodulation, making them a highly attractive frontier in the field of nanomedicine exploration. Autoimmune hepatitis (AIH) is a serious autoimmune liver disease caused by the disruption of immune balance, for which effective treatment options are still lacking. In this study, we initially identified glutathione (GSH)-protected AuNCs as a promising nanodrug candidate for AIH alleviating in a Concanavalin A (Con A)-induced mice model. However, to enhance treatment efficiency, liver-targeted delivery needs to be improved. Therefore, human serum albumin (HSA)-encapsulated AuNCs were constructed to achieve enhanced liver targeting and more potent mitigation of Con A-induced elevations in plasma aspartate transaminase (AST), alanine transaminase (ALT), and liver injury in mice. In vivo and in vitro mechanism studies indicated that AuNCs could suppress the secretion of IFN-γ by Con A-stimulated T cells and subsequently inhibit the activation of the JAK2/STAT1 pathway and eventual hepatocyte apoptosis induced by IFN-γ. These actions ultimately protect the liver from immune cell infiltration and damage caused by Con A. These findings suggest that bio-protected AuNCs hold promise as nanodrugs for AIH therapy, with their liver targeting capabilities and therapeutic efficiency being further improved via rational surface ligand engineering.

One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications.

N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

Colon-Accumulated Gold Nanoclusters Alleviate Intestinal Inflammation and Prevent Secondary Colorectal Carcinogenesis via Nrf2-Dependent Macrophage Reprogramming.

Inflammatory bowel disease (IBD) is one of the main factors leading to colitis-associated colorectal cancer (CAC). Therefore, it is critical to develop an effective treatment for IBD to prevent secondary colorectal carcinogenesis. M2 macrophages play crucial roles in the resolution phase of intestinal inflammation. However, traditional drugs rarely target intestinal M2 macrophages, and they are not easily cleared. Gold nanoclusters are known for their in vivo safety and intrinsic biomedical activities. In this study, a glutathione-protected gold nanocluster is synthesized and evaluated, namely, GA. Interestingly, GA specifically accumulates in the colon during IBD. Furthermore, GA not only promotes M2 differentiation of IL-4-treated peritoneal macrophages but also reprograms macrophage polarization from M1 to M2 in a pro-inflammatory environment. Mechanistically, this regulatory effect is exerted through activating the antioxidant Nrf2 signaling pathway, but not traditional STAT6. When applied in IBD mice, we found that GA elevates M2 macrophages and alleviates IBD in an Nrf2-dependent manner, evidenced by the abolished therapeutic effect upon Nrf2 inhibitor treatment. Most importantly, GA administration significantly suppresses AOM/DSS-induced CAC, without causing obvious tissue damage, providing critical evidence for the potential application of gold nanoclusters as nanomedicine for the treatment of IBD and CAC.

The role of m6A methylation in prenatal maternal psychological distress and birth outcome.

BACKGROUND: Prenatal maternal psychological distress (PMPD) is a known risk factor for adverse birth outcomes. N6-methyladenosine RNA (m6A) methylation is crucial in moderating RNA biology. This study aimed to evaluate the relationships between PMPD, birth outcomes, and placental m6A methylation. METHODS: This was a prospective cohort study. PMPD exposure was assessed by questionnaires about prenatal stress, depression, and anxiety. Placental m6A methylation was measured using a colorimetric assay. The relationships between PMPD, m6A methylation, gestational age (GA), and birth weight (BW) were analyzed using structural equation models (SEMs). Maternal weight gain during pregnancy and infant sex were included as covariables. RESULTS: The study included 209 mother-infant dyads. In an adjusted SEM, PMPD was associated with BW (B = -26.034; 95 % CI: -47.123, -4.868) and GA (B = -0.603; 95 % CI: -1.102, -0.154). M6A methylation was associated with PMPD (B = 0.055; 95 % CI: 0.040,0.073) and BW (B = -305.799; 95 % CI: -520.164, -86.460) but not GA. The effect of PMPD on BW was partially mediated by m6A methylation (B = -16.817; 95 % CI: -31.348, -4.638) and GA (B = -12.280; 95 % CI: -23.612, -3.079). Maternal weight gain was associated with BW (B = 5.113; 95 % CI: 0.229,10.438). LIMITATIONS: The study sample size was small, and the specific mechanism of m6A methylation on birth outcomes needs to be further explored. CONCLUSIONS: In this study, PMPD exposure negatively affected BW and GA. Placental m6A methylation was associated with PMPD and BW and partially mediated the effect of PMPD on BW. Our findings highlight the importance of perinatal psychological evaluation and intervention.

Fabrication of modified electrode by reduced graphene oxide (rGO) and polyaniline (PANI) for enhancing azo dye decolorization in bio-electrochemical systems (BESs).

Bio-electrochemical systems (BESs) have attracted wide attention in the field of wastewater treatment owing to their fast electron transfer rate and high performance. Unfortunately, the low electro-chemical activity of carbonaceous materials commonly used in BESs remains a bottleneck for their practical applications. Especially, for refractory pollutants remediation, the efficiency is largely limited by the cathode property in term of (bio)-electrochemical reduction of highly oxidized functional groups. Herein, a reduced graphene oxide (rGO) and polyaniline (PANI) modified electrode was fabricated via two-step electro-deposition using carbon brush as raw material. Benefiting from the modified graphene sheets and PANI nanoparticles, the rGO/PANI electrode shows highly conductive network with the electro-active surface area increased by 12 times (0.013 mF cm-2) and the charge transfer resistance decreased by 92% (0.23Ω) comparing with the unmodified one. Most importantly, the rGO/PANI electrode used as abiotic cathode achieves highly efficient azo dye removal from wastewater. The highest decolorization efficiency reaches 96 ± 0.03% within 24 h and the maximum decolorization rate is as high as 20.9 ± 1.45 g h-1·m-3. The features of improved electro-chemical activity and enhanced pollutant removal efficiency provide a new insight toward development of high performance BESs via electrode modification for practical application.

Observation of the protein expression level via naked eye: Pt clusters catalyze non-color molecules into brown-colored molecules in cells.

α v ß 3 is overexpressed in various tumor cells and plays a key role in tumor genesis, invasion, and metastasis. Therefore, it is of great significance to precisely detect the α v ß 3 level in cells via a simple method. For this purpose, we have constructed a peptide-coated platinum (Pt) cluster. Due to its bright fluorescence, well-defined Pt atom numbers, and peroxidase-like catalytic activity, this cluster can be used to evaluate α v ß 3 levels in cells by fluorescence imaging, inductively coupled plasma mass spectrometry (ICP-MS), and catalytic amplification of visual dyes, respectively. In this report, the expression level of α v ß 3 in living cells is well-detected by the naked eye under an ordinary light microscope when the Pt cluster binds to αvß3 in cells and catalyzes non-color 3,3'-diaminobenzidine (DAB) into brown-colored molecules in situ. Moreover, SiHa, HeLa, and 16HBE cell lines with different α v ß 3 expression levels can be visually distinguished by the peroxidase-like Pt clusters. This research will provide a reliable method for the simple detection of α v ß 3 levels in cells.

Peptide-Protected Gold Nanoclusters Efficiently Ameliorate Acute Contact Dermatitis and Psoriasis via Repressing the TNF-α/NF-κB/IL-17A Axis in Keratinocytes.

Immune-mediated skin diseases have a high prevalence and seriously affect patients' quality of life. Gold compounds have been considered promising therapeutic agents in dermatology, but the high incidence of adverse reactions have limited their clinical application. There is a great need to develop more effective and less toxic gold-based drugs. Gold nanoclusters fabricated by using peptides (pep-AuNCs) have appeared as potential biomedical nanomaterials because of their excellent biocompatibility, ease of fabrication and unique physicochemical properties. Glutathione (GSH) is an endogenous tripeptide and has been used for lightening the skin color. Therefore, we fabricated a well-defined gold nanocluster with GSH as an example to explore the immunomodulatory effect of AuNCs on a TNF-α-treated human keratinocyte cell line (HaCaT) in vitro, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis (ICD) model and the oxazolone (OXA)-induced psoriatic model in vivo. The results indicated that topically applied AuNCs successfully attenuated the severity of ICD and psoriasis-like lesions. In vitro and in vivo, AuNCs effectively inhibited the abnormal activation of the NF-κB pathway and the consequent overexpression of proinflammatory cytokines in keratinocytes. In particular, the transactivation of IL-17A, the most important cytokine in psoriasis pathology, was effectively inhibited by AuNCs treatment. In addition, AuNCs did not show any obvious cytotoxicity in HaCaT cells at doses even up to 100 µM and did not induce any irritation in the healthy skin and major organs, which indicated their favorable biosafety. These results indicate that biocompatible pep-AuNCs might be a promising gold-based nanomedicine for the treatment of inflammatory skin diseases.

Liver-Targeted Near-Infrared Fluorescence/Photoacoustic Dual-Modal Probe for Real-Time Imaging of In Situ Hepatic Inflammation.

Early diagnosis of hepatic inflammation is the key to timely treatment and avoid the worsening of liver inflammation. Near-infrared fluorescence (NIRF) probes have high sensitivity but low spatial resolution in lesion imaging, while photoacoustic (PA) imaging has good spatial location information. Therefore, the development of a NIRF/PA dual-modal probe integrated with high sensitivity and spatial location feedback can achieve an accurate early diagnosis of hepatic inflammation. Here, we report an activatable NIRF/PA dual-modal probe (hCy-Tf-CA) for the detection of the superoxide anion (O2·-) in early hepatic inflammation. hCy-Tf-CA showed high selectivity and sensitivity for detecting O2·- fluctuation in vitro. More importantly, by introducing hepatocyte-targeting cholic acid (CA), the probe successfully achieved accurate in situ imaging of acute inflammatory liver injury (AILI) and autoimmune hepatitis (AIH) in vivo. The introduced CA not only promotes the hepatic targeting accumulation of probes but also improves the performance of low background dual-modal imaging in vivo. Therefore, hCy-Tf-CA provides an effective strategy for significantly improving in situ imaging performance and holds great potential for early, sensitive, and accurate diagnosis of hepatic inflammation.

Cell-Penetrating Peptide Conjugated Au Nanoclusters Selectively Suppress Refractory Lymphoma Cells via Targeting Both Canonical and Noncanonical NF-κB Signaling Pathways.

Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is the most aggressive form of DLBCL, with a significantly inferior prognosis due to resistance to the standard R-CHOP immunochemotherapy. Survival of ABC-DLBCL cells addicted to the constitutive activations of both canonical and noncanonical NF-κB signaling makes them attractive therapeutic targets. However, a pharmaceutical approach simultaneously targeting the canonical and noncanonical NF-κB pathway in the ABC-DLBCL cell is still lacking. Peptide-conjugated gold nanoclusters (AuNCs) have emerged unique intrinsic biomedical activities and possess a great potential in cancer theranostics. Here, we demonstrated a Au25 nanocluster conjugated by cell-penetrating peptides that can selectively repress the growth of ABC-DLBCL cells by inducing efficient apoptosis, more efficiently than glutathione (GSH)-conjugated AuNCs. The mechanism study showed that the cell-penetrating peptides enhanced the cellular internalization efficiency of AuNCs, and the selective repression in ABC-DLBCL cells is due to the inhibition of inherent constitutive canonical and noncanonical NF-κB activities by AuNCs. Several NF-κB target genes involved in chemotherapy resistance in ABC-DLBCL cells, including anti-apoptotic Bcl-2 family members and DNA damage repair proteins, were effectively down-regulated by the AuNC. The emerged novel activity of AuNCs in targeting both arms of NF-κB signaling in ABC-DLBCL cells may provide a promising candidate and a new insight into the rational design of peptide-conjugated Au nanomedicine for molecular targeting treatment of refractory lymphomas.

A dimer-monomer transition captured by the crystal structures of cyanobacterial apo flavodoxin.

In cyanobacteria and algae (but not plants), flavodoxin (Fld) replaces ferredoxin (Fd) under stress conditions to transfer electrons from photosystem I (PSI) to ferredoxin-NADP+ reductase (FNR) during photosynthesis. Fld constitutes a small electron carrier noncovalently bound to flavin mononucleotide (FMN), and also an ideal model for revealing the protein/flavin-binding mechanism because of its relative simplicity compared to other flavoproteins. Here, we report two crystal structures of apo-Fld from Synechococcus sp. PCC 7942, one dimeric structure of 2.09 Å and one monomeric structure of 1.84 Å resolution. Analytical ultracentrifugation showed that in solution, apo-Fld exists both as monomers and dimers. Our dimer structure contains two ligand-binding pockets separated by a distance of 45 Å, much longer than the previous structures of FMN-bound dimers. These results suggested a potential dimer-monomer transition mechanism of cyanobacterial apo-Fld. We further propose that the dimer represents the "standby" state to stabilize itself, while the monomer constitutes the "ready" state to bind FMN. Furthermore, we generated a new docking model of cyanobacterial Fld-FNR complex based on the recently reported cryo-EM structures, and mapped the special interactions between Fld and FNR in detail.

Clusterbody Enables Flow Sorting-Assisted Single-Cell Mass Spectrometry Analysis for Identifying Reversal Agent of Chemoresistance.

Identifying effective reversal agents overcoming multidrug resistance with causal mechanisms from an efflux pump protein is of vital importance for enhanced tumor chemotherapy in clinic. To achieve this end, we construct a metal cluster-based probe, named clusterbody, to develop flow sorting-assisted single-cell mass spectrometry analysis. This clusterbody synthesized by biomimetic mineralization possesses an antibody-like property to selectively recognize an efflux pump protein. The intrinsic red fluorescence emission of the clusterbody facilitates fluorescence-activated high-throughput cell sorting of subpopulations with different multidrug resistance levels. Furthermore, based on the accurate formula of the clusterbody, the corresponding protein abundance at the single-cell level is determined through detecting gold content via precise signal amplification by laser ablation inductively coupled plasma mass spectrometry. Therefore, the effect of reversal agent treatment overcoming multidrug resistance is evaluated in a quantitative manner. This work opens a new avenue to identify reversal agents, shedding light on developing combined or synergetic tumor therapy.

Three-Dimensional Quantitative Coherent Diffraction Imaging of Staphylococcus aureus Treated with Peptide-Mineralized Au-Cluster Probes.

Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of ∼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.

Dual peptide nanoparticle platform for enhanced antigen-specific immune tolerance for the treatment of experimental autoimmune encephalomyelitis.

Current therapeutic strategies for autoimmune diseases such as multiple sclerosis (MS) are directed towards nonspecific immunosuppression, which has severe side effects. The induction of antigen-specific tolerance has become an ideal therapy for autoimmune diseases. In this study, we have constructed a dual peptide nanoparticle platform, including the antigen peptide of the primary signal and inhibitory peptide of the co-stimulatory signal, for T-cell activation and to trigger antigen-specific immune tolerance to treat experimental autoimmune encephalomyelitis (EAE), a murine model for MS. The peptide LABL binding with ICAM-1 was encapsulated in PLGA nanoparticles and the antigenic peptide MOG35-55-KKK was then covalently bonded to the surface of the PLGA nanoparticles. In this way, peptide-loaded PLGA nanoparticles (NPsLABL+MOG) were developed. When the dual peptide nanoparticles were administered intravenously either prophylactically or therapeutically to MOG35-55-immunized mice, it completely prevented the occurrence of EAE in the prophylactic therapy trial and decreased inflammatory cell infiltration and the demyelination of the nerve myelin in the spinal cord in both prophylactic and therapeutic trials. In therapeutic experiments especially, the dual peptide nanoparticles a showed stronger inhibitory effect on EAE than the MOG peptide nanoparticles alone. Mechanistically, the dual peptide nanoparticles reduced MHC II and the co-stimulatory molecule CD86 expression of dendritic cells (DCs) on the surface and induced abortive T-cell activation, which eventually led to a decreased infiltration of Th1 and Th17 cells in the central nervous system and showed antigen-specific immune tolerance. The dual peptide nanoparticles have great potential for the treatment of autoimmune diseases by inducing immune tolerance.

The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au25 Probes.

Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack the expression of the most common three receptors seen in other subtypes of breast cancers. The medium-term diagnosis of breast cancers is essential for development and prognosis. According to reports, patients with TNBC may be converted to a positive epidermal growth factor receptor 2(HER-2) after chemotherapy, and trastuzumab treatment will have a better prognosis. Therefore, it is important to accurately quantify the expression of HER-2 in breast cancer cells. Herein, we design a red fluorescent Au25 probe synthesized with BSA-biotin as the ligand, which is accurately quantified by HER-2 primary antibody-biotin using the avidin system. The quantitative detection of the expression of HER-2 in breast cancers is helpful for the companion diagnostic of breast cancer treatment and provides follow-up treatment.

A comparison of Remdesivir versus gold cluster in COVID-19 animal model: A better therapeutic outcome of gold cluster.

While gold compound have been approved for Rheumatoid arthritis treatment as it well suppresses inflammatory cytokines of patients, no such treatment is currently available for COVID-19 treatment in vivo . We firstly disclose gold cluster yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from gold cluster (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication and intrinsically down-regulates NFκB pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 hamsters.